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Modulation of immune response in Systemic Lupus Erythematosus by synthetic lipoxin analogue, NAP1051

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Modulation of immune response in Systemic Lupus Erythematosus by synthetic lipoxin analogue, NAP1051

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Content Copyright 2024 Priyal Dave
MODULATION OF IMMUNE RESPONSE IN SYSTEMIC LUPUS ERYTHEMATOSUS
BY SYNTHETIC LIPOXIN ANALOGUE, NAP1051.
by
Priyal Dave
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfilment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CLINICAL AND EXPERIMENTAL THERAPEUTICS)
December 2024

ii
Dedication
I dedicate this thesis to my family, friends and mentors who have always supported me
throughout my graduate school journey. To my parents, Samir and Meghna Dave for
encouraging me and always being there for me. To my partner, Josh, for his unwavering
support throughout this journey. Lastly, to my siblings, Shail, Shreya and Diya for their
unconditional love and encouragement.

iii
Acknowledgments
I would like to express my heartfelt gratitude to my PhD. advisor, Dr. Stan Louie, for
his unwavering support throughout my graduate studies. I feel incredibly fortunate to have had
an advisor who consistently encouraged me and helped me stay focused on my goals. This
journey would not have been possible without your invaluable guidance and mentorship. I am
deeply thankful for the knowledge you shared, the life lessons you imparted, and the fresh
perspectives you brought to every challenge I faced. Your motivation, wisdom, and willingness
to explore the unknown have been a source of inspiration. Thank you for guiding me through
obstacles, inspiring me to become a better researcher, and providing opportunities to develop
new skills. Your mentorship has left a lasting impact on my personal and professional growth.
I would also like to thank my other committee members, Dr. Isaac Asante and Dr.
William Stohl, for their close mentorship throughout this journey. I am very grateful to Dr.
Asante for mentoring me through the in-vivo studies and providing guidance on my
experimental designs, approaches to data analysis, and troubleshooting. I cannot thank you
enough for always looking out for me and giving me valuable life advice.
To Dr. Stohl, I am extremely grateful for the collaborations between our groups and the
incredible opportunities and results I’ve been afforded because of your mentorship. Your
commitment to collaborative lupus disease research has been truly inspiring and I thank you
for motivating me. I would also like to express my gratitude for generously providing the
animals for our experiments. I would also like to thank members of Dr. Stohl’s group including
Ning Yu and Ying Xin Wu for their support in our collaborations together.
I would like to thank my lab members and who have helped me throughout my PhD. in
every regard – it would not have been possible without your support. Thank you to Tiange
Dong, Eugene Zhao, Kabir Ahluwalia, Rita Li, Andrew Mead, Angela Lu, Brandon Ebright,
Catherine Chester, Dante Dikeman, Xiaohui Yu, Aditya Naik, Dr. You-Jung Ha, Wei Hu and

iv
Hua Pei for your friendship and support. Finally, I am very grateful to Ziyi Wang who
supported me throughout this journey and for broadening my exposure to flow cytometry and
newer techniques.

v
TABLE OF CONTENTS
Dedication ................................................................................................................................ ii
Acknowledgments.................................................................................................................. iii
List of Tables......................................................................................................................... vii
List of Figures....................................................................................................................... viii
Abstract....................................................................................................................................xi
CHAPTER 1: INTRODUCTION TO SYSTEMIC LUPUS ERYTHEMATOSUS,
IMMUNE CELL RELATED INFLAMMATION AND LIPOXIN A4..............................1
1.1 BACKGROUND OF SYSTEM LUPUS ERYTHEMATOSUS (SLE)..........................................................................1
1.2 EPIDEMIOLOGY AND RISK FACTORS ASSOCIATED SLE .................................................................................2
1.3 DIAGNOSIS, STAGING AND PROGNOSIS OF SLE .............................................................................................4
1.4 PATHOGENESIS OF SLE..................................................................................................................................7
1.5 SLE AND INFLAMMATION ..............................................................................................................................9
1.5.1 Apoptosis and damage associated molecular patterns (DAMPs).........................................................9
1.6 IMMUNE CELLS IN SLE.................................................................................................................................11
1.6.1 Effector cells of Innate Immune System...............................................................................................11
1.6.2 Effector cells of Adaptive Immunity ....................................................................................................17
1.7 CYTOKINES, TOLL-LIKE RECEPTORS, IMMUNOGLOBULOINS AND COMPLEMENT FACTORS .........................19
1.7.1 Cytokines .............................................................................................................................................19
1.7.2 Toll like receptors (TLRs): ..................................................................................................................19
1.7.3 Immunoglobulins and Complement Factors:......................................................................................21
1.8 POLYUNSATURATED FATTY ACIDS AND THEIR ROLE IN REGULATING IMMUNE ACTIVITY ............................21
1.9 BIOSYNTHESIS OF LIPOXIN A4 .....................................................................................................................25
1.10 METABOLISM OF LXA4 AND ATLA ..........................................................................................................29
1.11 HYPOTHESIS AND SPECIFIC AIMS...............................................................................................................31
1.12 OUTLINE OF THE DISSERTATION ................................................................................................................32
CHAPTER 1: BIBLIOGRAPHY.........................................................................................34
CHAPTER 2: IDENTIFICATION AND ALTERATIONS IN MACROPHAGE
PROFILES AND ITS CONTRIBUTION TO SYSTEMIC LUPUS
ERYTHEMATOSUS IN NZM2328 MICE. ........................................................................44
2.1 INTRODUCTION.............................................................................................................................................44
2.1.1 Understanding macrophage phenotype and their functions ...............................................................44
2.1.2 Role of Macrophages in SLE...............................................................................................................46
2.1.3 Impact of Lipoxin A4 on Macrophage Subsets....................................................................................47
2.2 METHODS.....................................................................................................................................................50
2.2.1 Mouse BMDM Collection....................................................................................................................50
2.2.2 BMDM differentiation and polarization..............................................................................................50
2.2.3 Flow cytometry ....................................................................................................................................50
2.2.4 Gating strategy ....................................................................................................................................51
2.2.5 RNA extraction and cDNA synthesis and Gene Expression of BMDM...............................................51
2.2.6 Efferocytosis Assay..............................................................................................................................52
2.2.7 Efferocytosis Imaging..........................................................................................................................53
2.2.8 Lipidomics sample extraction, processing and analysis .....................................................................54
2.2.9 Statistical Analysis...............................................................................................................................55
2.3 RESULTS.......................................................................................................................................................55
2.3.1 Characterization of Polarized Macrophages comparing NZM 2328 with C57BL/J:.........................55
2.3.2 SPMs Mediated Transition from M1 to M0 Phenotype.......................................................................57
2.3.4 Differences in efferocytic surface markers and profiles for NZM2328 versus C57BL/6J BMDMs....60

vi
2.3.5 SPMs stimulated macrophage mediated efferocytosis in NZM2328 and C57BL/J mice:...................63
2.3.6 Evaluating NZM2328 vs C57BL/6J BMDM, Kidney and Plasma Lipidome:.....................................66
2.4. DISCUSSION.................................................................................................................................................68
2.4.1 Macrophage-Based Interventions in Systemic Lupus Erythematosus.................................................68
CHAPTER 2: BIBLIOGRAPHY.........................................................................................71
CHAPTER 3: CHARACTERIZATION OF IN VIVO ACTIVITIES OF
NAP1051 IN SLE MURINE MODEL NZM2328 ...............................................................89
3.1 INTRODUCTION.............................................................................................................................................89
3.2 MATERIALS AND METHODS .........................................................................................................................92
3.2.1 NAP1051..............................................................................................................................................92
3.2.2 In vivo studies for NZM2328 mice.......................................................................................................92
3.2.3 Histologic evaluation...........................................................................................................................93
3.2.4 Immunofluorescence staining..............................................................................................................94
3.2.5 RNA extraction, cDNA preparation, and RT-PCR analysis................................................................95
3.2.6 Lipodomics Analysis of NZM2328 mouse Plasma and Kidney ...........................................................96
3.2.7 Statistical analysis...............................................................................................................................97
3.3. RESULTS......................................................................................................................................................97
3.3.1 Safety profile of NAP1051 evaluated using Cellular Blood Counts (CBC), Blood Chemistry
and liver function tests..................................................................................................................................97
3.3.2 Effect of NAP1051 on proteinuria and autoantibodies in NZM2328 female mice..............................98
3.3.3 Glomerular histology and IgG and C3 deposits in NZM2328 mice....................................................99
3.3.4 Effect of NAP1051 on Inflammatory Markers in Kidney ..................................................................100
3.3.5 NAP1051’s Effect on Immune cell infiltration and T Cell Infiltration..............................................102
3.3.6 Effect of NAP1051 on in-situ NETosis in NZM2328 Kidney.............................................................103
3.3.7 Effect of NAP1051 on the Kidney and Plasma Lipidome in NZM2328 mice....................................105
3.3.8 SPM Metabolite Ratios in Plasma and Kidney .................................................................................107
3.4 DISCUSSION................................................................................................................................................108
CHAPTER 3: BIBLIOGRAPHY.......................................................................................111
CHAPTER 4: CHARACTERIZATION OF IN VIVO ACTIVITIES OF NAP1051
IN SLE MURINE MODEL MRL.LPR .............................................................................114
4.1 INTRODUCTION...........................................................................................................................................114
4.2 MATERIALS AND METHODS .......................................................................................................................117
4.2.1 NAP1051............................................................................................................................................117
4.2.2 In- vivo studies...................................................................................................................................117
4.2.3 Proteinuria and Glomerular Histology .............................................................................................118
4.2.4 Immunofluorescence Staining ...........................................................................................................118
4.2.5 Mesenchymal stem cell isolation:......................................................................................................119
4.2.6 RNA extraction, cDNA preparation, and RT-PCR analysis..............................................................120
4.2.7 Lipidomics analysis of MRL.lpr mouse kidney and plasma ..............................................................120
4.2.8 Statistical analysis.............................................................................................................................121
4.3 RESULTS.....................................................................................................................................................122
4.3.1 Effect of NAP1051 on Clinical Markers in MRL.lpr mice ................................................................122
4.3.2 Effect of NAP1051 on Proteinuria, Glomerular Histology and T-cell infiltration ...........................123
4.3.3 Effect of NAP1051 on Opsonized Antibodies and Neutrophil Infiltration ........................................124
4.3.4 NAP1051 effect on NETosis Markers in Kidney ...............................................................................126
4.3.5 Effect of NAP1051 on the Kidney and Plasma Lipidome in SLE murine models .............................128
4.3.6 Effect of NAP1051on bone marrow mescenchymal stem cell (MSC) activation...............................130
4.4 DISCUSSION................................................................................................................................................132
CHAPTER 4: BIBLIOGRAPHY.......................................................................................135
CHAPTER 5: CONCLUDING REMARKS .....................................................................139
CHAPTER 5: BIBLIOGRAPHY.......................................................................................148
COMPLETE BIBLIOGRAPHY........................................................................................151

vii
List of Tables
Table 1.1: SLE stratification based on disease severity and treatment criteria.........................6
Table 1.2: Comparison of surface signals between phagocytosis versus efferocytosis..........16
Table 1.3: List of TLR ligands and drugs that alter the downstream activation signals.........21
Table 1.4: Lipoxins and their effects on immune cells...........................................................25
Table 2.1: RT-PCR run method with temperature and times that were used to run
NZM2328 and C57BL/6J mouse BMDM samples. ................................................................52
Table 3.1: Proteinuria ranks based on the absolute proteinuria values in mg/dL...................93
Table 3.2: List of primary and secondary antibodies used along with dilutions
factors for histologic staining of NZM2328 kidney tissue .....................................................94
Table 3.3: List of forward and reverse mouse primer sequences used for RT-PCR...............95
Table 3.4: RT-PCR run method with temperatures and times used to run NZM2328
mouse kidney samples .............................................................................................................96
Table 4.1: Study design for MRL.lpr mice to test oral efficacy of NAP1051......................118

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List of Figures
Figure 1.1: Overview of SLE pathogenesis highlighting the roles of NETs,
autoantibody production, inflammatory cytokines, and immune cell interactions
that lead to tissue damage, particularly in the kidneys. ...........................................................11
Figure 1.2: Role of Lipoxins in suppressing inflammatory responses by regulating
immune cells............................................................................................................................23
Figure 1.3: Biosynthesis pathways for lipoxins and aspirin triggered lipoxins......................28
Figure 1.4: LXA4 and its main pathways of enzymatic metabolism......................................29
Figure 1.5: NAP1051 and its structure, the box highlights modifications between
LXA4 and NAP1051 chemical structure .................................................................................30
Figure 2.1: Disrupted macrophage polarization profiles in SLE-prone mice BMDMs
contributing to autoimmunity. .................................................................................................47
Figure 2.2: Gating strategy for BMDMs to identify F4/80+ subset population and
gated at F4/80+CD86+ for M1 macrophages, F4/80+CD206+ for M2 macrophages. ...........51
Figure 2.3: CD86 and CD206 identify M1 and M2-like cells respectively in stimulated
mouse BMDM.. .....................................................................................................................556
Figure 2.4: Schematic view of M0, M1 and M2 activation and the effect of
ATLA at 1µM-10 µM and NAP1051 1µM-10 µM effect on polarization.
BMDMs were labeled with CD86, CD206 antibodies. ...........................................................58
Figure 2.5: Macrophage mRNA characterization in NZM2328 and C57BL/6J with
and without stimulation............................................................................................................60
Figure 2.6: In vitro BMDM efferocytosis of apoptotic dHL-60s...........................................63
Figure 2.7: In vitro BMDM C57BL/6J efferocytosis of apoptotic dHL-60 cells,
where ATLA treated BMDM at 1 and 10 µM and 1 µM NAP1051 increased the
level of efferocytosis................................................................................................................65

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Figure 2.8: BMDM, Serum and Kidney lipidome analysis for NZM2328 mice....................67
Figure 3.1: Safety profile of NAP1051 evaluated using CBC markers..................................98
Figure 3.2: Change in proteinuria levels in NZM2328 mice urine after 28-days
of treatment with NAP1051.....................................................................................................99
Figure 3.3: NZM2328 kidney sections stained with H&E, immunofluorescent
IgG and C3 after 28 days treatment with NAP1051................................................................11
Figure 3.4: Genetic expression of inflammatory markers associated with inflammtion
in SLE-prone NZM2328 mice kidney ...................................................................................101
Figure 3.5: Perivascular infiltration and T-cell infiltration in NZM2328 female kidney.....103
Figure 3.6: In-situ deposition of neutrophils was accessed using immunoflurescent
staining and genetic expression of neutrophil and NETosis related markers........................104
Figure 3.7: Analysis of Plasma and Kidney Lipidome for NZM2328 mice that
were orally treated with NAP1051 for 28 days .....................................................................106
Figure 3.8: Plasma and Kidney PUFA metabolite ratios. Heatmaps displaying PUFA
metabolite ratios in relation to vehicle treated animals .........................................................108
Figure 4.1: Lupus like rash in MRL.lpr model after treatment with dose escalation of
NAP1051 for 28 days.............................................................................................................123
Figure 4.2: Blood urea nitrogen levels and proteinuria levels for MRL.lpr mice after
treatment with orally dosed NAP1051...................................................................................124
Figure 4.3: IgG and C3 deposists staining and quantification in MRl.lpr mice kidneys......125
Figure 4.4: RT-PCR markers associated with SLE pathogenesis. Genetic expression
of inflammatory markers such as TNF-a, CXCL-2 and IFN-a .............................................127
Figure 4.5: Lipidomic analysis of MRL.lpr plasma and kidney levels after oral
treatment with NAP1051 for 28 days ....................................................................................129

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Figure 4.6: Correlation of individual lipids between plasma and kdney analytes
that play an important role in the pro-resolving pathways in SLE and can be used
as a tool to predict kidney disease state by analyzing the circulating plasma lipids ............130
Figure 4.7: Gating stratergy for analysis of MSCs in bone marrow in MRL.lpr mice...........98
Figure 5.1: The role of B-cells and its receptors in SLE pathogenesis...................................98

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Abstract
Systemic Lupus Erythematosus (SLE) is a multifaceted autoimmune disease marked
by widespread inflammation, excessive autoantibody production, and organ damage, with
lupus nephritis being a frequent and severe manifestation. Current therapies, often limited in
their efficacy and associated with significant side effects, highlight the need for novel treatment
approaches. This dissertation explores the therapeutic potential of NAP1051, a synthetic
lipoxin analogue designed to mimic the pro-resolving properties of endogenous lipoxin A4
(LXA4) while overcoming its rapid metabolism and short half-life.
NAP1051 was evaluated in murine models of SLE, including NZM2328 and MRL.lpr
strains, to assess its immunomodulatory and anti-inflammatory effects. Studies revealed that
NAP1051 significantly reduced proteinuria, autoantibody levels, and renal immune complex
deposition. Additionally, histological and immunofluorescence analyses demonstrated
decreased glomerular damage and reduced infiltration of inflammatory cells. Upon probing the
mechanisms involved by which NAP1051 exerted its therapeutic efficacy we see that
NAP1051 treatment modulated macrophage polarization, promoting a shift from proinflammatory M1 to anti-inflammatory phenotypes, and enhanced macrophage-mediated
efferocytosis of apoptotic cells. These effects were linked to improved clearance of apoptotic
cells, reduced neutrophil extracellular trap formation (NETosis), and the attenuation of
cytokine-driven inflammatory cascades in vivo.
Building on these findings, we conducted a detailed analysis of polyunsaturated fatty
acids (PUFAs) and their bioactive lipid mediator metabolites in both murine models to
elucidate the mechanisms by which dysregulated lipid metabolism contributes to SLE
progression. This investigation also aimed to understand the impact of NAP1051 on these
processes. NAP1051 effectively restored lipid mediator homeostasis by reducing the levels of
pro-inflammatory lipids, including leukotrienes and prostaglandins, while enhancing the

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production of pro-resolving mediators such as resolvins. Mechanistic studies identified the
ALX/FPR2 receptor as a critical pathway through which NAP1051 exerted its effects,
modulating immune cell activity and significantly reducing the production of key proinflammatory cytokines, including IL-1β, IL-6, and TNF-α. Notably, NAP1051 demonstrated
a strong safety profile, with no observed adverse effects on hematologic, hepatic, or renal
functions, highlighting its potential as a promising candidate for further therapeutic
development.
Collectively, this dissertation provides compelling evidence for the use of NAP1051 as
a novel therapeutic approach for SLE. By directly addressing the molecular and cellular
abnormalities underlying SLE, including defective apoptotic cell clearance, dysregulated
macrophage activity, and unresolved inflammation, NAP1051 offers a targeted and effective
solution to mitigate disease progression. These findings lay the groundwork for future research
and clinical translation of synthetic lipoxin analogues in autoimmune diseases.

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Chapter 1: Introduction to Systemic Lupus Erythematosus, Immune cell
related Inflammation and Lipoxin A4.
1.1 Background of System Lupus Erythematosus (SLE)
Systemic Lupus Erythematosus (SLE) is a complex autoimmune disorder involving the
production of excessive autoantibodies that can lead to significant damage across multiple
organ systems. The clinical manifestations of SLE are diverse, with involvement of the skin,
joints, kidneys, blood, and central nervous system (CNS). If the disease is not adequately
managed, progression can result in severe complications and increased morbidity. Common
complications include lupus nephritis (LN), cardiovascular disease, neurological symptoms,
infections, and psoriasis-like conditions. LN is one of the most frequent manifestations,
affecting up to 74% of patients (Ramos et al., 2010). It typically arises from immune complex
deposition in the renal glomeruli, triggering complement activation and chronic inflammation,
ultimately leading to renal impairment (Walport, 2002)
The course of SLE is highly variable, with symptoms that may fluctuate in severity, ranging
from mild to life-threatening. A key molecular feature of the disease is the breakdown of selftolerance, resulting in the production of autoantibodies targeting native DNA and other cellular
components. SLE pathogenesis involves several molecular processes, including excessive
production of type I and II interferons (IFNs), dysregulated apoptosis and impaired clearance
of cellular debris, and the accumulation of neutrophil extracellular traps (NETs or NETosis).
These disruptions contribute to the generation of autoantibodies targeting nucleic acids and
associated proteins (Miyagawa, 2023).
In addition to these molecular mechanisms, dysregulation of B lymphocytes and
inflammatory cytokines, driven by elevated IFN activity, plays a critical role in the disease
process. Recent research has highlighted the importance of the innate immune system,
specifically abnormalities in neutrophil and macrophage function, in the early stages of SLE

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development. These immune cells contribute to excessive autoantibody production,
exacerbating the autoimmune response. A variety of autoantibodies are found in SLE,
including anti-Scl-70 (seen in systemic sclerosis), anti-La and anti-Ro (associated with
Sjögren's syndrome or cutaneous lupus), as well as anticardiolipin and antiphospholipid
antibodies. The presence of these antibodies suggests a close association between SLE and
other autoimmune conditions (Hernández-Molina et al., 2011). Although the types of
autoantibodies vary, a shared feature across cases is their targeting of nuclear and cytoplasmic
antigens, reflecting the systemic nature of the immune dysregulation in SLE.
1.2 Epidemiology and Risk Factors Associated SLE
SLE affects an estimated 2.78 to 2.90 per 100,000 individuals, with a notable higher
prevalence in women, particularly during childbearing years. Studies indicate that women are
approximately nine times more likely than men to develop SLE, with the disease typically
emerging between ages 15 and 45 (Jarukitsopa et al., 2015; Justiz Vaillant et al., 2024; Rider
et al., 2018). The onset and progression of SLE are influenced by a combination of
environmental and genetic factors that interact to trigger the production of autoantibodies,
central to the disease’s pathogenesis.
Environmental factors linked to SLE include exposure to silica, tobacco smoke, air
pollutants, ultraviolet radiation, pesticides, heavy metals, and specific vaccines such as
COVID-19 and hepatitis B (Salleh et al., 2022), all of which may increase the likelihood of
autoimmune responses in genetically predisposed individuals (Parks et al., 2017).
Lifestyle factors like chronic stress, fatigue, and alcohol use have also been associated
with SLE risk (Xiao et al., 2023). Hormonal influences play a significant role, with oral
contraceptives and hormone replacement therapy further elevating SLE risk, highlighting the
complex interactions between immune and endocrine systems (Parks et al., 2017).

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Genetic predisposition has also been linked to increase risk for SLE. There is indirect
evidence in support genetic factors in relations to increased predisposition for SLE. Twin
studies have estimated a 24% to 35% concordance between monozygotic twins, which is
drastically higher than dizygotic twin pairs where the concordance was only 2% to 5% (S. R.
Block, 2006; Deapen et al., 1992). Specific genetic factors associated with increased risk for
SLE include genetic variation found in the human leukocyte antigen (HLA). In particular, the
class II alleles such HLA-DR2 (DRB1*1501) and HLA-DR3 (DRB1*0301) have been most
consistent genetic risk factors linked SLE in Caucasian populations. The presence of these
genetic factors has overall 2- to 3-fold increased risk for SLE (Tsao, 2003). In addition, there
is evidence pointing to susceptible ethnicities at increased risk for SLE such as those of African,
Asian, and Hispanic ethnicity when compared to Caucasians.
The genetic architecture of SLE includes mutations across various genes, which differ
among demographic groups and can complicate the identification of specific genetic
contributors due to linkage disequilibrium (LD), where certain alleles are inherited together
more often than statistically expected. Copy number variants (CNVs) in genes like FCGR3B
and complement component C4 also contribute to SLE susceptibility, impacting immune
complex clearance and autoantibody production (Barbosa et al., 2018). Low copy numbers in
these genes are linked to impaired clearance of immune complexes by neutrophils, a key
factor in SLE pathogenesis, with emerging evidence suggesting neutrophil and macrophage
dysfunction play an active role in disease progression (Tay et al., 2020).
Certain medications, including methyldopa, hydralazine, and procainamide, have been
linked to drug-induced lupus erythematosus (DILE). This is likely due to their capacity to
induce epigenetic changes, including DNA methylation and histone modification, which may
initiate autoimmune responses.

4
Patients with SLE often exhibit hypomethylation, leading to the overexpression of genes
sensitive to methylation, including IFN-regulated genes (Richardson et al., 1990). Immune
complexes involving autoantibodies and self-antigens are recognized by plasmacytoid
dendritic cells (pDCs), activating type I and II interferons through Toll-like receptor 7
(TLR7) (S. Chen et al., 2019). These findings highlight that genetic and environmental
interaction which can initiate and sustain the chronic inflammatory responses characteristic of
SLE. Further, they highlight the importance of targeting immune complex clearance as a
therapeutic approach to mitigate SLE progression and associated inflammation.
1.3 Diagnosis, Staging and Prognosis of SLE
Currently, there is no single test that can definitively diagnose SLE. Instead, diagnosis
involves a comprehensive evaluation of clinical symptoms, laboratory tests, and imaging
studies. Diagnosis and management of SLE has evolved significantly, particularly with the
development of unified classification criteria by the European League Against Rheumatism
(EULAR) and the American College of Rheumatology (ACR). These criteria were jointly
established to improve the consistency and accuracy of SLE diagnosis by providing a
structured approach that accounts for both clinical and immunological features of the disease.
The EULAR/ACR 2019 classification criteria for SLE are based on a point-based system that
requires an initial positive antinuclear antibody (ANA) test, a hallmark of SLE, followed by
the assessment of clinical manifestations and laboratory abnormalities (Aringer et al., 2019).
While single ANA test does not suffice for diagnosis because ANA can also be present in
healthy individuals, additional clinical criteria are set forth by ACR including the presence of
photosensitivity, oral ulcers, malar rash, discoid rash, arthritis, serositis, nephritis, and
neurologic symptoms can be used in combination with ANA positivity to provide a SLE

5
diagnosis. These criteria prioritize weighted clinical signs across multiple organ systems and
immunologic markers, aiming for greater sensitivity and specificity compared to previous
standards (Aringer et al., 2019).
The EULAR/ACR criteria include both clinical and immunologic features that span
across nine organ systems, with each criterion assigned a weighted score. To qualify for an
SLE diagnosis, a score at least 10 points, with symptoms ranging from constitutional issues
like fever, mucocutaneous lesions, arthritis, and serositis, to more severe manifestations, such
as renal or neurological involvement (Petri et al., 2012). This scoring approach assists in the
identification and classification of SLE based on the severity of organ involvement.
Accordingly, these classification guide in treatment strategy. For example, nephritis and
neurological symptoms are heavily weighted for their association with higher morbidity and
the need for more aggressive treatment (Aringer, 2019).
This classification system also reflects a deeper understanding of SLE’s immunologic
effects. It includes distinct markers like low complement levels, anti-double-stranded DNA
(anti-dsDNA) antibodies, anti-IgG antibodies, and other autoantibodies specific to SLE.
These immunologic criteria are given significant weight due to their predictive value for
disease progression and flares. The updated criteria have proven beneficial in reducing the
likelihood of false-positive diagnoses, particularly by ruling out individuals with positive
ANA but lacking other systemic features of SLE (Aringer et al., 2019; Petri et al., 2019).
Furthermore, the EULAR/ACR criteria also guide disease staging and management by
aligning the SLE Disease Activity Index (SLEDAI) with classification scores, thus offering a
continuum for treatment planning as shown in Table 1.1. Based on the EULAR/ACR criteria
and SLEDAI scores, clinicians can implement treatment plans to address specific levels of
disease activity and organ involvement. This allows for tailoring SLE treatments to manage

6
mild symptoms to targeting severe organ manifestations with immunosuppressive or biologic
therapies (Fava & Petri, 2019).
Active SLE disease is often referred to as a “Flare-up” can occur even after achieving
disease remission. This clinical finding is correlated to increased levels of ANA, thus close
monitoring should be continued even when disease remission has been achieved and
sustained. To track remission effectively, indices like Definition of Remission in SLE
(DORIS) and Lupus Low Disease Activity State (LLRS) are employed to reach treatment
goal (Parra Sánchez et al., 2023). Currently, treatment goals are summarized in relations
SLEDAI score and stratification in Table 1.1. These guidelines are to promote sustained
remission periods without signs and symptoms, improve quality of life, and reduce flares
episode. Ongoing research is essential to further refine diagnostic criteria, staging methods,
and treatment strategies, ultimately enhancing the overall prognosis for individuals with SLE.
Stratification Key Features SLEDAI
Score
Treatment (at any time during
disease course) Additional Notes
Mild SLE
Constitutional
symptoms; skin
manifestations
(e.g., rashes)
0-1
Oral glucocorticoids (GC) ≤
10 mg/day (prednisone equivalent) or
intramuscular GC and/or
hydroxychloroquine (HCQ)
Aim to taper GCs to
≤5 mg/day
(prednisone
equivalent).
Moderate SLE
Organ
involvement
without severe
damage
1-2
HCQ and/or Immunosuppressants
(e.g., methotrexate, azathioprine) and
GCs
Use
immunosuppressants
if disease is not
controlled with
HCQ.
Severe SLE
Significant organ
damage from at
least one organ
(e.g., lupus
nephritis, CNS
involvement)
2-3
Intravenous (IV) Cyclophosphamide
or high-dose methylprednisolone and
biologics (rituximab, belimumab)
Consider rituximab
for refractory cases.
Table 1.1: SLE stratification based on disease severity and treatment criteria. (Thanou et al., 2019)
(Zucchi et al., 2023). SLE Disease Activity Index: SLEDAI Score; GC: Glucocorticoids; SLE Disease
Activity Index: SLEDAI Score.

