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Characterization of changes in metabolism and inflammation in the lacrimal gland in dry eye disorders
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Characterization of changes in metabolism and inflammation in the lacrimal gland in dry eye disorders
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Content
Characterization of changes in metabolism and inflammation
in the lacrimal gland in dry eye disorders
by
Minchang Choi
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)
August 2024
ii
Acknowledgements
First of all, I would like to thank my PI, Dr. Sarah Hamm-Alvarez for her constant patience
and guidance. During the 6 years that I devoted to the PhD degree, she has had numerous occasions
where she showed leadership and humility to not only myself but also to the whole lab. She always
gave the appropriate push when I was going through both scientific and personal struggles and was
open to spend her own resources for my own professional development. I am truly grateful to have
been given the opportunity to be in an environment where every lab member is open to help, and
I believe this is a true portrayal of her leadership character which has set an example for my future
endeavors. I would also like to thank Dr. Andrew J. MacKay in giving numerous suggestions
during collaborative meetings and presentations. Dr. Bangyan Stiles has been a constant reminder
for me to think of different aspects that may be in play and be open to new ideas through my
interactions with her and classes that I had taken.
Secondly, I would like to thank all the lab members past and present that have contributed
to my development: Dr. Maria Edman-Woolcott who gave me professional and personal advice,
Srikanth Reddy Janga, who taught me to be flexible in my thoughts and experiments, Dr. Hao Guo
who served as the examplary scientist for all, Dr. Yaping Ju who never shut down my forever
questions, Dr. Christina Fu who helped me through my initial projects and helped me during my
downs, Dr. Changrim Lee for the openness to take care of my Korean personality, Cindy Toscano
Xiaoyang Li and Carlos Delgado for keeping me sane throughout my journey. I would like to also
thank Dr. Anh Truong who would give me personal advice as a PharmD graduate and Dr. Jason
Junge and Dr. Cintia de Paiva for their valuable advice.
Finally, I would like to thank my wife, Dr. Seoyoung Choi, who sacrificed so much and
provided me with support no matter what. I would not have been able to get through without her.
iii
Table of Contents
Acknowledgements………………………………………………………………………………..ii
List of Figures…………………………………………………………………………...………. iv
List of Tables……………………………………………………………………………………...vi
Abstract…………………………………………………………………………………………..vii
Chapter 1: Introduction to the ocular surface, lacrimal gland and dry eye disorders………….….1
Chapter 2. Modulation of PP2A, TTP and P38 MAPK for CTSS Regulation…………….….….4
1.1 Introduction.………………………………………………………………………….….…....4
1.2 Methods……………………………………………………………………………………….8
1.3 Results………….…………………………………………………….………………………14
1.4 Discussion……………………………………………………………………………………28
Chapter 3: Multinucleate Macrophages in the aged lacrimal gland.…………………………….31
2.1 Introduction ………………………………………………………………………………….31
2.2 Materials and Methods……………………………………………………………………….33
2.3 Results..………………………………………………………………………………………40
2.4 Discussion……………………………………………………………………………………57
Chapter 4: Fluorescence Lifetime Imaging of aged lacrimal glands …………………………...63
3.2 Introduction …………………………………………………………………………….……63
3.3 Materials and Methods…………………………………………………………………….…72
3.3 Results………………………………………………………………………………………..85
3.4 Discussion……………………………………………………………………………………86
Conclusion……………………………………………………………………………………….88
References……………………………………………………………………………………….89
iv
List of Figures
Chapter 2
Figure 1. Diagram showing interaction of PP2A and TTP to regulate CTSS mRNA stability and
translation.…………………………………………………………………………………………8
Figure 2. Tristetraprolin (TTP) is increased at the protein level while the gene expression is
decreased in male NOD LG versus BALB/c LG ……………….………………….….……...…16
Figure 3. Western Blotting of p38 and phospho-p38 in 12 wk male NOD and BALB/c LG
Lysates…………………………………………….…………………….…………………..……17
Figure 4. Real time qRT-PCR and Western Blotting data of components of the PP2A regulatory
pathway in 12-16 wk male BALB/c and NOD LG.....……………………………..……….……18
Figure 5. CTSS activity is increased in Okadaic Acid (OKA) treated HCET and ex vivo
LG…....………………….….……..…….….……….….……….….……….….……….….……19
Figure 6. p38 Inhibitor and SMAP effects on CTSS mRNA abundance in HCET cells exposed
to IFN-γ….….……..…….….……….….……….….……………….…….….……….….……...20
Figure 7. Maximum Tolerated Dose study design and toxicity measurement...………….……...22
Figure 8. Effects of SB202190 on indications of autoimmune dacryoadenitis in 14wk male
NOD mice.….……..…….….……….….……….….……….….……….………………..……...25
Figure 9. Efficacy of higher dosage SB202190 on indications of autoimmune dacryoadenitis
In 14 week male NOD mice.….……….….…….….…..…….…………………………………...26
Figure 10. Western Blotting of phospho-P38 and P38 from SB202190 treated LG lysates at
different time-points. A. Top membrane (phospho-P38) and bottom membrane (p38) shows
the LG lysates from vehicle treated with SB202190.…….............................................................27
Figure 11. Western Blotting of phospho-P38 and P38 from SB202190 treated spleen lysate
at different time-points....…..…..……..….……….….……….….……………………….……...28
Chapter 3
Figure 1. RT-qPCR of Ctsl of young and old aged female C57 mice………………….………...41
Figure 2. NPC1 and active CTSL are increased with age in B6 mouse LG lysates…………..…42
Figure 3. Analysis of NPC1 and total protein stain including outlier..………….………..……...43
v
Figure 4. LED photobleaching of LG tissue sections reduces endogenous
autofluorescence…………...…..…..…..……..….……….….……….….……….……....……...44
Figure 5. Photobleaching parameters associated with reduction of tissue autofluorescence……47
Figure 6. NPC1 and NPC2 are enriched in extra-acinar structures in intermediate and old B6
mouse LG………………...…..…..…..……..….……….….……….….……….……....……......49
Figure 7. NPC1 and NPC2 are enriched in apparent F4/80+ multinucleated macrophages in
aging LG……...……………….....…..……..….……….….……….….……….……....……......51
Figure 8. Mononuclear and multinuclear F4/80+ macrophage comparisons in old and young
LG……...………………...…..…..…..……..….……….….……….….……….……....……......53
Figure 9. CTSL is enriched with age in F4/80+ multinucleated macrophages containing NPC1.55
Figure 10. Cd11b and F4/80 enrichment in mononuclear and multinucleated macrophages in
LG.....………………...…..…..…..……..….……….….……….….……….……....……............57
Chapter 4
Figure 1. Phasor approach to FLIM analysis and NAD(P)H-enzyme binding trends in the
phasor……………...…..…..…..……..….……….….……….….……….……....……................66
Figure 2. Immunoprecipitated NOX2 from LPS stimulated RAW 264.7 cells FLIM image and
phasor……………...…..…..…..……..….……….….……….….……….……....……................74
Figure 3. Metabolism FLIM imaging of young and old LG..……….……....…….......................77
Figure 4. Pathological Signal FLIM imaging of young and old LG. ..……….……....……….…79
Figure 5. Old LG shows NOX2 and lipofuscin signal in multinucleated macrophages………….81
Figure 6. Old LG shows increased phosphor-p47phox in macrophages…..……....…………..…83
Figure 7. LPS stimulated RAW 264.7 cells have higher p47phox and phosphorylated p47phox..85
vi
Abstract
Dry eye disease (DED) occurs when the ocular surface system cannot make enough
tears or can’t preserve the tears that are already made. Among the many organs
components of the complex system that provide and maintain the tears, the lacrimal gland
(LG) is the exocrine organ that produces the tears. Through autoimmune diseases such
as Sjögren’s Syndrome (SS) and also through aging, the function of the LG deteriorates
substantially which exacerbates the DED. As most treatment procedures alleviate the
symptoms and not the etiology of DED, a further understanding of the pathological
mechanism is essential. I present through my thesis a possible mechanism involving the
p38 MAP Kinase and tristetraproline contributing to the increase in Cathepsin S, a
cysteine protease involved in antigen presentation that is elevated in SS patients and
murine models of the disease in the LG and tears. Also, I present an increase in a novel
multinucleate macrophage in the aging LG that is involved in lipid metabolism and has
pro-inflammatory characteristics. Finally, I present an application, fluorescence lifetime
imaging microscopy (FLIM), to understand changes in metabolism and cell composition
in the LG. These discoveries may help enhance the understanding of the development
of dry eye disease in relation to the lacrimal gland.
1
Chapter 1: Introduction to the ocular surface, lacrimal gland, and dry eye
disorders
1.1 Introduction
The ocular surface system includes the components that are essential for vision.
Unlike all other wet-surfaced epithelia of the body, this system is open to the outside world
and subject to drying, injury and pathogens. The cornea, conjunctiva, lacrimal gland (LG),
and the meibomian gland are all major machineries of the ocular surface system that work
to provide, protect, and maintain a smooth refractive surface on the cornea.1 While the
corneal and conjunctival epithelia produce the hydrophilic mucins that help keep the tears
on the eye surface, the meibomian gland provides the tear lipid layer that prevents tear
evaporation. The LG is an exocrine gland responsible for producing the aqueous layer
of the tear film, which provides moisture, lubrication, and nutrients to the ocular surface.2,3
Changes in LG function have been linked to aqueous tear deficiency, which is associated
with dry eye disease.
Dry eye disease (DED) is a common ocular disorder that affects 5 to 50 percent of the
global population.4 In the US, DED prevalence is 2.7% in individuals aged 18 to 34-years
compared to 18.6% in individuals >75-years. DED prevalence is 2-fold higher in women
than in men.5 With the aging of the US population, an alarming increase of 60% in DED
prevalence is expected by 2030.6,7 DED is the leading cause of visits to eye care
specialists, creating an estimated $3.8 billion/year in direct US healthcare costs with over
$55.4 billion estimated in societal costs.5,8 Given the steep rise in patient numbers and
ensuing societal burden, a deeper understanding of DED is essential.
The focus of my research has been on the changes in the LG associated with SSassociated dry eye and age-related dry eye. Sjögren’s Syndrome (SS) is a chronic,
2
systemic autoimmune disease that manifests a lymphocytic infiltration and gradual loss
of function of the LG and the salivary glands (SG) 9. This leads to dry symptoms of both
the eyes and the mouth, also known as keratoconjunctivitis sicca and xerostomia
respectively. Other systemic symptoms may include meningitis, lymphoma, visceral organ
inflammation and chronic pain and fatigue also exist causing a serious decrease in quality
of life for the patients 10. With a prevalence of 2.2 to 11.0 per 10,000 inhabitants, SS is
estimated to have 4 million patients in the US 11,12. Currently the US-FDA approved
treatment option for SS are for symptomatic treatment of dry eye with Restasis. Also, the
only FDA approved medications are for symptomatic treatment of dryness: Cevemiline
and Salagen. Cevimeline’s and Salagen’s FDA approved indication is to treat the
symptoms of dry mouth in patients with SS 13,14. There are immunomodulatory treatments,
such as cyclosporine, that are used off-label for SS-associated dry eye, and
investigational drugs (RO5459072, Iscalimab) trials that are ongoing 15,16. There are
currently no treatments that help deter the progress as the pathogenesis and etiology are
not fully understood.
The US prevalence of DED is over 6 times higher in individuals over 75 years
compared to 18 to 34 years individuals (18.6% vs. 2.7%), and 2-fold higher in women
than in men.5 Age-related changes in the LG such as decreased tear secretion17,18 and
altered glandular structure19 may contribute to the development of age-related DED.
Similar changes are seen in C57BL/6J mice by 6-9 months, with changes in corneal
surface irregularity, corneal barrier disruption, and tear volume compared to 8-week mice
in both sexes.20 The aging mouse LG also exhibits increased lymphocytic infiltration and
formation of ectopic lymphoid structures similar to that of humans.21,22 With the C57BL/6J
3
aging model, increased oxidative stress in the LG and its amelioration with an anti-oxidant
diet has been demonstrated.23 Consistent with this, superoxide dismutase knockout mice,
with a reduced ability to reduce oxygen radicals, show accelerated infiltration of immune
cells, higher fibrosis, and apoptosis in the LG.24
As the following three chapters illustrate, I have explored changes in the LG in
murine models of SS-associated DED as well as age-related DED.
4
Chapter 2. Modulation of PP2A, TTP and P38 MAPK for CTSS Regulation
2.1. Introduction
Cathepsin S (CTSS) is a cysteine protease, mostly expressed in the lysosomes of
immune cells, such as macrophages, APCs (antigen processing cell), B-cells, dendritic
cells, and lacrimal gland acinar cells.25-30 CTSS has many physiological functions, of
which degradation of the extracellular matrix, protein catabolism and processing of the
MHC II–associated invariant chain (Ii) (Figure 1).