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1.4 Pathogenesis of SLE
The heterogenous clinical nature of SLE have made it difficult to pinpoint the precise
molecular pathogenesis (Choi et al., 2012). More recently, SLE pathogenesis have been linked
to both innate and adaptive immune systems. While high levels of autoantibodies are clinical
evidence supporting the concept of B-cell dysfunction, however more recently, dysfunction in
the innate immunity have also been implicated in the complex molecular pathology.
Current SLE molecular pathogenesis include disruption in IFN pathways, excessive
autoantibody production, immune complex deposition in tissues, and imbalanced activity of
various immune cells, such as B cells, T cells, neutrophils, and macrophages. In SLE,
overproduction of type I and type II IFN (e.g., IFN-α and IFN-g) by pDCs in response to nucleic
acid-containing immune complexes (Rönnblom & Leonard, 2019). Chronic IFN signaling
amplifies inflammation and promotes the activation of B and T lymphocytes.
B-lymphocyte dysfunction is well established pathological feature of SLE, where
overproduction of autoantibodies targeting dsDNA has been linked to immune complex
formation. Immune complexes accumulation in tissues (e.g., kidneys) can promote
complement component recruitment and assembly to further promote inflammation. In
parallel, failure in T-lymphocytes to regulate these aberrant responses, can allow inflammation
to persist, which contributes to the loss of self-tolerance (Lasorsa et al., 2023).
Despite establish role of B-lymphocytes in SLE, there is emerging evidence supporting
aberrant innate immune response participating in SLE pathogenesis. Neutrophils sequestered
in the inflamed foci can promote neutrophil extracellular traps (NETs) which expose nuclear
antigens and further stimulate IFN production to exacerbate autoimmunity (Melbouci et al.,
2023).
In addition, defective macrophage efferocytotic activity will lead to less efficient removal
of apoptotic cells (ACs) and cellular debris has been described in SLE (Tas et al., 2006). This

8
dysfunction perpetuates chronic inflammation, leading to interferonopathy, activation of the
B-lymphocyte to produce autoantibodies, which ultimately increase tissue damage. Altogether,
these molecular abnormalities create a self-amplifying cascade of immune activation,
autoantibody production, and inflammation.
Women are more prone to developing SLE suggesting a hormonal influences, where women
of child-bearing ages appear to be more susceptible to SLE (Solhjoo et al., 2024). This disparity
has led to investigation as to the role of altered sex hormones in SLE. Estrogen, progesterone,
testosterone and their metabolites have been implicated in the pathogenesis of SLE. Estrogen
has been shown to increase the survival and proliferation of bone marrow derived progenitor
B-cell. One study showed estradiol (E2) can enhance production of anti-dsDNA and IgG levels
in SLE patients when compared to a non-diseased control group. E2 treatment can also to
enhance IFN-a signaling by inhibiting Ikb kinase-e (Ikke) (Kim et al., 2022). Additionally,
progesterone causes Th2 polarization capable of stimulating production of IL-10 and IL-6 in
SLE when compared to healthy patients (Gröndal et al., 2000).
In contrast, testosterone can dampen the SLE autoimmunity, where androgen
supplementation in lupus-prone female NZB x NZW F1 mice was found to have benefits
(Gröndal et al., 2000). Testosterone treated mice had lower levels of anti-dsDNA and
glomerulonephritis as compared to the non-treated controls. In one clinical study, low plasma
testosterone in female SLE patients have been identified to contribute to SLE severity (Lahita
et al., 1987). While in men, hypogonadism is a risk factor for SLE in men. There is evidence
that androgen receptor polymorphism altering the length of a CAG repeats in exon 1 have been
associated as risk factor with men developing humoral autoimmunity.

9
1.5 SLE and Inflammation
1.5.1 Apoptosis and damage associated molecular patterns (DAMPs)
Emerging data suggest that SLE is an autoimmune disorder that arises from improper
cell death signaling and a failure in clearing apoptotic cells (ACs), leading to activation of
chronic inflammation. When apoptotic cellular debris is not adequately removed, secondary
necrosis occurs, resulting in the release of intracellular contents such as DNA and neoantigens.
Apoptosis, a programmed cell death mechanism, maintains tissue balance by eliminating
damaged or aged cells. In healthy individuals, macrophages and other phagocytes swiftly clear
these ACs through efferocytosis, preventing the escape of nuclear material into the
extracellular space and avoiding unnecessary immune responses. However, in SLE, there is
impaired apoptosis and defective efferocytosis of immune cells, allowing apoptotic cells to
accumulate and release nuclear antigens, including DNA, histones, and other nuclear proteins,
which then become autoantibody targets(Abdolmaleki et al., 2018). This impaired macrophage
clearance can lead to the formation of immune complexes, triggering inflammation,
particularly in organs like the kidneys, which can contribute to lupus nephritis (Gaipl et al.,
2006).
The continuous release of intracellular components, including DAMPs, intensifies the
autoimmune response by promoting the production of pro-inflammatory cytokines such as
interleukin-1β (IL-1β), IL-6, IL-8, IFN-α, and tumor necrosis factor-a (TNF-α). These
cytokines maintain an inflammatory microenvironment, further driving immune-mediated
tissue damage (Ohl & Tenbrock, 2011). Circulating immune complexes can bind to tissues and
activate the complement system, which recruits neutrophils and macrophages, amplifying
inflammation and causing progressive organ damage. Defective neutrophil apoptosis in SLE
perpetuates this inflammatory cycle through releasing nuclear antigens to fuel the autoimmune
response. Dysfunction of neutrophils and macrophages is emerging as an important molecular
pathology associated with sustained pro-inflammatory activity in SLE, where understanding of

10
this interaction will help to dissect the molecular pathology of SLE and other autoimmune
diseases.
Inadequate removal of post-apoptotic cells exposes multiple clearance issues, including
defective phagocytosis, improper opsonization by IgG of autoantigens, and deficiencies in
enzymes such as DNase, which are essential for breaking down nuclear material. Alongside
macrophages, other phagocytes, including dendritic cells and neutrophils, display similar
efferocytosis defects, escalating the autoimmune reaction as summarized below in Figure 1.1
(Lande et al., 2011). These efferocytosis impairments have been associated with both genetic
predispositions and environmental factors (Sheng et al., 2024). Moreover, chronic exposure to
inflammatory cytokines like TNF-α and IFN-g can reduce the efferocytic efficiency of
macrophages, contributing to an accumulation of apoptotic debris and driving disease
progression (Gaipl et al., 2006).

11
Figure 1.1: Overview of SLE pathogenesis highlighting the roles of NETs, autoantibody production,
inflammatory cytokines, and immune cell interactions that lead to tissue damage, particularly in the
kidneys. Key immune processes include NET formation, B-cell activation, monocyte differentiation,
and macrophage polarization, collectively driving chronic inflammation and autoimmunity in SLE.
(created using Biorender.com)
1.6 Immune cells in SLE
1.6.1 Effector cells of Innate Immune System
As shown in Figure 1.1, there are a number of different immune cells that play a role
in SLE pathogenesis. In this section we will review the role of each cell types and their
contribution to SLE pathology.
Neutrophils: Neutrophils are the most abundant leukocyte in blood, playing a critical role in
host defense by functions including phagocytosis of pathogens, releasing antimicrobial
granules, and forming neutrophil extracellular traps (NETosis) as overviewed in Figure 1.1
(Pan et al., 2020). Recruitment of neutrophils to the site of tissue damage and/or infection is
critical for the wound reparative process. However, activated neutrophil infiltration and

12
persistence can sustain chronic inflammation leading to additional tissue damage. While SLE
was traditionally attributed to the breakdown of tolerance and sustained autoantibody
production due to adaptive immune system dysfunction. Current insights emphasize an
imbalance in the generation and elimination of apoptotic cells, particularly involving
neutrophils. The inflammatory responses mediated by neutrophils include cellular adhesion to
vascular endothelium, migration into affected tissue, and elimination, involving the
production and secretion of reactive oxygen species (ROS) and cytoplasmic granules (H.
Block et al., 2022; Vorobjeva & Chernyak, 2020). However, persistently activated
neutrophils, particularly through the formation of NETs or NETosis, have been implicated as
potential contributors to SLE pathogenesis.
Once infiltrated into the target tissue, sterilization processes are initiated through the
upregulation of NADPH oxidase (NOX2) and the formation of ROS (Figure 1.1). This oxygen
burst triggers neutrophil elastase (NE) translocation into the nucleus, promoting chromatin
decondensation (Brinkmann, 2018) as shown in Figure 1.1. Additionally, ROS can also
activate ERK phosphorylation, further supporting chromatin disassembly and release (El Kebir
et al., 2007). These processes can also be triggered by peptidyl arginine deiminase-4 (PAD-4),
which facilitates the deimination of arginine residues on histone proteins, leading to chromatin
decondensation and expulsion (Serhan et al., 2007).
This vicious cycle of persistent inflammation promotes the production of additional
inflammatory mediators, subsequently encouraging additional leukocyte influx. Consequently,
promoting the clearance of NETosis emerges as a potential novel strategy for managing
autoimmune diseases such as SLE and rheumatoid arthritis. Moreover, NETs have been shown
to activate plasmacytoid dendritic cells, leading to the production of Type I and Type II IFNs,
which further amplify the autoimmune response in SLE (X. Zhang et al., 2021). The
accumulation of NETs in tissues, including the kidneys and skin, contributes to tissue damage

13
and inflammation, particularly in lupus nephritis. NETs have also been shown to induce
endothelial damage, promote thrombosis, and contribute to vasculitis, all of which are common
complications in SLE (Salemme et al., 2019).
In addition to their role in promoting inflammation, NETs impair the resolution of
inflammation by interfering with efferocytosis. Studies have shown that the presence of NETs
inhibits the ability of macrophages to clear ACs, thereby perpetuating the cycle of
inflammation in SLE (Kaplan, 2011). Thus, NETosis and defective clearance mechanisms
work synergistically to drive disease progression in SLE.
Macrophages: Normally, neutrophil activation is followed by rapid elimination.
However, the persistence of activated neutrophil or NETosis can sustain immune activation
and further trigger IFN production. Macrophages play an important role in eliminating of
apoptotic neutrophils and other cellular debris. However, macrophage dysfunction or inability
to adequately remove DAMPs has been implicated in SLE (Gerasimova et al., 2022).
The importance of functional macrophages is predicated on its vast and highly versatile
biological properties including antigenic elimination and cellular debris clearance. The
elimination of foreign intrusion is mediated through macrophage mediated phagocytosis
leading to activation of the inflammatory pathway. However removal of apoptotic cellular
debris is governed by non-phlogistic phagocytosis or efferocytosis which is further elaborated
in Table 1.2.
Macrophages are pivotal players in the innate immunity, where their biological activities
include identifying and reacting to invading pathogens. This process is initiated by
phagocytosis of the foreign antigen or pathogen, where the processing of the foreign antigen
will result in T-lymphocyte-mediated activation of the immune cascade. This illustrate the
crucial link between innate and adaptive immunity, which is facilitated by the presentation of
both endogenous and exogenous antigens such as the major histocompatibility complex (MHC

14
II) and CD86 (Guerriero, 2019). The ability to distinguish endogenous versus exogenous
antigen can dictate the type of response. This process is vital to activate adaptive immune
responses against diseases such as infections, cancer, and autoimmune conditions.
Other participating systems include the family of toll-like receptors (TLRs) and NOD-like
receptors (NLRs) which are both found on macrophages. Macrophage mediated phagocytosis
is also tightly regulated by pattern recognition receptors (PRRs) can detect pathogens and
endogenous signals. PRRs associated with macrophages including TLRs, C-type lectin
receptors (CLRs), NLRs, RIG I like receptors (RLRs), and scavenger receptors (SRs). TLRs
are classified as either membrane-bound or cytosolic receptors capable of detecting PAMPs
and DAMPs, where their binding can activate immune responses (Wicherska-Pawłowska et
al., 2021).
Macrophage can be classify according to their biological phenotypes as either M1
(classically activated) or M2 (alternatively activated) as shown in Figure 1.1. M1 macrophages
promote an inflammatory cascade through secretion of pro-inflammatory cytokines such as
TNF-α, IL-1β, and IL-6 and are involved in the early immune response to infection or tissue
damage (Doran et al., 2020). In contrast, M2 macrophages are typically associated with tissue
repair, wound healing, and anti-inflammatory processes, secreting cytokines like IL-10, IL-13
and TGF-β. These functional phenotypes are influenced by various signals, which determine
the macrophage phenotypes which is distinguished by its transcriptional and metabolic profiles.
Despite this classification, recent studies have shown that macrophage phenotype is not a fixed,
but rather these phenotypes are dynamic and plastic process (Yunna et al., 2020).
These phenotypes will also dictate macrophage role in antigen presentation to Tlymphocytes and facilitating T cell proliferation through the surface co-expression of
molecules like CD86 and MHCII. In SLE, there is a shift toward M2 macrophages, which
paradoxically promotes chronic inflammation rather than resolving it. In lupus nephritis, M2

15
macrophages are associated with fibrotic changes and impaired clearance of immune
complexes, contributing to tissue damage. Instead of functioning as anti-inflammatory cells,
M2 macrophages in SLE fail to clear apoptotic cells effectively and secrete pro-inflammatory
mediators through mechanisms such as NLRP3 inflammasome activation (Oliveira et al.,
2021). This paradox of M2 macrophages in SLE that promote inflammation instead of proresolution properties may highlight their central SLE pathogenesis.
As aforementioned, a key function of macrophages is the removal of pathogens and cellular
debris. This process entails engulfing and digesting pathogens, dead cells, and debris. Beyond
eliminating the DAMPs and PAMPs, it also plays a vital role in maintaining tissue homeostasis
by clearing cellular remnants. Phagocytosis and efferocytosis are macrophage-driven processes
responsible for the ingestion and breakdown of pathogens and cellular debris, respectively.
While both processes involve the internalization of particles or cells into a vacuole, the major
difference is macrophage phagocytosis leads to antigen presentation and immune activation. It
plays a crucial role in host defense and inflammation, helping to eliminate pathogens and
trigger immune responses (Table 1.2).
In contrast, efferocytosis is often referred to removal of apoptotic cells and debris which
is essential for maintaining tissue homeostasis through pro-resolving properties. Efferocytosis
is a specialized form of phagocytosis that involves the recognition and clearance of apoptotic
cells by macrophages without triggering inflammation and is often considered pro-resolving.
Macrophages possess receptors that recognize specific signals present on apoptotic cells, in
this case neutrophils, allowing them to recognize and engulf these dying cells efficiently. Some
of these signals include T-cell immunoglobulin and mucin domain-containing protein 4 (TIM4) which is a phosphatidylserine (PS) receptor involved in the recognition and clearance of
ACs. Signals such as aVb3 integrin, MerTK and AXL and Tyro3 are also important signals

16
macrophages use to engulf apoptotic cells recognizing PS expressed on stressed or apoptotic
cells regulating tissue homeostasis (Grabiec et al., 2018).
Once engulfed, macrophages break down the ACs and prevent the release of potentially
harmful intracellular contents, while also releasing anti-inflammatory mediators to resolve
inflammation. However, in autoimmune diseases, the clearance of apoptotic neutrophils by
macrophages is impaired. This failure may occur due to various reasons, such as defects in the
recognition and binding of surface signals, altered phagocytic capacity of macrophages (Doran
et al., 2020), or an excessive load of dying cells overwhelming the clearance capacity of
macrophages (Mistry & Kaplan, 2017). As a result, the apoptotic neutrophils and their
remnants may accumulate in tissues and can initiate and sustain inflammation through releasing
its cellular contents through the process of NETosis (Meng et al., 2022). It should be noted that
the clearance of apoptotic neutrophils by macrophages triggers the phenotypic transition from
M1 to M2 (Ahamada et al., 2021) which is the critical difference between phagocytosis and
efferocytosis as highlighted in Table 1.2.
Phagocytosis Efferocytosis
Target Pathogens (e.g., bacteria), debris, or
damaged cells
Apoptotic (dying) cells
Primary "Eat-Me"
Signals
Pathogen-associated molecular patterns
(PAMPs) (e.g., lipopolysaccharides,
peptidoglycan) and damage-associated
molecular patterns (DAMPs) (e.g.,
HMGB1, ATP)
Phosphatidylserine (PS), oxidized
lipids, calreticulin
Receptors Involved Toll-like receptors (TLRs), scavenger
receptors, complement receptors
Receptors specific for apoptotic
cells: Tim-4, MerTK/AXL, BAI1
Immune Response Pro-inflammatory: Activates immune
cells, induces cytokine production to
fight infections
Anti-inflammatory: Promotes
tissue repair and resolution of
inflammation
Outcome Elimination of pathogens or foreign
material, triggering inflammation
Clearance of apoptotic cells
without inflammation, maintaining
tissue homeostasis
Context of
Recognition
"Danger" or infection cues; targets often
release inflammatory signals
Silent clearance: apoptotic cells,
non-threatening removal
Table 1.2: Comparison of surface signals between phagocytosis versus efferocytosis (Ge et al., 2022;
Yin & Heit, 2021).

17
1.6.2 Effector cells of Adaptive Immunity
T-Lymphocytes: The innate immune system and adaptive immune system are
interlinked through cytokines, complements and immune complexes. The adaptive immune
system has been implicated as one of the root causes for SLE and its manifestations. T cells
particularly play a pivotal role in the pathogenesis of SLE, contributing to inflammation
through the secretion of pro-inflammatory cytokines, stimulation of B cells to produce
autoantibodies, and sustaining the disease through autoreactive memory T cells. However, in
patients with SLE, there are abnormalities in the ratios and functions of certain T cell subsets,
mainly an imbalance between T helper cells (Th). Th cells orchestrate immune responses
against pathogens which typically express CD4+ on their cell surface and are classified as Th1
and Th2 cells.
Th1 cells typically secrete TNF-a, IFN-g and IL-2 and activate macrophages and CD8+
cells. In contrast, Th2 cells secrete IL-4, IL-13, IL-6 and promote B-cells activation and
differentiation into plasma cells (Raphael et al., 2015). In SLE the balance between Th1 and
Th2 is disrupted, where greater influence of Th2 (IL-13 and IL-4) on hyperactivation of B-cells
contributing to generation of autoantibodies and SLE progression (Horwitz et al., 1998).
Additionally, critical to SLE pathogenesis are T follicular helper (Tfh) cells. These cells also
produce the cytokine IL-21, which promotes B cell differentiation into memory B cells and
antibody-generating plasma blasts. In SLE, the pathological expansion of Tfh cells is driven
by their interaction with TLR7 and activation to circulating immune complexes. In LN, Tfh
aggregate in renal tissue with B cells and the resultant abnormal increase contributes to
heightened antibody production and the breakdown of self-tolerance in SLE patients.
Regulatory T (Treg) cells constitute a distinct T cell subset that suppresses the immune
response and upholds self-tolerance, inhibiting autoreactive lymphocytes in healthy individuals
(Sakaguchi et al., 2009). Treg cell in SLE are often imbalanced. T cell cytokine profile
characterized by reduced IL-2 and reduced FOXP3 which leads to compromised Treg cell

18
development and function. Additionally, IL-2 plays a role in constraining the expression of IL17 (Yadav et al., 2013), a pro-inflammatory cytokine found to be elevated in SLE. Several
studies have pointed to Th17 cells that playing an important role in SLE pathogenesis. IL-17
levels in SLE patients have correlated with SLEDAI scores and baselines levels were higher in
patients with active nephritis (X. Y. Yang et al., 2013). This is due to IL-17 and BAFF having
synergistic effects on B-cell proliferation and differentiation causing a pro-inflammatory
response and tissue injury. CD8 T cells also play a crucial role in recognizing peptide antigens
presented by MHC class I molecules (López et al., 2016). These cells are involved in infection
control, anti-tumoral responses, and autoimmunity. However, patients with SLE exhibit defects
and abnormalities in CD8+ T cells which contribute to autoimmunity in SLE and can
potentially explain the predisposition of SLE patients to infections, especially when using
immunosuppressive drugs (Paredes et al., 2021).
B-Lymphocytes: B-lymphocytes or B cells play a significant role in the pathogenesis of
SLE by responding to antigens and producing autoantibodies. The pathways implicated in the
aberrant activation of B cells include the TLR pathway, stimulation via B cell-activating factor
(BAFF), and B-cell receptor (BCR)-mediated activation, all of which contribute to the
breakdown of tolerance (Karrar & Cunninghame Graham, 2018). Transitional B cells, which
are susceptible to TLR9 stimulation, contribute to the production of autoreactive marginal zone
B cells observed in SLE patients. Impairment of B cell tolerance can also result from cytokine
stimulation, particularly BAFF. SLE patients with elevated BAFF levels exhibit significantly
higher levels of antibodies against dsDNA, histone, and cardiolipin (Nashi et al., 2010).
Moreover, the BCR plays a pivotal role in regulating negative and positive selection, with
continuous BCR sensing being crucial for B cell survival in healthy individuals. The prosurvival signaling of BAFF serves to counteract the pro-apoptotic signals triggered by BCR.
Nevertheless, an imbalance in these signals within SLE leads to a breakdown in tolerance and

19
the generation of autoantibodies, ultimately contributing to the development of the disease
(Yap & Chan, 2019).
1.7 Cytokines, Toll-like receptors, Immunoglobuloins and Complement
Factors
1.7.1 Cytokines
The hallmark of SLE is the presence of autoantibodies and immune complex disposition
into affected organs like the kidney and skin. Elevated apoptosis, coupled with impaired
clearance of apoptotic cells, leads to the accumulation of high levels of autoantibodies (Božič
& Rozman, 2006). The dysregulated production of cytokines further contributes to immune
dysfunction, fostering tissue inflammation, and causing organ damage. For SLE, pivotal
cytokines such as type I and type II IFNs, IL-6, IL-1b, TNF-α, along with immunomodulatory
cytokines like IL-4 and TGF-β, play crucial roles as aforementioned above. Neutrophils
undergo NETosis following activation by inflammatory cytokines such as TNF-α. This can
lead to the exposure of self-antigens and triggering an immune response against these selfcomponents. The immune system recognizes these self-antigens as foreign and mounts an
immune response, generating autoantibodies and activating immune cells, such as T-cells. This
immune response leads to inflammation and tissue damage, characteristic of autoimmune
diseases. Recent research has also shed light on the significance of IL-21 and IL-17 in
autoimmunity (Zídek et al., 2009), while renewed attention has been given to IL-2 in the
context of SLE (La Cava, 2023).
1.7.2 Toll like receptors (TLRs):
TLRs are a family of PRRs that are able to detect pathogens and endogenously
produced cellular debris. Differentiating the two types of mechanism are associated with the
resource of antigens. Foreign intrusion will activate PAMPs, while endogenous cellular debris
releasing DAMPs. Present on various immune cells like macrophages, dendritic cells, and B

20
cells, TLRs play a pivotal role in detecting potential pathogens, initiating immune responses in
the form of cytokines, chemokines, and antimicrobial molecules (Goulopoulou et al., 2016).
In SLE, aberrant TLR activation has been reported, particularly TLR7/9, can lead to
neutrophil activation, diverting them from phagocytosis to exacerbated NETosis, depending
on the stimuli received (Yu & Su, 2013). TLR-7/8, recognizing single-stranded RNA (ssRNA)
from viruses, and can also be activated by ROS, DAMPs, and mitochondrial DNA, triggering
NETosis. While TLR activation is often associated with inflammation and autoimmunity,
recent findings show specific TLR binding, or its absence can modulate SLE pathogenesis. For
instance, increased TLR7 expression, is linked to a higher SLE risk (G. J. Brown et al., 2022).
TLR8 activation may inhibit TLR7 through various proposed mechanisms, offering therapeutic
potential in reducing SLE inflammation (T. Chen et al., 2021). Negative feedback loops, such
as TGFβ and IL-10, can counter TLR-mediated inflammation by suppressing TLR signaling
pathways and B-cell activation, disrupting the proinflammatory cytokine cycle (Schülke,
2018). TLR9, recognizing CpG DNA motifs, contributes to autoantibody production in SLE.
Inhibiting CpG DNA binding to TLR9 may reduce autoantibody production and hasten clinical
remission (Nündel et al., 2015).
TLR2/4 recognition of microbial components in neutrophils contributes to ROS and
pro-inflammatory cytokine production, causing tissue damage and inflammation in SLE.
Impaired TLR4 signaling might be beneficial, reducing autoantibody and cytokine production.
TLR2 may have a protective role, potentially influenced by certain gene variations. TLR3,
expressed on neutrophils and other immune cells, is activated by double-stranded RNA
(Devarapu & Anders, 2018). Its activation contributes to inflammation and glomerulonephritis
in SLE.

21
TLR Ligands Treatment Targets
TLR1/2 Bacterial lipoproteins No specific drugs
TLR3 Double-stranded RNA Polyinosinic Polycytidylic acid (Poly(I:C))
TLR4 Lipopolysaccharide (LPS) TAK-242 (Resatorvid)
TLR7 Single-stranded RNA (ssRNA) Imiquimod, R848
TLR8 Single-stranded RNA (ssRNA) Imiquimod, R848
TLR9 Unmethylated CpG DNA or ssDNA CpG oligonucleotides
Table 1.3: List of TLR ligands and drugs that alter the downstream activation signals. (Bunting et al.,
2011; Elloumi et al., 2022; Fekonja et al., 2012; Patinote et al., 2020; Tam et al., 2021).
1.7.3 Immunoglobulins and Complement Factors:
Damage to organs and tissues occurs in the areas where IgG accumulates, which can
recruit immune cells expressing constant fragment receptor (FcR) such as neutrophils and
macrophages. Infiltration of inflammatory cells can disrupt the normal tissue structure. This
severe damage to multiple organs is a leading cause of mortality among individuals with SLE
(Cuadrado et al., 2019). Commonly affected organs include the kidneys, skin, joints, liver,
spleen, lungs, and brain. While patients with SLE generate IgA, IgE, IgG, and IgM
autoantibodies, IgG are the most predominant (Chong et al., 2012). The foundation of organ
and tissue damage is associated with localized accumulation of IgG and immune complexes
(ICs) accumulation. The development of SLE is closely linked to elevated levels of
autoreactive IgG. Nonetheless, the precise mechanism by which IgG leads to organ and tissue
damage remains unclear. Tissue-deposited ICs, formed by the interaction of autoantibodies and
autoantigens, activate immune cells, prompting the production of inflammatory cytokines by
binding to Fc gamma receptors (FcγRs) (Tsokos, 2011). Recent research has revealed that IgG
deposited in organs triggers local inflammation by binding to FcγRs found on the surface of
monocytes/macrophages, resulting in damage to multiple organs and tissues (E. E. Brown et
al., 2007).
1.8 Polyunsaturated fatty acids and their role in regulating immune activity

22
Polyunsaturated fatty acid (PUFA) are known to be important modulator of
inflammation, where imbalance in omega-6 (n-6) with omega-3 (n-3) can shift the balance to
inflammation. Importantly, arachidonic acid (AA) which is a n-6 fatty acid is the precursor to
cyclooxygenase (COX) and 5-lipooxygenase (5LOX) metabolites to form prostanoids which
are classified as inflammatory bioactive lipids (Simopoulos, 2002). AA can also be
metabolized by cytochrome p450 (CYP) and other LOX isoforms such as 12- and 15-LOX. In
contrast to COX metabolites formed through CYP, 12- or 15-LOX metabolites are primarily
pro-resolving in nature. This highlights the delicate balance between the various AA
metabolites.
Given the central role of defective clearance, macrophage dysfunction, and NETosis in
SLE pathogenesis, targeting these pathways represents a promising therapeutic strategy. The
strong correlation between SLE and pro-inflammatory responses directs us to the discovery of
endogenous molecules that can suppress persistent inflammation and resolve the inflammatory
responses associated with SLE. The resolution of inflammation is a complex process and
involves many different cell types and signaling molecules. As shown in Figure 1.2 the
DAMPs/PAMPs recruiting neutrophils to the site of injury. This is followed by
monocytes/macrophages. In SLE, a pro-inflammatory environment leading to M1
macrophages exerting a phagocytic response.

23
Figure 1.2: Role of Lipoxins in suppressing inflammatory responses by regulating immune cells
(Jaén et al., 2021).
For the resolution to begin, neutrophils must undergo apoptosis and be subsequently
removed from the microenvironment by macrophages. Importantly, both neutrophils and
macrophages express ALXR and other SPM receptors. This highlights the importance of this
understudied role of bioactive lipids in inflammation. As mentioned above, macrophages are
responsible for removal of the apoptotic neutrophils. Upon apoptotic cell engulfment,
macrophages switch from the pro-inflammatory state to pro-resolving state. Anti-inflammatory
cytokines including TGF-β and IL-10 are produced by these pro-resolving macrophage to
inhibit the production of inflammatory mediators (Korns et al., 2011). In the past few decades,
a class of molecules named specialized pro-resolving mediators (SPMs), including lipoxins
(LXs), resolvins (Rvs), protectins (PD), and maresins (MaRs), has been characterized with an
important role in resolving inflammation (Corminboeuf & Leroy, 2015; Q. Zhang et al., 2017).
SPMs are biosynthesized in response of inflammatory signals, and in turn exert their antiinflammatory and pro-resolving activities to restore tissue homeostasis. LXA4 receptor is the
formyl peptide receptor 2 (FPR2/ALXR), a promiscuous G-protein coupled receptor highly
expressed on neutrophils and phagocytes (Zhu et al., 2021). As shown in Figure 1.2, via

24
FPR2/ALXR, LXA4 is responsible for reducing expression of pro-inflammatory cytokines like
IL-1β, IL-6, IL-8 and IFN-γ and upregulating anti-inflammatory proteins such as IL-10 and IL4 in several cell types.
Functionally, LXA4 can of neutrophil recruitment to tissue sites (Dong et al., 2021). It
does so because neutrophils (Table 1.4) express the aspirin-triggered lipoxin receptor (ALXR)
or FPR2. The binding of N-formyl-l-methionyl-l-leucyl-phenylalanine (fMLP) onto ALXR
initiates neutrophil chemotaxis to the affected site, LXs compete with fMLP and bind onto
ALXR/FPR2 receptors preventing neutrophil migration (Yuen et al., 2016). Maddox et al.
revealed that LXA4 and its stable analogs, such as aspirin triggered lipoxin (ATLs), which will
be discussed further in the upcoming sections, enhance the migration of monocytes and their
adhesion to laminin, without altering the cytotoxic function of these monocytes (Maddox et
al., 1997). Moreover, studies have shown that LXA4 facilitates the phagocytosis of apoptotic
neutrophils by macrophages derived from monocytes (Godson et al., 2000) and prevents
apoptosis in macrophages triggered by stimuli such as LPS and IFNγ, thereby supporting the
resolution of inflammation (Prieto et al., 2010). LXA4 also plays a role in modulating memory
B-cell responses through an FPR2-dependent mechanism as shown in Table 1.4 (Ramon et al.,
2014).
Additionally, both LXA4 and aspirin trigger lipoxin-A4 (ATLA) have been widely
studied in various disease models, including skin, gastrointestinal, and pulmonary disorders, as
well as in the context of angiogenesis. Key in vivo activities of these molecules are summarized
in Table 1.3. Notably, the cellular functions of LXA4 and ATLA—such as reducing neutrophil
migration, promoting the clearance of apoptotic neutrophils by macrophages, and suppressing
pro-inflammatory cytokine production—align with in vivo findings, which demonstrate these
molecules’ ability to promote resolution of inflammation (Chiang et al., 2006). In SLE
research, LXA4 and its analogs have shown the ability to suppress inflammatory cytokines and

25
LXA4 along with LXA4/creatinine ratios can be used as effective biomarkers for lupus
nephritis and other manifestations of SLE. This is due to SLE patients having lower levels of
lipoxins in their urine and plasma compared to healthy controls while having higher levels of
leukotrienes (LTs) (Das, 2011).
Target Cell Lipoxins Effect Reference
Neutrophil Reduces AP1, NFkB activity and lowers IL-8
expression.
Fiore & Serhan, 1995; József et
al., 2002; Levy et al., 1999;
Papayianni et al., 1996; C. N.
Serhan et al., 1995
Monocyte Increases chemotaxis without promoting
inflammation, reduces IL-8 activity especially in
asthma related inflammation.
Bonnans et al., 2002; József et
al., 2002; Maddox et al., 1997
Macrophage Efferocytosis of inflammatory cells without
promoting inflammation.
Godson et al., 2000; Mitchell et
al., 2002
T-cell Boosts CCR5 expression, limits TNF production
by inhibiting ERK signaling.
A. Ariel, Chiang, Arita, Petasis,
& Serhan, 2003; Amiram Ariel
et al., 2006
B-cells Decreases human memory B cell antibody
production via ALX/FPR2-dependent pathway.
Ramon S, Bancos S, Serhan
CN, Phipps RP. Lipoxin A₄
modulates adaptive immunity
by decreasing memory B-cell
responses via an ALX/FPR2-
dependent mechanism. Eur J
Immunol. 2014;44(2):357-369.
doi:10.1002/eji.201343316
Leukocytes
(whole
blood)
Inhibits CD11b expression, also regulates Lselectin expression on cell surface.
Filep, Zouki, Petasis, Hachicha,
& Serhan, 1999; József, Zouki,
Petasis, Serhan, & Filep, 2002
Table 1.4: Lipoxins and their effects on target immune cells.
1.9 Biosynthesis of Lipoxin A4
Lipoxin A4 (LXA4) is produced and released by activated found in the inflammatory
site (Maderna & Godson, 2009). They are formed through metabolism of cellular membrane
phospholipids like phosphatidylcholines (PC), phosphatidylethanolamines (PE), and
phosphatidylserines (PS), where phospholipase A2 (PLA2) isoforms hydrolyze phospholipids
to liberate free PUFAs. Currently there are three major types of isoforms of PLA, which
include 1) calcium-dependent cytosolic phospholipase A2 (cPLA2), 2) calcium independent
PLA2 (iPLA2), and 3) soluble PLA2 (sPLA2). cPLA2 hydrolyzes phospholipids like PC can

26
liberated arachidonic acid (AA) and a lysophospholipid. In contrast, calcium-independent
phospholipase A2 (iPLA2) hydrolyses phospholipids to release eicosapentanoic acid (EPA),
docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA).
AA is an omega-6 (n-6) lipid precursor where its metabolism form inflammatory
bioactive lipids which are group as prostanoids. These cyclooxygenase (COX) metabolites of
AA prostaglandins, leukotrienes (LTs) and thromboxanes (TXs) which are potent regulators
of inflammation in both systemically and locally in the tissue/cell. PUFA metabolites are
classified as either pro-inflammatory or pro-resolving based on the type of immunologic
response they induce. Pro-inflammatory lipid mediators promote leukocyte recruitment and
cytokine production while generating ROS. On the other hand, pro-resolving lipids attenuate
inflammatory responses by inducing leukocyte apoptosis and facilitating the clearance of
cellular debris through monocyte-mediated efferocytosis (Grazda et al., 2023). Most proinflammatory eicosanoids are derived from AA metabolism. These include prostaglandins
(PGs) and thromboxanes (TXs) and LTs. Patients in the early stages of SLE showed an
increased biosynthesis of PGE2 and LTB4 which in turn mobilizes neutrophils migration and
activation (Godson et al., 2000).
LX on the other hand are pro-resolving mediators capable of counteracting the effects
of LTs while reducing the chemotaxis and activation of neutrophils (Dong et al., 2021). The
biosynthesis of lipoxins (LXs) involves transcellular metabolism via distinct pathways
depending on the cellular context. In human cells, two primary lipoxygenase (LOX)-
mediated pathways contribute to LX formation. The first pathway involves sequential
lipooxygenation of AA via 15-LOX found in epithelial cells and monocytes, and 5-LOX in
neutrophils, resulting in an inverse relationship between LX and LT formation (Gilbert et al.,
2021).

27
The second route involves platelet/leukocyte interactions promoting LX formation
through transcellular conversion of the 5-LOX epoxide product, LTA4 to LXA4 and LXB4
mediated by the LX-synthetase activity found in 12-LOX in platelets (Chiang et al., 2006).
Beyond transcellular routes, another recognized LX biosynthesis source involves the
esterification of 15-HETE in inositol-containing phospholipids within the membranes of
human neutrophils. This suggests that, during disease or host defense, The complexity of LX
formation signaling networks is heightened by potential regulation of biosynthetic enzymes
by specific cytokines as well. Interleukin 4 (IL-4) and IL-13 act as negative regulators.
Cytokines such as granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-13
up-regulate 5-LOX transcripts (Shim et al., 2006), while pro-inflammatory cytokines like IL1β, IL-6, and TNF-α induce cyclooxygenase-2 (COX-2), potentially contributing to ATL
formation (Miyamoto et al., 2000).Impaired LX biosynthesis may correlate with an inability
to resolve acute inflammatory reactions, contributing to a more chronic inflammatory
phenotype, as the one seen in SLE (Podolska et al., 2015).
Besides LXs, n-3 PUFAs such as EPA, DHA and DPA are intermediates that can
form pro-resolving mediators. The hydroxylation of DHA and DPA mediated by 15-LOX
leads to the formation of oxidized intermediates and these can undergo further metabolism to
generate three classes of lipid mediators: D-series resolvins (RvDs) via 5-LOX, MaRs via
soluble epoxide hydrolase (sEH) and additionally, there is a group of resolvins derived from
EPA known as E-series resolvins (RvEs) (López-Vicario et al., 2016).
Resolvins (Rvs) are produced by leukocytes and platelets in response to
inflammation, which promotes inflammation resolution and promote tissue repair. This may
partially explain why SLE patients with higher levels of EPA and DHA had a better
prognosis and attenuated nephritis, where higher levels of these n-3 PUFA may increase
formation of E- and D-series Rvs, respectively (Colas et al., 2014).

28
Figure 1.3: Biosynthesis pathways for lipoxins and aspirin triggered lipoxins (Chiang et al., 2006).
As aforementioned, LX has several isoforms and enantiomers. One of isoforms is
synthesized through aspirin-mediated alternative biosynthetic pathway for AA metabolism
(Figure 1.3) (Romano et al., 2015). As one of the most commonly used non-steroidal antiinflammatory drugs (NSAIDs), aspirin is renowned for its effectiveness in alleviating fever,
pain, and inflammation through inhibition of COX isoform activity, responsible for
prostaglandin synthesis (Giménez-Bastida et al., 2019). Aspirin can irreversible acetylate
serine residue on COX-1, preventing the formation of both prostaglandins and thromboxanes
(Serhan & Chiang, 2008). However, the impact of this acetylation varies between the two
COX isoforms: COX-1 and COX-2. In the case of COX-1, which is constitutively expressed,
aspirin acetylation leads to permanent deactivation of the enzyme. Conversely, in inducible
COX-2, which plays a more prominent role in inflammatory responses, acetylation of COX-2
on the serine residue converts it to have 15R-lipoxygenase-like activity (Serhan et al., 1984).

29
The acetylation of COX-2 converts the enzyme to be able to synthesis 15 epi-LXA4
or aspirin trigger lipoxin A4 (ATLA) and 15 epi-LXB4 (Chiang et al., 2006). The acetylation
of COX-2 alters its enzymatic function, mimicking that of a 15R-lipoxygenase, and enables
the conversion of AA into 15R-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15RHETE) (Chiang & Serhan, 2006; Chiang et al., 2006; (Smith et al., 1987). Although the
precise mechanism behind this activity is not fully understood, it is suggested that the Lshaped binding of AA within the active site of acetylated COX-2 may play a crucial role in
this process.
1.10 Metabolism of LXA4 and ATLA
Figure 1.4: LXA4 and its main pathways of enzymatic metabolism (Owen et al., 2022).
LXA4 undergoes several metabolic modifications to regulate its bioactivity and
promote clearance. One of the drawbacks of LXA4 is that it has a very short half-life of a few
seconds and is rapidly metabolized by 15-hydroxyprostaglandin dehydrogenase; 12-
hydroxydehydrogenase and prostaglandin reductase/leukotriene B4 dehydrogenase
(PGR/LTB4DH) as seen in Figure 1.4. LXA4 is oxidized or reduced on the C15-C20 chain
by all the enzymes listed above. Reduction of the tetraene core disrupts its conjugated

30
double-bond system, diminishing receptor affinity and signaling potency. C-15 hydroxyl
oxidation converts it to a ketone, influencing interactions with ALXR/FPR2 and modulating
anti-inflammatory effects (Araújo et al., 2018). Beta-oxidation cleaves two-carbon fragments
from the carboxyl end, systematically shortening the molecule, a typical pathway in lipid
catabolism (Clish et al., 2000). Additionally, omega-oxidation introduces a hydroxyl group at
the terminal methyl group, enhancing polarity and facilitating subsequent phase I and phase
II metabolism, which prepares it for renal excretion. ATLA on the other hand is said to be
more biologically active than LXA4 and has a longer half-life of a few minutes. However,
this limitation compromises its viability as a potential anti-inflammatory therapeutic for
managing chronic inflammatory diseases.
Figure 1.5: NAP1051 and its structure, the box highlights modifications between LXA4 and
NAP1051 chemical structure (Dong et al., 2021).
As mentioned above, unfortunately, the systemic half-life of LXA4 is short and thus
is rapidly cleared (Chandrasekharan & Sharma-Walia, 2015). This greatly limits its ability to
be a potent anti-inflammatory drug to combat sustained inflammation. To navigate these
challenges, we have synthesized and characterized a biomimetic analog of LXA4, NAP1051
as shown in Figure 1.5. NAP1051 had increased stability with the benzene ring along with
the methyl ester group increasing its pharmacological activity making it a pro-drug.
NAP1051 was found to preserve key in vitro LXA4 activities such as induction of neutrophil

31
apoptosis and promote macrophage clearance of apoptotic neutrophils leading to tissue
resolution (Saas et al., 2022). NAP1051 can also mediate resolution of NETosis by
preventing neutrophil chemotaxis and macrophage efferocytosis (Dong et al., 2021). Here we
further explored the pharmacological properties of NAP1051. They are further highlighted in
the sections below.
1.11 Hypothesis and Specific Aims
LXA4 is a powerful anti-inflammatory agent; however, its rapid metabolism by various
enzymes limits its therapeutic potential. To address this, we developed an analog, NAP1051,
designed to retain the essential functional groups necessary for cellular interactions. In this
analog, the hexatriene system is replaced with an aromatic ring to improve molecular stability,
and the carboxylic acid group is substituted with a methyl ester to further enhance the
molecule’s stability.
This project aims to investigate the molecular and pharmacological mechanisms
involved in SLE with and without NAP1051, a small-molecule analog of the endogenous proresolving lipid mediator LXA4. In this thesis, I will focus on characterizing the molecular
pathology of SLE using two SLE-prone murine models and investigate the mechanism(s) by
which NAP1051 can disrupt the cycle of autoimmunity.
I propose that NAP1051 will modulate inflammation in bone marrow-derived
macrophages (BMDMs) by altering macrophage polarization profiles, influencing biolipid
balance systemically and in tissues. I will dissect whether NAP1051 can mediate clearance of
neutrophils by macrophages, which may be an effective way to treat SLE. In addition, I will
also probe whether NAP1051 can enhance lysosomal acidification allowing macrophages to
better accommodate excessive cellular debris.

32
The findings from these studies will provide further provide critical insight into the
therapeutic potential and feasibility of utilizing an anti-inflammatory approach targeting two
distinct SLE murine models, NZM2328 and MRL.lpr. This central hypothesis led to the pursuit
of the following specific aims (SA):
SA 1: To assess NAP1051’s ability to mediate macrophage transitions from an inflammatory
state to enhance efferocytosis for apoptotic cell clearance.
SA 2: Determine whether NAP1051 can promote autoimmune disease resolution using two
distinct SLE murine models (e.g., NZM2328 and MRL.lpr)
SA 3: Characterize alterations in the bioactivity of PUFA metabolites in BMDMs, serum and
kidneys for SLE murine models.
1.12 Outline of the Dissertation
This dissertation offers the hypothesis SLE is activated through both genetic and
environmental factors. The dysregulation in SLE persists due to the failure to resolve
inflammation and restore homeostasis. To demonstrated this, we will compare the difference
in BMDMs and bioactive lipids between SLE prone NZM2328 females and aged matched and
sex matched C57BL/6 mice. The macrophage phenotypes (e.g., M1 and M2) of differentiated
macrophage will be compared. These BMDM will undergo in vitro stimuli, and then evaluated
for their phenotypes when NAP1051 and ATLA treatment is added. The impact of SPMs and
their ability to immunomodulate efferocytosis is interrogated (Chapter 2). In subsequent
chapters, the molecular and pharmacological impact will be assessed in-vivo using NZM2328
mice that are treated with these SPMs systemically. We use the SLE prone NZM2328 for its
well characterized SLE disease initiation and progression to LN by 4-4.5 months of age (Ge et
al., 2013). To this end, we compared the impact of NAP1051 to determine whether its
administration can alter disease course. Histological and biochemical and molecular dissection

33
showed disease attenuation corresponding with plasma and kidney biolipid profiles (Chapter
3). To affirm our findings using NZM2328, MRL.lpr was also used to determine whether our
findings work through promoting macrophage mediated elimination of ACs and cellular debris.
In addition, we evaluated whether treatment with NAP1051 can realign mesenchymal stem
cells in the bone marrow to allow them to promote kidney regenerations. To evaluate these
audacious hypotheses, we used MRL.lpr prone SLE where disease onset is seen by 8-10 weeks
of age. Unlike NZM2328, MRL.lpr mice clinical presents with cutaneous lesions. This study
also allowed us to determine the optimal dosing strategy and the impact of NAP1051 on
proteinuria, glomerulonephritis, lymphocyte infiltration and inflammatory gene expression
was closely assessed (Chapter 4). In Chapter 5, I will overview our findings and point to
next steps in hopes to develop effective therapy for patients with SLE.

34
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Chapter 2: Identification and Alterations in Macrophage Profiles and its
Contribution to Systemic Lupus Erythematosus in NZM2328 mice.
Sections of this chapter contain figures and language that were copied and pasted or adapted
from one of my own first-authored original research article currently in preparation (Dave. P
et al., 2025)
2.1 Introduction
2.1.1 Understanding macrophage phenotype and their functions
The origin of all circulating and tissue macrophages are derived from bone marrow
hematopoietic stem cells (J. Yang et al., 2014). The formation of mature macrophages is
governed by a highly coordinated cytokine-mediated differentiation and maturation. This
highly regulated process is key in maintaining systemic sterility and homeostasis, where
dysfunction has been linked to a number of chronic diseases (Belchamber & Donnelly, 2017;
Gerasimova et al., 2022).
The importance of functional macrophages is predicated on its vast and highly versatile
biological properties including antigenic elimination and cellular debris clearance. The
elimination of foreign intrusion is mediated through macrophage mediated phagocytosis,
where antigen presentation with MHC II will lead to activation of the inflammatory pathway.
However removal of macrophage removal of apoptotic cellular (AC) debris is governed by
non-phlogistic phagocytosis or efferocytosis (Doran et al., 2020). In this process, the
phagocytosis does not activate the immune cascade, but rather promotes restoration back to
homeostasis.
Macrophages are pivotal players in the innate immunity, where their biological
activities include identifying and reacting to invading pathogens. This process is initiated by
phagocytosis of the foreign antigen or pathogen, where the processing of the foreign antigen
will be presented to T-lymphocyte, where co-stimulation with stimulator can promote immune

45
cascade activation (Guerriero, 2019). This illustrate the crucial link between innate and
adaptive immunity, which is facilitate by the presentation of both endogenous and exogenous
antigens. The ability to distinguish endogenous versus exogenous antigen can dictate the type
of response, which highlight the importance of the MHC II and co-stimulating signals. This
ability to distinguish foreign antigen is vital for activating appropriate adaptive immune
responses against diseases such as infections while preventing autoimmunity to emerge (Pan
et al., 2020).
Following macrophage processing of foreign antigens, and presentation to Tlymphocytes along with co-stimulation of B7/CD28 can unleash a barrage of responses. This
antigen presentation can promote release of antimicrobial agents to combat pathogens,
chemokines to signal immune cells to the site of inflammation, and pro-inflammatory cytokines
to amplify the inflammatory cascade, ultimately paving the way for the induction of adaptive
immune responses tailored to the specific invading pathogen. Interestingly, DAMPs binding
onto toll like receptors (TLR) can promote both inflammatory and pro-resolving properties
(Piccinini & Midwood, 2010). Secondary DAMPs such as oxidized phosphatidylserine or
annexin A1 activation of macrophages can shift macrophages to a pro-resolving phenotype,
leading to termination of inflammatory signals and promote tissue resolution (McArthur et al.,
2020).
These type of macrophage responses have led some to classify macrophages according
to their biological phenotypes as either M1 or M2. M1 macrophages promote inflammation
cascade through elaboration and secretion of pro-inflammatory cytokines promoting proinflammatory activities (Meng et al., 2022). In contrast, M2 macrophage promotes cellular
debris uptake and elimination, thus promote pro-resolving properties through elaboration of
IL-4 and IL-10 (Doran et al., 2020).

46
These functional phenotypes are influenced by various signals, which determine the
macrophage phenotypes which is distinguished by its transcriptional and metabolic profiles.
Despite this classification, recent studies have shown that macrophage phenotype is not a fixed,
but rather these phenotypes are dynamic and plastic process (Shapouri-Moghaddam et al.,
2018). These phenotypes will also dictate macrophage’s role in antigen presentation to Tlymphocytes and facilitating T cell proliferation through the surface co-expression of
molecules like CD86 and MHCII (Bandola-Simon & Roche, 2023). Given its pivotal role in
bridging the innate and adaptive immune systems, macrophages offer promising avenues for
disease prevention, relapse reduction, and the formulation of novel therapeutic interventions.
2.1.2 Role of Macrophages in SLE
Recent studies have highlighted the pivotal role of macrophages in the pathogenesis of
SLE. In SLE patients and mouse models, macrophages exhibit pro-inflammatory M1
phenotype or an inefficient levels of anti-inflammatory M2 phenotype (Yunna et al., 2020).
This altered macrophage polarization contributes to a persistent inflammatory environment and
defective tissue repair. M1 macrophages in SLE contribute to the production of proinflammatory cytokines and promote the activation of autoreactive lymphocytes, leading to the
formation of immune complexes that deposit in tissues such as in lupus nephritis (S. Yang et
al., 2023). Macrophages with Fc receptors bind onto immune complexes stimulating the release
of additional pro-inflammatory mediators, thereby exacerbating tissue injury (Dalby et al.,
2020). In contrast, M2 macrophages in SLE appear to lose their ability to resolve inflammation
effectively. While they retain their ability to clear apoptotic cells, they fail to transition into a
fully pro-resolving state, and thus perpetuating chronic inflammation instead of promoting
healing (Ahamada et al., 2021).

47
The imbalance between M1 and M2 macrophages contributes to the long-term tissue
damage observed in SLE, highlighting the importance of restoring macrophage homeostasis
for disease management as overviewed in Figure 2.1. Our hypothesis is that there is
paradoxical features in the bone marrow derived macrophages (BMDMs) from SLE prone mice
model. Macrophages exhibit immune activation despite expressing anti-inflammatory surface
biomarkers.
Figure 2.1: Disrupted macrophage polarization profiles in SLE-prone mice BMDMs contributing to
autoimmunity. (created using Biorender.com)
2.1.3 Impact of Lipoxin A4 on Macrophage Subsets
Most of the inflammatory research has been focused on the role and interaction of
inflammatory cytokines. While these are important molecules important in regulating the
immune system, however, emerging evidence of additional inflammatory mediator include the
bioactive lipids as important players in the pathogenesis of SLE.