31-33 The Ii peptide plays a pivotal role in
acting as a placeholder within the MHC II complex‘s antigenic peptide groove during its
assembly in the endoplasmic reticulum. This prevents the endogenous peptides from
being presented as potential antigens. Within the lysosome of mononuclear phagocytic
origin cells, CTSS cleaves the li peptide in the MHC II groove into the CLIP peptide (class
II invariant chain-associated peptide), during a later phase of the antigen presentation
process. After the li peptide cleavage by CTSS, the CLIP peptide is able to be replaced
for other antigenic peptides with higher affinity for the MHC II complex.31,34,35 Thus, CTSS
plays a crucial role in MHC class II–mediated antigen presentation, and conversely,
higher levels of cathepsin S have been linked to the generation of autoreactive CD4+ T
cells due to increased MHC II presentation time.35,36 Due to its distinct role in antigen
presentation, CTSS is implicated in various autoimmune diseases including rheumatoid
arthritis, systemic lupus erythematosus and multiple sclerosis.37
Although our lab has identified CTSS as a tear biomarker and a therapeutic target
for SS, and shown that it is elevated in both infiltrating immune and acinar cells in the
lacrimal gland (LG),38 there have been limited studies identifying the ways in which CTSS
5
is distributed and released from LG acinar cells (LGAC) in SS specifically. CTSS is
synthesized as a pre-proenzyme, in which the pre-peptide works as the signaling peptide
to direct the protein to the rough endoplasmic reticulum and the propeptide acts to keep
it in the proteolytically inactive state.
39 In the Golgi, CTSS is post-translationally modified
by being mannose 6-phosphorylated and is recognized by the cation-dependent mannose
6-phosphate receptor (CD-MPR). After being sorted into clathrin-coated transport
vesicles, CTSS, like other procathepsins, is activated during its transportation through the
endo/lysosome and the maturation of the late endosomes.
39-41
The male non-obese diabetic mouse (NOD) is commonly used as a murine model
of the autoimmune dacryoadenitis (inflammation of the LG) and ocular surface
inflammation, characteristic of SS. 42 Sex differences are evident in the NOD mice. Both
Male and Female NOD mice develop type 1 diabetes at 12 wks (female) and 20 wks
(male)43 but the male mice show the ocular manifestations of SS spontaneously from 8-
10 weeks, and develop a LG lymphocytic infiltration at 6-12 weeks but exhibit little SG
inflammation 44. Female NOD mice develop a relatively later salivary gland (SG)
lymphocytic infiltration at 20 - 24 wks, but less LG inflammation 45. The male NOD mice
share similar ocular surface system manifestations to SS patients such as lymphocytic
infiltration of the LG, reduced tear flow, generation of a proteolytic tear film, reduced
myoepithelial cells and loss of extracellular matrix 46-49 and elevated cytokines (IL-1β,
TNF-α, IFN-γ, ) in LG and tears.50-54
Similar to studies in the male NOD model,
55 CTSS activity was demonstrated as
significantly elevated in SS patient tears relative to tears of patients with non-SS dry eye
or other autoimmune diseases.56 In vitro studies suggest that elevated tear CTSS can
6
affect ocular surface homeostasis, since it is able to induce expression and secretion of
pro-inflammatory cytokines and matrix metallopeptidase 9 (MMP-9) in a corneal cell
line.57 In the male NOD mouse, CTSS activity and protein are increased not only in tears
but also in the protein-secreting acinar cells of the LG, suggesting additional actions.49,50
With knowledge of the relationship of other protease-sensitive pathways to inflammation,
we sought out other potential targets of CTSS that might be implicated in the etiology of
SS.
Protein phosphatase 2A (PP2A) and tristetraprolin (TTP) are upstream
components that regulate the stability of CTSS mRNA and subsequently its protein
expression (Figure 1). With three different subunits A (scaffold), B (regulatory), and C
(catalytic), PP2A exists in dimeric (A and C) and trimeric forms (A, B, and C). PP2A activity
has been used as a target in various cancers including lung cancer, prostate cancer, and
pancreatic cancer 58-61 due to its tumor suppressor activity 62. PP2A activates TTP, which
binds to unique sequences present in untranslated regions of mRNA of pro-inflammatory
cytokines (IL-6, IL-8, IL-17, TNF), CTSS, and MMP 9 and deter their translation 63-68 as in
Figure 1. These pro-inflammatory cytokines and MMP9 have been shown to be highly
expressed in SS patient tears 68-72. In contrast, TTP can be phosphorylated by p38/ MAPK
and subsequently become inactivated and thus increase the aforementioned mRNA
translation 73. PP2A itself is regulated by multiple proteins and small molecules including:
SET (SE Translocation), Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A),
Okadaic Acid (OKA) and small molecular activator of PP2A (SMAP) 58,63,74. Erlotinib, an
EGFR-targeted tyrosine kinase inhibitor, prevents CIP2A expression and thus increases
PP2A activity 63. While SET can be inhibited by the sphingosine analog, Fingolimod
7
(FTY720) 75, PP2A can be directly activated by SMAPs and inhibited by OKA 58,76. There
are other inhibitors that are commercially available for PP2A inhibition, such as calyculin
A, microcystin-LR, and fostriecin. However, OKA is the preferred inhibitor due to its
stability (years for OKA, two or 3 months for Fostriecin at -20 °C) and relative specificity
for PP2A against other protein phosphatases 60. Also, SB202190 is a p38 MAPK inhibitor,
that has been tested in cells and mice.
77 The linkage between PP2A and CTSS has been
pursued in asthma and chronic obstructive pulmonary disease, where cigarette smoke
was shown to increase CIP2A expression and thereby decrease PP2A activity in lung
epithelial cells 78.
The p38 mitogen-activated protein kinases (MAPK) are evolutionarily conserved
serine/threonine protein kinases that convert the extracellular signals to the intracellular
machinery to regulate a variety of cellular processes, including inflammation, cell
differentiation, cell growth and death.79,80 While there are four p38 kinase families, p38α,
p38β, p38γ, and p38δ, p38α and p38β are ubiquitously expressed, whereas p38γ and
p38δ are expressed in a tissue-specific manner.
79,80 A wide range of environmental
stresses (redox stress, ultraviolet irradiation, cytokines, heat shock and osmotic shock)
79-81 induces the activation of p38 which stimulates an inflammatory response, a key part
of the host defense system. Excessive inflammation contributes to the pathogenesis of
multiple human diseases, making the p38 pathway inhibitors potential drugs for
inflammation-related diseases.
79,80 Thus, targeting p38 for the development of novel
therapeutics against multiple chronic and acute pathologies is being tested. Here, we
hope to elucidate the involvement of the p38, TTP and PP2A pathway in the pathogenesis
of SS and possible modulation of the disease by P38 inhibition.
8
Figure 1. Diagram showing interaction of PP2A and TTP to regulate CTSS mRNA stability
and translation. The activation (DT-061) and inhibition (CIP2A, SET) of PP2A leads to the
increase and decrease in de-phosphorylation of TTP respectively. There are other agents
such as SB202190 that inhibits the phosphorylation of P38 and RO5461111 which is a
CTSS inhibitor. The phosphorylated TTP would render TTP inactive and hinder it from
binding to the mRNA of pro inflammatory cytokines and CTSS. The CTSS mRNA gets
translated into the proenzyme of CTSS, which matures while it travels in the late
endosome and lysosome.
2.2 Methods
2.2.1 Reagents
Keratinocyte Serum Free Medium (KSFM) with human recombinant epidermal growth
factor and bovine pituitary extract (#17005042) were from Life Technologies (Carlsbad,
CA). Reverse transcription Reagents (N8080234, ThermoFisher Scientific Inc., Waltham,
MA). TaqMan Universal Mix II, no UNG (4440040), TaqMan Reverse Transcription
Reagents (N8080234) are also from ThermoFisher. Tris-Buffered Saline with 0.1%
Tween® 20 Detergent (TBST) and 4-methylumbelliferyl N-acetyl-β-d-glucosaminide
SB202190
Extracellular matrix degradation
MHCII invariant chain processing
9
(CAS no. 37067-30-4) were from Sigma-Aldrich (St. Louis, MO, USA). Bovine serum
albumin (#2905) was from Calbiochem (Billerica, MA). Hank’s Balanced Salt Solution
(HBSS) without Mg2+ and Ca2+ was from Lonza Group Ltd. (Basel, Switzerland).
ProLong Gold Antifade Mounting Medium was from Invitrogen (Grand Island, NY, USA).
Bio-Rad protein assay dye (#5000006) was from Bio-Rad (Hercules, CA, USA). 10%
Triton X-100 (#NC0478124), 10% tris-glycine gels (#XP0010C), nitrocellulose iBlotTM 2
transfer stacks (#IB23001), and TaqMan reverse transcription kits (#N8080234) were
from Thermo Fisher Scientific (Waltham, MA, USA). Revert™ 700 total protein staining
kit (#926-11010) was from LI-COR (Lincoln, NE, USA). RNeasy Universal Kits (73404)
was from Qiagen (Germantown, MD, USA). Fluorescent blocking buffer was from
Rockland Immunochemical Inc. (Limerick, PA, USA). VWR Superfrost. Plus microslides
were from VWR (Radnor, PA, USA). GAPDH (Hs99999905_m1), and CTSS
(Hs00175407_m1) are from ThermoFisher.
2.2.2 Mice
Male NOD/ShiLtJ mice (Stock #: 001976) and BALB/cJ mice (Stock #: 000651) were
purchased from the Jackson Laboratory (Bar Harbor, ME) and were aged to more than
12 weeks of age. Animal use was in compliance with policies approved by the University
of Southern California Institutional Animal Care and Use Committee and in accordance
with the Guide for the Care and Use of Laboratory Animals 8th edition/ the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research.
10
2.2.3 Western Blotting of LG lysate
For gel electrophoresis, one whole LG was used per sample. LG were
homogenized using Beadbug prefilled tubes in RIPA buffer containing protease inhibitor
cocktail. The supernatant was recovered after centrifugation at 8000 × g at 4°C for 10 min.
After measuring each sample’s protein concentration with the Pierce BCA protein assay
kit, samples were incubated in reducing dye and b-mercaptoethanol (6x) at 95°C for 5
min. 60 µg or 20 µg of each sample in sample buffer were loaded onto precast 10% TrisGlycine gels and resolved by SDS-PAGE at 120 V at 4°C for 2 hr. Proteins on gels were
transferred to nitrocellulose membranes using an iBlot™ 2 gel transfer machine.
Membranes were stained for total protein using the Revert™ 700 Total Protein Stain Kit
for Western Blot Normalization and the signal was read using the LiCor Odyssey Fc
machine. The signal for each lane was used to normalize the signal of the protein of
interest to total protein loaded. Total protein stain was rinsed away per the manufacturer’s
protocol, and membranes were blocked for 1 hr with blocking buffer at room temperature
while shaking. Membranes were washed 3 times for 5 min with TBS-T (Tris Buffered
Saline- Tween), then incubated with either rabbit anti-mouse TTP (1:50) or rabbit antimouse Phospho-p38 (1:1000) or rabbit anti-mouse p38 (1:1000) in blocking buffer at 4°C
overnight. After 3 washes, 5 min each with TBS-T, membranes were incubated with
secondary donkey anti-Rabbit IR 680 (1:4000), in blocking buffer at room temperature for
1 hr. After another 3 washes for 5 min each with TBS-T, membranes were imaged with
the LiCor Odyssey Fc machine. Signal quantification was done with Image Studio Version
5.2. Controls included blots processed without exposure to primary antibody.
11
2.2.4 Ex Vivo treatment of LG and HCET cells with Okadaic Acid and measurement of
CTSS activity
LG were harvested from BALB/c mice and cut in half. The OKA in DMSO was
dissolved in HBSS media at 1 µM concentration. The LG was either treated with the
vehicle (DMSO) in HBSS media or OKA in HBSS media for 2 hours in 37°C cell incubator.
The LG were lysed with cell lysis buffer and, HCET cells were plated in 12 well plates and
were given either DMSO (vehicle) or OKA (1 µM) for 4 hours in the 37°C cell incubator.
CTSS activity was measured according to the kits’ manufacturer protocol and previously
published articles from our lab.
82
2.2.5 Effects of SMAP and p38 Inhibitor in HCET cells on CTSS mRNA
HCET cells were grown in T75 flasks to 70 – 80 % confluency and were transferred to 3
12-well plates. These cells were starved for approximately 18 hours with cell media
without human recombinant growth factors and bovine pituitary extract. Then, the cells
were treated to either full medium (KSFM media with human recombinant growth factors
and bovine pituitary extract), 1 to 1000 DMSO in full medium, or 10 µM SB202190 (HY10295, MedchemExpress) in full medium for 2 hours. After the exposure, the media was
taken out and washed with sterile PBS two times. Then the cells were treated with either
full medium or full medium containing 1 to 1000 DMSO, recombinant human IFN- γ
(1 µg/mL) or recombinant human IFN- γ plus 10 µM SB202190 for 8 hours. Each group
included 3 wells and each experiment was repeated 3 times. The procedure was also
repeated for 5 µM SMAP instead of 10 µM SB202190. mRNA was extracted after the cell
12
lysis buffer using the RNeasy Plus Mini Kit. The buffer was transferred from one well into
the other well in the same treatment group. The mRNA was reverse transcribed using the
TaqMan Reverse transcription reagents and the gene expression levels were measured
using the TaqMan gene assays on QuantStudio™ 6 Flex Real-Time PCR system.
2.2.6 Add LG qPCR for NOD BALB/cJ method
RNA from the LG were extracted using the RNeasy Plus Universal Mini Kit. The
reverse transcription reaction used the reverse transcription kit, with 4 μg total RNA from
LG for each 50 μL reaction. cDNA was obtained using GeneAmp PCR System 9700 with
incubation cycles of 25 °C (10 min), 48 °C (30 min) and 95 °C (5 min). Real-time qPCR
was carried out with the QuantStudio 12K Flex Real-Time PCR System with GAPDH as
an internal control. Human and mouse primers to p38 alpha, CTSS, PP2A, CIP2A, ZFP36
(TTP gene), IL1-beta, and IFN-γ were used.