48
These lipids are being studied for their vast role in contributing to inflammation.
Arachidonic acid (AA) is an omega-6 (n-6) polyunsaturated fatty acid (PUFAs) which is
precursor that can be metabolized by cyclooxygenases (COX 1/2) and 5-LOX to form
prostanoids, pro-inflammatory mediators. Prostaglandins (PGs), leukotrienes (LTs) and
thromboxanes are some of the pro-inflammatory lipids that have been associated with a wide
range of physiological processes such as pain processing, blood clot regulation, altering
vascular permeability and have been implicated in numerous inflammatory disease states
(Cuzzo & Lappin, 2024; Ricciotti & FitzGerald, 2011).
In contrast there is a pro-resolving lipid downregulates inflammation and restores tissue
homeostasis. These pro-resolving bioactive lipids are known as specialized pro-resolving
mediators (SPMs) that include lipoxins (LXs), resolvins (Rvs), neuroprotectins (NPs) and
maresins (MaRs). As discussed in Chapter 1, lipoxin A4 (LXA4), is one of the SPMs that
promotes pro-resolution mechanisms, including tissue homeostasis and regenerative processes.
LXA4 can bind onto formyl peptide receptor-2 (FPR2/ALXr) to promote macrophage
mediated efferocytosis of apoptotic cells to promote pro-resolving properties (Ge et al., 2020;
Saqib et al., 2018). A study done on RAW264.7 showed that LXA4 was able to successfully
able to mitigate polarization to M1 macrophages in-vitro (Sanchez-Garcia et al., 2023). LXs
also regulate neutrophil chemotaxis, adhesion and transmigration. Similarly, along with LXA4,
an alternate SPM can form via aspirin-mediated alteration of COX-2 (Tsai et al., 2021). Aspirin
is a non-steroidal anti-inflammatory drug (NSAIDs) and is renowned for its effectiveness in
alleviating fever, pain, and inflammation by inhibiting the COX enzymes responsible for PG
synthesis (Vane & Botting, 2003). This inhibition occurs through the irreversible acetylation
of a serine residue on COX-1, preventing the formation of both PGs and TXs (Serhan &
Chiang, 2008). However, aspirin acetylation of COX-2 can convert it into lipoxygenase like
enzyme capable of metabolizing AA to 15R-HETE. Further metabolism of 15R-HETE can

49
form 15-epi-lipoxin or aspirin triggered lipoxin A4 (ATLA) and 15-epi-lipoxin B4 (Khoshbin
et al., 2023). While ATLA is considered to be more potent and longer acting than LXA4,
ATLA also has a short half-life of a few minutes which would make it unsuitable to be used as
a therapeutic agents against chronic inflammation (Liu et al., 2018).
Unfortunately, the systemic half-life of LXA4 is short and thus is rapidly cleared as
mentioned in Chapter 1 (Chandrasekharan & Sharma-Walia, 2015). This greatly limits its
ability to be a potent anti-inflammatory drug to combat sustained inflammation. To navigate
these challenges, our group synthesized and characterize a biomimetic analog of LXA4,
NAP1051. NAP1051 had increased stability with the benzene ring along with the methyl ester
group increasing its pharmacological activity making it a pro-drug.
NAP1051 was found to preserve key in vitro LXA4 activities such as induction of
neutrophil apoptosis and promote macrophage clearance of apoptotic neutrophils leading to
tissue resolution (Saas et al., 2022). NAP1051 can also mediate resolution of NETosis by
preventing neutrophil chemotaxis and macrophage efferocytosis (Dong et al., 2021). Herein,
we explored the protective roles of NAP1051 in interferon gamma (IFN-γ) +
lipopolysaccharide (LPS) or IL-4+IL-10 stimulated macrophages from both NZM2328 and
C57BL/6J models. We chose NZM2328 mice, as female NZM2328 mice begin to manifest
histologic features of renal disease such as crescentic glomerulonephritis, lymphocyte
infiltration into the kidney and proteinuria by 4-5 months in age compared with healthy age
and sex matched C57BL/6J mice. NZM2328 mice with proteinuria levels higher than
100mg/dL were chosen for the experiments conducted in section 2.3. These models were
employed to compare the macrophage polarization profiles upon stimulation with LPS + IFNg or IL-4+IL-10. Additionally, since we have mentioned that SLE is driven by the accumulation
of apoptotic cells and DAMPs, we also evaluated efferocytic profiles and compared diseased
NZM2328 BMDM with healthy age and sex-matched animals.

50
2.2 Methods
2.2.1 Mouse BMDM Collection
NZM2328 and C57BL/6 mice were euthanized and collected hindlimb bones in 1xPBS
at room temperature for ex vivo BMDM derivatization. The mouse was sprayed with 70%
ethanol, thoroughly. A small incision was made under the belly and the skin was peeled away.
The tibia and femur were collected put into a 1.5mL tube with proximal end facing
down. The bone marrow cells were separated from the bones by using tabletop centrifuge at
max speed for 5 seconds into the 1.5mL tube. The bone marrow was inspected visually, and
the bones appeared white and there was a large red pellet in the tube. The cells were then
resuspended in 1mL RBC lysis buffer and left in the dark for 1 minute. The cells were then
passed thoroughly through 70um filter using a syringe plunger and neutralized using 39mL
1xPBS. The cells were then spun down at 300xg for 5 minutes and counted.
2.2.2 BMDM differentiation and polarization
The cells were resuspended at 1 x 10 cells/mL in IMDM media + M-CSF at 100ng/mL
and plated into a 10-cm cell culture treated plate to begin BMDM differentiation (Day 0). Each
plate received 10 mL of cells totaling to 10 million cells per plate. On Day 6 of differentiation
the BMDM polarization for M0, M1, and M2 cells was continued. The IMDM media was
slowly aspirated and new pre-warmed BMDM polarization media was added to the cells.
To stimulate M1, 20 ng/mL LPS and IFN-g of each was added and incubated for 24 hours. To
stimulate M2 phenotype, 20 ng/mL of IL-4 and IL-10 of each were added and allowed to be
incubated for 24h. Stimulated BMDM were then harvested and processed for analysis.
2.2.3 Flow cytometry
Cells undergoing flow cytometric measurements were centrifuged and cell culture
removed and washed with pre-warmed flow staining buffer (PBS+ 10% FBS) and 0.01%
sodium azide. The cells were scrapped if need and preincubated with Human BD Fc Block™

51
purified anti-human CD16/CD32 mAb at 4˚C for 5 minutes. Then the antibody of interest
directly to preincubated cells in the presence of Human BD Fc Block™ (Mouse BD Fc Block™
need not be washed off before staining cells). Final antibody concentrations (10 µg/mL). The
cells were labelled with F4/80+CD86+ for M1 macrophages, F4/80+CD206+ for M2
macrophages. The cells were incubated in the dark for 30 min at 4˚C. The cells were washed
twice and then resuspended in flow staining buffer and run on FACS Fortessa X-20 benchtop
flow cytometer (Becton, Dickson and Company, Franklin Lakes, NJ). These samples were then
analyzed using the FlowJo software V9 (FlowJo, LLC).
2.2.4 Gating strategy
The cells were gated as summarized in Figure 2.2. The cells were gated for live cells,
followed by F4/80+ subset population, and then gated for F4/80+CD86+ to quantify the M1
macrophages. In contrast, F4/80+CD206+ was used to quantify the number M2 macrophages.
Figure 2.2: Gating strategy for BMDMs to identify F4/80+ subset population and then gated at
F4/80+CD86+ for M1 macrophages, F4/80+CD206+ for M2 macrophages.
2.2.5 RNA extraction and cDNA synthesis and Gene Expression of BMDM.
RNA was extracted according to the TRIzol Kit (Thermo Fisher Scientific, Waltham,
MA) instructions and the and concentration was determined via the NanoDrop™

52
spectrophotometer (Thermo Fisher Scientific, Waltham, MA). cDNA was prepared using the
RevertAid™ First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA)
following the manufacturer’s protocol. The RT-qPCR master mix was prepared by mixing
PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Foster City, CA) according to
manufacturer’s instruction. The RT-PCR master-mix was prepared for each gene by mixing
SYBR Green master-mix (Applied Biosystems, Foster City, CA) with forward and reverse
primers. The run method is described in below in Table 2.1. Samples were run in quadruplicate, and results were
analyzed using data assist software (Invitrogen, Carlsbad, CA). The ΔΔCt values were
calculated and plotted as fold changes using GraphPad Prism (GraphPad, San Diego, CA).
Cycle Stage Temperature and Time
Initial Stage 50°C for 2 minutes
Intermediate Stage 95°C for 2 minutes
PCR Amplification 40 cycles
Melting Step 95°C for 1 second
Annealing Stage 60°C for 30 seconds
Melt Curve
Stage/Dissociation Stage
1.6°C to 95°C for 15 seconds; 1.6°C to 60°C for 1 minute; 0.15°C
to 95°C for 15 seconds
Table 2.1: RT-PCR run method with temperature and times that were used to run NZM2328
and C57BL/6J mouse BMDM samples.
2.2.6 Efferocytosis Assay
Mouse BMDM cells were seeded and differentiated in 12-well tissue culture plates
using IMDM culture media with 10% Fetal Bovine Serum (FBS), 1X Glutamax and 1X Nonessential amino acids, at a density of 0.5 x 106 cells per well. On day 5-7, the differentiated
BMDMs were pre-incubated with designated treatments for 1 hour in serum-free RPMI-1640.
Apoptotic dHL-60 cells were produced by incubating cells 4.5 x 106 cells/3mL in 1 µM
staurosporine (STS) for 3 h at 37 °C, 5% CO2, followed by labeling with Hoechst 33342
solution (20 mM, 1:10000 dilution) for 30 min. This method consistently yields more than 90%

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Annexin V+ apoptotic dHL-60 cells. Hoechst-labeled apoptotic cells were then co-incubated
with BMDMs at 5:1 and 15:1 AC: BMDMs ratio for 1 h at 37 °C, 5% CO2.For flow cytometrybased quantification, the BMDMs were washed with 1X DPBS 3-4 times to remove unengulfed
apoptotic cells, lifted using cell stripper (Corning, NY), stained with PE-conjugated F4/80
antibody (Biolegend, CA), and prepared as single-cell suspensions in flow staining buffer.
2.2.7 Efferocytosis Imaging
dHL-60 cells were collected, and the cell concentration was adjusted to 1 × 10⁶
cells/mL. The cells were then incubated in 1 µM Staurosporine at 37°C with 5% CO₂ for 18
hours in IMDM media as mentioned above. For DiI staining of apoptotic dHL-60 cells, the
cells were collected and adjusted to a concentration of 1.65 × 10⁶ cells/mL in serum-free
DMEM/F12 media. Five microliters of the cell-labeling solution were added per mL of cell
suspension, mixed gently, and incubated for 20 minutes at 37°C. The cells were then washed
and added to the BMDMs. For treatments on BMDM, the medium was aspirated, and the cells
were washed. Then, 500 µL of warm serum-free RPMI was added to each well, followed by
the addition of treatments in quadruplicate wells. Apoptotic dHL-60 cells were then added at a
target-to-effector ratio of 5:1. The slides were covered and incubated for 90 minutes at 37°C
with 5% CO₂, followed by two washes with pre-warmed PBS to remove unbound cells. The
cells were fixed with 10% neutral buffered formalin (NBF) for 15 minutes at room temperature
and washed twice with warm PBS. For actin and DAPI staining, a staining solution was
prepared by diluting 0.5 µL of DMSO stock in 200 µL of PBS for one well, and 5% goat serum
with 0.1% Triton X was added to reduce non-specific background staining. The cells were
incubated in the staining solution for 30 minutes at room temperature in the dark, followed by
the addition of 1 µg/mL DAPI in PBS and a 10-minute incubation at room temperature in the
dark. This was followed by mounting sections, the chamber was carefully removed from the

54
wells, and excess water was blotted while keeping the specimen moist. VECTASHIELD
Vibrance Antifade Mounting Medium was dispensed onto the specimen and coverslip, with a
recommended drop volume of approximately 25 µL per 22 mm x 22 mm coverslip. The
mounted samples were placed on a flat, dry surface in the dark to allow the medium to cure.
Slides were viewable after 30 minutes, but optimal antifade performance was achieved after 2
hours, with a refractive index of 1.47 upon full curing (24 hours). For long-term storage, slides
were stored at room temperature or refrigerated.
2.2.8 Lipidomics sample extraction, processing and analysis
Lipidomic profiling of BMDM, serum, and kidney used a modified validated targeted
liquid chromatography-mass spectrometry (LC-MS) based lipidomics assays as shown by
(Ebright et al., 2022). Approximately 50–100 mg of each tissue sample was weighted and
information recorded. Lipids were extracted by adding 250-500 μL methanol. Internal
standards (d5-RvD2, d8-5S-HETE, d4-PGE2, d5-LXA4, and d4-LTB4) were added, and the
samples were homogenized using a TissueLyser (Qiagen) and homogenized using three bursts.
After centrifugation, the supernatant was diluted with water to a final concentration of (1:9
ratio) 10% methanol and further extracted using pre-conditioned Strata X 33μm Polymeric
Reverse Phase cartridges. The samples were washed with 1 column volume of water and the
lipid components were eluted with methanol. The collected lipid faction was evaporated to
dryness and reconstituted in 50% methanol to a total volume of 50 µL. The analytes were
separated and quantified using an Agilent 1290 UPLC linked to a QTRAP Sciex API6500+
LC-MS/MS system, with a Poroshell 120 EC-C18 column and a specified gradient of water +
0.01% formic acid (mobile phase A) and methanol + 0.01% formic acid (mobile phase B). The
flow rate was 0.5 mL/min, and the column temperature was maintained at 40°C. Analytes of
interest included pro-inflammatory lipid metabolites such as leukotrienes, prostaglandins, and

55
thromboxanes, as well as specialized pro-resolving mediators like lipoxins, resolvins,
neuroprotectins, and maresins.
Peak selections were manually reviewed and selected where the area under the curve
(AUC) values were normalized by protein concentration or sample volume. These values were
further adjusted for batch-to-batch variability and sample loss using internal standards.
Statistical analysis and figure generation were performed using R and GraphPad Prism, with
group comparisons made using one-way ANOVA or two-tailed unpaired t-test when
appropriate.
2.2.9 Statistical Analysis
Results were expressed as percentage for BMDM M1/M2 classification, efferocytosis
and as fold change for RT-PCR heatmaps and graphs. The programs R and GraphPad Prism
(GraphPad, San Diego, CA) were used to perform the statistical analysis and generate the
figures. Statistical significant threshold was a priori set with an alpha of 0.05.
2.3 Results
2.3.1 Characterization of Polarized Macrophages comparing NZM 2328 with C57BL/J:
To characterize macrophage phenotypes following differentiation with IFN-γ + LPS,
IL-4, and IL-10, we conducted surface marker analysis using flow cytometry as shown in
Figure 2.3 B–D. Macrophage marker F4/80+ cells were not different between NZM2328
versus C57BL/6 in unstimulated BMDMs (Figure 2.3B).
NZM2328 mice exhibited a distinct difference in macrophage profile compared to
C57BL/6J mice. LPS+IFN-γ stimulation showed that NZM2328 had less CD86+ cells (~40%)
as compared to CD86+ from BMDM C57BL/6 (~80%) (Fig 2.3C, ****p-value>0.0001). The
NZM2328 BMDM were less like to be stimulated by LPS and IFN-γ treatment to form M1
phenotype.

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When exposed to IL-4 and IL-10, BMDM tend to undergo differentiation towards the
M2 phenotype, marked by the expression of CD206 (Xu et al., 2020) (Azad et al., 2014; Makita
et al., 2015). Notably, NZM2328 mice exhibit a more robust response to these stimuli
compared to C57BL/6J mice (Figure 2.3 D, ***p<0.001). This observation emphasizes the
influence of the disease state on inter-species differences, with C57BL/J mice showing a more
subdued response to the IL-4+ IL-10 stimuli. This once again highlights the involvement of
macrophage dysfunction and activation in the manifestation of disease disparities between
species.
Figure 2.3: CD86 and CD206 identify M1- and M2-like cells respectively in stimulated mouse
BMDM. (A) For in-vitro polarization; mouse BMDM were differentiated for 6 days with M-CSF and
then stimulated with LPS+IFN-g or IL-4+IL-10. After 24 hours cells were analyzed by flow
cytometry and (B) M0 BMDM for all unstimulated control macrophages to observe differences
between NZM2328 and C57BL/J mice (C) M1-like macrophages stimulated with LPS+IFN-g (D)
M2-like cells were quantified after stimulation with IL-4+IL-10. Unpaired two-tailed t-test was used
in (B, C, D) ***p < 0.001, ****p < 0.0001.

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2.3.2 SPMs Mediated Transition from M1 to M0 Phenotype
To determine what effect, if any, would NAP1051 treatment have on macrophage
phenotypes. These effects were compared between BMDM derived from NZM2328 and
compared to C57BL/6J stimulated with either LPS+IFN-g or IL-4+ IL-10 stimulation. Figure
2.4 A gives us an overview of all the different cell types present based on the type of stimuli.
Stimuli included LPS + IFNg treated with NAP1051with treatment and ATLA without and we
indeed saw a reduction in CD86+ cells (‡p<0.0001). The co-administration of 1 and 10 µM
NAP1051 reduce percentage of CD86+ from ~80% to about ~35% and 31%, respectively, in
C57BL/6 BMDM. Similarly, ATLA 1 and 10 µM was able to reduce C57BL/6 CD86+ cells to
~29% for both ALTA concentrations. This demonstrates that the effect of NAP1051 was
comparable to the outcomes reported for LXA4 in previous studies (Dong et al., 2021).
Surprisingly, upon testing these SPMs with IL-4 and IL-10 “alternatively activated” M2
macrophages, we observed that the macrophages retained their M2 or CD206 signature for
C57BL/6J mice, however when ATLA and NAP1051 were added at a 1µM concentration to
the NZM2328 BMDMs stimulated with IL-4 and IL-10, these macrophages reverted to M0
phenotype. This showed that SPMs were only active/had pharmacological effect on diseased
mice BMDMs. Investigating the mechanisms of how it does this would be a future endeavor
and a current limitation of this study.

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Figure 2.4: (A-F) Schematic view of M0, M1 and M2 activation and the effect of ATLA at 1µM-10
µM and NAP1051 1µM-10 µM effect on polarization. BMDMs were labeled with CD86, CD206
antibodies and then analyzed via flow cytometry. (A) LPS and IFN-g followed by treatment with (B)
NAP1051 1uM for 24 hours. (C) ATLA 1uM for 24 hours. Both drugs showed transition from M1 to
M0 phenotype. (D) IL-4 and IL-10 treated macrophages were treated with (E) NAP1051 and (F)
ATLA at 1µM concentration for 24 hours. (n=6) Data was analyzed using unpaired t-test (**p < 0.01,
****p < 0.0001).
2.3.3 mRNA Profiling of Bone Marrow Derived Macrophages.
To further characterized the difference between NZM2328 and C57BL/6J, gene expression
of proinflammatory marker and pro-resolving markers were compared, where C57BL/6J
served as reference. Proinflammatory genes were upregulated while pro-resolving genes were
downregulated in the NZM2328 (*p<0.05, **p<0.01). Notably iNOS was upregulated, while
Arginase-1 was downregulated in NZM2328 which is a maker for macrophage polarization
states shifting more towards the pro-inflammatory state. This is further confirmed by proinflammatory markers such as TNF-α, IL-6, IL-1β, and IFN-γ were upregulated in NZM2328
mice (Figure 2.5 A), while pro-resolving cytokines like IL-10, TGF-β, and PPAR-γ were
downregulated. PPAR-γ has been well established to activate the M2 macrophage phenotype
(Abdalla et al., 2020). This indicates a heightened disease state in NZM2328 mice. While
proteins and cytokines have been associated with inflammation. Less is known as to the role

59
of inflammatory mediators in SLE. Targeted genes such as cPLA2 regulate the level of
arachidonic acid (AA), the precursor of inflammatory lipids like prostaglandins and
thromboxanes (TBX2). AA is metabolized by COX isoforms such as COX-1/2, only COX-2,
the inducible enzyme was upregulated but not statistically significant. Further downstream
enzyme such as PGE2 synthetase were upregulated (**p<0.01, Figure 2.5), while PGD2
synthetase was downregulated (*p<0.05, Figure 2.5). We also evaluated the n-3 fatty acid
synthesis pathway where iPLA2 was upregulated (**p<0.01, Figure 2.5 A). However,
lipoxygenase enzymes such as ALOX-5 and -15, where ALOX-15 was statistically reduced
(p<0.001). ALOX-15 has been shown to be anti-inflammatory due to its role in biosynthesis of
resolvins. Lastly resolvin E2 receptor ChemR23 or CMKLR1 in mice, reprograms
macrophages to an anti-inflammatory phenotype (Lavy et al., 2023), was downregulated
compared to the healthy C57BL/6J counterpart. Figure 2.5 C shows the bar graph illustrating
renal disease z-scores (-log2(p-value)) in NZM2328 versus C57BL/6J. Ingenuity pathway
analysis predicted the propensity for NZM2328 mice to have the listed disease conditions as
opposed to its healthy counterpart.
Interestingly, the group treated with NAP1051 at 1uM exhibited similar characteristics
to that of M2 macrophages treated with IL-4 and IL-10, aligning with NAP1051's known antiinflammatory properties. Post-treatment with 1uM NAP1051 following LPS and IFN-g
exposure demonstrated efficacy, as evidenced by significant downregulation of TNF-a, IL-1b,
and IL-6 when compared with only LPS and IFN-g stimulated groups. Notably, IFN-g levels
remained unchanged post-treatment due to prior external treatment with IFN-g. Levels of IL10 post-NAP1051 treatment at 1uM were significantly upregulated, resembling those of IL4+IL-10 treated groups. Additionally, upregulation of TFG-b and FPR2 was observed
compared to the LPS+IFN-g treated group, with FPR2 being a receptor for lipoxins like

60
NAP1051, suggesting its role in modulating inflammation. These findings collectively
establish a distinct M1 vs. M2 macrophage phenotype profile, demonstrating NAP1051's
ability to mitigate some effects of LPS and IFN-g when administered as a post-treatment for
24 hours.
Figure 2.5: Macrophage mRNA characterization in NZM2328 and C57BL/6J with and without
stimulation. (A) Unstimulated BMDMs. Triplicates were subjected to qPCR to determine expression
of pro-inflammatory cytokines (IL-1b, IL-6, TNF-a and IFN-g, iNOS and CXCR2) and pro-resolving
cytokines (IL-10 and TGF-b, Arginase-1 and PPAR-g). Lipoxygenase enzymes (ALOX-5, ALOX-15,
sEH, COX-1 and COX-2) and lipoxin and resolvin E receptors (FPR2/3 and ChemR23) were
evaluated as well. Log10 fold change was plotted where reference was C57BL/6J control vs NZM2328
BMDMs. Data was analyzed using unpaired t-test (*p<0.05; **p<0.01; ***p < 0.001;
****p < 0.0001). (B) Triplicates were subjected to qPCR to determine expression of genes from
NZM2328 mice after addition of NAP1051. One way ANOVA with Dunnett’s multiple comparisons
test was used for statistical analysis **p < 0.01, ***p < 0.001, ****p < 0.0001. NZM2328
unstimulated BMDM was used as control. (C) IPA analysis showing a bar graph illustrating renal
disease z-scores (-log10(p-value)) in NZM2328 versus its healthy counterpart C57BL/6J.
2.3.4 Differences in efferocytic surface markers and profiles for NZM2328 versus
C57BL/6J BMDMs

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Next to check if there was truly M2 dysfunction macrophages we probed the efferocytic
potential of unstimulated macrophages. In SLE there is macrophage (M1/M2 subsets)
dysfunction leading to impaired clearance resulting in inflammation. Our results, using 1
BMDM to 5 apoptotic cells (ACs) showed that BMDMs from NZM2328 mice exhibited a
higher level of apoptotic cell engulfment compared to BMDMs from C57BL/6J mice
(***p < 0.001). The high efferocytic activity observed in NZM2328 mice across the board
suggests that their macrophages are pre-primed for engulfment, as these macrophages were
observed to engulf multiple cells our image analysis (Figure 2.6 C). Knowing that SLE
patients often have an increased AC burden we increased the BMDM: AC ratio to 1:10 to
mimic SLE conditions. As expected, the NZM2328 macrophages could not engulf ACs and
reduced efferocytosis rate by. half (@16%). Remarkably C57 BMDMs continued engulfing
ACs at the same rate regardless of AC burden (Figure 2.6 B).
To confirm if these BMDM were able to “digest” the apoptotic cells after efferocytosis
we probed the lysosomal pH. pHrodo red was used to probe pH after efferocytosis. C57BL/6J
vs NZM2328 pHrodo+ staining reveals that the NZM2328 mice have a higher level of
acidification with 1:5 ACs and almost equal acidification at 1:10 AC ratio, indicating that it
may not be a phagolysosome dictated disease. We also chose to look at the efferocytic cell
surface signals (Figure 2.6 G), on the macrophages to examine what is causing the
significant decreases in efferocytosis for the diseased state at 1:10 ACs. We see that alpha V
beta integrin (aVb3), myeloid-epithelial-reproductive tyrosine kinase (MerTK) and AXL
(TAM family) were all downregulated (Figure 2.6 G) further evidencing the disease state in
NZM2328 mice.
In addition, we evaluated the signals that promotes efferocytosis. T-cell
immunoglobulin and mucin domain-containing protein 4 (TIM-4) is a phosphatidylserine
receptor involved in the recognition and clearance of apoptotic cells (efferocytosis).

62
Upregulation of TIM-4 suggests NZM2328 BMDM can more efficiently engaged in
efferocytosis through recognition of apoptosis which may promote clearance cellular debris
and helping to resolve inflammation. We see that the traditional efferocytosis signals such as
aVb3 integrin, MerTK and AXL were all downregulated and could explain why NZM2328
BMDMs could not engulf more cells at a 1:15 AC burden. Additionally, sex-hormone binding
globulin (SHBG) is upregulated significantly (Figure 2.6 G). Estrogen tends to push
macrophages toward the anti-inflammatory/M2 phenotype, which under normal circumstances
is beneficial for tissue repair (Enright & Werstuck, 2024). However, in the context of SLE,
these M2 macrophages may fail to resolve inflammation properly, contributing to chronic
inflammation and tissue damage. In NZM2328 mice, it has been noted that TIM4 and SHBG
are significantly upregulated, suggesting a link between these molecules and the disease's
progression. Additionally, NZM2328 BMDM were found to have downregulation of Signal
Regulatory Protein Alpha (SIRP-α) in macrophages which may indicated changes in
phagocytosis activity. It acts as an inhibitory receptor and interacts with its ligand, CD47
functions as an "immune checkpoint," essentially sending a "don't eat me" signal to
macrophages to inhibit the phagocytosis of healthy or self-cells (Alblas et al., 2005). This
suggests that NZM2328 macrophages are overly permissive to apoptotic cell clearance, further
fueling the inflammatory environment (Figure 2.6 G).

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Figure 2.6: (A) In vitro BMDM efferocytosis of apoptotic dHL-60s (1:5). Levels for NZM2328
diseased mice were significantly higher (>2x) compared to their healthy counterpart. (B) In vitro
BMDM efferocytosis of apoptotic dHL-60s (1:10). Levels for NZM2328 diseased mice significantly
reduced from 1:5, C57 mice remained relatively stable. (C) Macrophage mediated efferocytosis of ACs
images showing the Green: macrophages engulfing the Red: ACs. (D) pHrodo+ cells showing
lysosomal acidification of the macrophage phagosome after engulfment of apoptotic dHL-60s in
NZM2328 BMDM compared to C57BL/6J. (E) pHrodo+ cells showing lysosomal acidification of the
macrophage phagosome after engulfment of apoptotic dHL-60s (1:10) (n=4) (F-G) RT-qPCR of
macrophage cell surface receptors that facilitate AC uptake. Receptors were significantly
downregulated. Data was analyzed using unpaired two tailed t-test (**p < 0.01, ***p < 0.001,
****p < 0.0001). Pictures were taken with a 20X objective (scale bar 100 μm)
2.3.5 SPMs stimulated macrophage mediated efferocytosis in NZM2328 and C57BL/J
mice:
Recent studies have highlighted the potential of SPMs such as Lipoxin A4 (LXA4) and
naturally occurring ATLA in promoting pro-resolution mechanisms, including tissue
homeostasis and regenerative processes. LXA4 binds to the formyl peptide receptor-2
(FPR2/ALXR) to enhance macrophage-mediated efferocytosis of apoptotic cells (ACs).

64
However, the short systemic half-life of LXA4 limits its therapeutic potential (Saqib et al.,
2018). To address this limitation, a small molecule biomimetic of LXA4, NAP1051, was
developed to extend its elimination half-life. NAP1051 retains key activities of LXA4, such as
inducing neutrophil apoptosis and promoting macrophage clearance of apoptotic neutrophils,
thereby facilitating tissue resolution (Saas et al., 2022).
Additionally, NAP1051 enhances efferocytosis, similar to ATLA in a dose dependent
manner (Dong et al., 2021). Efferocytosis, the process by which apoptotic cells are engulfed
and cleared by phagocytes, is crucial for maintaining tissue homeostasis and immune
regulation. Mouse BMDMs were cultured and exposed to apoptotic dHL-60 cells and our
results demonstrate that treatment with NAP1051 and ATLA significantly enhances the
efferocytic capacity of mouse BMDMs compared to untreated controls. Based on this we also
studied efferocytic potential of pre-treated BMDMs with ATLA and NAP1051 at similar
concentrations. First, we used 1:5 BMDM to AC ratio (Data not shown). BMDMs from
NZM2328 mice pre-treated with either NAP1051 or ATLA did not show an increase in
efferocytosis of apoptotic cells compared to untreated controls; in fact, their efferocytic ability
tended to decrease or stay the same for dHL-60 cells. This led us to further investigate the 1:10
AC burden ratio and we see an increase in efferocytic potential for both 1 and 10µM NAP1051
treated groups in C57BL/6J and NZM2328 mice (Figure 2.7A and 2.7B). Lysosomal pH
during efferocytosis with and without SPM treatment was probed as well and we see that with
1µM and 10 µM ATLA the pH significantly increases (***p < 0.001, ****p<0.0001) for
C57BL/6J mice (Figure 2.7E) along with NAP1051 treatments at both concentrations. 1 and
10 µM NAP1051 and 10µM ATLA both increased pH for NZM2328 mice (Figure 2.7F) as
well suggesting that these phagolysosomes may have engulfed several cells that need to be
‘digested’ and the higher threshold and acidification is achieved with addition of these SPMs.

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Flow cytometry analysis confirmed the enhanced uptake of apoptotic cells, indicating
a greater efficiency of efferocytosis in the presence of these lipoxin analogues. These results
suggest a potential therapeutic strategy for SLE and like inflammatory conditions characterized
by impaired efferocytosis.
Figure 2.7: Using BMDM:AC 1:10 ratio for all (A) In vitro BMDM efferocytosis of apoptotic dHL60 cells in C57BL/6J BMDMs. Here we see significant changes with treatment. (B) In vitro BMDM
efferocytosis of apoptotic dHL-60 cells in NZM2328 BMDMs. Here we see significant increases with
treatment. Representative efferoytosis images taken for (C) C57BL/6J mice and (D) NZM2328 mice.
Green: Actin staining for macrophages, Red: Dil staining for apoptotic dHL-60s and Blue: DAPI
staining. (E) pHrodo+ cell staining to observe lysosomal acidification in apoptotic dHL-60 cells in
C57BL/6J mice and (F) NZM2328 mice. 10µM ATLA and 1 and 10µM NAP1051 increased
acidification significantly for both animal models. Analysis was done using unpaired two-tailed t-test.
(n=4) (*p < 0.05, **p<0.01) Pictures were taken with a 20X objective (scale bar 100 μm)

66
2.3.6 Evaluating NZM2328 vs C57BL/6J BMDM, Kidney and Plasma Lipidome:
Figure 2.8 provides a comparative lipidomic analysis across different tissues—BMDM, serum,
and kidney—in two distinct mouse strains, C57BL/6J (represented in blue) and NZM2328
(represented in red). Each tissue type's data is illustrated in a dot plot, capturing the log10 fold
change of various lipid metabolites. These metabolites are primarily eicosanoids and related
bioactive lipids, which play essential roles in inflammatory and immune responses. Our
findings reveal that pro-inflammatory lipids were significantly upregulated, while proresolving lipids such as EPA and DHA were downregulated in NZM2328 mice, indicating that
the disease had manifested and disrupted lipid regulation. Enzymes associated with these lipids,
such as those responsible for the production of HEPEs and HDPAs—key precursors for
downstream resolvins—were also reduced in the NZM2328 mice compared to their healthy
counterparts. This was also backed by our RT-qPCR findings which showed a shift to cPLA2
metabolites. Unfortunately, we were unable to detect resolvins in these samples, potentially
due to the lower limit of detection in our method. In BMDMs from NZM2328 mice, we
observed elevated levels of PGE2 and PGD2, hydroxyeicosatetraenoic acids (HETE) and
thromboxanes (TBX2), which correlated with the disease state. This is further supported by the
increased expression of COX-1 and COX-2, which are responsible for the synthesis of these
pro-inflammatory lipids. 11,12 EET, which is known to be a pro-resolving lipid, its levels were
upregulated in all the compartments. Additionally, the serum lipidome of NZM2328 mice
showed a downregulation of pro-resolving lipids, including LXA4, EPA, and DPA. This
enhanced variation between C57BL/6J and NZM2328 in the serum lipidome suggests that
NZM2328 may undergo a broader systemic lipid remodeling or an intensified inflammatory
signalling cascade, as serum lipids often reflect systemic physiological states and inflammatory
responses (Dennis & Norris, 2015). Interestingly, in the kidneys of NZM2328 mice, all lipids
were elevated compared to the healthy controls, suggesting heightened disease activity. This

67
increase included not only pro-inflammatory lipids such as prostaglandins, thromboxanes, and
leukotrienes but also pro-resolving lipids like DHA, resolvins, and DPA, hinting at an
immunocompensatory response aimed at balancing inflammation and resolution. Notably,
EPA was the only lipid that did not show elevation. Given that EPA is known to inversely
correlate with disease activity, its absence of upregulation further suggests a state of high
disease activity in NZM2328 mice (Serhan & Levy, 2018). All of this tied with high levels of
HETE, TXB2, and PGD2 in NZM2328 kidney tissue further emphasizes a potential propensity
for increased inflammatory or oxidative stress responses, as these lipids are known mediators
in renal pathophysiology.
Figure 2.8: (A) Kidney lipidome comparison between C57BL/6J and NZM2328 SLE-prone mice. (B)
Serum lipidome comparison between C57BL/6J and NZM2328 SLE-prone mice. (C) BMDM
lipidome comparison between C57BL/6J and NZM2328 SLE-prone mice. Data was analyzed and
plotted as Log10 fold change compared to C57BL/6J mice. (n=3)

68
2.4. Discussion
2.4.1 Macrophage-Based Interventions in Systemic Lupus Erythematosus
The observed dysregulation in macrophage function and activation in SLE highlights the
pivotal role of these immune cells in the pathogenesis of the disease. Our study aimed to
elucidate the potential therapeutic efficacy of NAP1051, a LXA4 analogue, in modulating
macrophage polarization and attenuating inflammatory responses in the context of SLE. The
findings of this study reveal critical insights into macrophage behavior and the role of SPMs
in modulating immune responses in SLE. The comparative analysis between NZM2328 and
C57BL/6J mice highlights key differences in macrophage polarization, efferocytosis
efficiency, and lipid metabolism, emphasizing the extent to which the chronic inflammatory
state in SLE influences immune cell function. NZM2328 BMDMs demonstrated impaired
responsiveness to LPS and IFN-γ stimulation, achieving only 40-45% M1 polarization
compared to 80% in C57BL/6J controls, which suggests that the chronic inflammatory
environment in SLE primes these macrophages. We hypothesize that this resistance may stem
from the disease state, which could affect the differentiation of these pre-primed cells already
exposed to a similar stimulus, thus diminishing their responsiveness. This resistance to
polarization indicates a fundamental alteration in macrophage plasticity which is a dynamic
feature typically essential for modulating immune responses. Conversely, the same
NZM2328 macrophages exhibited strong differentiation toward the M2 phenotype when
stimulated with IL-4 and IL-10, but despite this shift, their anti-inflammatory functions were
not sufficient to resolve inflammation effectively, perpetuating tissue damage and chronic
immune activation. M2 macrophages play a pivotal role in the abnormal immune response
characteristic of the condition. M2 macrophages constitute a specific subset of macrophages
known for their anti-inflammatory properties and ability to promote tissue repair. In SLE, the
imbalance in macrophage polarization towards the M2 phenotype disrupts the equilibrium
between pro-inflammatory and anti-inflammatory reactions, thereby fueling the chronic

69
inflammation and tissue injury typical of the disease. Further, our RT-qPCR analysis revealed
that NAP1051 treatment resulted in a shift towards an anti-inflammatory M2-like phenotype
in NZM2328 macrophages, characterized by downregulation of pro-inflammatory cytokines
and upregulation of anti-inflammatory markers. This is consistent with the observed
reduction in inflammatory cytokine secretion and enhanced expression of markers associated
with tissue repair and resolution of inflammation. NAP1051 along with ATLA enhanced the
macrophage mediated efferocytosis of the mouse BMDM for both NZM2328 and C57BL/J
mice in their M0 resting state which further helped prove our hypothesis that NAP1051
treatment can help resolve SLE induced inflammation and clear apoptotic cell debris
preventing the inflammatory cascade progression.
The administration of NAP1051 and ATLA provided promising insights into the therapeutic
potential of lipoxin analogues in modulating macrophage plasticity. Both agents
demonstrated the ability to promote the transition of macrophages from an M1 phenotype to
an M0-like state, particularly in diseased NZM2328 mice, suggesting that these lipoxin
analogues could serve as pharmacological tools to mitigate excessive inflammation. The
impact of NAP1051 and ATLA on efferocytosis in NZM2328 BMDMs indicates that these
compounds enhance lysosomal acidification and apoptotic cell uptake suggesting that in
diseases like SLE, the macrophages’ prior exposure to inflammatory stimuli and altered
polarization profiles, is a therapeutic target for SPM-based therapies, requiring further
investigation into how these therapies can be optimized. The lipidomic analysis further
corroborates the role of disrupted lipid metabolism in SLE, as NZM2328 mice exhibited
significantly elevated levels of pro-inflammatory lipids, including PGE2, PGD2, and
thromboxanes, which amplify immune responses and sustain inflammation. At the same time,
there was a marked downregulation of key pro-resolving lipids, such as EPA and DHA,
particularly in the serum and BMDMs of NZM2328 mice, signalling a systemic disruption in

70
the balance between inflammatory and pro-resolving mediators. Interestingly, while some
pro-resolving lipids such as DPA and resolvins were elevated in the kidneys, reflecting an
attempt at immunocompensation, the absence of EPA upregulation in diseased mice aligns
with severe disease activity, given that EPA levels are known to inversely correlate with
inflammation.
Overall, our results support the hypothesis that NAP1051 can modulate macrophage
polarization and attenuate inflammatory responses in SLE, offering a promising therapeutic
strategy for the management of this complex autoimmune disorder. Further investigation into
the molecular mechanisms underlying the immunomodulatory effects of NAP1051 and its
potential clinical efficacy in murine/human SLE is merited.

71
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Chapter 3: Characterization of in vivo Activities of NAP1051 in SLE
Murine Model NZM2328
3.1 Introduction
Systemic lupus erythematosus (SLE) is a chronic inflammatory disease with
autoimmune characteristics. SLE is an autoantibody-associated, multi-system disease rooted
in the loss of self-tolerance to nuclear antigens. Although dysfunction of the adaptive
immune system has long been viewed as central in SLE pathogenesis, recent discoveries have
pointed to a role for dysfunction of the innate immune system. There is an imbalance
between generation of apoptotic cells and their elimination, especially with regards to
increased apoptotic neutrophils and inability to be removed through macrophage mediated
clearance. Neutrophils, the most abundant innate effector cells, serve as first-line defenders
against foreign antigenic intrusion (Lee et al., 2003). Normally, activated neutrophils are
recruited and sequestered in infected or damaged tissues, where they undergo apoptosis once
they have performed their function, and are cleared by macrophages through non-phlogistic
phagocytosis (Tecchio et al., 2014). Although neutrophils are vital in protecting against
microbial pathogens and tissue reparative processes, the inability to induce neutrophil
apoptosis or its elimination will extend neutrophil lifespan through proinflammatory cytokine
secretion. This is one of the many mechanisms that can sustain inflammation, promote tissue
injury and autoimmunity (Yu & Su, 2013).
Upon appropriate stimulation by antigenic or cytokine signals, neutrophils can expel
their DNA contents as neutrophil extracellular traps (NETs), via a process known as NETosis
(Papayannopoulos, 2018). The elevation of NADPH oxidase (NOX-2) (Röhm et al., 2014)
alongside increased activity of toll-like receptor 7 (TLR7) (G. J. Brown et al., 2022),
contributes to the production of reactive oxygen species (ROS) (Paladhi et al., 2024).
Notably, TLR7 is implicated in the pathogenesis of systemic lupus erythematosus (SLE),

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highlighting its role in autoimmune responses associated with increased oxidative stress
(Lood et al., 2017).
This oxidative burst leads to the nuclear translocation of neutrophil elastase (NE) and
myeloperoxidase (MPO) along with activation of peptidyl arginine deiminase-4 (PAD-4).
PAD-4 is activated intracellularly by calcium which leads to structural changes that activate
this protein (PAD-4, encoded by the PADI4 gene in humans and the Padi4 gene in mice) (J.
Zhang et al., 2016). PAD-4 catalyzes the deiminization of arginine residues found on histones
within the nucleus to form citrulline residues, thereby promoting chromatin decondensation
and unwinding (Rabadi et al., 2016) . Moreover, unmethylated CpG-DNA released, as a
consequence of chromatin decondensation and unwinding, activates TLR7 and TLR9
(Takeshita et al., 2001) which, as mentioned above, are also implicated in SLE pathogenesis
(Mallavia et al., 2020; Wen et al., 2023). Taken together, NETosis-driven inflammation is
emerging as an attractive therapeutic target for SLE.
Besides cytokines and chemokines, certain bioactive lipids, such as arachidonic acid
(AA) and its metabolites prostaglandins (PGs) and leukotrienes (LTs), also contribute to the
inflammatory process as mentioned previously in Chapter 1. Contrary to these inflammatory
bioactive lipids there are specialized pro-resolving mediators (SPMs), such as lipoxins (LXs),
which mediate resolution of inflammation. LXs do so by promoting non-phlogistic
phagocytosis (efferocytosis) via macrophage uptake of apoptotic neutrophils and cellular
debris (Doran et al., 2020). Lipoxin A4 (LXA4), one of the most studied LXs, is a potent
modulator of inflammation by decreasing neutrophil chemotaxis, adhesion, and
transmigration and prevents apoptosis of macrophages triggered by stimuli such as LPS and
IFN-γ, thereby supporting the resolution of inflammation (O'Meara & Brady, 1997; Prieto et
al., 2010). In contrast, LXA4 deficiency is associated with immunologic diseases such as
asthma, glomerulonephritis, rheumatoid arthritis and even SLE (Kieran et al., 2004;

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McMahon et al., 2001). In SLE research, LXA4 and its analogs have shown the ability to
suppress inflammatory cytokines. Urine LXA4/creatinine ratios can be used as effective
biomarkers for lupus nephritis and other manifestations of SLE. This is due to SLE patients
having lower levels of lipoxins in their urine and plasma compared to healthy controls while
having higher levels of LTs (Das, 2011; McMahon et al., 2001).
Unfortunately, LXs are rapidly metabolized and inactivated in vivo by eicosanoid
oxido-reductase enzymes, thereby limiting their potential therapeutic utility (Maderna &
Godson, 2009). To circumvent this problem, we have developed a LXA4 analogue,
NAP1051. The hexatriene structure of LXA4 is replaced by an aromatic ring which enhances
molecular and metabolic stability. The carboxylic acid is substituted by a methyl ester to
further stabilize the molecule and elicit a sustained anti-inflammatory effect.
To test the in-vivo effects of NAP1051 we employed the NZM232 SLE-prone murine
model. The NZM2328 mouse strain is an inbred model commonly used to study SLE. This
mouse model was accidentally generated when a New Zealand White (NZW) mouse strain
was crossbred with a New Zealand Black (NZB) F1 hybrid mice resulting in the mixed New
Zealand Mixed (NZM) strain (Li et al., 2017). In NZM2328 mice particularly, there were
four SLE genes that were identified. Cgnz1 and Agnz1, which are located on the telomeric
end of chromosome 1, are significantly linked to chronic glomerulonephritis. Two genetic
intervals on chromosome 17 were also linked to acute glomerulonephritis such as H-2-Tnf
complex and Agnz2. Lastly, a single locus Adaz1 identified on chromosome 4 in NZM2328
mice was linked to IgG and anti-dsDNA autoantibodies plasma levels. These genomic
differences between NZM strains and C57BL6J strains account for the SLE-like symptoms in
these mice (Waters et al., 2001). NZM2328 mice display aberrant production of
autoantibodies, chronic glomerulonephritis, and immune complex deposition which closely
mimic human SLE pathology. Specifically, female NZM2328 mice tend to develop more

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severe symptoms compared to their male counterparts. This could be due to the effects of
estrogen on macrophage and B-cell activation as mentioned in Chapter 1. These symptoms
range from proteinuria, edema and nephritis leading to early mortality. Key pathogenic
mechanisms identified in NZM2328 mice include defects in B-cell activation and survival,
abnormal cytokine signaling, such as increased interferon gamma (IFN-g), and the
involvement of a TNF family receptor, B-cell Activating Factor (BAFF) in mediating SLE
pathology and its pathways. The NZM2328 strain's versatility makes it valuable for
understanding the genetic and immune contributions to lupus-like autoimmunity.
In this chapter, in-vivo activities of NAP1051 in NZM2328 mouse model for SLE
were characterized. Here we will explore histological and immunological methods to further
explore the pharmacological activities by which NAP1051 exerts its therapeutic effects.
NAP1051 enhanced neutrophil clearance from the affected site while modulating SLE- and
NETosis-associated inflammatory cytokines and molecular markers. These molecular
changes translated to dramatic reductions in renal pathology and improved renal function.
3.2 Materials and Methods
3.2.1 NAP1051
The active pharmaceutical ingredient was synthesized, purified, and made into an
orally bioavailable formulation using pharmaceutically accepted excipients such as hydroxypropyl b-cyclodextrin. The process has been elaborated in patent US7683193B2.
3.2.2 In vivo studies for NZM2328 mice
All animal studies were approved by the University of Southern California IACUC.
SLE-prone female NZM2328 were used at 4-5 months of age (Waters et al., 2001). Mice
were randomized to their NAP1051 treatment groups (vehicle only, 5 or 10 mg/kg/day) and
treated by oral gavage once daily for 28 consecutive days. Proteinuria levels were monitored
three times each week using Albustix (Siemens, Munich, Germany). These mice were given a

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proteinuria score as listed in Table 3.1. On day 29, mice were anesthetized, and blood was
collected by cardiac puncture followed by cervical dislocation. Part of the blood (500µL) was
collected in potassium EDTA tubes and sent to Antech Diagnostics (Oak Brook, IL) for
blood chemistry and liver function tests to evaluate the safety profile of NAP1051.
Additional blood was allocated to the testing for serum autoantibody levels such as IgG, antidsDNA and anti-histone IgG levels. One kidney was cut in half, with one section placed in
10% formalin solution (VWR International, Radnor, PA) for histologic evaluation (Anguiano
et al., 2020) and the other section placed into TRIzol (Invitrogen, Carlsbad, CA) for RNA
extraction. The second kidney was flash-frozen in liquid nitrogen for immunofluorescence
and lipidomic studies.
Proteinuria Levels (mg/dL) Proteinuria Scores
Trace/0 1
30+ 2
100++ 3
300+++ 4
>2000++++ 5
Table 3.1: Proteinuria ranks defined based on the absolute proteinuria values in mg/dL for
NZM2328 mice.
3.2.3 Histologic evaluation
Sections of formalin-fixed kidneys were stained with hematoxylin and eosin and
assessed by light microscopy for histologic features as previously described (Anguiano et al.,
2020). To evaluate T cell and neutrophil infiltration into the kidneys, 4-µm sections of
paraffin-embedded formalin-fixed kidneys were deparaffinized and rehydrated by immersing
the slides through a series of xylene, ethanol, and PBS solutions. Heat-induced antigen
retrieval was done using a citrate buffer (pH = 6.0) in a pressure cooker. After the antigen
retrieval, slides were moved onto a humid chamber and washed in PBS three times. Tissue
sections were blocked with 2.5% (v/v) normal goat serum in PBS with 0.1% Triton X-100 for

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30 min at room temperature and then stained with primary antibody solution as shown in
Table 3.2.
Table 3.2: List of primary and secondary antibodies used along with dilution factors for
histologic staining of NZM2328 kidney tissues.
After overnight incubation at 4˚C with the primary antibodies listed in Table 3.2, slides were
mounted with a secondary antibody solution (Table 3.2) prepared in blocking solution in the
dark for 45 min at room temperature. After washing with PBS, DAPI counterstain was added
(1 µg/mL in PBS) for 10 min at room temperature. Slides were washed and coverslips were
secured. Images were taken on an Olympus BX43 microscope and Zeiss LSM880 + Airyscan
fast Confocal microscope. 10X and 40X eyepiece objectives were used.
3.2.4 Immunofluorescence staining
Sections (5-µm) from flash-frozen kidneys were bathed in cold acetone (-20˚C) for 15
min, blocked with 10% horse serum, and developed for IgG and C3 staining through addition
of FITC-conjugated goat anti-mouse IgG antibody (Southern Biotechnology Associates,
Birmingham) and FITC-conjugated goat anti-mouse C3 antibody (MP Biomedicals, Solon,
OH), respectively. The slides were counterstained with DAPI-containing Fluoro-gell II
(Electron Microscopy Sciences, Hatfield, PA), dried under a coverslip, and stored at -20˚C
until imaged. IgG and C3 depositions were quantified as mean fluorescence per glomerulus.
Images were analyzed using ImageJ, and the total IgG and C3 areas were measured for each
field with Otsu algorithm and exported as % area. Images were taken using an Olympus
BX43 microscope (Olympus America Inc.). At least five fields per slide were taken randomly
with 10X objective.

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3.2.5 RNA extraction, cDNA preparation, and RT-PCR analysis
Kidney tissues were placed in TRIzol and were homogenized using the TissueLyser II
(Qiagen LLC, Germantown, MD) for 10 minutes at 30Hz. RNA was extracted according to
the TRIzol kit instructions. cDNA was prepared using the GoScript Reverse Transcription
System (Promega, Madison, WI) according to manufacturer’s instruction. The RT-PCR
master-mix was prepared for each gene by mixing SYBR Green master-mix (Applied
Biosystems, Foster City, CA) with forward and reverse primers (Table 3.3)
Table 3.3: List of forward and reverse mouse primer sequences used in RT-PCR analysis.
The run method is described in Table 3.4. Samples were run in quadruplicate, and
results were analyzed using data assist software (Invitrogen, Carlsbad, CA). The ΔΔCt values
were calculated and plotted as fold changes using GraphPad Prism (GraphPad, San Diego,
CA).
Cycle Stage Temperature and Time
Initial Stage 50°C for 2 minutes
Intermediate Stage 95°C for 2 minutes
PCR Amplification 40 cycles

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Melting Step 95°C for 1 second
Annealing Stage 60°C for 30 seconds
Melt Curve
Stage/Dissociation Stage
1.6°C to 95°C for 15 seconds; 1.6°C to 60°C for 1 minute; 0.15°C
to 95°C for 15 seconds
Table 3.4: RT-PCR run method with temperature and times used to run NZM2328 mouse
kidney samples.
3.2.6 Lipodomics Analysis of NZM2328 mouse Plasma and Kidney
Lipids were extracted and detected from BMDM, serum, and kidney tissue using liquid
chromatography-mass spectrometry (LC-MS) at the University of Southern California.
Approximately 50–100 mg of each sample was used, and lipids were extracted by adding 250-
500 μL methanol. Internal standards: d5-RvD2, d8-5S-HETE, d4-PGE2, d5-LXA4, and d4-
LTB4 (Cayman Chemical Company) were added, and the samples were homogenized using a
TissueLyser II (Qiagen LLC, Germantown, MD) programmed at 30 Hz for 5-min intervals.
After centrifugation, the supernatant was diluted with water to a final concentration of 10%
methanol and further extracted using Strata X 33μm Polymeric Reverse Phase cartridges
(Phenomenex, CA). The lipid components were eluted with methanol, evaporated to dryness,
and reconstituted in 50% methanol. The analytes were separated and quantified using an
Agilent 1290 UPLC linked to a QTRAP Sciex API6500+ LC-MS/MS system, with a Poroshell
120 EC-C18 column and a specified gradient of water + 0.01% formic acid (mobile phase A)
and methanol + 0.01% formic acid (mobile phase B). The flow rate was 0.5 mL/min, and the
column temperature was maintained at 40°C. Analytes of interest included pro-inflammatory
lipid metabolites such as leukotrienes, prostaglandins, and thromboxanes, as well as specialized
pro-resolving mediators like lipoxins, resolvins, and maresins.
Peak selections were manually reviewed, and area under the curve (AUC) values were
normalized by protein concentration or sample volume. These values were further adjusted for
batch-to-batch variability and sample loss using internal standards. Statistical analysis and

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figure generation were performed using GraphPad Prism, with group comparisons made using
a two-tailed unpaired t-test.
3.2.7 Statistical analysis
Results are expressed as the mean (range) unless specified otherwise. GraphPad Prism
(GraphPad, San Diego, CA) was used to conduct statistical analyses on quantitative data, and
image data were analyzed with ImageJ.
3.3. Results
3.3.1 Safety profile of NAP1051 evaluated using Cellular Blood Counts (CBC), Blood
Chemistry and liver function tests
We evaluated the safety profile of NAP1051 in a dose escalation study for 28 days.
We accessed levels of white blood cells (WBCs) and differentials, red blood cells (RBCs)
and platelets. All these CBC values were within the range of vehicle treated animals. A slight
reduction of neutrophil counts was seen with NAP1051 but these values are with within
normal limits (Raabe et al., 2011). Interestingly, platelets were also reduced in the NAP1051
treatment where these reductions were still within the normal limits.
In Figure 3.1 B, the liver function tests (LFTs) such as serum glutamic pyruvic
transaminase (SGPT/ALT) and alkaline phosphatase (ALP) levels were not different. There
was high variability in ALP in the group receiving 10 mg/kg/day. NAP1051 which was orally
gavaged did not show any signs of toxicity even at the highest 10 mg/kg/day group. Figure
3.1 C shows the trend in blood urea nitrogen levels in NZM2328 mice decreasing after
treatment with NAP1051. Interestingly Figure 3.1 D shows us the cholesterol levels in the
serum decrease significantly when compared to NAP1051 restoring total cholesterol into
normal levels. They findings suggest at a high dosage of 10 mg/kg/day, the NAP1051
appears to be safe when given as oral gavage into NZM2328 female mice.

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Figure 3.1: (A) Safety profile of NAP1051 evaluated using CBC markers (B) Liver function tests. All
values are plotted as mean ± standard deviation (C) Total Blood Urea Nitrogen in the NZM2328 mice
serum. The trend shows a decrease in BUN levels after 28 days of treatment with NAP1051. (D) Total
Cholesterol was reduced after treatment with NAP1051 for 28 days. Analysis was done using oneway ANOVA using Dunnett’s multiple comparison test. *p < 0.05 (n=6).
3.3.2 Effect of NAP1051 on proteinuria and autoantibodies in NZM2328 female mice
Proteinuria is a common symptom associated with SLE correlating with lupus
nephritis (LN) (Musa et al., 2024). NZM2328 mice treated for 28 days with NAP1051 (5 and
10 mg/kg) showed a dose-dependent proteinuria reduction (*p< 0.05) (Figure 3.2 A).
However, there was no significant changes in blood urea nitrogen was shown in this study.
Further, circulating antibodies such as IgG, anti-dsDNA IgG and anti-histone IgG
levels in SLE have been linked to active disease renal disease. In Figure 3.2 no significant
changes in the serum antibody levels for IgG, anti-dsDNA or anti-histone IgG were detected.