2.2.7 Dose determination of SB202190 in male BALB/c mice
BALB/cJ Mice were aged to 12 wks and were injected intraperitoneally with either vehicle
only (DMSO + Normal Saline) or 5mg/kg, 2mg/kg, 800 µg/kg, 320 µg/kg, 128 µg/kg or
51.2 µg/kg SB202190 in DMSO (in Normal Saline Solvent) daily for 14 days. There were
3 mice per group. After the 14th injection, cardiac puncture was conducted under
anesthesia and then mice were sacrificed. The lung, LG, kidneys, liver and spleen weight
were measured. Also, the total body weight before first injection and after the last injection
13
was compared. Serum was separated and the AST, ALT for liver function and BUN,
Creatinine for kidney function was measured.
2.2.8 Effects of p38 Inhibition in male NOD mice with SB202190
Male NOD mice aged between 12–14 wks were treated with either vehicle (DMSO
+ Normal Saline), 800 µg/kg SB202190 or 128 µg/kg SB202190 with intraperitoneal
injection once daily for 14 days. The mice organs such as lung, LG, kidneys, liver and
spleen were gathered and measured. The LG was harvested, and RT-qPCR was
conducted. Further study with 5mg/kg was conducted as to confirm whether the higher
toxic dose would have an effect due to concerns that lower dosages would not reach the
LG. One last study with SB202190 (5 mg/kg) was conducted with different durations of
exposure to the drug (2 weeks, 1 week, 3 days) to confirm whether there was a temporal
effect on kinase activity from the systemic injections. Spleen lysates were chosen as an
alternative organ to LG to assess bioavailability.
For measuring tear production, tear production was assessed using a basal thread
test. As described, a ZoneQuick phenol red-embedded thread was applied in both eyes
at the canthus of the ocular surface for 10 sed, while the animal was under light
anesthesia with isoflurane.82 Basal tear volume was recorded as the length of thread
wetting by basal tears in mm.
The lymphocytic infiltration of the LG was calculated from 3 different sections
within the gland (25, 50, and 75% cut) and labeled with hematoxylin and eosin (H & E)-.
14
The Zeiss Axioscan 7 slide scanner was used to quickly scan all the slides and the area
measurement was done using the QuPath program.
2.3 Results
2.3.1 PP2A, tristetraprolin, and P38 MAPK in NOD mice
As aforementioned, CTSS activity has been observed to be increased in the NOD
mice tear and lacrimal gland compared to those BALB/c mice. Figure 1 shows the
hypothesized effects of manipulation of TTP phosphorylation/dephosphorylation of either
the p38 MAPK or PP2A. This pathway was investigated in the SS murine model, male
NOD mice. We observed the phosphorylation of TTP to be significantly increased in the
12 wks male NOD mice LG lysate compared to lysates from the age and sex matched
BALB/c LG lysate. This was apparent in the single extra signal at the membrane in Figure
2A. Quantification shows more than 400% relative signal in the NOD LG lysates when
normalized to the total protein signal of the whole lane (Figure 2B). Interestingly, the gene
expression was significantly decreased in the NOD LG by approximately 50% possibly
showing a downstream inhibition of the gene expression while the TTP is in an inactive
(phosphorylated) state.
The phosphorylated state of the p38 was explored with LG lysate from NOD and
BALB/cJ mice. Figure 3A shows the Western blots of the phospho-p38 and p38 signals
on the same membrane from the same sample. Figure 3B-D shows the phospho-p38
and p38 protein signals compared to total protein signal. P38 and phospho-p38 protein
amount were all increased in the NOD mouse LG and the phospho-p38 to p38 ratio was
15
also higher in the NOD LG compared to BALB/cJ LG lysates. For gene expression of p38,
there was no significant difference between the LG of the two strains. The phosphorylation
state of the p38 is in accordance with the TTP phosphorylation status as it would be
phosphorylated by the p38. Once inactive, CTSS mRNA and activity could be increased
in the NOD mice LG.
The PP2A and CIP2A gene expression showed significant changes between NOD
and BALB/cJ LG lysates (Figure 4). PP2A was significantly decreased 50% while CIP2A
showed a trend towards a 50% increase in the NOD mouse LG. There was a similar trend
seen in the CIP2A protein, increased significantly by 2 fold in the NOD mice LG. As CIP2A
would inhibit PP2A, this would enhance the phosphorylated TTP that is detected.
16
Figure 2. Tristetraprolin (TTP) is increased at the protein level while the gene expression
is decreased in male NOD LG versus BALB/c LG. A. BALBc and NOD LG lysates were
Western blotted for TTP. (n=5 mice each, 12-wks age matched). The phosphorylated
inactive form, which is the top band of the doublet, is seen in the NOD LG lysate samples
but not in the BALB/c LG lysates. B. Quantification of phosphorylated TTP protein
abundance is shown. Total phosphorylated tristetraprolin protein amount in male NOD LG
lysate is 40 times higher than the BALB/c LG lysate (n=5 12-week age matched,
p=0.0028). Each lane’s signal was normalized to total protein staining intensity. All error
bars are SD. C. Gene expression of Zfp36 (Mice TTP gene) is decreased in the male
NOD LG relative to the BALB/c mice LG (n= 4 mice, 12-week age matched).
BALB/c NOD
P-TTP
BALB/c NOD
0
2000
4000
6000
Relative Intensity (%)
TTP Protein Abundance
**
TTP Gene Expression
BALB/c NOD
0.0
0.5
1.0
1.5
Relative Quatification (RQ)
*
37
25
A
B C
17
Figure 3. Western Blotting of p38 and phospho-p38 in 12 wk male NOD and BALB/c LG
lysates A. 60 ug of lysate protein was loaded into each lane. Alternating lanes of 12 wk
male BALB/c (B) and NOD (N) LG lysates were loaded. The estimated molecular weight
of p38 is 40 kD and pp38 is 42kD. The top blot is of p38 and bottom is of phospho-p38.
B. p38 protein amount significantly increased in NOD vs. BALB/c LG. (n=3, p=0.0224). C.
pP38 protein amount significantly increased in NOD vs. BALB/c LG (n=3, p=0.0111), D.
phospho-P38 to P38 signal ratio was compared. NOD was significantly higher than
BALB/c. (n=3, p=0.0460). Each lane’s signal was normalized to total protein staining
intensity. All error bars are SD.
BALBc NOD
0
200
400
600
pP38 to P38
% Signal compared to Control
✱
BALBc NOD
0
500
1000
1500
pP38
% Signal compared to Control
✱
BALBc NOD
0
100
200
300
400
P38
% Signal compared to Control
✱
P38
pP38
A
B C D
20 Ctrl
20 Ctrl
P38 alpha Gene Expression
Relative Quatification (RQ)
Balb/c NOD
0.0
0.5
1.0
1.5
E
50
37
50
37
50
37
50
37
18
Figure 4. Real time qRT-PCR and Western Blotting data of components of the PP2A
regulatory pathway in 12-16 wk male BALB/c and NOD LG. A, B. Real time qRT-PCR
data of the PP2A catalytic subunit and CIP2A mRNA, respectively, in age matched male
BALB/c and NOD LG. PP2A catalytic subunit mRNA was significantly lower in the NOD
vs BALB/c (n=4, p=0.0054). CIP2A mRNA mRNA was trending higher in NOD vs BALB/c
(n=6, p=0.0663). C. 20 ug of lysate protein was loaded in each lane. Lanes 1 to 5 are
male NOD LG lysates and lane 6-9 are male BALB/c LG lysates. Estimated CIP2A
molecular weight is 90 kDa. D. Each lane’s signal was normalized to total protein staining
intensity. (P=0.006) All error bars are SEM.
2.3.2 CTSS Activity and Okadaic Acid in NOD LG and HCET
For the HCET that were treated with OKA, the media was shown to have a
significant 3 fold increased CTSS activity compared to the control. OKA is known to inhibit
PP2A which would in turn keep the TTP phosphorylated and inactive. Under these
conditions, the proinflammatory cytokines and CTSS mRNA are more likely to be
CIP2A
Balb/c NOD
0.0
0.5
1.0
1.5
2.0
2.5
Relative Quatification (RQ)
p=0.0663
PP2A
Relative Quantification (RQ)
Balb/c NOD
0.0
0.5
1.0
1.5
**
A B
C
100
75
NOD BALB/c
19
translated and this is shown in Figure 5A. Figure 5B shows a similar significant increase
in the OKA treated BALB/cJ LG. The ex-vivo OKA treatment, albeit a 2-hour treatment,
showed 2 times increase in the activity.
Figure 5. CTSS activity is increased in Okadaic Acid (OKA) treated HCET and ex vivo
LG. A. HCET cells exposed to OKA (1 uM) for 4 hours showed that OKA elicited
significantly increased release of CTSS activity into culture medium relative to DMSO.
(n=3, p=0.0297) B. OKA treatment (1 µM, 2 hr) increased (Left) CTSS activity in BALB/c
LG fragments incubated ex vivo (n=5) (** P<0.001)
2.3.3 CTSS mRNA RT-qPCR in SMAP and P38 inhibitor treated HCET
HCET were initially treated for 2 hours with media only, DMSO (vehicle), SMAP, or
p38 inhibitor (SB202190). This was done to test as a pre-treatment for interferon-gamma
exposure, which is known to increase CTSS mRNA, protein, and activity in HCET cells.
After this 2-hour exposure, the cells were either treated 8 hours with media only, DMSO,
interferon-gamma, interferon-gamma plus p38 inhibitor, or interferon-gamma plus SMAP.
Observing Figure 6A and 6B, the 2-hour exposure to either p38 inhibitor or SMAP did
not decrease CTSS mRNA. However, as can be seen with Figure 6A, the p38 inhibitor
had reduced CTSS mRNA abundance while SMAP did not have an effect.
BALB/c LG CTSS Activity
DMSO OKA
0
100
200
300
CTSS Activity (% of Control)
**
A A B
20
Figure 6. p38 Inhibitor and SMAP effects on CTSS mRNA abundance in HCET cells
exposed to IFN-γ. In a 12 well plate HCET cells were seeded and left to grow for 4 hours.
A B
120 100 80 60 40 20 0
2Hr P38 8 Hr IFN v + P38
2Hr DMSO 8HR IFN v + P38
2Hr M 8 HR IFN v + P38
2Hr P38 8 Hr IFN v
2Hr DMSO 8 Hr IFNv
2Hr M 8 Hr IFN v
2Hr M 8 HR DMSO
2Hr M 8 Hr M
2Hr P38
2Hr DMSO
2Hr M
OHr M
Fold Change
CTSS mRNA
* *
*
Red = 8 Hr IFN v
Blue = 8 Hr IFN v + P38 Inh.
Shaded = 2 Hr P38 Inh. Pre
140 120 100 80 60 40 20 0
2Hr SMAP 8Hr INFv + SMAP
2Hr DMSO 8Hr INFv + SMAP
2Hr M 8Hr INFv + SMAP
2Hr SMAP 8Hr INFv
2Hr DMSO 8Hr INFv
2Hr M 8Hr INFv
2Hr M 8Hr DMSO
2Hr M 8Hr M
2Hr SMAP
2Hr DMSO
2Hr M
0Hr M
CTSS mRNA
Fold Change
Red = 8Hr INFv
Blue = 8Hr INFv + SMAP
Shaded = 2HR SMAP Pre.
A B
120 100 80 60 40 20 0
2Hr P38 8 Hr IFN v + P38
2Hr DMSO 8HR IFN v + P38
2Hr M 8 HR IFN v + P38
2Hr P38 8 Hr IFN v
2Hr DMSO 8 Hr IFNv
2Hr M 8 Hr IFN v
2Hr M 8 HR DMSO
2Hr M 8 Hr M
2Hr P38
2Hr DMSO
2Hr M
OHr M
Fold Change
CTSS mRNA
* *
*
Red = 8 Hr IFN v
Blue = 8 Hr IFN v + P38 Inh.
Shaded = 2 Hr P38 Inh. Pre
140 120 100 80 60 40 20 0
2Hr SMAP 8Hr INFv + SMAP
2Hr DMSO 8Hr INFv + SMAP
2Hr M 8Hr INFv + SMAP
2Hr SMAP 8Hr INFv
2Hr DMSO 8Hr INFv
2Hr M 8Hr INFv
2Hr M 8Hr DMSO
2Hr M 8Hr M
2Hr SMAP
2Hr DMSO
2Hr M
0Hr M
CTSS mRNA
Fold Change
Red = 8Hr INFv
Blue = 8Hr INFv + SMAP
Shaded = 2HR SMAP Pre.
21
Then were exposed to a 18 hr starvation period (KSFM media without growth factors).
Then they were given a 2 hr pretreatment of Media, DMSO (Vehicle), or 10 uM p38
Inhibitor (SB202190) in A and 5 uM SMAP in B. After 2 hrs, the cells were washed with
PBS and then treated for 8 hr with either interferon γ or interferon γ plus 10 uM p38
Inhibitor (A)/ 5uM SMAP (B). The mRNA was isolated from the cells and was used for
Real-time RT-qPCR for CTSS mRNA level analysis. n=3 different repeats, (* p<0.05)
2.3.4 P38 inhibitor maximum tolerated dosage
To explore the maximum tolerated dose, BALB/cJ mice were treated with different
dosages of SB202190 versus vehicle by daily intraperitoneal injection for 14 days as can
be seen in Figure 7A. The serum chemistry shows a sudden increase in the liver
enzymes (AST and ALT) by the 800 µg/kg dosage. The kidney enzymes did not show a
difference among the different doses. Also, the weight of the different organs including
the liver, kidney, LG, spleen and lung did not change.
A
22
Non
Veh
51.2ug/kg
128ug/kg
320ug/kg
800ug/kg
2mg/kg
5mg/kg
0
500
1000
35
IU/L
ALT
Non
Veh
51.2ug/kg
128ug/kg
320ug/kg
800ug/kg
2mg/kg
5mg/kg
0
500
1000
45
IU/L
B AST Non Veh 51.2ug/kg 128ug/kg 2mg/kg 320ug/kg 800ug/kg 5mg/kg
0
20
40
60
80
BUN
mg/dL
Non
Veh
51.2ug/kg
128ug/kg
320ug/kg
800ug/kg
2mg/kg
5mg/kg
0.0
0.2
0.4
0.6
0.8
Creatinine
mg/dL
Vehicle Control
51.2ug/kg
128ug/kg
320ug/kg
800ug/kg
2mg/kg
5mg/kg
-5
0
5
10
Body Weight Difference (g)
Difference in Body Weight
Vehicle Control
51.2ug/kg
128ug/kg
320ug/kg
800ug/kg
2mg/kg
5mg/kg
0.0
0.1
0.2
0.3
0.4
0.5
LG weight to Body Weight
(%)
LG % BW
Vehicle Control
51.2ug/kg
128ug/kg
320ug/kg
800ug/kg
2mg/kg
5mg/kg
0.0
0.2
0.4
Spleen weight to Body Weight
(%)
Spleen % BW
Vehicle Control
51.2ug/kg
128ug/kg
320ug/kg
800ug/kg
2mg/kg
5mg/kg
0.0
0.2
0.4
0.6
0.8
1.0
Lung weight to Body Weight
(%)
Lung % BW
Vehicle Control
51.2ug/kg
128ug/kg
320ug/kg
800ug/kg
2mg/kg
5mg/kg
0
2
4
6
8
10
Liver weight to Body Weight
(%)
Liver % BW
Vehicle Control
51.2ug/kg
128ug/kg
320ug/kg
800ug/kg
2mg/kg
5mg/kg
0
1
2
3
Kidney weight to Body Weight
(%)
C Kidney % BW
Non
Veh
51.2ug/kg
128ug/kg
320ug/kg
800ug/kg
2mg/kg
5mg/kg
0
500
1000
35
IU/L
ALT
Non
Veh
51.2ug/kg
128ug/kg
320ug/kg
800ug/kg
2mg/kg
5mg/kg
0
500
1000
45
IU/L
B AST Non Veh 51.2ug/kg 128ug/kg 2mg/kg 320ug/kg 800ug/kg 5mg/kg
0
20
40
60
80
BUN
mg/dL
Non
Veh
51.2ug/kg
128ug/kg
320ug/kg
800ug/kg
2mg/kg
5mg/kg
0.0
0.2
0.4
0.6
0.8
Creatinine
mg/dL
Vehicle Control
51.2ug/kg
128ug/kg
320ug/kg
800ug/kg
2mg/kg
5mg/kg
-5
0
5
10
Body Weight Difference (g)
Difference in Body Weight
Vehicle Control
51.2ug/kg
128ug/kg
320ug/kg
800ug/kg
2mg/kg
5mg/kg
0.0
0.1
0.2
0.3
0.4
0.5
LG weight to Body Weight
(%)
LG % BW
Vehicle Control
51.2ug/kg
128ug/kg
320ug/kg
800ug/kg
2mg/kg
5mg/kg
0.0
0.2
0.4
Spleen weight to Body Weight
(%)
Spleen % BW
Vehicle Control
51.2ug/kg
128ug/kg
320ug/kg
800ug/kg
2mg/kg
5mg/kg
0.0
0.2
0.4
0.6
0.8
1.0
Lung weight to Body Weight
(%)
Lung % BW
Vehicle Control
51.2ug/kg
128ug/kg
320ug/kg
800ug/kg
2mg/kg
5mg/kg
0
2
4
6
8
10
Liver weight to Body Weight
(%)
Liver % BW
Vehicle Control
51.2ug/kg
128ug/kg
320ug/kg
800ug/kg
2mg/kg
5mg/kg
0
1
2
3
Kidney weight to Body Weight
(%)
C Kidney % BW
23
Figure 7. Maximum Tolerated Dose study design and toxicity measurement. A. BALB/cJ
Mice were aged to 12 wks and were injected intraperitoneally with either vehicle only
(DMSO + Normal Saline) or 5 mg/kg, 2 mg/kg, 800 µg/kg, 320 µg/kg, 128 µg/kg or 51.2
µg/kg SB202190 in DMSO (in Normal Saline Solvent) daily for 14 days. Serum chemistry
and vital organ weight measurements were done. B. Serum chemistry of AST, ALT, BUN,
and creatinine was conducted. C. Differences in body weight before and after injection as
well as liver, kidney, LG, spleen and lung weight per whole body weight were compared.
(n=3 mice per group)
2.3.5 P38 inhibitor efficacy in NOD mice
Vehicle, lower dose (128 µg/kg), and higher dose (800 µg/kg) were given to 14 wks
aged male NOD mice 14wks intraperitoneally for 14 days. The unstimulated tear amount
did not change with either dose versus the control. Also, the % infiltration of the LG did
not change significantly but showed a decreasing trend (Figure 8A). Also, the LG gene
expression of p38 and CTSS did not change, while the ZFP36 (TTP gene) increased at
the lower dose. One of the toxicities that was shown here was the Liver weight compared
to the body weight of the individual mice (Figure 8C).
We pursued additional experiments to see if there was an effect of the SB202190
at a higher dosage of 5 mg/kg as in Figure 9. The unstimulated tear amount did not
change significantly but showed an increasing trend with the 5mg/kg dosage (Figure 9A).
Another non-significant trend was seen with the % infiltration with 5mg/kg showing a lower
infiltration mean. However, CTSS activity did not show any trend with the different
dosages in the LG lysates. Interestingly, the CTSS gene showed a significant increase in
at 5 mg/kg. Also, the TTP gene showed an increasing trend with the higher dosage, and
no other genes showed a trend.
24
Observing the increase in CTSS at the higher dosage, we wanted to see if there
was a temporal effect of the p38 inhibitor from 2 wks, 1 wk, and 3 days treatment, which
might cause a compensatory increase in CTSS after an initial downregulation. Compared
to the 2 wk vehicle exposure, no effect on the phosphorylated p38 to P38 ratio was seen
in any of these treatments. The 2 week and 1 week LG showed a minor trend to an
increase in the ratio (Figure 10). Looking at the spleen data (Figure 11), the
phosphorylated p38 signal decreased at the 3 day exposure. However, this was not
enough of a difference to elicit a change in the phosphorylated p38 to p38 ratio.
Vehicle 128ug/kg 800ug/kg
0
10
20
30
% Infiltration
% Infiltration
Vehicle 128ug/kg 800ug/kg
-10
-5
0
5
10
Unstimulated Tear Amount Difference
mm Difference after Treatment
A
Vehicle 128ug/kg 800ug/kg
0
100
200
300
400
% Expression
ZFP36 Gene Expression
✱
Vehicle 128ug/kg 800ug/kg
0
50
100
150
200
CTSS Gene Expression
% Expression
Vehicle 128ug/kg 800ug/kg
0
50
100
150
200
p38 Gene Expression
% Expression
B
25
Figure 8. Effects of SB202190 on indications of autoimmune dacryoadenitis in 14wk male
NOD mice. A. Unstimulated tear amount and % lymphocytic infiltration of the LG of vehicle,
128 µg/kg, or 800 µg/kg SB 202190 treated mice were compared. (n= 8, 8, 9 mice each
for vehicle, 128 µg/kg, or 800 µg/kg group respectively) B. p38, CTSS, and ZFP36 (TTP
gene) were compared for each group. (* P<0.05) C. Differences in body weight before
and after injection start and end and liver, kidney, LG, spleen and lung weight per whole
body weight was compared. (n=3 mice per group) (* P<0.05, ** P<0.001)
Vehicle Control
128ug/kg
800ug/kg
-1
0
1
2
3
4
Difference in Body Weight
Body Weight Difference (g)
Vehicle Control
128ug/kg
800ug/kg
0.00
0.05
0.10
0.15
LG weight to Body Weight
(%)
LG % BW
Vehicle Control
128ug/kg
800ug/kg
0
2
4
6
8
10
Liver weight to Body Weight
(%)
Liver % BW
✱✱
✱
Vehicle Control
128ug/kg
800ug/kg
0.0
0.2
0.4
Spleen weight to Body Weight
(%)
Spleen % BW
Vehicle Control
128ug/kg
800ug/kg
0.0
0.2
0.4
0.6
0.8
1.0
Lung weight to Body Weight
(%)
Lung % BW
Vehicle Control
128ug/kg
800ug/kg
0.0
0.5
1.0
1.5
2.0
2.5
Kidney % BW
Kidney weight to Body Weight
(%)
C
26
Figure 9. Efficacy of higher dosage SB202190 on indications of autoimmune
dacryoadenitis in 14 week male NOD mice. A. % Lymphocytic infiltration, unstimulated
Vehicle 800ug/kg 5mg/kg
0
10
20
30
% Lymphocytic Infiltration
% Infiltration
Vehicle 800ug/kg 5mg/kg
-10
-5
0
5
10
15
Unstimulated Tear Amount Difference
mm Difference after Treatment
Vehicle 800ug/kg 5mg/kg
0
50
100
150
200
CTSS Activity in LG Lysate
% CTSS ACTIVITY
Vehicle 800ug/kg 5mg/kg
0
100
200
300
400
500
Relative Quatification (RQ)
CTSS Gene Expression
✱
Vehicle 800ug/kg 5mg/kg
0
50
100
150
200
IFNv Gene Expression
Relative Quatification (RQ)
Vehicle 800ug/kg 5mg/kg
0
50
100
150
200
p38a Gene Expression
Relative Quatification (RQ)
Vehicle 800ug/kg 5mg/kg
0
100
200
300
Zfp36 Gene Expression
Relative Quatification (RQ)
Vehicle 800ug/kg 5mg/kg
0
50
100
150
200
IL1b Gene Expression
Relative Quatification (RQ)
Vehicle 800ug/kg 5mg/kg
0
50
100
150
200
PP2Ac Gene Expression
Relative Quatification (RQ)
A
B
27
tear amount, and CTSS activity of the LG of vehicle, 128 µg/kg, or 800 µg/kg SB 202190
treated mice were compared. (n= 21, 18, 23 mice each for vehicle, 800 µg/kg, or 5 mg/kg
group respectively for both % lymphocytic infiltration, and unstimulated tear amount, n=6,
3, and 9 for CTSS activity red and black dots represent two separate assay results.) B.
RT-qPCR results of CTSS, Ifng, p38a, zfp36, Il1b, and PP2Ac were compared. (n=6, 4,
and 11 for vehicle, 800 µg/kg, or 5 mg/kg group respectively, * P<0.05)
Figure 10. Western Blotting of phospho-P38 and P38 from SB202190 treated LG lysates
at different time-points. A. Top membrane (phospho-P38, red arrow) and bottom
membrane (p38) shows the LG lysates from vehicle treated with SB202190 5mg/kg
treated for 2 weeks, 1 week and 3 days and Vehicle for 2 weeks. (n=4 for vehicle and
2wk, and n=5 for 1wk, 3d. The membrane shown is one of two total membranes.) B.
Quantification of the protein signal normalized to total protein stain is shown.
Vehicle 2 wk 1 wk 3 d
0
100
200
300
LG pP38 Signal Intensity
% Signal Intensity to Vehicle
Vehicle 2 wk 1 wk 3 d
0
100
200
300
LG P38 Signal Intensity
% Signal Intensity to Vehicle
Vehicle 2 wk 1 wk 3 d
0
50
100
150
200
LG pP38/P38 Signal Intensity
% Signal Intensity to Vehicle
P38
pP38
A
B
28
Figure 11. Western Blotting of phospho-P38 and P38 from SB202190 treated spleen
lysate at different time-points. A. Top membrane (phopho-P38) and bottom membrane
(p38) shows the spleen lysates from vehicle treated with SB202190 5mg/kg treated for 2
weeks, 1 week and 3 days and Vehicle for 2 weeks. (n=4 for vehicle and 2wk, and n=5
for 1wk, 3d. The membrane shown is one of two total membranes.) B. Quantification of
the protein signal normalized to total protein stain is shown.
2.4 Discussion
The PP2A, TTP, p38, and CTSS regulatory network has not been investigated in
regard to the SS pathology and possible treatment mechanisms in mice. However, there
have been prior studies in the p38 MAPK pathway in SS by another group with another
P38 inhibitor. 83 Stimulation of LG ex vivo with IL-1 beta, which is elevated in SS,84
activates p38/MAPK in BALB mice, and injection of a p38/MAPK inhibitor (SB203580)
into LG of MRL/lpr mice improved tear flow.