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Figure 3.2: (A) Change in proteinuria levels in NZM2328 mice urine after 28-days of treatment with
NAP1051. (B-D) Total serum IgG, anti-dsDNA IgG and anti-histone IgG levels, all seemed to remain
relatively similar. Analysis was done using one-way ANOVA using Dunnett’s multiple comparison
test. *p < 0.05 (n=6).
3.3.3 Glomerular histology and IgG and C3 deposits in NZM2328 mice
The impact of NAP1051 treatment have on kidney was histologically evaluated where
kidney sections from NZM2328 treated with NAP1051 were compared to vehicle (Figure 3.3
A & D; *p<0.05). The presence of glomerular crescents is indicative of rapidly progressing
renal disease, which can be readily detected in vehicle-treated mice. In contrast, NZM 2328
treated with NAP1051 at the 5 or 10 mg/kg/day revealed a reduction in crescent glomeruli in
mice treated with 10 mg/kg/day (Figure 3.3 A). The reduction of crescent glomeruli
correlated with a dosage dependent reduction IgG deposition in the kidney (Figure 3.3 B &
F; **p<0.01). In parallel, robust glomerular deposition of C3 developed in vehicle-treated

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NZM 2328 mice (Figure 3.3 C & F, p<0.05), whereas C3 deposition was reduced in mice
treated with 5.0 or 10 mg/kg/day NAP1051 when compared with vehicle treated mice.
Figure 3.3: (A) NZM2328 kidney sections stained with H&E from vehicle, 5.0 mg/kg/day, and 10
mg/kg/day of NAP1051 treatment. Arrows indicate glomerular crescents and lobules present in
vehicle-treated mice but not in NAP1051-treated mice. (B-C) Immunofluorescence staining of IgG
and C3 on kidney sections from NZM 2328 mice. Considerable IgG deposition is seen in vehicletreated mice but has significant reduction in NAP1051-treated mice. Green: IgG staining and C3
staining. C3 deposits were reduced in animals treated with NAP1051 in a dose dependent manner
when compared to the vehicle-treated group. (D) Glomerular crescents found as a percentage of total
glomeruli count in the kidneys for NZM2328 mice. (E) Quantification of IgG and (F) C3 probe
fluorescence is presented as fold change relative to vehicle, respectively. NAP1051 treated samples
were compared to vehicle treated group by one-way ANOVA followed by Dunnett’s multiple
comparison test. Pictures were taken with a 40X objective (scale bar is 20 μm). n = 6/group. *:
p<0.05; **: p<0.01.
3.3.4 Effect of NAP1051 on Inflammatory Markers in Kidney
Given the decreased glomerular deposition of IgG and C3 and reduced crescent
formation in the glomeruli, which corresponded to preserved renal histology in NAP1051-
treated mice, we reasoned that expression of pro-inflammatory genes in kidneys treated with

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NAP1051 mice. Accordingly, kidney expression of the Tnf, Cxcl2, and Ifng genes were
reduced in a dosage dependent manner (Figure 3.4, p<0.5, **p<0.01). In particular, since
NAP1051 can reduce peripheral neutrophils, we evaluate the expression of CXCL2, the
homologue for IL-8 in mice was evaluated since mice do not express human IL-8 gene
(Asfaha et al., 2013). In each case, expression was lower in NAP1051-treated mice than in
vehicle-treated mice (Figure 3.4).
In addition to expression of these pro-inflammatory genes, we assessed expression of
the TLR since they play a role in innate immunity activity. Specifically, SLE is associated
with overexpression of TLR7, whereas up-regulation of TLR9 has been described to be
protective in SLE. NAP1051-treated mice revealed decreased Tlr7 expression which was
accompanied by increased Tlr9 expression when compared to vehicle- treated mice (Figure
3.4).

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Figure 3.4: Genetic expression of inflammatory markers associated with inflammation, Tnf-a, CXCL2 and IFN-g in the kidney were analyzed using RT-PCR. A significant decrease was observed for the
listed markers along with TLR-7 and TLR-9 which are known to further exacerbate disease conditions.
GAPDH and β-actin were used as reference genes. (n=6/group) Data is graphed using one way
ANOVA and multiple comparison tests. (* p<0.05; **: p<0.01; ***: p<0.001)
3.3.5 NAP1051’s Effect on Immune cell infiltration and T Cell Infiltration
Immune cell infiltration plays a crucial role in kidney inflammation and damage
observed in lupus nephritis (LN) (Cao et al., 2019). Among these infiltrating immune cells
are T-cells, monocytes, and neutrophils, each contributing to the progression of renal injury.
In Figure 3.5 A, the presence of a distinct purple hue observed in H&E staining highlights
the infiltration of lymphocytes within the kidney tissue. Notably, a substantial accumulation
of infiltrating lymphocytes was evident in the group treated with the vehicle control, whereas
this infiltration was markedly reduced in animals administered NAP1051.
To further investigate the extent of immune infiltration, T-cell infiltration within the
kidney was evaluated given their known role in LN pathology. Specifically, the presence of
CD3e+ cells as a marker of T-cell which revealed a dose-dependent decrease in CD3e+ cell
numbers in mice treated with NAP1051 compared to those receiving only the vehicle control.
The quantification of CD3e+ used fluorescent labelled CD3e+ in kidney sections, where the
quantification used an image J analysis (Figure 3.5 D to G; **** p<0.0001). This reduction
in T-cell infiltration aligns closely with the H&E findings showing T-cell infiltration which
was reduced with NAP1051 treatments. As previously discussed in Chapter 1, T-cells are
key contributors to the pathogenesis of SLE, driving both systemic inflammation and organspecific damage. The ability of NAP1051 to attenuate T-cell infiltration in the kidney offers
promising insights into its potential to mitigate the immune-mediated damage associated with
LN.

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Figure 3.5: Perivascular Infiltration (PVI) in NZM2328 Female mice Kidney. (A) Vehicle, (B) 5.0,
and (C) 10 mg/kg/day. (D-F) NZM2328 kidney sections stained with CD3e+ staining. Arrows indicate
T-cell infiltration present in vehicle-treated mice. (G) Quantification of T-cells infiltrating the
perivascular region of the kidney, compared to vehicle treated animals. Analysis was done using oneway ANOVA and Dunnett’s multiple comparison test (****: p<0.0001). Blue: DAPI and Red: CD3e+
staining. Pictures were taken with a 10X objective (scale bar 100 μm) n=6.
3.3.6 Effect of NAP1051 on in-situ NETosis in NZM2328 Kidney
We previously showed that NAP1051 can reduce neutrophil formyl peptide mediated
chemotaxis and promote macrophage-mediated efferocytosis of apoptotic neutrophils in vitro
and in mouse xenograft colorectal cancer models (Dong et al., 2021). To determine whether
NAP1051 can attenuate NETosis in NZM2328 kidneys, in situ NETosis in kidney tissue used
co-staining of anti-neutrophil elastase and anti-PECAM1 to identify neutrophils penetration
from the vasculature and into the kidneys parenchymal. The kidneys (cortex and medulla) of
NAP1051-treated mice harbored fewer neutrophils than did the kidneys of vehicle-treated
mice (Figure 3.6 A to E). Moreover, the co-staining documented the release of neutrophils
into the extravascular space and migration of these neutrophils into the kidneys of the
vehicle-treated mice (Figure 3.6). In contrast, reduced kidney neutrophil infiltration was
observed in NAP1051-treated mice.

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Secondly, we probed the effect of NAP1051 in NZM2328 mice to study the
expression of innate immunity-associated genes in the kidneys. Expression of the NETosisassociated genes, Cybb and Padi4, was determined in the kidneys. Expression of Padi4 in the
NAP1051-treated cohorts was substantially lower than that in vehicle-only treated cohorts
(Figure 3.6), and expression of Cybb in NAP1051-treated NZM2328 mice was also
considerably lower than in vehicle-only-treated NZM2328 mice. All this tied together
supported our hypothesis that NAP1051, like its analogue, LXA4 can indeed reduce in-situ
neutrophil migration and NETosis in the kidneys of SLE prone NZM2328 mice.
Figure 3.6: (A-M) In-situ deposition of neutrophils was accessed using a co-staining of Green:
neutrophil elastase, Red: platelet and endothelial cell adhesion molecule 1 (PECAM1) and Blue:
DAPI. The white arrows point to the intravascular neutrophils. (N-P) Neutrophils identified using
Green: neutrophil elastase, Red: citH3 and Blue: DAPI in extravascular spaces of the kidneys treated
with the vehicle but were not as abundant in the treated animals. White arrows point to the
extravascular neutrophils which were localized in the renal cortex and medulla. (Q) NOX-2 and (R)
PAD-4 was accessed after 28 days of treatment with NAP1051. These genes are associated with the

105
process of NETosis and we observe a significant decrease in the PAD-4 gene NZM2328 mouse model
(n=6/group) GAPDH and β-actin were used as reference genes. *: p<0.05 using one way ANOVA
and Dunnett’s multiple comparison tests.
3.3.7 Effect of NAP1051 on the Kidney and Plasma Lipidome in NZM2328 mice
NAP1051, a bioactive analogue LXA4, is engineered to regulate inflammation by
promoting anti-inflammatory and pro-resolution pathways. In a dose escalation study, 4-
month-old female NZM2328 mice received NAP1051 via oral gavage daily for 28 days,
starting at disease induction. Using LC-MS analysis, we quantified a range of bioactive lipids
derived from AA, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and
docosapentaenoic acid (DPA) to understand how the drug influences their biosynthesis, crucial
in modulating innate inflammatory responses. In the kidney (Figure 3.7B), treatment groups
with 5 and 10mg/kg/day of NAP1051 displayed decreased levels of PGE2, PGD2
prostaglandins, TXB2, and 5-HETE, all well-known contributors to inflammation.
Prostaglandins, by promoting vasodilation and increasing vascular permeability, play a
significant role in inflammation. In SLE-related kidney inflammation, they may exacerbate
renal damage by fostering inflammatory responses (Pellefigues et al., 2018). Interestingly, LX
levels in the kidney decreased dose-dependently with NAP1051 treatment, possibly indicating
resolution of inflammation and restoration of kidney homeostasis, or due to downregulation of
5-HETE precursors. EPA levels, including precursors like 15-HEPE and 12-HEPE, were
upregulated with higher NAP1051 doses, holding promise for managing SLE due to their antiinflammatory properties. DHA, serving as a precursor for specialized pro-resolving lipid
mediators (SPMs) like Resolvin D1 (RvD1) and Resolvin D2 (RvD2), is pivotal in resolving
inflammation. In SLE, where chronic inflammation and immune dysregulation are prevalent,
DHA-derived resolvins offer therapeutic potential by dampening excessive immune responses
and promoting tissue repair. Notably, DHA and its metabolites, including RvD1 and RvD2,
were significantly upregulated in groups receiving 5 and 10 mg/kg/day of NAP1051. In plasma,
analytes like PGE2, PGD2, and TXB2 were upregulated in treatment groups, while lipoxins,

106
DHA, and resolvins were upregulated in the 5 and 10 mg/kg/day treatment groups. This
suggests a potential localized action of NAP1051 in the kidney without affecting circulating
plasma lipid levels (Figure 3.7), highlighting its efficacy and specific targeting properties
against lupus nephritis.
Figure 3.7: Analysis of Plasma and Kidney Lipidome for NZM2328 mice that were orally treated
with NAP1051 for 28 days. (A) Plasma lipidome analysis (B) Kidney lipidome analysis. Data was

107
analyzed and plotted using a mean expression dot plot. The size of the dots represents the p-value and
the color represents the fold changes compared to vehicle (n=6).
3.3.8 SPM Metabolite Ratios in Plasma and Kidney
To further analyze tissue-specific lipidomic changes, we calculated the 5/12/15-LOX
metabolite ratios from the lipidomics data and visualized them using a heatmap (Figure 3.8).
This heatmap illustrates the fold-change ratios of 12-HETE to 15-HETE metabolites across
plasma and kidney in NZM2328 murine model. In the plasma lipidome, the 12/15 LOX ratio
was elevated, with a significant increase in 15-HETE levels relative to AA, suggesting a shift
towards a pro-resolving state in macrophages. (*p < 0.05). The 12/15 LOX ratio in plasma was
elevated across the lipids and interestingly the 5-LOX ratio, particularly for DPA RvD1/17-
HDPA was significantly recued in the 5 and 10mg/kg day treatment group. 5-LOX has been
associated with a heightened inflammatory state and immune dysregulation in autoimmune
diseases. Here NAP1051 treatment managed to reduce 5-LOX ratio while elevating 12/15-
LOX in NZM2328 plasma. However, these changes did not reach the levels observed in the
kidney lipidome, highlighting tissue-specific differences in lipid metabolism and inflammatory
responses. The kidney lipidome demonstrated a more balanced 12/15 LOX ratio, with modest
changes in both EPA and DHA levels. Notably, in certain experimental groups, a decrease in
15-HETE was observed, which may indicate a diminished capacity for the need for
inflammation resolution, as the levels of pro-resolving lipids in the kidney lipidome were
significantly elevated as compared to the plasma lipidome. This reduced 12/15-HETE level
correlated with a minor increase in the 12/15 LOX ratio, suggesting a shift in the circulating
lipid profile where the inflammation have been resolved as previously indicated by the reduced
crescentic glomeruli, T-cell infiltration and increase in resolvins.

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Figure 3.8: (A) Plasma and (B) Kidney PUFA metabolite ratios. Heatmaps displaying PUFA
metabolite ratios in relation to vehicle treated animals at Day 28. Ratios were analyzed via the
Kruskal–Wallis’s test with Dunn’s correction (*p< 0.05).
3.4 Discussion
The outcomes from this study reinforce the potential of NAP1051 as a therapeutic
agent for SLE, particularly in modulating inflammatory responses and improving kidney
pathology. A primary goal was to evaluate NAP1051’s effects on proteinuria, autoantibody
levels, immune cell infiltration, and NETosis within the kidneys as all these markers were
severely displayed by NZM2328 mice. Findings from the 28-day treatment with NAP1051
reveal several key insights.
First, NAP1051 demonstrated a favorable safety profile, as evidenced by consistent
liver function and hematological parameters, with no significant toxicity observed even at
higher dosages such as 10mg/kg/day. Notably, the drug led to a significant reduction in BUN
and serum cholesterol levels, suggesting its potential to improve renal function and systemic
lipid profiles. This points toward NAP1051 not only being non-toxic but also capable of

109
addressing metabolic disturbances associated with SLE. Another notable outcome was the
significant reduction in proteinuria among treated mice, especially at higher doses.
Proteinuria is a critical marker of renal involvement LN, and this reduction indicates that
NAP1051 directly impacts kidney function. Although circulating autoantibodies such as IgG,
anti-dsDNA, and anti-histone IgG remained unchanged, this aligns with the understanding
that renal impairment in NZM2328 mice develops before substantial changes in these
antibodies. Therefore, the therapeutic effect of NAP1051 may involve mechanisms
independent of antibody modulation.
Histological examination further corroborated the efficacy of NAP1051, with a clear
reduction in glomerular crescents and immune complex deposits (IgG and C3) in treated
mice. The attenuation of crescentic glomeruli and complement activation suggests that
NAP1051 mitigates kidney damage by dampening immune complex-mediated inflammation.
Moreover, the drug’s ability to reduce perivascular immune cell infiltration highlights its
immunomodulatory role, likely reducing T-cell and neutrophil activity, both of which are
implicated in the pathology of LN (Tilstra et al., 2018). The study also sheds light on the
influence of NAP1051 on key inflammatory markers. Reduced expression of Tnf-α, Cxcl2,
and Ifn-γ genes in treated mice demonstrates NAP1051's capacity to suppress inflammatory
cytokines and chemokines associated with SLE. This effect is particularly significant because
TNF-α and CXCL2 play vital roles in recruiting neutrophils to sites of inflammation and
injury; CXCL2, specifically, is a homologue of IL-8 in mice and serves as a critical mediator
of neutrophil chemotaxis (Asfaha et al., 2013). These findings align with the observed
decrease in neutrophil migration and NETosis within the kidneys, indicated by lower NOX-2
(encoded by Cybb) and Padi4 expression along with the immunofluorescence staining for NE
and citrullinated histones in the perivascular region of the kidney. This suggests that
NAP1051 can inhibit neutrophil migration and subsequent NETosis, a crucial process in SLE

110
pathogenesis, thereby preventing excessive neutrophil-driven inflammation and tissue
damage.
Lastly, the lipidomic analysis revealed that NAP1051 modulates both proinflammatory and pro-resolving lipid mediators. A decrease in prostaglandins and
leukotrienes in the kidneys, along with an increase in resolvins and lipoxins, reflects a shift
toward resolving inflammation (Ricciotti & FitzGerald, 2011). This selective modulation of
lipid pathways suggests that NAP1051 promotes tissue repair and homeostasis, potentially
offering long-term benefits in managing SLE and its associated symptoms. In summary, the
findings suggest that NAP1051 holds promise as an effective therapeutic agent by improving
kidney function, reducing inflammation, and mitigating immune responses associated with
SLE. Its ability to modulate both cytokine activity and lipid metabolism presents a
comprehensive approach to controlling disease progression. These findings will further be
confirmed in another SLE murine model in Chapter 4.

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Chapter 4: Characterization of in vivo Activities of NAP1051 in SLE
Murine Model MRL.lpr
4.1 Introduction
Systemic lupus erythematosus (SLE) is a progressive autoimmune disease capable of
adversely affecting a wide spectrum of organ systems (Wenzel, 2019). A hallmark of SLE is
excessive autoantibodies targeting self-antigens such as DNA and extractable nuclear antigens.
Current understanding suggests both the innate and adaptive immune pathways are activated
in SLE. Unfortunately, the heterogeneity of SLE has prevented clear understanding as to the
molecular pathogenesis of SLE. Currently, SLE treatment options are limited and associated
with significant side effects and reduced quality of life (G. Murphy & Isenberg, 2019).
The aberrant innate immune response against self-antigens results in the
activation of CD3e+ T-cells, triggering the release of inflammatory cytokines that cause
tissue damage. This response is further amplified by autoantibodies, which enhance
opsonization and activate B-lymphocytes (Choi et al., 2012), perpetuating the immune
response. Successfully resolving this cycle of chronic inflammation can help restore
homeostasis and protect tissues from further damage. Recent studies suggest that a key
factor in this pathology is the imbalance between the generation of apoptotic cells and
their efficient clearance, particularly in relation to neutrophils (Dong et al., 2021).
Neutrophils, the most abundant cells in the innate immune system, are the first
responders to sites of injury, releasing reactive oxygen species (ROS) and cytoplasmic
granules to combat pathogens. However, persistent activation of neutrophils has been
identified as a significant factor in systemic lupus erythematosus (SLE) pathogenesis
(Arneth, 2019). A major aspect of this involvement is through the process known as
neutrophil extracellular trap formation, or NETosis. NETs, which consist of web-like
chromatin structures embedded with cytotoxic granules such as myeloperoxidase
(MPO), cathepsin G (CPG), elastase, and eicosanoids, are increasingly recognized as

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contributors to SLE (Gupta & Kaplan, 2016). NETosis interferes with immune complex
clearance, enhances nucleic acid sensing, activates lymphocyte signaling, and promotes
interferon production—all of which drive the progression of SLE.
In normal conditions, neutrophil sequestration in tissues is followed by
resolution of the inflammatory lesions. However, when proinflammatory signals are
present, neutrophil lifespan can extend significantly, resulting in sustained
inflammation (Kaplan, 2011). This is consistent with what is seen in SLE patients, where
elevated circulating levels of neutrophil chemokines, including tumor necrosis factor-a (TNFa) and interleukin-8 have been reported (H. S. Murphy et al., 1998). This persistent
inflammation continues to produce inflammatory mediators, which attract additional
leukocytes to the site (Carestia et al., 2016). In contrast, the resolution of inflammation
involves halting leukocyte migration, down-regulating cytokine production, inducing
apoptosis in inflammatory cells, and promoting efferocytosis of apoptotic cells by
macrophages (McCracken & Allen, 2014). Together, these mechanisms enable
inflammation resolution and tissue recovery. Evidence suggests that inducing
neutrophil apoptosis and enhancing macrophage-mediated efferocytosis can reduce
autoimmune disease severity, as seen in experimental models of antigen-induced
arthritis (Hrycek et al., 2013).
Neutrophils express formyl peptide receptor 2 (FPR2), also known as ALXR,
which, when bound by N-formyl-l-methionyl-l-leucyl-phenylalanine (fMLP), triggers
migration and chemotaxis to inflamed areas (Serhan et al., 2007). Once neutrophils
enter tissue, they engage sterilization mechanisms by activating NADPH oxidase
(NOX-2), producing ROS. This oxygen burst activates neutrophil elastase (NE), which
translocates to the nucleus to initiate chromatin decondensation. Furthermore, ROS can
activate Mek/Erk phosphorylation, supporting chromatin disassembly and release (Ariel

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& Serhan, 2012). Peptidyl arginine deiminase-4 (PAD-4) also facilitates chromatin
decondensation by deiminating histone proteins, embedding MPO, NE, and CPG into
the chromatin structure. Therapeutic strategies aiming to promote the clearance of NETs
offer potential in treating autoimmune diseases (Lopes et al., 2011).
Lipoxins (LX), a group of bioactive lipid mediators, bind to the ALXR receptor.
Unlike traditional immune activation, LX binding to ALXR induces neutrophil
apoptosis and stimulates macrophage efferocytosis, leading to the removal of apoptotic
neutrophils and resolution of acute inflammation (Yuen et al., 2016). Specifically,
lipoxin A4 (LXA4) has been shown to mediate inflammation resolution by regulating
leukocyte trafficking and promoting neutrophil apoptosis (Brinkmann, 2018;
Papayannopoulos, 2018). Reduced LXA4 levels have been linked to persistent
inflammation (El Kebir et al., 2007), while its administration enhances macrophagemediated clearance of apoptotic cells and prevents macrophage apoptosis, facilitating
resolution of inflammation. LX's relevance to glomerulonephritis is significant. It
prevents kidney inflammation by inducing neutrophil apoptosis and supporting
macrophage-mediated efferocytosis of apoptotic cells (Godson et al., 2000). Studies
have shown that decreased LXA4 biosynthesis correlates with increased neutrophil
infiltration in models of nephritis, while elevated levels of 15-lipoxygenase (15-LO)
support LXA4 biosynthesis and contribute to the resolution of poststreptococcal
glomerulonephritis (Prieto et al., 2010; Wang et al., 2013). These findings highlight
LX's potential therapeutic role in SLE, as its administration may mitigate disease
progression.
However, natural LX compounds lack chemical stability, being vulnerable to
oxidation, and has a short plasma half-lives. To address these limitations, we have
developed a stable LXA4 analogue, NAP1051, which is over 80% bioavailable and is

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chemically stable. Structurally similar to LXA4, NAP1051 retains essential functional
groups for cellular interaction. It includes an aromatic ring in place of the hexatriene
system to enhance stability, and the carboxylic acid group has been replaced with a
methylester as shown in Chapter 1. Our research investigates the potential of NAP1051
as a novel approach for treating SLE by promoting inflammation resolution through
ALXR activation.
Our evaluation of NAP1051’s efficacy utilizes the MRL.lpr mouse model, which
is homozygous for the lymphoproliferation mutation (Faslpr). These mice exhibit
characteristics akin to human SLE, including systemic autoimmunity, extensive
lymphadenopathy due to abnormal T-cell proliferation, arthritis, and immune complex
glomerulonephritis. The pathophysiology in MRL.lpr mice is driven by mutations
affecting genes such as Fas, contributing to defective apoptosis and immune
dysregulation (Katzav et al., 2001). In this study, we assessed the effect of NAP1051
on inflammatory and NETosis-related markers in NM2328 mice, observing significant
reductions in renal pathology and enhanced renal function. Additionally, we
investigated NAP1051’s role in stem cell mobilization, extracting bone marrow cells to
assess regeneration potential.
4.2 Materials and Methods:
4.2.1 NAP1051
The active pharmaceutical ingredient was synthesized, purified and made into an orally
bioavailable formulation using pharmaceutically accepted excipients. The process has been
elaborated in patent US7683193B2.
4.2.2 In- vivo studies
All animal studies were approved by the University of Southern California IACUC. MRL.lpr
mice (Jackson Laboratory, stock number 000485) were used at 4-5 months and 10 weeks of
age, respectively. Mice were randomized to their NAP1051 treatment groups (vehicle only, 5,

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10, 20 mg/kg/day), and treated by oral gavage once daily for 28 consecutive days. Body weight
and proteinuria levels were monitored three times each week using Albustix. (Siemens,
Munich, Germany). On day 29, mice were anesthetized, and blood was collected by cardiac
puncture followed by cervical dislocation. The kidneys were harvested, and one kidney was
sectioned, with one section placed in 10% formalin solution (VWR International, Radnor, PA)
for histologic evaluation and the other section placed into TRIzol (Invitrogen, Carlsbad, CA)
for RNA extraction. The second kidney was flash-frozen in liquid nitrogen for
immunofluorescence studies.
Mouse
Strain No. Dose
(mg/kg/day) Route Time
(days) Monitoring and Measurements
MRL.lpr 8 Vehicle Oral 28 In Life: QMWF weight, behavioral
changes, rash and proteinuria
EOS: Blood for blood chemistry, CBC,
Kidney for IF/RT-PCR (NETosis markers),
bone marrow derived MSC isolation
MRL.lpr 8 5 Oral 28
MRL.lpr 8 10 Oral 28
MRL.lpr 8 20 Oral 28
Table 4.1: Study Design of MRL.lpr mice to test the oral efficacy of NAP1051.
4.2.3 Proteinuria and Glomerular Histology
Proteinuria measurements were performed three times a week and recorded in an unbiased
blind trial. The proteinuria levels were measured by AlbustixÒ and manufactures instructions
were followed. (Siemens, Munich, Germany). Sections of formalin-fixed kidneys were stained
with hematoxylin and eosin and assessed by light microscopy for histologic features (Anguiano
et al., 2020).
4.2.4 Immunofluorescence Staining
Flash-frozen kidneys sections (5-µm) from were bathed in cold acetone (-20˚C) for 15 min,
were blocked with 10% horse serum, and were developed for IgG and C3 staining through
addition of FITC-conjugated goat anti-mouse IgG antibody (Southern Biotechnology
Associates, Birmingham) and FITC-conjugated goat anti-mouse C3 antibody (MP
Biomedicals, Solon, OH), respectively. The slides were counterstained with DAPI-containing

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Fluoro-gell II (Electron Microscopy Sciences, Hatfield, PA), covered with a coverslip, allowed
to dry, and stored at -20˚C until imaged. IgG and C3 depositions were quantified fluorescence
intensity per glomerulus. Images (what magnification?) were analyzed using ImageJ, and the
total IgG and C3 areas were measured for each field (how many fields) with Otsu algorithm
and exported as % area. Images were taken using an Olympus BX43 microscope (Olympus
America Inc.). At least five fields per slide were taken randomly with 10X objective.
4.2.5 Mesenchymal stem cell isolation:
Isolated mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) were obtained
from mouse bone marrow of MRL.lpr mice and adjusted to a concentration of 1 × 107 cells/mL.
For cell counts, cells from all samples were pooled evenly, including control tubes from all
samples. Tubes were then prepared according to the experiment schema, comprising unstained
cells, single-color compensation controls (one for each color) with pooled cells, fully stained
cells, and FMO (fully stained minus one fluorochrome) controls. Zombie Violet dye staining
was performed by washing the cells with PBS buffer (protein-free), diluting the dye at 1:500
in PBS, and resuspending 1 × 106 cells in 100 µL of diluted Zombie Violet solution. After
incubation at room temperature in the dark for 20 minutes, the cells were washed once with 2
mL flow staining buffer (with FBS) before proceeding with the antibody staining procedure.
Cells could optionally be fixed with paraformaldehyde prior to permeabilization or analyzed
without fixation. For cell surface staining with Fc block, cells were added to appropriate tubes
with a volume adjustment to 100 µL (containing 1 × 106 cells/tube/100 µL). The cell
suspension was preincubated with Mouse BD Fc Block™ purified anti-mouse CD16/CD32
mAb 2.4G2 (1 μg/million cells in 100 μL) at 4˚C for 5 minutes (2 μL). Fc block was also added
to compensation controls. Antibodies of interest were then added directly to preincubated cells
in the presence of Mouse BD Fc Block™, with final antibody concentrations of 10 µg/mL or

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1 µg per 100 µL. The specific antibodies and their concentrations were CD45-PerCP/Cy5.5
(0.2 mg/mL, 1.25 μL), Ly-6A/Ly-6E-APC-Cy7 (0.2 mg/mL, 1.25 μL), CD-29-FITC (0.5
mg/mL, 1 μL), and CD105-APC (0.2 mg/mL, 1.25 μL). Following a 30-minute incubation at
4˚C in the dark, the cells underwent two wash steps by adding 2 mL staining buffer,
centrifuging for 5 minutes at 400×g and 4°C, removing the supernatant, vortexing the pellet,
and resuspending it in 2 mL staining buffer. After the second wash, the cells were resuspended
in 400 μL staining buffer and kept at 4°C in a fridge until the scheduled time for analysis. The
samples were analyzed on BD FACS Fortessa (BD Franklin Lakes, NJ).
4.2.6 RNA extraction, cDNA preparation, and RT-PCR analysis
Kidney tissues were placed in TRIzol and were homogenized using the TissueLyser II (Qiagen
LLC, Germantown, MD) for 10 minutes at 30Hz. RNA was extracted according to the TRIzol
kit instructions. cDNA was prepared using the GoScript Reverse Transcription System
(Promega, Madison, WI) according to manufacturer’s instruction. The RT-PCR master-mix
was prepared for each gene by mixing SYBR Green master-mix (Applied Biosystems, Foster
City, CA) with forward and reverse primers samples were run in quadruplicate, and results
were analyzed using data assist software (Invitrogen, Carlsbad, CA). The ΔΔCt values were
calculated and plotted as fold changes.
4.2.7 Lipidomics analysis of MRL.lpr mouse kidney and plasma
The plasma and kidneys from MRL.lpr were profiled using a quantitative function
lipidomic assay. Plasma samples on ice and transferring 100 µL aliquots into fresh tubes.
Following this, an Internal standard Mix (50ng/mL) was added to each aliquot. Subsequently,
200 µL of pure MeOH was added to each aliquot to facilitate the extraction of lipid
components. The samples were vortexed and then centrifuged at 10,000 rpm for 5 minutes.