83 While SB203580 inhibits p38 MAPK by
binding to the ATP pocket and it does not inhibit the phosphorylation of P38 MAPK,
85
P38
pP38
A
B
Vehicle 2 wk 1 wk 3 d
0
50
100
150
200
250
% Signal Intensity to Vehicle
Spleen pP38 Signal Intensity
✱
Vehicle 2 wk 1 wk 3 d
0
50
100
150
200
Spleen P38 Signal Intensity
% Signal Intensity to Vehicle
Vehicle 2 wk 1 wk 3 d
0
50
100
150
Spleen pP38/P38 Signal Intensity
% Signal Intensity to Vehicle
Veh vs 3d
p=0.0821
29
SB202190, used in my study, inhibits p38 by competing with the ATP and stops the
phosphorylation of the P38.
It is interesting that the systemic p38 inhibition did not result in reduced p38
phosphorylation in the LG despite the high dosage and long duration of treatment. While
a pharmacokinetic study of the drug inside the blood and the LG was not conducted, a
need for a targeted delivery approach might have benefited this study. This is clearly
observed in the SB203580 injection study in which the researchers did an exorbital LG
direct injection into the anesthetized mice.83 An intra-LG, or supra-LG injection which has
been conducted in the lab with different drug compounds might have produced a more
pronounced tear secretion and % infiltration decrease as observed in the dosage studies
conducted (Figure 8A, 9A). One limitation to the local delivery study may have been the
formulation of SB202190 which is not as soluble in normal saline and necessitated the
initial dilution into pure DMSO as was in the formulation of the intraperitoneal injection.
Another possible explanation of SB202190 not having an inhibitory effect on the
phosphorylation of P38 in the LG may have been a temporal reason. In the
pharmacokinetic and pharmacodynamic study of Cathepsin S inhibitor LY3000328 in
healthy humans, the plasma CTSS activity was initially decreased, while after 48 hours
at one-time doses of different dosage strengths, the CTSS activity actually was increased
more than basal. 86 Another similar trend was observed in another CTSS inhibitor study
using RO5459072, where the plasma CTSS mass increased after week 2 during a study
giving the drug by oral intake twice a day. 87 This high level was then sustained during the
whole 12 week treatment period. As observed in Figure 11, the short term (3d) p38
decreased phosphorylation, while there was a trend towards the increase of the pP38 to
30
p38 ratio in the longer time points (1wk, and 2wk). If the systemic delivery was to be
pursued, an observation of the shorter time point may be beneficial.
31
Chapter 3: Multinucleate Macrophages in the aged lacrimal gland
3.1 Introduction
Lipid dysregulation is a hallmark of aging, and changes in lipid metabolism are
implicated in the pathogenesis of age-related diseases including DED.88 The cholesterol
transporters, Niemann-Pick disease type C1 (NPC1), and Niemann-Pick disease type C2
(NPC2), are dysregulated in aging.89-91 NPC1 is a transmembrane protein that mediates
intracellular cholesterol trafficking, while NPC2 is a soluble protein that functions in
egressing and recycling of lipoprotein-derived cholesterol.92-94 Dysregulation of these
enzymes is linked to lipid accumulation in atherosclerosis and age-related macular
degeneration.95,96 Cathepsins are lysosomal proteases that are involved in antigen
processing during immune responses, degradation of proteases and chemokines for
cellular homeostasis, autophagy, proliferation, and metastasis among many other
functions. 30,41,97 Essential for lysosomal function and catabolism, the cathepsin proteases
are also linked to aging and lipid dysregulation. Increased cathepsins are documented in
atherosclerotic lesions,98,99 with cathepsin B, D, and L enriched in macrophage-derived
foam cells localized in necrotic cores of lipid plaques.100 Cathepsin L (CTSL) is specifically
linked to macrophage function in lipid degradation.101,102 While in vitro and mice in vivo
studies suggest CTSL’s role in the reduction of body weight gain and adipogenesis
through fibronectin, insulin receptor, and insulin-like growth factor 1 receptor degradation,
103,104 little is known about its role in lipid metabolism and possible dysregulation of NPC1,
NPC2, and CTSL in aged LG.
Macrophages, part of the innate immune system, also play key roles in tissue
development, homeostasis, and repair of damaged tissue.105 Different macrophages are
32
known, depending on their tissue location, function, and phenotype, with some implicated
in lipid clearance. Foamy macrophages accumulate an abnormal amount of lipids and
cholesterol esters and are hallmarks of early atherosclerotic plaques.106 These foam cells
can aggregate into multinucleated giant cells named Touton giant cells in areas of high
lipid concentration.107,108 Other multinucleated macrophages are reported in different
tissues, with osteoclasts in bone,109 foreign body giant cells in implants,110 and Langhans
giant cells in lungs.111
Previous studies on mouse LG have highlighted the increased accumulation of
lipofuscin in tissues with age.19,112-114 Lipofuscin is a mixture of oxidized proteins, lipids,
and metals that are found in the lysosomes or cytosol of aging postmitotic cells.115 This
nondegradable and insoluble product has an autofluorescent 400-700 nm emission
spectrum that significantly hinders application of immunofluorescence to tissue at useful
visible light emission wavelengths. Multiple products such as Sudan Black B or reducing
agents have been tested to overcome the problem.116 While the need to reduce lipofuscin
autofluorescence signal in samples labeled with histological dyes may not be necessary,
this modification was pivotal in our ability to discretely visualize the distribution of proteins
of interest by immunofluorescence at higher magnification. Photobleaching with an LED
light has previously been used as an approach for eliminating lipofuscin-related
autofluorescence in brain samples 117, an approach which we utilize here in LG.
Here, we investigate the possible dysregulation of lipids and lipid-metabolizing
enzymes, and their relationship to macrophage infiltration, in the aged LG. We report here
that lipids accumulate with age in the LG. Using a newly developed photobleaching
regimen to reduce endogenous autofluorescence, we identify a novel multinucleated
33
macrophage population enriched in F4/80, CTSL, NPC1, and NPC2 and increased in
aged LG that may participate in clearance of tissue lipids.
3.2 Methods
3.2.1 Mice
Female C57BL/6J (B6) mice were from the Jackson Laboratory (Bar Harbor, ME)
and aged up to > 2 years or received from the NIH/National Institute of Aging. Animal use
complied with policies approved by the University of Southern California and Baylor
College of Medicine Institutional Animal Care and Use Committees and was in
accordance with the Guide for the Care and Use of Laboratory Animals 8th edition and the
ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Mice were
aged to young (2-3 months), intermediate (10-14 months), and old (>24 months) ages.
After intraperitoneal injection of 100 mg/kg ketamine + 10 mg/kg xylazine anesthesia and
euthanasia, female mouse LG were isolated and processed immediately or stored at –
80°C. Mice were housed at facilities at either Baylor College of Medicine, University of
Southern California, or the National Institutes of Health with ad libitum access to food and
water. Because DED is more frequent in women7,118 and aged male mice do not develop
corneal barrier disruption (a hallmark of DED),119 the study used only female mice.
3.2.2 Materials and Reagents
Ketamine (NDC:13985-54-10) and xylazine (NDC:13985-704-10) were from
VetOne (Boise, ID). Oil-red-O staining solution (O0625), Beadbug prefilled tubes
(Z7637800) and tris-buffered saline containing tween® 20 (TBS-T), pH 8.0 (T9039) were
from Sigma Aldrich (St. Louis, MO). Phosphatase inhibitor cocktail (5872S) was from Cell
34
Signaling Technology (Danvers, MA). 10% Tris-Glycine gels (XP00105BOX) and iblot2
NC regular stack (IB23001) were from Invitrogen (Waltham, MA), and the Revert™ 700
Total Protein Stain Kits for Western Blot Normalization (P/N 926-11016) was from LiCor
(Lincoln, NE). The Pierce BCA Protein Assay Kit (23225) was from Thermofisher
(Waltham, MA). Blocking Buffer for Fluorescent Western Blotting (MB-070) was obtained
from Rockland Immunochemicals (Pottstown, PA). Superfrost plus microscope slides
(48311-703) were from VWR (Radnor, PA). ProLong Gold antifade mounting medium
(P36934) was from Invitrogen (Waltham, MA). Optimal cutting temperature compound
(O.C.T.) (25608-930) was from VWR (Suwanee, GA), and the RNeasy Plus Mini RNA
(74134) isolation was from Qiagen (Hilden, Germany). Ready-To-Go You-Prime FirstStrand kit (27-9263-01) was from GE Healthcare (Chicago, IL). Primers for Real-time
PCR and the TaqMan Universal PCR Master Mix AmpErase UNG were from Thermofisher
(Waltham, MA). 3% glutaraldehyde (#01909-10), osmium tetroxide (OsO4; #0972A-20),
Poly/Bed 812 plastic resin (#08792-1), and toluidine blue (#01234-25) were purchased
from Polysciences (Warrington, PA). All antibodies used for immunofluorescence,
Western blotting and flow cytometry are in Table 1.
Target Species Category Number RRID number Manufacturer Concentration
Primary Ab (WB/IF) NPC1 Rabbit anti-mouse ab134113 AB_2734695 Abcam 1:4000 (WB) 1:100 (IF)
NPC2 Rabbit anti-mouse 19888-1-AP AB_10639363 Proteintech 1:100
F4/80 Rat anti-mouse MA1-91124 AB_2277854 Invitrogen 1:50
CTSL Goat anti-mouse AF1515 AB_2665930 R&D Systems 1mg/mL (WB)1:40 (IF)
CD11b Rabbit anti-mouse PA5-90724 AB_2806205 Invitrogen 1:100
Phalloidin A-22287 AB_2620155 Thermofisher 1:200
DAPI D-1306 AB_2629482 Thermofisher 1:1000
Species Fluorophore Category Number RRID number Manufacturer Concentration
Secondary Ab Donkey anti rabbit IR680 926-68073 AB_2716687 Licor 1:4000
Donkey anti goat IR 680 926-68074 AB_2650427 Licor 1:4000
Donkey anti rat AF 488 A-21208 AB_2535794 Invitrogen 1:200
Donkey anti goat AF 568 A-11057 AB_2534104 Invitrogen 1:200
Donkey anti rabbit AF 594 A-21207 AB_141637 Invitrogen 1:200
Target Filter Fluorophore RRID number Category Number Manufacturer
Flow Cytometry Ly-6C (Ly6C) B14 RB780 AB_394656 568739 BD Pharmingen
CD64 (Fc-γ receptor 1,B) YG3 PE-CF594 AB_2566558 139320 Biolegend
F4/80 YG5 PE-Cyanine 5 AB_893494 123112 Biolegend
CD11b (Integrin Alpha M, ITGAM) YG9 PE-Cyanine 7 AB_469588 25-0112-82 Invitrogen
CD45 R4 AF700 AB_493715 103127 Biolegend
Viability (Live/Dead, Live-Dead) R7 Near IR L34993 Invitrogen
35
Table 1. Antibody Information for Western blotting, immunofluorescence, and flow
cytometry. Primary and secondary antibody information including the target, species,
category number, research resource identifier number (RRID), manufacturer, and
concentrations used for respective purposes (WB=Western Blotting, IF=
Immunofluorescence) are included. Also, the flow cytometry antibodies have information
on target, filter, fluorophore, RRID, category number, and manufacturer.
3.2.3. qRT-PCR of LG
Total RNA from LG was extracted using a RNeasy Plus Mini RNA isolation kit
following the manufacturer’s protocol. After isolation, RNA concentration was measured,
and cDNA was synthesized using the Ready-To-Go You-Prime First-Strand kit. Real-time
PCR was performed using specific TaqMan primer for ctsl (Mm00515597_m1), and
TaqMan Universal PCR Master Mix AmpErase UNG in a commercial thermocycling
system according to the manufacturer’s recommendations. The Gapdh
(Mm99999915_g1) gene was used as an endogenous reference for each reaction.
Quantitative PCR results were analyzed by the comparative Ct method and normalized
to the Ct value of Gapdh. The young group served as calibrators.
3.2.4. Western Blotting of LG lysate
For gel electrophoresis, one whole LG was used per sample. LG were
homogenized using Beadbug prefilled tubes in RIPA buffer containing protease inhibitor
cocktail. The supernatant was recovered after centrifugation at 8000 × g at 4°C for 10 min.
After measuring each sample’s protein concentration with the Pierce BCA protein assay
kit, samples were incubated in reducing dye and b-mercaptoethanol at 95°C for 5 min. 40
µg of each sample in sample buffer were loaded onto precast 10% Tris-Glycine gels and
resolved by SDS-PAGE at 120 V at 4°C for 2 hr. Proteins on gels were transferred to
36
nitrocellulose membranes using an iBlot™ 2 gel transfer machine. Membranes were
stained for total protein using the Revert™ 700 Total Protein Stain Kit for Western Blot
Normalization and the signal was read using the LiCor Odyssey Fc machine. The signal
for each lane was used to normalize the signal of the protein of interest to total protein
loaded. Total protein stain was rinsed away per the manufacturer’s protocol, and
membranes were blocked for 1 hr with blocking buffer at room temperature while shaking.
Membranes were washed 3 times for 5 min with TBS-T, then incubated with either antirabbit NPC1 (1:4000) or anti-goat CTSL antibodies (1 mg /mL) in blocking buffer at 4°C
overnight. After 3 washes, 5 min each with TBS-T, membranes were incubated with
secondary donkey anti-Rabbit IR 680 (1:4000) or donkey anti-Goat IR 680 (1:4000),
respectively, in blocking buffer at room temperature for 1 hr. After another 3 washes for 5
min each with TBS-T, membranes were imaged with the LiCor Odyssey Fc machine.
Signal quantification was done with Image Studio Version 5.2. Controls included blots
processed without exposure to primary antibody.
3.2.5. Tissue processing for photobleaching, immunofluorescence, and confocal
fluorescence microscopy
LG were fixed in 4% paraformaldehyde and 4% sucrose in PBS for 3 hr at room
temperature and then in 30% sucrose PBS solution at 4°C overnight. O.C.T. embedding
was done the following day, and samples were frozen on dry ice, and stored at – 80 °C.
Tissues were sectioned at 5 µm thickness and mounted on Superfrost plus microscope
slides. For photobleaching of autofluorescence, procedures were adapted from a study
in brain tissue.117
37
For LG photobleaching, LG sections mounted on microscope slides were placed in
a rectangular plastic container and submerged in sterile PBS. An LED desk lamp (Rotary
LED Light Model Q1, 200-240V, 7W 50/60Hz) was placed ~6 cm from the slides. The
plastic cover covering the LED light source on the LED desk lamp was removed for direct
exposure and higher lux. Light emission characteristics were measured with a Light
Passport Pro Standard (Allied Scientific Pro) spectrometer before exposure and
intermittently throughout photobleaching to confirm the consistency of light lux and
wavelength. The entire assembly was covered in aluminum foil to shield slides from
ambient light. The whole apparatus was regularly monitored to ensure PBS remained
sufficient to cover the slides, and to ensure lack of contamination. Photobleaching
occurred at constant illumination for at least 7 days and slides were periodically checked
to see if autofluorescence remained using confocal microscopy with a 20x objective,
Numerical Aperture (NA) 0.8 on a Zeiss LSM 800 with the 488 nm laser line.
Once photobleaching was complete, tissue sections on slides were further
quenched with 50 mM NH4Cl in PBS for 20 min and permeabilized with 0.3% Triton X100 for 30 min. Slides were washed twice with PBS for 15 min each at room temperature
with shaking. Slides were then blocked with 5% BSA in 0.3% Triton X-100 for 3 hr at room
temperature. Slides were incubated with primary antibodies as follows: goat anti-mouse
to CTSL (1:40), goat anti-mouse NPC1 (1:100), rabbit anti-mouse NPC2 (1:100), rat antimouse to F4/80 (1:50), or rabbit anti-mouse CD11b (1:100) in blocking buffer overnight at
4°C. On day 2, slides were washed with PBS 3 times for 15 min each at room temperature
with shaking. The tissue was then incubated with secondary antibodies as follows: Alexa
Fluor (AF) 488 goat anti-rabbit (1:200) and AF 568 donkey anti-goat (1:200). DAPI and
38
Phalloidin AF 647 were added with secondary antibodies and incubated for 1 hr at 37°C.
Slides were washed again with PBS 3 times, then mounted with ProLong Gold antifade
mounting medium and a glass coverslip and left to dry overnight. Images were acquired
with either a Zeiss LSM 800 with Airyscan processing or a Zeiss LSM 880 with Airyscan
processing, both using a 63x oil 1.4 NA objective. Images were subjected to equivalent
processing with brightness and contrast of the proteins of interest conserved on the
QuPath program version 0.4.2. Images labeled Mouse 1 and Mouse 2 indicate images
from separate mice, but Mouse 1 and 2 are not necessarily the same mice across each
of the Figures.
For counting of nuclei per F4/80-positive (F4/80+) macrophages in young and old
(photobleached) LG sections, 5 µm cut sections were labeled to detect F4/80, actin and
nuclei as above and were scanned using the Keyence BZ-X810 microscope with 60x
objective using DAPI (nuclei), GFP (F4/80) and Cy5 (actin) fluorescence filters. The
images were stitched using the BZ-H4A Advanced Analysis Software in the
uncompressed option with no shading auto-correction and converted to tiff. F4/80+ cells
were delineated using the QuPath program and nuclei were counted manually.
Calculation of LG area was with Fiji version 2.14.0/1.54f and the lens scale bar from
Keyence.
3.2.6. Tissue autofluorescence
For assessment of autofluorescence to optimize photobleaching protocols, tissues
from intermediate and old LG were put in containers with sterile PBS and exposed to
photobleaching at 4oC or kept at 4oC covered in aluminum foil (mock photobleaching) as
39
aforementioned. After 7 days, slides were quenched and blocked as described above and
labeled with DAPI. After mounting, images were acquired with a Keyence BZ-X810
microscope with 20x objective using DAPI, GFP, TRITC, and Cy5 fluorescence filters.
Images acquired were stitched using the BZ-H4A Advanced Analysis Software in the
uncompressed option, no shading auto-correction, and converted to tiff.
To measure autofluorescence excitation and emission profiles of aged LG sections,
a lambda scan, which acquires the emission spectrum of the same specimen at different
excitation wavelengths. Sections from young, intermediate, and old LG on superfrost plus
slides subjected to photobleaching or mock-photobleaching at 4oC as described in
Methods were quenched with NH4Cl in PBS for 20 min and permeabilized with 0.3%
Triton X-100 for 30 min. After 3 washes of 15 min with PBS, the slides were blocked for 3
hr with 5% BSA in 0.3% Triton X-100. The tissues were either incubated with blocking
buffer, or 1:100 rabbit anti-mouse NPC1 plus 1:200 AF 488 goat anti-rabbit, or incubated
with 1:200 AF 488 goat anti-rabbit only. The slides were then mounted with ProLong
Antifade mounting medium and covered with glass coverslips. Lambda scans were
acquired using a 63x 1.4 NA objective on a Zeiss LSM 880 with Airyscan. Excitation laser
wavelengths were 405 nm, 458nm, 488nm, 514nm, 561nm, 594m, and 633nm. Each
relative fluorescent intensity value was taken on the same field of view and the emission
wavelength range was from 422nm to 682nm.
3.2.7. Statistical analysis
For statistical analysis of gene and protein signals, GraphPad Prism 9.5.1 software
(GraphPad Software Inc, San Diego, CA) was used. A Kruskal-Wallis test was used to
40
compare the protein signals and gene expression from mice at 3 different ages. A MannWhitney U test was used to investigate the effects of age on different immune cells and
to observe the changes in the abundance of single and multinucleated F4/80+
macrophages in aged and young mice by immunofluorescence. The criterion for
significance was set at P < 0.05.
2.3 Results
3.3.1. Gene expression of lipid metabolism-related proteins is increased with age
To determine if lipid deposition in aged LG was associated with changes in gene
expression, we performed qRT-PCR evaluation of genes encoding proteins involved in
lipid metabolism. NPC1 and NPC2 (encoded by Npc1 and Npc2, respectively) function in
cholesterol trafficking and metabolism, and their dysregulation is linked to lipid
accumulation in other age-related diseases. Mucolipin 2 (encoded by Mcoln2),120,121 and
lipase (encoded by Lipa)
122 are also involved in lipid transport and degradation. Npc1,
Npc2, Lipa, and Mcoln2 were observed to be increased gene expression with age in
LG.123 When we assessed Ctsl expression in samples of young and old LG, its gene
expression was not significantly affected with age (Fig. 1).
41
Figure 1. RT-qPCR of Ctsl of young and old aged female C57 mice. Gene expression
was unchanged between young and old LG. (n=3 mice).
3.3.2. NPC1 protein abundance is significantly increased in aged mouse LG
To further investigate the changes in gene expression of proteins implicated in
lipid metabolism with aging, we evaluated NPC1 and CTSL levels by Western blotting of
LG lysates. NPC1 was significantly increased in old LG by ~3 fold compared to young LG
(Fig. 2A, C). The signal was normalized by total protein stain signal as shown in Figure
3A. The outlier shown in the Figure 3B was omitted in the final analysis after the signal
being identified as an outlier using the ROUT method. Although not statistically significant,
Young (11-13wk)
Old (93 wk)
0
50
100
150
Ctsl
relative fold
42
there was a trend to increased active CTSL (cleaved from pro-CTSL) with increasing age
(Fig. 3B, D).
Figure 2. NPC1 and active CTSL are increased with age in B6 mouse LG lysates. A.
Young, Intermediate, and Old (n = 3-8) female B6 LG lysates show NPC1 signal by
Western blotting. * indicates outlier as determined by the Rout method using GraphPad
software. B. Young, intermediate (Int), and old (n = 3-8) female B6 LG lysates show Active
(cleaved from the pro-CTSL) CTSL signal by Western blotting. Bands from the same blot
were rearranged to reflect the aging order. C. NPC1 signal in Old LG shows a 3-fold
increased protein compared to that expressed in young LG (* indicates significance, p =
0.0263). D. There was no statistically significant difference in active CTSL signal between
each group. Each dot represents a right LG from one mouse. A Kruskal Wallis test was
used to compare the relative expression of genes among the different age groups.
43
Figure 3. Analysis of NPC1 and total protein stain including outlier. A. The outlier
that was omitted in the analysis in Fig. 3 is shown, including both NPC1 signal by
Western blotting and Total protein stain of the same blot. The asterisk indicates the lane
of the outlier. B. NPC1 protein expression relative to respective total protein analysis
including the outlier and the relative signal of the outlier lane with respect to total protein
is shown. The relative protein expression was calculated based on comparison to the
average signal for the young, and the blue color indicates the outlier sample’s data. A
Kruskal-Wallis test was used to compare the protein signals of each group and the
outlier was identified using the ROUT method.
44
3.3.3. Assessment of tissue autofluorescence with age
We sought to expand our analysis of NPC1, CTSL and NPC2 using
immunofluorescence rather than Western blotting, which required a significant amount of
these precious aged LG samples without the ability to provide spatial information. The
aging LG is a challenging choice for immunofluorescence since strong autofluorescence
from lipofuscin-like structures is reported in aged BALB/c mouse LG.112 Lipofuscin
autofluorescence has a wide emission wavelength profile from 450 – 700 nm, overlapping
with emission wavelengths of most commonly used fluorophores. Imaging of old B6 LG
sections using a Keyence microscope (Fig. 4, top row) verified strong autofluorescence.
Specifically, non-photobleached (NP) old LG showed emission through FITC (Emission
488 – 540 nm), TRITC (Emission 540 – 620 nm), and Cy5 (Emission 620 – 700 nm) filters,
although samples were labeled only with DAPI.
45
Figure 4. LED photobleaching of LG tissue sections reduces endogenous
autofluorescence. Non-Photobleached or Photobleached tissue sections from old B6
mouse LG labeled with DAPI were imaged at 20x magnification using a Keyence
Fluorescence Microscope with emission collected through Cy5, FITC, TRITC, and DAPI
filters. Autofluorescence was scattered throughout the tissue in old non-photobleached
samples detected through Cy5, FITC, and TRITC emission filters, while consecutive
sections of old samples that were photobleached exhibited a greatly reduced signal. The
top and bottom panels images show consecutive sections of the same LG. (*
=lymphocytic infiltration, scale bar : 500 μm)
To explore the potential of photobleaching to reduce endogenous tissue
autofluorescence, we used lambda scanning to determine the emission profile of the
sample with varying excitation with each primary laser line. The autofluorescence lambda
scan profile for unlabeled non-photobleached samples and non-photobleached samples
labeled with primary anti-NPC1 plus AF488-labeled secondary antibody, or AF488
secondary antibody alone are shown in Fig. 5A (top row). Excitation at 561 nm elicited
the strongest emission, which was roughly comparable to the sample incubated with
AF488-labeled secondary antibody. The same signal was detectable, overlapping, and
commensurate in magnitude to that obtained with the sample incubated with primary antiNPC1 plus AF488-labeled secondary antibody. A small distinct peak of fluorescence
elicited at 488 and 514 nm excitation could be detected that was poorly resolvable from
autofluorescence.
Utilizing a commercial desk lamp LED for photobleaching (Fig. 5B), 7 days of
treatment was established as sufficient to reduce tissue autofluorescence. As shown in
photobleached images in Fig. 4 (bottom row) of a section of old LG cut consecutively
after the non-photobleached section from the same mouse LG above, the photobleached
sample showed fluorescence associated only with DAPI. This lack of endogenous
46
autofluorescence was verified using lambda scanning. The photobleached samples in Fig.
5A (bottom row) showed essentially no autofluorescence with 561 nm excitation,
associated with the most autofluorescence in non-photobleached samples (top row). 488
nm and 514 nm excitation likewise showed minimal autofluorescence emission. These
results show that photobleaching of freshly cut LG sections with an LED light permits use
of immunofluorescence. The emission spectrum/relative intensity values for the
illuminating light (Fig. 5C) and energy (e.g., flux) Fig. 5D were determined to relate
photobleaching capability with energy input received by each tissue sample.
47
Figure 5. Photobleaching parameters associated with reduction of tissue
autofluorescence. A. Lambda scanning was carried out on non-photobleached (top row)
and photobleached (bottom row) tissue sections incubated with either blocking buffer,
secondary antibody alone labeled with AF488, or primary antibody to NPC1 plus
secondary antibody labeled with AF488. While non-photobleached samples exhibited
emission throughout the different conditions and laser wavelengths, photobleached
samples showed signal only with the primary plus secondary antibody combination. B. An
LED Desk lamp was used to photobleach slides. An apparatus was set up with sufficient
sterile PBS to cover the slides in a plastic container. With aluminum foil covering the whole
apparatus, slides were photobleached under constant illumination at 4°C for 7 days. C.