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The supernatant was carefully transferred to fresh tubes and diluted with water to achieve a
10% MeOH solution. Strata X 33 µm polymeric reverse phase column was used for solid phase
extraction (Phenomenex, Torrance, CA). The column was conditioned with 1 mL MeOH
followed by 1 mL water. The sample in 10% MeOH was then added to the column, washed
with 500 µL pure MeOH, and the flow-through collected. After centrifuging the flow-through
and drying it under N2 gas, the sample was reconstituted to 50 µL with 50% MeOH. Twentyfive microliters were transferred to LC/MS vials, and the remaining sample was stored at -80°C
until further analysis using the lipidomics assay on the Sciex 6500 QTOF (Sciex, Foster City,
CA).
Kidney lipidomic analysis and NAP1051 measures were evaluated. A single intact
kidney excised from each animal was weight for normalization. Similar to the plasma
lipidomic procedure, an Internal standard Mix (50ng/mL) was added to each sample,
followed by the addition of 500 µL pure MeOH for lipid extraction. The samples were then
homogenized using metal beads in 5-minute intervals at 30 Hz (TissueLyser, Qiagen). After
vortexing and centrifugation at 10,000 rpm for 5 minutes, the supernatant was transferred to
fresh tubes and diluted with water to achieve a 10% MeOH solution. The subsequent steps,
including conditioning the extraction column, washing, centrifuging, drying, and
reconstitution of the sample, were identical to those described in the plasma lipidomic
profiling procedure. Analysis was carried out using the same lipidomics assay on the Sciex
6500 QTOF (Sciex, Carlsbad, CA). (Sciex, Foster City, CA).
4.2.8 Statistical analysis
Results are expressed as the mean and the error bars represent the standard deviation
unless specified otherwise. GraphPad Prism (GraphPad, San Diego, CA) was used to conduct
statistical analyses on quantitative data, and image data were analyzed with ImageJ.

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4.3 Results
4.3.1 Effect of NAP1051 on Clinical Markers in MRL.lpr mice
In this study, the oral administration of NAP1051 was evaluated against a vehicle
treatment using hydroxypropyl beta cyclodextrin (HpβCD) as a control. The results
demonstrated a highly significant reduction (p < 0.001) in the development of SLE-like
cutaneous lesions in MRL.lpr mice treated with either 10 or 20 mg/kg/day of NAP1051.
Typically, MRL.lpr mice, which serve as a model for human SLE, are prone to developing
cutaneous lesions that mirror the skin manifestations observed in SLE patients. In the
vehicle-treated group, by Day 27, all (100%) of the animals had developed these
characteristic lesions, underscoring the aggressive nature of the disease in this model and the
rapid onset of dermatologic symptoms.
Remarkably, NAP1051 treatment at both dosages (10 and 20 mg/kg/day) led to a
dramatic reduction in the prevalence of cutaneous lesions as seen in Figure 4.1. By Day 27,
less than 25% of the animals in NAP1051-treated group displayed these skin lesions. This
indicates that NAP1051, even at the lower dose, has a substantial protective effect against the
development of SLE-like dermatologic symptoms in MRL.lpr mice.
These findings suggest that NAP1051 may effectively modulate the inflammatory
pathways that contribute to SLE-related skin damage. The dose-dependent effect observed
here opens pathways for further research into optimal dosing and the broader impact of
NAP1051 on other SLE-related manifestations.

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Figure 4.1: (A) Showing lupus like rash in the MRL/lpr model after treatment with NAP1051 for 28
days the mice treated (B and C) seem to develop a rash later into the treatment or not at all (D) as
opposed to the vehicle treated group, that developed a rash at day 7. (E) The skin rash survival
probability between all the groups treated with NAP1051 daily. p < 0.001 as calculated by log-rank
(Mantel-Cox) test. N=8/group
4.3.2 Effect of NAP1051 on Proteinuria, Glomerular Histology and T-cell infiltration
By 8-10 weeks of age, female MRL.lpr mice begin to manifest histologic features of
renal disease, whereas clinically apparent renal disease (impaired renal function, severe
proteinuria) does not ensue until months later. MRL.lpr mice treated for 28 days with NAP1051
or with vehicle alone, a trend pointing to a dose-dependent reduction in BUN was observed
(Figure 4.2A), and a similar trend pointing to reduction in proteinuria was also observed
(Figure 4.2B). Given the paucity of overt clinical disease (elevated BUN, severe proteinuria)
in MRL/lpr mice at 8-10 weeks age (the age of the mice at the conclusion of treatment), it is
not surprising that the differences between vehicle-treated and NAP1051-treated mice in
parameters of clinical renal disease were modest. Nevertheless, at the histologic level, dramatic
differences were observed. Whereas glomerular crescents, indicative of rapidly progressing
renal disease, were readily detected in vehicle-treated mice (Figure 4.2C), they were reduced
in mice treated with NAP1051 at the 10 mg/kg/day dosage and were essentially absent in mice
treated with NAP1051 at the 20 mg/kg/day dosage. T cell infiltration into the kidneys also

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paralleled the histologic findings, with a dose-dependent reduction in the number of CD3e+
cells in NAP1051-treated mice than in the vehicle-only-treated mice (Figure 4.2 D-E)
Figure 4.2:(A) After treatment with NAP1051 for 28 days the Blood Urea Nitrogen (BUN) (mg/dL)
levels along with (B) Change in proteinuria levels from Day 1 to Day 28 were measured in MRL.lpr
mice. (C) H&E staining was done for the kidney sections from vehicle, 5.0 mg/kg/day, and 10
mg/kg/day and 20mg/kg/day after treatment with NAP1051. Arrows indicate glomerular crescents
and lobules present in vehicle-treated mice but not in NAP1051-treated mice. (D) Quantification for
the glomerular crescents for the NAP1051 treated animals analyzed by one-way ANOVA and
multiple comparison test (***p<0.001). (E) CD3e+ staining done for MRL.lpr. Arrows indicate T-cell
infiltration present in vehicle-treated mice and quantified in Figure 2E (**p<0.01; ****p<0.0001)
Blue: DAPI and Red: CD3e+ infiltrating cells. Pictures were taken with a 10X objective (scale bar 100
μm)
4.3.3 Effect of NAP1051 on Opsonized Antibodies and Neutrophil Infiltration
In SLE, IgG and C3 can aggregate and bind to the surfaces of apoptotic cells,
promoting their opsonization and potentially contributing to the development of nephritis
(Kelley et al., 2010). This process is especially relevant in SLE-related kidney inflammation,
where these immune deposits can accumulate and exacerbate disease progression. NZM2328
treated with NAP1051 significantly reduces tubulointerstitial infiltrates as well as glomerular

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IgG deposits within the kidneys, highlighting its potential therapeutic effects in reducing
renal inflammation as shown in Chapter 3. However, in studies conducted with MRL.lpr
mice, NAP1051 did not significantly reduce glomerular C3 deposition, suggesting a selective
effect of this treatment on certain immune pathways. This difference in response unveils the
complex mechanisms underlying immune deposition and renal involvement in SLE,
suggesting that while NAP1051 may effectively limit IgG-related immune activity, its impact
on C3 deposition remains limited in certain models.
NAP1051 blocks NETosis at the site of organ damage (kidneys) of MRL.lpr mice, in
situ NETosis was assessed, we co-stained for citrullinated histones and neutrophil elastase to
identify neutrophils infiltrating the kidneys. The renal cortex and medulla in NAP1051
harbored fewer neutrophils than did the kidneys of vehicle-treated mice (Figure 4.3 J-K).
Figure 4.3: (A-C) IgG staining and (D) quantification (E-G) C3 staining for MRL.lpr mice showing
(H) quantification of C3 probe fluorescence is presented as fold change relative to vehicle,
respectively. Green: IgG staining and C3 staining. IgG and C3 deposits were reduced in animals
treated with NAP1051 in a dose dependent manner when compared to the vehicle-treated group
(A&E). (D&H) Quantification of fluorescent area respectively compared using one-way ANOVA
analysis and analyzed using multiple comparison tests. *: p<0.05. (I-K) Neutrophils identified using
Green: neutrophil elastase, Red: citH3 and Blue: DAPI in extravascular spaces of the kidneys treated
with the vehicle but were not as abundant in the treated animals. White arrows point to the

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extravascular neutrophils which were localized in the renal cortex and medulla. Pictures were taken
with 40X magnification. N=8/group.
4.3.4 NAP1051 effect on NETosis Markers in Kidney
The crosstalk between NET’s and interferons posed to be an interesting pathway since
a variety of stimuli including IFN Type I/II and TLR7 trigger the process of NETosis (Regli et
al., 2020). Enhanced TLR-7 signalling was also shown to drive an aberrant B and T-cell
response further promoting the SLE pathogenesis (G. J. Brown et al., 2022). TLR9 deficiency
in B cells was sufficient to exacerbate nephritis while extinguishing anti–nucleosome
antibodies. In addition, impaired TLR9 expression has been found in lymphocytes derived from
SLE patients (Dieudonné et al., 2019). One study has shown that SPMs such as epi-LXA4 and
epi-resolvins were able to restore TLR9-mediated macrophage uptake of impaired neutrophils
(Sekheri et al., 2020). This finding was further affirmed by NAP1051 treatment in MRL.lpr,
which demonstrated upregulation of TLR9 expression (Figure 4.4). Gene expression of
CXCL-2 and TNF-a both decreased significantly with increase in treatment. CXCL-2 is the
homologue for IL-8 in mice and along with TNF-a is a powerful chemoattractant for
neutrophils. Expression of TLR-7 was downregulated with increasing doses as compared to
the vehicle treated animals. This suggests that TLR-7 does indeed decrease with increasing
concentration of NAP1051. As stated earlier, an enhanced TLR7 signaling drives aberrant
survival of B cell receptor (BCR)-activated B cells and enhances the disease state.
TLR-9 upregulation has been shown to preserve kidney function and prevent nephritis.
We show that with increasing doses of NAP1051 the expression is increased and trending
upwards which could indicate preventing accelerated nephritis. PAD-4 expression decreased
significantly for 5mg/kg/day and 10mg/kg/day as well, but expression increased for the
20mg/kg/day group. This could suggest that we ate hitting a threshold with the increasing
doses. NOX-2 expression was preserved among all the dosage groups. With 20mg/kg/day

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trending upward. Type I interferon (IFN) is critical for host defence. Spontaneous and sustained
IFN induction, however, is a hallmark of and causal factor in lupus, Sjogren’s syndrome and
other autoimmune diseases like SLE.
Type I IFN, in this case IFN-a is essential for the pathogenesis of autoimmune
diseases and can induce ds-DNA receptor activation leading to downstream cGAS-STING
pathway activation. This pathway leads to mitochondrial oxidative stress and increased
cytosolic ds-DNA and autoantibody production. This indicates that our treatment reduced the
activation of IFN-a and this was inversely correlated with immunity related GTPase family
M1 (IRGM1) gene expression which is shown in the heat map (Figure 4.4). IRGM1
expression increased with increasing doses of NAP1051. This would indicate that the drug is
reversing some of the oxidative damage generated due to the disease state.

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Figure 4.4: RT-PCR of makers associated with SLE pathogenesis. Genetic expression of
inflammatory markers associated with inflammation, Tnf, CXCL-2 and IFN-a in the kidney were
analyzed using RT-PCR. A significant decrease was observed for the listed markers along with TLR-7
and TLR-9 which are known to further exacerbate disease conditions. Genetic expression of NOX-2
and PAD-4 was also accessed after 28 days of treatment with NAP1051. These genes are associated
with the process of NETosis and we observe a significant decrease in the MRL/lpr mouse model. N=8
4.3.5 Effect of NAP1051 on the Kidney and Plasma Lipidome in SLE murine models
NAP1051, a LX analogue, is usually synthesized via the metabolism of AA, which is the
precursor for prostaglandins (PG’s), leukotrienes (LT’s). In contrast, omega-3 (n-3) PUFA like
DHA and EPA are produced through iPLA2 metabolism of phospholipids to liberate these antiinflammatory fatty acids. Studies have shown higher levels of DHA in SLE patients had
lowered severity staging with lower levels of lupus nephritis (Kobayashi et al., 2021). Given
that PUFA metabolites serve as potent mediators of inflammation, understanding their
involvement in the progression SLE may give us better understanding of how NAP1051 exerts
its effect on murine SLE models. In Figure 4.5, there is a noticeable trend indicating a dosedependent decrease in lipoxin levels in the plasma with NAP1051 treatment. One possible
interpretation of this observation is that NAP1051 might be effectively resolving inflammation
and restoring the plasma to a state of homeostasis, consequently reducing the need for
endogenous production of lipoxins. Alternatively, the decline in lipoxins could be attributed to
a decrease in the production of AA and hydroxyeicosatetraenoic acid (HETE) precursors, as it
appears that these are downregulated with higher doses of NAP1051. This decrease in lipoxins
could result from either reduced metabolism of the precursors or simply a reduction in
precursor availability due to inflammation resolution. The dose-dependent decrease in
epoxyeicosatrienoic acids (EETs), which are derived from AA by cytochrome P450 (CYP)
enzymes, could similarly be a consequence of decreased AA availability. Notably, all three
metabolites closely mirror the pattern observed for AA levels in terms of both inter- and intragroup variability. However, it is intriguing that the dihydroxy metabolites of 11,12 and 14,15

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EET increase with higher doses of NAP1051. These EET dihydroxy metabolites (DiHETrEs),
although traditionally considered to be biologically inactive due to their rapid conjugation
outside the cell, exhibit an unexpected increase. Interestingly, there is no significant alteration
in plasma levels of docosahexaenoic acid (DHA) observed in Figure 4.5. Notably, Resolvin
D3 and LXA5 were the only lipids significantly upregulated in the plasma within the
10mg/kg/day NAP1051 treatment group.
Figure 4.5: (A) Lipidomic Analysis on MRL.lpr mouse plasma after 28 days treatment with
NAP1051. Plasma levels of AA-derived lipoxins and their 5-HETE precursors including
Leukotrienes. (B) Lipidomic Analysis on MRL.lpr mouse kidney after 28 days treatment with
NAP1051. Kidney levels for PGE2 and PGD2 were upregulated with lower doses of NAP1051
treatment but downregulated with 20mg/kg/day. There were outliers identified using ROUT (Q = 1%)
and removed from the lipid analyte being accessed. Comparisons plotted using one-way ANOVA and
analyzed using Dunnett’s multiple comparison tests. *: p<0.05; ***p<0.001. N=8.

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A correlational analysis between the lipid mediator levels in plasma and kidney (Figure 4.9)
were analyzed and it showed a strong positive correlation (r2
=0.8063). This opens the prospects
of using the liquid biopsy to predict what may be occurring in the tissues or organs. These
analytes were accessed using their area under the curve and correlated these lipids to identify
potential biomarkers that aid the disease progression and pathology. The potential biomarkers
are defined above as those demonstrating strong correlations with SLE pathology and renal
function decline (R2 ≥ 0.80). This can help us identify disease progression and patient outcomes
using only the plasma sample to visualize the kidney lipid changes. In the correlation plots
shown above we identified AA, RvD1 and 5,6 EET as some of the analytes that had a R2 ≥
0.80. More importantly, we identified DHA, LXB4 and LXB5 to have a R2 ≥ 0.90. This could
mean that these 3 lipid mediators could potentially be used as biomarkers for SLE where we
could predict the disease state with accessing the plasma levels. Further testing will help us
understand the pathway these lipids are metabolized/oxidized and how the lipid profiles
overlap between plasma and kidney
Figure 4.6: Correlation of individual lipid analytes between the plasma and kidney lipids that play an
important role in the pro-resolving pathways in SLE and can be used as a tool to predict kidney
disease state by analyzing plasma lipids.
4.3.6 Effect of NAP1051on bone marrow mescenchymal stem cell (MSC) activation
Mesenchymal stem cells (MSCs) are versatile progenitor cells that can be isolated from
various tissue sources, including bone marrow, adipose tissue, kidney tissues, skin, salivary

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glands, limb buds, menstrual blood, and perinatal tissues. These cells are notable for their
ability to differentiate into adipocytes, osteoblasts, and chondrocytes, highlighting their
multipotent nature. MSCs originating from bone marrow have been shown to play a role in
regulating the self-renewal, maturation, and recruitment of hematopoietic stem cells (HSCs),
as well as promoting tissue repair. Given these regenerative capabilities, MSCs have been
explored as a potential therapeutic option for chronic kidney disease (CKD) and acute kidney
injury (AKI) due to their paracrine secretion of factors that can modulate immune responses
and reduce inflammation (Sagaradze et al., 2020). Previous findings support this regenerative
role of MSCs, as evidenced by a study in which transplantation of human umbilical-derived
MSCs (up to 1.2 x 10^6 cells per 10 g body weight) led to inflammation reduction and partial
remission of kidney injury in MRL.lpr mice (Le Thi Bich et al., 2020).
Here we evaluated MSCs isolated from MRL.lpr mice treated with the bioactive lipid
NAP1051, which is designed to modulate inflammatory responses. Bone marrow cells (BM)
treated with NAP1051 showed an increase in BM MSC%, where at 20 mg/kg there was
significant increased BM MSC (p<0.05) of CD105+. The level of MSC elevation in
NAP1051 treated animals are similar to those transplanted by (Guo et al., 2023). These
findings suggest that NAP1051 can expand BM MSC and contribute This study
demonstrated a dose-dependent increase in MSCs from bone marrow, suggesting that
NAP1051 treatment may not only reduce inflammation but also mobilize MSCs to assist in
kidney repair. This mobilization indicates the potential of NAP1051 to support kidney
regeneration by leveraging MSCs. Dysregulated immune cell production, differentiation, and
function can lead to chronic inflammation that weakens MSCs' capacity for tissue repair, thus
limiting kidney regeneration. These results are reflective of inflammatory cytokine gene
earlier shown in Figure 4.5.

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Figure 4.7: (A) Gating strategy for the analysis of MSCs in BM. At necropsy BM cells were isolated
and then stained with MSC cell markers (CD29, Sca-1 and CD105) and immune cell marker (CD45).
MSCs were characterized as ZombieDye-CD45-CD29+Sca-1+CD105+. Zombie Dye is a dye used to
identify dead cells, which could be a result of processing and storage conditions. (B) Results showing
percentage of bone marrow cells identified as MSC’s after applying gating strategy as stated above.
The groups treated with NAP1051 have a higher absolute cell count of bone marrow MSC’s which
could indicate that the drug aids the process of mobilizing stem cells in diseased animals which could
help prevent the chronic inflammatory disease state. Analysis done using one-way ANOVA and
multiple comparisons tests. *p< 0.05, n=8.
4.4 Discussion
The results of our study demonstrate that NAP1051 holds significant promise as a
therapeutic agent for SLE by targeting multiple inflammatory pathways and promoting the
resolution of chronic inflammation. Our findings reveal that NAP1051, a LX analogue, not
only reduces immune-mediated tissue damage through neutrophil regulation and NETosis
inhibition but also enhances MSC mobilization, potentially aiding tissue repair and
regeneration. These mechanisms address key aspects of SLE pathology and align with recent
insights from other studies on autoimmune and inflammatory diseases(Gupta & Kaplan, 2016).
NAP1051’s ability to inhibit NETosis is particularly noteworthy, given that NETs are
implicated in exacerbating SLE through immune complex deposition, sustained inflammation,
and lymphocyte activation. Our results align with Gupta & Kaplan (2016), who highlighted the
contribution of NETs to SLE pathology by demonstrating that chromatin webs embedded with
cytotoxic granules promote autoimmunity. By limiting NET formation, NAP1051 disrupts this
pathogenic loop, thereby reducing downstream inflammatory signaling. This approach to
targeting neutrophil-mediated tissue damage is increasingly recognized in autoimmune

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research which emphasizez the role of neutrophils and NETs in perpetuating chronic
inflammation and tissue injury in SLE (Bissenova et al., 2022).
Further, our study also revealed decreased TNF-α and CXCL-2 expression in
NAP1051-treated mice, suggesting that the analogue modulates key inflammatory cytokines
that recruit neutrophils to inflamed tissue sites. These findings are consistent with those of (de
Oliveira et al., 2016), who demonstrated that halting neutrophil migration and downregulating
inflammatory cytokines are critical steps in resolving inflammation. NAP1051’s ability to
regulate cytokine levels thus appears to interrupt the cycle of immune cell recruitment and
chronic inflammation, creating a more favorable environment for tissue protection and repair.
The observed increase in TLR-9 expression and concurrent reduction in TLR-7
expression in NAP1051-treated mice supports the role of this analogue in modulating the
immune response. TLR-9 deficiency can worsen nephritis in SLE, while studies also showed
that restoring TLR-9 functionality enhances macrophage uptake of impaired neutrophils
(Bossaller et al., 2016; Wen et al., 2023). This ability of NAP1051 to upregulate TLR-9
suggests that it may facilitate the clearance of apoptotic cells and damaged tissues, thereby
preventing the accumulation of autoantigens that can drive SLE pathology. In contrast, the
downregulation of TLR-7, known to promote B- and T-cell aberrations in autoimmune
contexts, aligns with the therapeutic goals of mitigating maladaptive immune responses.
Our findings also revealed that NAP1051 promotes MSC mobilization, as evidenced
by the dose-dependent increase in bone marrow-derived MSCs. This aligns with Guo et al.
(2023), who demonstrated that MSC mobilization could support tissue regeneration and repair
by supplying progenitor cells capable of differentiating into multiple cell types. MSCs’
potential role in treating kidney injury, a common complication in SLE, is supported by studies
that found that MSCs could modulate immune responses and reduce inflammation in kidney
disease models. Given that SLE is often characterized by renal involvement, NAP1051’s ability

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to increase MSC levels may provide regenerative support to damaged kidney tissues,
suggesting that this lipoxin analogue could serve a dual role in both inflammation resolution
and tissue repair (F. Chen et al., 2023).
The lipidomic analysis revealed dose-dependent decreases in arachidonic acid (AA) and related
metabolites in plasma, which may indicate a shift toward a less inflammatory lipid profile.
These changes align with findings by Kobayashi et al. (2021), who reported that elevated DHA
levels correlated with reduced SLE severity and lupus nephritis. Interestingly, our study
showed increased levels of DHA, LXB4, and LXB5, lipids with anti-inflammatory properties,
in plasma, particularly in the higher dose NAP1051 group. This lipid shift may signify that
NAP1051 modulates lipid metabolism in favor of pro-resolving pathways, further supporting
its therapeutic potential for SLE. Additionally, the strong correlation (R2 = 0.8063) observed
between plasma and kidney lipid profiles suggests that non-invasive plasma sampling could be
used as a biomarker to monitor disease state and treatment efficacy, as indicated by similar
findings in studies of inflammatory biomarkers (Afshinnia et al., 2018; Baek et al., 2022)
This study positions NAP1051 as a promising therapeutic candidate for SLE by addressing
critical pathological mechanisms, including NETosis inhibition, immune regulation, and MSC
mobilization. By modulating inflammatory lipid mediators and cytokine expression, NAP1051
may offer a multifaceted approach to managing SLE, potentially alleviating symptoms and
slowing disease progression. Future research should investigate the long-term effects of
NAP1051 on immune modulation and MSC mobilization in clinical contexts to validate its
efficacy for SLE treatment.

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Chapter 5: Concluding Remarks
Systemic lupus erythematosus (SLE) is a complex and chronic autoimmune disease that
can clinically manifest in various organs. SLE disproportionately impacts women at the
reproductive age, which suggest a potential sex hormone effect.
The pathogenesis of SLE has pointed to immune dysregulation, including the
production of autoantibodies against nuclear components and inflammatory cytokines such as
IL-6, TNF-a, and IFN-g. Inflammatory dysregulation is accompanied by excessive
autoantibodies able to form immune complexes that further induced IFN secretion, which
trigger inflammatory cascade leading to additional tissue damage (Katsiari et al., 2010).
Despite advances in the understanding of immune aspect of the disease, effective therapies
capable of disease control or mitigation co-morbidities continue to be evasive.
The progression of SLE is linked to a perpetual cycle of chronic inflammation, which
leads to persistent activation of immune cells, such as plasmacytoid dendritic cells,
macrophages and neutrophils, which contribute to ongoing tissue injury and impair the
restorative arm of wound-healing (Banchereau et al., 2016). Moreover, during immune
activation the role of neutrophil extracellular traps (NETs) is emerging as a factor in SLE
pathogenesis, fueling inflammation and promoting autoimmunity (Knight & Kaplan, 2012).
This thesis highlighted macrophage dysfunction in SLE. In the context of excessive
cellular debris and damage associated molecular patterns (DAMPs), the inability to remove
these cellular byproducts is an important factor in tissue resolution and ability to maintain
homeostasis. We have shown that macrophage inability to process cellular debris may lead to
macrophage senescence, where the consequence is reduction in DAMPs and cellular
elimination. We proposed that the ability to promote macrophage mediated clearance may be
one strategy to reduce DAMPs and thus allow the system to realign this chronic inflammatory
condition.