The relative intensity for each wavelength from the LED light is shown obtained by
Lighting Passport Pro. D. Additional parameters for LED light characterization are shown.
48
3.3.4. NPC1 and NPC2 immunofluorescence are increased with age in non-acinar cells
in the old LG
NPC1 and NPC2 staining in photobleached LG sections from young, intermediate,
and old B6 mouse LG were then evaluated (Fig. 6A). NPC1 was detected in intermediate
and old but not young LG, with expression largely seen at the periphery of acinar clusters.
Little expression in acinar or ductal cells was detectable. As with NPC1, NPC2 was
increased in LG from intermediate and old mice and exhibited an extra-acinar distribution
comparable to NPC1 (Fig. 6B). Thus, both increased NPC1 (confirmed by gene and
protein expression) and increased NPC2 (confirmed by gene expression) are increased
in extra-acinar cell populations with age in the LG. In Fig. 6 (and subsequent figures)
rows labeled Mouse 1 and Mouse 2 are images from different mice for all age groups.
Mouse 1 and 2 are not necessarily the same across different panels or figures.
49
Figure 6. NPC1 and NPC2 are enriched in extra-acinar structures in intermediate
and old B6 mouse LG. A. Photobleached B6 LG sections from young, intermediate, and
old LG were labeled with primary and secondary antibodies to NPC1, as well as
rhodamine phalloidin and DAPI. NPC1 was seen in intermediate and old LG in proximity
to but not within acinar cells. B. Photobleached B6 LG sections from young, intermediate,
50
and old LG were labeled with primary and secondary antibodies to NPC2, as well as
rhodamine phalloidin and DAPI. Similar to NPC1, NPC2 was seen in intermediate and
old LGs in proximity to but within acinar cells. Scale bars: 20 µm. Each row labeled Mouse
1 and Mouse 2 are images from different mice for all age groups. Mouse 1 and 2 are not
necessarily the same across different panels or figures.
3.3.5. NPC1, NPC2, and CTSL are enriched in LG in a novel multinucleated macrophage
population that increases with aging
The cell type associated with enrichment of NPC1 and NPC2 in the aged LG was
determined in photobleached specimens. Previously, we had detected an apparent lipidrich multinucleated cell population at the periphery of acinar clusters in old LG stained
with Oil-Red-O and hematoxylin (Fig. 7A), consistent with the detection of the toluidine
blue-labeled apparent multinucleated cells in Fig. 1C. These cells were similar
morphologically and in their location to a macrophage population labeled with F4/80 that
can be seen in Fig. 7C, D. in intermediate and old LG sections. These macrophages,
detected using an antibody to F4/80, appeared multinucleated in nature and were situated
in proximity to the locations where lipid droplets were present (Fig. 1) and where NPC
proteins were enriched (Fig. 6). Localization of NPC1 (Fig. 7C) and NPC2 (Fig. 7D) in
the cytoplasm of these multinucleated macrophages was confirmed by
immunofluorescence. Supplemental Video 1 provides a z-stack video of a
representative F4/80+ multinuclear cell containing 3 nuclei without (1A) and with (1B) the
cell of interest outlined. We also quantified the average number of nuclei per
multinucleated F4/80+ cell. The majority of multinucleated cells (~60%) had 2-3 nuclei
(Fig. 7B).
51
Figure 7. NPC1 and NPC2 are enriched in apparent F4/80+ multinucleated
macrophages in aging LG. A. Oil-Red-O and hematoxylin-stained old LG shows
multinucleated lipid-containing cells, also shown in magnification. Scale bars: 25 µm B.
The number of nuclei per multinucleate F4/80+ macrophages as a percentage of total
multinucleate macrophages in old LG were quantified. (n=3 old mouse LG) C.
Photobleached LG sections from young, intermediate, and old B6 mice were labeled with
primary and secondary antibodies to NPC1 (green), F4/80 (pink), rhodamine phalloidin
C Young Int Old Old no actin
*
* * *
*
*
*
*
*
*
*
*
*
*
*
D Young Int Old Old no actin
NPC1
F4/80
Mouse
1
NPC2
F4/80
NPC2
F4/80
A
Old
NPC1
F4/80
*
*
* *
Old
*
*
*
* *
* *
Mouse
2
Mouse
1
Mouse
2
B
25 μm 25 μm
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
0
10
20
30
40
50
Nuclei (n)
% of total multinucleate cell
Nuclei per F4/80 positive multinucleate macrophages
52
(red, actin) and DAPI (blue, nuclei). NPC1 is detected in F4/80-enriched multinucleated
cells adjacent to acinar clusters. C. Photobleached LG from young, intermediate, and old
B6 mice were labeled with primary and secondary antibodies to NPC2 (green), F4/80
(pink), rhodamine phalloidin (red, actin) and DAPI (blue, nuclei). NPC2 shows a similar
distribution to NPC1 within F4/80+ multinucleated cells. These cells are outlined in white
with asterisks indicating the position of the nuclei in panels B and C. Scale bars in C and
D, 20 µm. Each row labeled Mouse 1 and Mouse 2 are images from different mice for all
age groups. Mouse 1 and 2 are not necessarily the same across different panels or figures.
We further evaluated whether multinucleated F4/80+ macrophages were present
to the same extent in young and old LG. Fig. 8A shows a representative section from old
LG used to identify F4/80+ cells for the analysis. As shown in Fig. 8B, F4/80+
macrophages were present in both young and old LG. Approximately equal numbers of
mononuclear F4/80+ cells were present per area of the tissue; however, in old LG the
number of multinuclear macrophages as well as total macrophages was dramatically
increased. In accord with this increase, the percentage of multinucleated F4/80+
macrophages as a percentage of total F4/80+ macrophages was found to be ~5% in
young LG but was increased to approximately 45% in old LG.
53
Figure 8. Mononuclear and multinuclear F4/80+ macrophage comparisons in old
and young LG. A. Whole LG image of F4/80 (green), actin (red), and DAPI (blue) is
shown in “Old LG”. “Old LG Counted” shows the “Old LG” image with the counted cells
highlighted with a yellow border, highlighting the areas that were positive for F4/80
labeling. “Old LG 2nd Control” shows a consecutive section labeled with secondary
antibody alone, and shows no green signal. Scale bar: 100 µm B. Quantification of
mononuclear and multinuclear F4/80+ macrophages is shown. The total number of
macrophages (mononuclear and multinuclear macrophages) and the relative abundance
of multinuclear macrophages are compared. (n=3 mice for old and young)
Old LG Old LG Counted Old LG 2nd Control
F4/80 Actin DAPI
A
B
Young Old
0
50
100
150
Mononuclear Macrophage
# of cells /200,000µm2
Young Old
0
50
100
150
200
Multinuclear Macrophage
# of cells /200,000µm2
Young Old
0
10
20
30
40
50
60
% of total multinucleate cell
Relative Abundance
Multinuclear Macrophage
Young Old
0
100
200
300
Total Macrophage
# of cells /200,000µm2
54
Given previous findings that CTSL was enriched in macrophages localized within
atherosclerotic plaques, we examined CTSL distribution with respect to macrophages and
NPC1. As shown in Fig. 9, CTSL was also enriched in this multinucleated macrophage
population (Fig. 9A) and colocalized with NPC1 (Fig. 9B). Evaluation of the intensity of
these proteins within apparent multinucleate macrophages in Fig. 9B shows that while
NPC1 and CTSL were enriched in multinucleate macrophages, that the relative
abundance of these proteins varied across individual macrophages. Collectively, Fig. 6-
9 demonstrate the enrichment of NPC1, NPC2, and CTSL with each other and within an
F4/80+ multinucleate macrophage population increased with age in the LG.
55
Figure 9. CTSL is enriched with age in F4/80+ multinucleated macrophages
containing NPC1. A. Photobleached LG sections from B6 mice at young, intermediate,
and old ages were labeled with primary and secondary antibodies to CTSL (pink), F4/80
(green), rhodamine phalloidin (red, actin) and DAPI (blue, nuclei). An F4/80+
multinucleated macrophage population colocalizes with CTSL in intermediate and old LG.
B. Photobleached LG sections from B6 mice at young, intermediate, and old ages were
labeled with primary and secondary antibodies to CTSL (pink), NPC1 (green), rhodamine
phalloidin (red, actin) and DAPI (blue, nuclei). NPC1 and CTSL are enriched in F4/80+
multinucleated cells adjacent to acini in the intermediate and old sections. (Multinucleated
cells are outlined in white, while * depicts the position of nuclei inside these cells). Scale
bars: 20 µm. Each row labeled Mouse 1 and Mouse 2 are images from different mice for
all age groups. Mouse 1 and 2 are not necessarily the same across different panels or
figures.
A Young Int Old Old no actin
B
F4/80
CTSL
Young Int Old
NPC1
CTSL
Old no actin
* *
*
* *
* *
*
* *
*
*
* *
*
*
F4/80
CTSL
NPC1
CTSL
*
*
Mouse
1
Mouse
2
Mouse
1
Mouse
2
56
2.3.7. Multinucleate macrophages in old LGs have varying levels of Cd11b
CD11b+ macrophages are present in proximity to acini as single mononuclear cells
in LG from all three age groups (Fig. 10). Mononuclear F4/80+ macrophages were also
observed in all three age groups. In the intermediate and old LG, F4/80+ macrophages
are also present in proximity to acini as multinucleated cells with mixed CD11b positivity.
Taken together, our findings suggest that with aging, not only does macrophage
expression of lipid-metabolizing enzymes increase, but there is an increase in specialized
macrophages that express different markers including F4/80.
57
Figure 10. Cd11b and F4/80 enrichment in mononuclear and multinucleated
macrophages in LG. Photobleached LG sections from young, intermediate and old B6
mice were labeled with primary and secondary antibodies to detect CD11b (magenta),
F4/80 (green), rhodamine phalloidin (red, actin), and DAPI (blue, nuclei). Cells with
CD11b (indicated with magenta arrows), F4/80 (green arrows), and both CD11b and
F4/80 (white arrows) are seen in the young LG. Multinucleated and mononuclear
macrophages are observed in both intermediate and old LG. Acini and possible
ducts/blood vessel are outlined in the images with white dotted lines. Scale bar: 20 μm
2.4. Discussion
In Chapter 2, we demonstrate that lipid deposition related NPC1 increases with
age in the LG. The limited tissue availability and small size of the aged LG made it
CD11b
F4/80
Actin
DAPI
Young Int Old
58
unfeasible to probe all potential gene expression hits using Western blotting. After
validating increased NPC1 expression by Western blotting, confirmation of the
enrichments utilizing immunofluorescence, which also provided essential information
about the location of proteins within the tissue. A method for reproducibly photobleaching
tissue autofluorescence within the LG advanced this effort. Further examination of the
distribution and enrichment of NPC proteins and CTSL with age in the LG revealed an
apparent multinucleated macrophage population (labeled with F4/80) situated at the base
of the acinar cells, adjacent to the sites of extracellular lipid deposits identified in the LG.
Macrophage infiltration in tissue plays many roles. As phagocytic cells, they are
responsible for infiltrating infected tissue and assisting in the clearance of
pathogens.124,125 In this capacity, they exhibit robust expression of lysosomal enzymes to
aid in rapid degradation of phagocytosed material. This ability enables them to metabolize
lipids through lipolysis. In diseases of lipid deposition such as atherosclerosis and obesity,
macrophage numbers are increased.126 However, the macrophages identified in the aging
LG are atypical with respect to foam cells in that they have a multinucleated phenotype.
These LG macrophages are also labeled with an antibody to F4/80, a protein enriched in
murine macrophages that is frequently used for their detection. The enrichment of NPC1,
NPC2 and CTSL in these macrophages and their location in the tissue at sites prone to
lipid accumulation with age make it tempting to speculate that these cells may function to
aid the clearance of excess lipid that might otherwise create cellular stress and degrade
LG function. Thus, these macrophages may represent a compensatory response to aging
that may have benefit to the tissue.
59
Macrophages can be detected using multiple markers including MerTK, CD68,
CD64, CD11b, LY6C and F4/80. Flow cytometry of the old LG using these markers
showed a similar expression of the CD11 positive macrophages in the old LG compared
to the young LG,123 while the immunofluorescence in Fig 10 show an increase of these
cells following aging. A possible explanation for this may be the big size of the
multinucleate macrophage and the 100 µm filter used for the flow cytometry. The
multinucleate macrophages with the larger size may not survive the isolation procedure
using the 100 µm filter before the flow cytometry and this may be a reason for the
apparent discrepancy between the flow cytometry data showing no increase in F4/80+
macrophages with age 123 and the clear demonstration of the increased multinucleate
F4/80+ macrophages with age by immunofluorescence (Fig. 8). Related to the possibility
of F4/80+ macrophages being excluded from flow as above, it is of note that mononuclear
F4/80+ macrophages are unchanged between young and old LG (Fig. 8), similar to
findings for F4/80+ macrophages by flow cytometry. Future studies using beads of known
sizes and imaging cytometer techniques will be required to fine-tune our observations.