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While there are drug treatments capable of manage the symptoms associated with SLE,
which include corticosteroids, antimalarials, and immunosuppressants, there are adverse events
associated with chronic use of these treatment. These include increased infection risk,
cardiovascular complications, metabolic dysregulation, and ocular and systemic toxicity,
where these effects have limited their long-term use (Dörner & Furie, 2019; Ruiz-Irastorza et
al., 2010; Tektonidou et al., 2017). All of these treatments focus on inhibiting the immune
response through inducing apoptosis of the immune cells like lymphocytes, neutrophils.
However, these strategies also reduce efferocytosis of cellular debris due to indiscriminate
targeting by these immunosuppressive agents.
There is also gap in knowledge as to the role of bioactive lipids in SLE. Although their
role in inflammation is well-established. Changes in the PUFAs and their metabolites are less
understood in autoimmune diseases like SLE. This thesis addresses the dearth of knowledge
of these important bioactive lipids like PGs, LTs and SPMs. Understanding the difference in
non-disease (C57BL/6J) versus NZM2328 unveiled strategies as how to mitigate SLE disease
progression.
We not only unveiled hidden molecular pathogenesis but was able to develop an
effective strategy to reduce SLE associated adverse events. Our disease interrogation of SLE
initially focused on immune mediated pathology within the bone marrow-derived cells.
Specialized pro-resolving mediators (SPMs), a class of lipid mediators, promoting proresolving mechanisms to restore homeostasis. Lipoxin (LXA4) is a metabolite of Arachidonic
Acid (AA) and the metabolism is mediated by 5-Lipoxygenase (5-LOX) activity. This
pathway forms leukotrienes (LTs), a proinflammatory prostanoid. However upon additional
metabolism of LTA4 via 15-LOX in leukocytes or 12-LOX in platelets can form LXA4,
which is classified as a SPM. This highlights the biological mechanism(s) as how cellular

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resolution of inflammation occurs. However, in SLE, this progressive metabolism of LTA4
to form LXA4 is disrupted and thus unable to promote formation of LXA4.
We have shown in neurodegenerative disease such as retinitis pigmentosa and
Alzheimer’s disease, that 15-LOX activity has been disrupted. In these publications, we found
that administration of NPD1 or LXA4 may be able to mitigate these dysfunctions. In this
thesis, we propose to administer an LXA4 analogue, NAP1051, to determine whether this can
alter SLE disease progression in SLE-prone NZM2328 and MRL.lpr. We have previous
characterized the biological activity of NAP1051which can inhibit neutrophil chemotaxis and
while promoting macrophage efferocytosis of apoptotic cell which are features of our
hypothesis that elimination of immune activation and promoting the elimination of DAMPs
can help to mitigate SLE pathology (Dong et al., 2021). We used the chemical probe to address
our hypothesis that resolving SLE inflammation using SPMs can attenuate SLE pathology. In
Chapter 2-4, we demonstrated that NAP1051, 1) Enhanced bone marrow derived macrophage
(BMDMs) efferocytosis and increased lysosomal acidification, facilitating the clearance of
apoptotic cells while reducing inflammatory phenotype of macrophages/ M1 macrophages in
NZM2328 SLE prone murine model. 2) Reduced crescent formation in the glomeruli of
NZM2328 and MRL.lpr murine SLE models while also reducing renal perivascular infiltration
of immune cells. 3) Protected against renal damage by reducing immune complex deposition
(IgG and C3) and T-cell infiltration (CD3e+) into the kidneys, which is particularly important
in lupus nephritis. 4) Decreasing levels of pro-inflammatory lipids such as prostanoids which
have all been associated with SLE and its progression (Baig et al., 2022). Increased the levels
of omega-3 PUFA lipids like eicosapentaenoic acid (EPA) in the kidney. There are reports
that higher serum EPA correspond to improved prognosis in SLE patients (Oh et al., 2023).
Docosahexaenoic acid (DHA) resolvins were also dramatically increased in the kidney and
serum of NZM2328 and MRL.lpr mice when treated orally with NAP1051 for 28 days. These

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findings suggest that NAP1051 activation can promote resolvin biosynthesis. We reason that
this may be due in part to increase expression of LOX pathways. While our findings in
NZM2328 are exciting, we were able to confirm our findings in MRL.lpr mouse model, where
in addition to the above listed pharmacological activities, NAP1051 also reduced the
occurrence of cutaneous lesions which closely resembles human SLE symptoms.
Figure 5.1: The role of B cells and its receptors in SLE pathogenesis (made with Biorender.com).
While this thesis focused on the innate arm of the immune system, we are cognizant of
the role of B-cells and B-cell receptors like BAFF (B-cell activating factor), TACI
(transmembrane activator and calcium modulator and cyclophilin ligand interactor), APRIL (A
proliferation-inducing ligand), and BCMA (B-cell maturation antigen), in the pathogenesis of
SLE has been well defined (Figure 5.1). The dysregulation of these receptors in SLE results in
B-cell hyperactivation, autoantibody production, and subsequent tissue damage, particularly in
lupus nephritis (Salazar-Camarena et al., 2016).
In particular, elevated BAFF levels support the survival of autoreactive B cells, which
leads to the formation of immune complexes that deposit in the kidneys and other organs,
driving inflammation. Our findings that suggest that SLE increases type II IFN expression

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which can induce B-lymphocyte production of BAFF. While BAFF is a key player in the
overall SLE pathogenesis, there are three receptors—TACI, BCMA, and APRIL—play distinct
roles in immune regulation, with TACI and APRIL being involved in class-switch
recombination and plasma cell survival, and BCMA being essential for mature B-cell survival
as shown in Figure 5.1. Increased levels of BAFF were also observed in NZBW. F1 and
MRL.lpr murine models for SLE (Gross et al., 2000; Salazar-Camarena et al., 2019).
Correspondingly patients with SLE also have elevated BAFF levels often correlated to
increased SLEDI scores and increased circulating anti ds-DNA levels (Petri et al., 2008). Our
findings further correlate with known information pertaining to SLE pathogenesis.
While targeting the BAFF pathway with therapies such as belimumab has shown some
clinical success, the response remains modest in many patients. The drug has been approved
by the FDA for the treatment of lupus nephritis following phase-III clinical trials. However,
despite these approvals, the response rates were modest, with only 43% and 41% of drugtreated patients achieving renal response compared to 32% and 23% in placebo groups (Furie
et al., 2020; Rovin et al., 2021). These modest clinical efficacy highlights the need for more
effective treatment for SLE.
Reduced kidney pathology in SLE-prone NZM 2328 and MLR.lpr, which are two
distinct models of SLE, using NAP1051 show the importance of SPM restoration in mitigating
SLE disease. While there is promising clinical responses, there is still a large number of
unanswered questions as to the viability of this approach. The team has already shown that the
safety of NAP1051, however these studies need to be affirmed in humans.
Other unaddressed molecular question is the role of kidney-resident macrophages
(KRMs) and spleen macrophages in the development and progression of SLE. We have shown
that there is significant difference between BMDM versus systemic effect versus kidney and
plasma lipidomics. To further dissect the region effect of sequestered macrophages, it would

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be important to understand the role KRM in disease progression and what dysregulation they
would have in animal models. More importantly, critical information such as the ability of
SPM administration in modulation the kidneys and other organs found in SLE. We and others
have shown dysregulated in lupus nephritis, contributing to persistent inflammation (L. Zhang
et al., 2020). Similarly, macrophages in the spleen, a critical organ in filtering blood and
managing immune responses, were found to support B-cell hyperactivity in SLE by promoting
the survival and activation of autoreactive B cells. Together, these findings indicate that
targeting the combined dysfunction of B-cell receptors, particularly in the kidneys and spleen,
could provide a more comprehensive therapeutic approach to treating SLE.
This thesis presents important insights into the role of BMDMs dysfunction in SLE, but
further exploration is needed in several areas to advance our understanding and develop more
effective therapies. Mechanistic studies on BAFF and APRIL signaling pathways are crucial,
as these pathways play a pivotal role in B-cell survival and autoimmune responses as
mentioned above (Figure 5.1). BAFF and APRIL maintain the survival of autoreactive plasma
cells by binding to BCMA and TACI and promoting cell survival by upregulation of the
PI3K/AKT pathway (Vincent et al., 2013).In addition to promoting survival of plasma cells,
BAFF is also recognized as an important co-stimulator of T-cells. Human T-cells secrete IFNg and IL-2 in response to BAFF signaling and promotes survival of T-cells exacerbating the
Th1 mediated inflammatory responses. Further studies done on T-cells showed that T-cell
responsiveness was impaired in only BAFF and BAFF-R deficient mice and not in TACI
deficient mice. This tells us that BAFF-R is the only receptor that mediates these T-cell
responses (Ng et al., 2004). In B-cells LXA4 has been shown to suppress the effect of
PI3K/AKT pathway and decreased the production and proliferation of antibodies in memory
B-cells thus preventing an inflammatory response (Ramon et al., 2014). Exploring how
NAP1051 is able to model this behavior and what effects it has on BAFF and BAFF-R activity

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in B-cells and T-cells will give us great insight as to how NAP1051 exerts its effect on the
adaptive immune system and how it can be used to module autoantibody production in SLE.
KRMs play a vital role in maintaining renal homeostasis, yet they become dysregulated
in lupus nephritis, contributing to chronic inflammation and fibrosis (L. Zhang et al., 2020).
Future research should investigate the phenotypic shifts between pro-inflammatory and proresolving states in KRMs to identify if the KRMs mimic the results we see with BMDMs.
NAP1051 and other pro-resolving mediators offer promise for resolving inflammation, and
identifying molecular markers specific to KRMs may facilitate identification of biomarkers for
disease progression.
The spleen, is another key organ in SLE pathology, is central to immune regulation
through its role in B-cell maturation and antigen presentation (Lisak & Ragheb, 2012). Spleen
macrophages support the survival of autoreactive B cells by facilitating immune complex
formation and BAFF/APRIL-mediated activation (Katsiari et al., 2010). Future studies should
explore the interactions between spleen macrophages and B cells, particularly focusing on
BAFF receptor signaling, to determine whether targeting spleen macrophages can reduce Bcell activation and autoantibody production (Chalmers et al., 2015). Given the limited efficacy
of belimumab and other BAFF-targeting therapies, studies should evaluate the potential of
combining BAFF inhibitors with treatments such as NAP1051, to address immune activation
and chronic inflammation comprehensively (Accapezzato et al., 2023). This approach could
prove particularly effective in managing lupus nephritis and halting disease progression.
Sex-specific differences in SLE are primarily driven by the effects of estrogen and its
metabolites on immune function as SLE disproportionately affects women. Estrogen promotes
the survival and proliferation of autoreactive B cells by upregulating BAFF and aids
macrophage polarization to the inflammatory phenotype (Grimaldi et al., 2002). According to
Grimaldi et al., 17β-estradiol also enhances the secretion of interleukin-6 (IL-6) and type I and

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type II interferons (IFNs), which amplifies the inflammatory responses. This hormone further
modulates macrophage polarization by favoring a pro-inflammatory M1 phenotype over the
anti-inflammatory M2 phenotype (Markle & Fish, 2014). Further, estrogen metabolites such
as dehydroepiandrosterone and estradiol have been shown to impair T-cell tolerance
mechanisms, increasing the risk of autoimmunity (Zandman-Goddard et al., 2007). The impact
of estrogen is particularly evident during reproductive years, when hormonal surges correlate
with SLE flares and severity (Walters et al., 2009). These effects help explain the higher
prevalence and more severe disease course observed in women compared to men.
NAP1051 shows potential for broader clinical applications beyond SLE. Future
investigations should examine its efficacy in other autoimmune diseases characterized by Bcell and macrophage dysfunction, such as rheumatoid arthritis and multiple sclerosis to expand
its therapeutic utility. Optimizing NAP1051's dosing will be critical in the clinical setting to
ensure effective use.
This thesis highlights the critical role of macrophage dysfunction in relations to SLE
pathogenesis, particularly in lupus nephritis. The findings show administration of LXA4
analogue, NAP1051 can mitigate renal pathologies through elimination of DAMPs/PAMPs
and promoting increased expression of LOX mediated metabolism to form SPMs. While
ALXR expression is increased in the BMDM of SLE-prone NZM2328, their ability to take up
apoptotic cells are compromised when the number of targets is increased. The addition of
NAP1051 can increase their capacity to eliminate ACs via increasing lysosomal acidification.
These findings suggest that in SLE, dysfunction of macrophage-mediated are factors that
should be evaluated. In addition, SPM treatments appeared to be able to transition macrophage
phenotype. The promising therapeutic potential of NAP1051 could further be explored in its
ability to modulate both B-cell and its receptors function, particularly in the kidneys and spleen.

147
Combination therapies that incorporate pro-resolving mediators like NAP1051 alongside
BAFF inhibitors could offer more effective treatment options for patients with lupus
nephritis. Understanding sex-specific differences, developing advanced biomarkers, and
expanding NAP1051's clinical applications will be crucial in shaping future therapeutic
strategies for SLE and advancing it to human-trials.

148
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(2000). TACI and BCMA are receptors for a TNF homologue implicated in B-cell
autoimmune disease. Nature, 404(6781), 995-999. doi:10.1038/35010115
Katsiari, C. G., Liossis, S. N., & Sfikakis, P. P. (2010). The pathophysiologic role of monocytes
and macrophages in systemic lupus erythematosus: a reappraisal. Semin Arthritis
Rheum, 39(6), 491-503. doi:10.1016/j.semarthrit.2008.11.002
Knight, J. S., & Kaplan, M. J. (2012). Lupus neutrophils: 'NET' gain in understanding lupus
pathogenesis. Curr Opin Rheumatol, 24(5), 441-450.
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Lisak, R. P., & Ragheb, S. (2012). The role of B cell-activating factor in autoimmune
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Markle, J. G., & Fish, E. N. (2014). SeXX matters in immunity. Trends Immunol, 35(3), 97-
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C. R. (2004). B cell-activating factor belonging to the TNF family (BAFF)-R is the
principal BAFF receptor facilitating BAFF costimulation of circulating T and B cells.
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Oh, J., Oda, K., Brash, M., Beeson, W. L., Sabaté, J., Fraser, G. E., & Knutsen, S. F. (2023).
Systemic Lupus Erythematosus and the Ratio of Omega-3 to Omega-6 Fatty Acids
Consumption among Women in the Adventist Health Study-2. Lupus, 32(14), 1637-
1645. doi:10.1177/09612033231213145
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(2008). Association of plasma B lymphocyte stimulator levels and disease activity in
systemic lupus erythematosus. Arthritis Rheum, 58(8), 2453-2459.
doi:10.1002/art.23678
Ramon, S., Bancos, S., Serhan, C. N., & Phipps, R. P. (2014). Lipoxin A₄ modulates adaptive
immunity by decreasing memory B-cell responses via an ALX/FPR2-dependent
mechanism. Eur J Immunol, 44(2), 357-369. doi:10.1002/eji.201343316
Rovin, B. H., Teng, Y. K. O., Ginzler, E. M., Arriens, C., Caster, D. J., Romero-Diaz, J., . . .
Huizinga, R. B. (2021). Efficacy and safety of voclosporin versus placebo for lupus
nephritis (AURORA 1): a double-blind, randomised, multicentre, placebo-controlled,
phase 3 trial. Lancet, 397(10289), 2070-2080. doi:10.1016/s0140-6736(21)00578-x
Ruiz-Irastorza, G., Ramos-Casals, M., Brito-Zeron, P., & Khamashta, M. A. (2010). Clinical
efficacy and side effects of antimalarials in systemic lupus erythematosus: a systematic
review. Ann Rheum Dis, 69(1), 20-28. doi:10.1136/ard.2008.101766
Salazar-Camarena, D. C., Ortíz-Lazareno, P., Marín-Rosales, M., Cruz, A., Muñoz-Valle, F.,
Tapia-Llanos, R., . . . Palafox-Sánchez, C. A. (2019). BAFF-R and TACI expression
on CD3+ T cells: Interplay among BAFF, APRIL and T helper cytokines profile in
systemic lupus erythematosus. Cytokine, 114, 115-127. doi:10.1016/j.cyto.2018.11.008
Salazar-Camarena, D. C., Ortiz-Lazareno, P. C., Cruz, A., Oregon-Romero, E., MachadoContreras, J. R., Muñoz-Valle, J. F., . . . Palafox-Sánchez, C. A. (2016). Association of
BAFF, APRIL serum levels, BAFF-R, TACI and BCMA expression on peripheral Bcell subsets with clinical manifestations in systemic lupus erythematosus. Lupus, 25(6),
582-592. doi:10.1177/0961203315608254
Tektonidou, M. G., Lewandowski, L. B., Hu, J., Dasgupta, A., & Ward, M. M. (2017). Survival
in adults and children with systemic lupus erythematosus: a systematic review and
Bayesian meta-analysis of studies from 1950 to 2016. Ann Rheum Dis, 76(12), 2009-
2016. doi:10.1136/annrheumdis-2017-211663
Vincent, F. B., Saulep-Easton, D., Figgett, W. A., Fairfax, K. A., & Mackay, F. (2013). The
BAFF/APRIL system: emerging functions beyond B cell biology and autoimmunity.
Cytokine Growth Factor Rev, 24(3), 203-215. doi:10.1016/j.cytogfr.2013.04.003

150
Walters, E., Rider, V., Abdou, N. I., Greenwell, C., Svojanovsky, S., Smith, P., & Kimler, B.
F. (2009). Estradiol targets T cell signaling pathways in human systemic lupus. Clin
Immunol, 133(3), 428-436. doi:10.1016/j.clim.2009.09.002
Zandman-Goddard, G., Peeva, E., & Shoenfeld, Y. (2007). Gender and autoimmunity.
Autoimmun Rev, 6(6), 366-372. doi:10.1016/j.autrev.2006.10.001
Zhang, L., Zhang, M., Chen, X., He, Y., Chen, R., Zhang, J., . . . Shi, G. (2020). Identification
of the tubulointerstitial infiltrating immune cell landscape and immune marker related
molecular patterns in lupus nephritis using bioinformatics analysis. Ann Transl Med,
8(23), 1596. doi:10.21037/atm-20-7507

151
Complete Bibliography
Accapezzato, D., Caccavale, R., Paroli, M. P., Gioia, C., Nguyen, B. L., Spadea, L., & Paroli,
M. (2023). Advances in the Pathogenesis and Treatment of Systemic Lupus
Erythematosus. Int J Mol Sci, 24(7). doi:10.3390/ijms24076578
Baig, S., Vanarsa, K., Ding, H., Titus, A., McMahon, M., & Mohan, C. (2022). Baseline
Elevations of Leukotriene Metabolites and Altered Plasmalogens Are Prognostic
Biomarkers of Plaque Progression in Systemic Lupus Erythematosus. Front
Cardiovasc Med, 9, 861724. doi:10.3389/fcvm.2022.861724
Banchereau, R., Hong, S., Cantarel, B., Baldwin, N., Baisch, J., Edens, M., . . . Pascual, V.
(2016). Personalized Immunomonitoring Uncovers Molecular Networks that Stratify
Lupus Patients. Cell, 165(3), 551-565. doi:10.1016/j.cell.2016.03.008
Chalmers, S. A., Chitu, V., Ramanujam, M., & Putterman, C. (2015). Therapeutic targeting of
macrophages in lupus nephritis. Discov Med, 20(108), 43-49.
Dong, T., Dave, P., Yoo, E., Ebright, B., Ahluwalia, K., Zhou, E., . . . Louie, S. G. (2021).
NAP1051, a Lipoxin A4 Biomimetic Analogue, Demonstrates Antitumor Activity
Against the Tumor Microenvironment. Mol Cancer Ther, 20(12), 2384-2397.
doi:10.1158/1535-7163.Mct-21-0414
Dörner, T., & Furie, R. (2019). Novel paradigms in systemic lupus erythematosus. Lancet,
393(10188), 2344-2358. doi:10.1016/s0140-6736(19)30546-x
Furie, R., Rovin, B. H., Houssiau, F., Malvar, A., Teng, Y. K. O., Contreras, G., . . . Roth, D.
A. (2020). Two-Year, Randomized, Controlled Trial of Belimumab in Lupus Nephritis.
N Engl J Med, 383(12), 1117-1128. doi:10.1056/NEJMoa2001180
Grimaldi, C. M., Cleary, J., Dagtas, A. S., Moussai, D., & Diamond, B. (2002). Estrogen alters
thresholds for B cell apoptosis and activation. J Clin Invest, 109(12), 1625-1633.
doi:10.1172/jci14873
Gross, J. A., Johnston, J., Mudri, S., Enselman, R., Dillon, S. R., Madden, K., . . . Clegg, C. H.
(2000). TACI and BCMA are receptors for a TNF homologue implicated in B-cell
autoimmune disease. Nature, 404(6781), 995-999. doi:10.1038/35010115
Katsiari, C. G., Liossis, S. N., & Sfikakis, P. P. (2010). The pathophysiologic role of monocytes
and macrophages in systemic lupus erythematosus: a reappraisal. Semin Arthritis
Rheum, 39(6), 491-503. doi:10.1016/j.semarthrit.2008.11.002
Knight, J. S., & Kaplan, M. J. (2012). Lupus neutrophils: 'NET' gain in understanding lupus
pathogenesis. Curr Opin Rheumatol, 24(5), 441-450.
doi:10.1097/BOR.0b013e3283546703
Lisak, R. P., & Ragheb, S. (2012). The role of B cell-activating factor in autoimmune
myasthenia gravis. Ann N Y Acad Sci, 1274, 60-67. doi:10.1111/j.1749-
6632.2012.06842.x

152
Markle, J. G., & Fish, E. N. (2014). SeXX matters in immunity. Trends Immunol, 35(3), 97-
104. doi:10.1016/j.it.2013.10.006
Ng, L. G., Sutherland, A. P., Newton, R., Qian, F., Cachero, T. G., Scott, M. L., . . . Mackay,
C. R. (2004). B cell-activating factor belonging to the TNF family (BAFF)-R is the
principal BAFF receptor facilitating BAFF costimulation of circulating T and B cells.
J Immunol, 173(2), 807-817. doi:10.4049/jimmunol.173.2.807
Oh, J., Oda, K., Brash, M., Beeson, W. L., Sabaté, J., Fraser, G. E., & Knutsen, S. F. (2023).
Systemic Lupus Erythematosus and the Ratio of Omega-3 to Omega-6 Fatty Acids
Consumption among Women in the Adventist Health Study-2. Lupus, 32(14), 1637-
1645. doi:10.1177/09612033231213145
Petri, M., Stohl, W., Chatham, W., McCune, W. J., Chevrier, M., Ryel, J., . . . Freimuth, W.
(2008). Association of plasma B lymphocyte stimulator levels and disease activity in
systemic lupus erythematosus. Arthritis Rheum, 58(8), 2453-2459.
doi:10.1002/art.23678
Ramon, S., Bancos, S., Serhan, C. N., & Phipps, R. P. (2014). Lipoxin A₄ modulates adaptive
immunity by decreasing memory B-cell responses via an ALX/FPR2-dependent
mechanism. Eur J Immunol, 44(2), 357-369. doi:10.1002/eji.201343316
Rovin, B. H., Teng, Y. K. O., Ginzler, E. M., Arriens, C., Caster, D. J., Romero-Diaz, J., . . .
Huizinga, R. B. (2021). Efficacy and safety of voclosporin versus placebo for lupus
nephritis (AURORA 1): a double-blind, randomised, multicentre, placebo-controlled,
phase 3 trial. Lancet, 397(10289), 2070-2080. doi:10.1016/s0140-6736(21)00578-x
Ruiz-Irastorza, G., Ramos-Casals, M., Brito-Zeron, P., & Khamashta, M. A. (2010). Clinical
efficacy and side effects of antimalarials in systemic lupus erythematosus: a systematic
review. Ann Rheum Dis, 69(1), 20-28. doi:10.1136/ard.2008.101766
Salazar-Camarena, D. C., Ortíz-Lazareno, P., Marín-Rosales, M., Cruz, A., Muñoz-Valle, F.,
Tapia-Llanos, R., . . . Palafox-Sánchez, C. A. (2019). BAFF-R and TACI expression
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Salazar-Camarena, D. C., Ortiz-Lazareno, P. C., Cruz, A., Oregon-Romero, E., MachadoContreras, J. R., Muñoz-Valle, J. F., . . . Palafox-Sánchez, C. A. (2016). Association of
BAFF, APRIL serum levels, BAFF-R, TACI and BCMA expression on peripheral Bcell subsets with clinical manifestations in systemic lupus erythematosus. Lupus, 25(6),
582-592. doi:10.1177/0961203315608254
Tektonidou, M. G., Lewandowski, L. B., Hu, J., Dasgupta, A., & Ward, M. M. (2017). Survival
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Bayesian meta-analysis of studies from 1950 to 2016. Ann Rheum Dis, 76(12), 2009-
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Vincent, F. B., Saulep-Easton, D., Figgett, W. A., Fairfax, K. A., & Mackay, F. (2013). The
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Abstract (if available)

Abstract Systemic Lupus Erythematosus (SLE) is a multifaceted autoimmune disease marked by widespread inflammation, excessive autoantibody production, and organ damage, with lupus nephritis being a frequent and severe manifestation. Current therapies, often limited in their efficacy and associated with significant side effects, highlight the need for novel treatment approaches. This dissertation explores the therapeutic potential of NAP1051, a synthetic lipoxin analogue designed to mimic the pro-resolving properties of endogenous lipoxin A4 (LXA4) while overcoming its rapid metabolism and short half-life.
NAP1051 was evaluated in murine models of SLE, including NZM2328 and MRL.lpr strains, to assess its immunomodulatory and anti-inflammatory effects. Studies revealed that NAP1051 significantly reduced proteinuria, autoantibody levels, and renal immune complex deposition. Additionally, histological and immunofluorescence analyses demonstrated decreased glomerular damage and reduced infiltration of inflammatory cells. Upon probing the mechanisms involved by which NAP1051 exerted its therapeutic efficacy we see that NAP1051 treatment modulated macrophage polarization, promoting a shift from pro-inflammatory M1 to anti-inflammatory phenotypes, and enhanced macrophage-mediated efferocytosis of apoptotic cells. These effects were linked to improved clearance of apoptotic cells, reduced neutrophil extracellular trap formation (NETosis), and the attenuation of cytokine-driven inflammatory cascades in vivo.
Building on these findings, we conducted a detailed analysis of polyunsaturated fatty acids (PUFAs) and their bioactive lipid mediator metabolites in both murine models to elucidate the mechanisms by which dysregulated lipid metabolism contributes to SLE progression. This investigation also aimed to understand the impact of NAP1051 on these processes. NAP1051 effectively restored lipid mediator homeostasis by reducing the levels of pro-inflammatory lipids, including leukotrienes and prostaglandins, while enhancing the production of pro-resolving mediators such as resolvins. Mechanistic studies identified the ALX/FPR2 receptor as a critical pathway through which NAP1051 exerted its effects, modulating immune cell activity and significantly reducing the production of key pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. Notably, NAP1051 demonstrated a strong safety profile, with no observed adverse effects on hematologic, hepatic, or renal functions, highlighting its potential as a promising candidate for further therapeutic development.
Collectively, this dissertation provides compelling evidence for the use of NAP1051 as a novel therapeutic approach for SLE. By directly addressing the molecular and cellular abnormalities underlying SLE, including defective apoptotic cell clearance, dysregulated macrophage activity, and unresolved inflammation, NAP1051 offers a targeted and effective solution to mitigate disease progression. These findings lay the groundwork for future research and clinical translation of synthetic lipoxin analogues in autoimmune diseases.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Dave, Priyal (author)

Core Title Modulation of immune response in Systemic Lupus Erythematosus by synthetic lipoxin analogue, NAP1051

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Clinical and Experimental Therapeutics

Degree Conferral Date 2024-12

Publication Date 12/20/2024

Defense Date 12/09/2024

Publisher Los Angeles, California (original), University of Southern California (original), University of Southern California. Libraries (digital)

Tag autoimmune,efferocytosis,inflammation,innate immune immunity,lipoxin,macrophage,nephritis,NETosis,neutrophil,OAI-PMH Harvest,systemic lupus erythematosus

Format theses (aat)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Louie, Stan (committee chair), Asante, Isaac (committee member), Stohl, William (committee member)

Creator Email priyalda@usc.edu,priyaldavee@gmail.com

Unique identifier UC11399EZKX

Identifier etd-DavePriyal-13706.pdf (filename)

Legacy Identifier etd-DavePriyal-13706

Document Type Dissertation

Format theses (aat)

Rights Dave, Priyal

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Source 20241223-usctheses-batch-1230 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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