F4/80 glycoprotein, used as a primary target to identify macrophages by
immunofluorescence, is a widely used murine macrophage marker 127,128 that is a member
of the epidermal growth factor seven span transmembrane family.129 Macrophages may
express different markers at different stages of maturation. For instance, in apolipoprotein
E deficient (ApoE-/- ) mice which are prone to develop atherosclerosis, macrophages
obtained from the aorta in mice fed a 3-week or 12-week high fat diet expressed different
markers.130 In another study in liver, CD11b was associated with newly migrating
macrophages while F4/80 was associated with tissue-resident macrophages.131
60
The macrophages in aged LG are not only distinct from those in young LG by virtue
of enrichment in lipid-metabolizing enzymes, but also by their multinucleate morphology.
Multinucleated macrophages have been detected in bone as osteoclasts,109 in prostheses
and implants as foreign body giant cells,110 in the lung as Langhans giant cells,111 and in
ocular tissue as Touton giant cells.107,108 Of these four known types of multinucleated
macrophages, the Touton giant cell appears most similar to the multinucleated
macrophages in aged LG in terms of function. Touton giant cells are multinucleated giant
cells present in juvenile xanthogranulomas, necrobiotic xanthogranulomas, and other
types of xanthomas.132 Xanthomas are associated with lipid deposits within organs,
especially the skin, that usually show up as yellow deposits (Greek xanthos
=yellow).132,133 Touton giant cells however have morphology inconsistent with the
multinucleated cells present in old LG, exhibiting instead a central eosinophilic cytoplasm
and an annulus of nuclei with a lipid-filled outer ring. LG multinucleated macrophages are
most similar in morphology to foreign body giant cells which have heterogeneously
distributed nuclei.134 B6 mice are known to gain fat not only in the body but also in the LG
with age,135 and it may be that the multinucleated macrophages may function similarly to
Touton giant cells .
Multinucleated cells (e.g., myoblasts, trophoblasts, and monocytes) in general go
through three stages of fusion: fusion competency development, migration, and
intracellular adhesion/cytoplasmic sharing.136 To develop fusion competency, adhesion
molecules (fusogens) must be expressed in the cells. 137 Dendritic cell-specific
transmembrane protein is a fusogen involved in osteoclast and foreign body cell
formation.138,139 Other fusogens that are present are E-cadherin,140 CD206 (a mannose
61
receptor),141 and macrophage fusion receptor.142 The combination of these fusogens may
induce the formation of multinucleated cells, lowering the energy barrier between lipid
layers of individual cells, and allowing adjacent cells to merge. Future exploration of the
presence of these fusogens in the multinucleated macrophages present in old LG may
aid in characterizing their process of formation and maturation.
Previous use of fluorescence imaging techniques in organs with aging have been
limited due to autofluorescence. We demonstrate here that an inexpensive LED-based
apparatus can photobleach LG tissue sections while preserving fundamental cellularity
and tissue organization. The 7-day photobleaching process removes endogenous
immunofluorescence in the visible wavelength emission spectrum such that the
application of standard fluorescently-labeled probes and antibodies is possible. One
limitation in this approach may be some loss in the ability of actin filaments to be
consistently labeled by phalloidin after photobleaching. It is possible that slight sample
heating associated with constant illumination, or photodegradation, may result in the
deterioration of these filaments.
In summary, lipids accumulate in the LG within acinar cells and in extra-acinar
deposits with aging in female B6 mice, a model of DED. We have identified a macrophage
population enriched in lipid metabolizing proteins (NPC, NPC2, and CTSL) that is
localized to these same lipid-laden regions in the aging LG that may be responsible for
the clearance of excess lipid. Further investigation of the subtype of these multinucleated
macrophages, their phenotype (M1 versus M2), and their elaboration of other
macrophage markers and fusogens will enable determination of whether they play
62
beneficial or pathological roles in age-related DED or are simply a marker of age-related
lipid dysregulation in this gland.
63
Chapter 4: Fluorescence Lifetime Imaging of aged lacrimal glands
4.1. Introduction
1 in 6 people in the world will be aged 60 or over by 2030, and the population is
expected to increase from 1 billion in 2020 to 1.4 billion.143 The rate of growth of the elderly
population is estimated to reach 2.1 billion by 2050.
143 Many chronic disorders, such as
heart disease (hypertension, heart failure, ischemic heart disease), cancer, obesity, and
mental disorders, have subsequently followed the rising trend.144,145 While there are
chronic disease regarded as preventable with lifestyle modification (healthy diet, exercise,
quitting smoking, and limiting alcohol intake),146 such research is not available in the
development of a chronic disease of the eye: dry eye disease (DED).
The lacrimal gland (LG) is an exocrine gland that plays a pivotal role in producing
the aqueous layer of the tear film, which in turn contributes to provide moisture, lubrication,
and nutrients to the ocular surface.2,3 Age-related changes of the LG such as
lymphocytic147 and fatty infiltration,148 fibrosis,148 volume,149 and decreased tear
secretion17-19 which may contribute to the age-related DED development. C57BL/6J mice
observe similar changes by 6-9 months; corneal surface irregularity change, corneal
barrier disruption, and tear volume compared to 8-week mice in both sexes 20 Specifically,
the aging C57BL/6J LG exhibit the similar increased lymphocytic infiltration, ectopic
lymphoid structures formation to that of human LGs.21,22 Thus, the aging C57BL/6J mice
serve as a great model for the observing age related DED and are chosen here.
Macrophages, part of the innate immune system, also play key roles in tissue
development, homeostasis, and repair of damaged tissue.105 These phagocytic cells are
64
categorized into the M1 (classically activated pro-inflammatory) type and M2 (alternatively
activated anti-inflammatory) type which both differentiate from the monocyte with
lipopolysaccharide or interferon gamma and interleukin 4 or interleukin 13
respectively.150,151 A pivotal enzyme that is involved in the pro-inflammatory M1
macrophages is the Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase 2
(NOX2), which generates superoxide a precursor to reactive oxidase species.152,153 The
activation of NOX2 involves the flavocytochrome b558, the cytosolic protein subunits
(p47phox, p67phox, and p40phox), and small G-proteins. The phosphorylation of
p47phox by phosphokinase C initiates the translocation of the cytosolic protein subunits
to gp91phox, a transmembrane catalytic protein.154 Activation of NOX2 will oxidate the
NADPH to NADP+ and this in turn generates the H2O2, which enables macrophage
differentiation and survival.155 The relationship between NOX2 and macrophages are
pivotal in the differentiation of the type of macrophage and are indicative of the status of
the immune system.
Macrophages are specialized in their function and phenotype, dependent on their
location, with some implicated in lipid clearance. Foamy macrophages accumulate an
abnormal amount of lipids and cholesterol esters and are hallmarks of early
atherosclerotic plaques.106 In areas of high lipid concentration, foam cells aggregate into
multinucleated giant cells named Touton giant cells.107,108 Other multinucleated
macrophages are reported in different tissues, with osteoclasts in bone,109 foreign body
giant cells in implants,110 and Langhans giant cells in lungs.111 We have also identified a
special multinucleate population in the aged LG that contain cholesterol transporters
65
Niemann-Pick disease type C1, Niemann-Pick disease type C2, and Cathepsin L, which
is involved in lipid metabolism.123
Here, I use fluorescence lifetime imaging microscopy (FLIM) to compare the
metabolic status of young and aged LG. I image NADH as the intrinsic fluorescent marker
and measure its fluorescence decay times (lifetime) as a reflection of cellular energy
metabolism. Shorter fluorescence decay lifetime is correlated with lower percentage of
bound NADH indicating higher free NADH concentration which both point to dependence
on glycolysis for energy production. Longer lifetimes correspond to higher percentages of
NADH bound to enzymes indicating cells are more metabolically active and rely on
Oxidative Phosphorylation (OxPhos) to produce ATP.156-162 The FLIM phasor analysis is
a powerful tool for interacting with live-cell imaging data and for extracting meaning out
of metabolic imaging.
Phasor analysis begins with pixel-by-pixel Fourier transformation of FLIM decays
into real (G) and imaginary (S) parts which are plotted in the phasor.158 Accumulation and
distribution of pixels within the phasor displays a FLIM fingerprint of the cells. There are
many enzymes to which NADH can bind, and each enzyme has a particular effect on the
NADH lifetime and phasor position.163
The phasor has two G-intercepts at 0 and 1, theoretically, where infinite lifetime
(as well as fluorescence uncorrelated to the excitation laser pulses) and instantaneous
lifetimes reside, respectively (Fig 1A, black arrows). These theoretical points cannot be
measured due to the response function of the microscope. The phasor is a 2D
representation of a 3D histogram with a rainbow lookup table which serves as a heat map
of pixel density. The FLIM phasor semi-circle demarcates where single-component
66
exponential decays are found. The phasor semi-circle is a continuous plot from
instantaneous to infinite lifetimes with dilation of lifetimes in the clockwise direction and
constricting in the counter-clockwise direction. Any pixels residing within the semi-circle
are mixtures of decays. The location is a linear combination of components falling on a
chord in the semi-circle. A pixel’s position along the chord is a ratiometric analysis of
lifetime contributions. 100% unbound NADH (0.0 Bound/Total ratio) has a lifetime of 0.4
ns and a single exponential decay on the semi-circle at the blue tip of the rainbow cone
(Fig 1B). By extending a line from 100% unbound NADH through the centroid of a phasor
distribution, ratiometric analyses are performed by measuring the fractional distance from
unbound NADH to the centroid along the chord. The rainbow scale bar shows how the
fractions change as chord length changes (Fig 1A).
Figure 1. Phasor approach to FLIM analysis and NAD(P)H-enzyme binding trends
in the phasor and the NOX2 interaction with NAD(P)H. (A) Fluorescence decays are
recorded for each pixel in images of NADH in PBS (B) and low or high concentrations of
Glycerol-3-phosphate dehydrogenase (G3PDH) (C & D). The resulting decay curves in
each pixel are transformed using Fast Fourier algorithms as Cosine and Sine functions.
Fourier coefficients for each image-pixel are plotted as G and S coordinates (G,S)
respectively such that each image-pixel has a corresponding pixel in the phasor plot,,
(phasor plot A is a combination of phasors B-D). The location of each phasor-pixel relates
to the percentage of bound NADH and the combination of enzymes present in the imagepixel. The rainbow look-up table (A) reflects the percentage of bound NADH and is the
color coding for images (B-D). (E) NAD(P)H Oxidase 2 (gp91phox) will oxidate the
NAD(P)H to NADP+ and this in turn generates the H2O2. The phosphorylation of p47
initiates the NOX2 oxidation. Adapted with permission from Cell Mol Immunol 12, 5–23
(2015).
0% bound
NADH
100% Free
NADH in
solution
G3PDH in
200uM NADH
Maximum
NADH
bound to
G3PDH
98% bound
NADH
30-95%
bound
NADH
A C
. Metabolic Scale D.
G
S
B E
67
With the FLIM image and phasor analysis of old and young LG, we investigate the
metabolic properties of the LG at different ages. We report here the unique profile of
activated NOX2 in a multinucleated macrophage population from old mouse LG, which
lays the ground for future research on age-related DED treatments.
4.2. Methods
4.2.1 Mice
Female C57BL/6J (B6) mice were either aged for up to two years or were received
from the NIH/National Institute of Aging, or purchased from the Jackson Laboratory (Bar
Harbor, ME). Following compliance with the animal use policies approved by the
University of Southern California, we used intraperitoneal injection of 100 mg/kg ketamine
+ 10 mg/kg xylazine for mice anesthesia and euthanasia. After euthanasia, the LG was
recovered and processed immediately or stored at -80°C for future experiments.
4.2.2 Materials and Reagents
F4/80-AF561 (505-4801-82, ThermoFisher), SiR-DNA (CY-SC007, Cytoskeleton, Inc)
Acridine Orange (A 1301,Thermo Fisher), TBS, Tris, NaCl, Acrylamide, Ammonium-persulfate, Tetramethylethylenediamine, rabbit anti-mouse phospho-p47phox antibody (PA5-
36863), goat anti-mouse p47phox antibody (PA1-9073), rat anti-mouse F/480 (MA1-
91124, Invitrogen), donkey anti-rat AF488 (A-21208, ThermoFisher), donkey anti-goat
AF568 (A-11057, ThermoFisher), Donkey anti-rabbit AF594 (A-21207, ThermoFisher),
68
DAPI (D-1306, ThermoFisher), Phalloidin- AF647 (A22287, ThermoFisher)
Lipopolysaccharide (LPS). Goat anti-mouse NOX2 Polyclonal Antibody (PA5-142646,
ThermoFisher), Protein G Plus-Agarose Suspension (IP04-1.5mL, Sigma Aldrich),
IGEPAL CA-630 (18896-50mL, Sigma Aldrich), Normal Mouse IgG (12-371, Sigma
Aldrich), rabbit anti-mouse gp91phox antibody (ab310337, Abcam), rat anti-mouse F4/80-
AF561 antibody (505-4801-82, ThermoFisher).
4.2.3 Immunoprecipitation of p47 from human macrophage cells
The RAW 264.7 cells were seeded in the 12 well plates and 3 hours were given wait
for them to settle on the wells. Then they were either exposed to LPS (100ng/mL) or
complete media (DMEM/F-12 complete media with 10% FBS) for 24hrs. Next day the cell
media was disposed and lysis media was added to the wells. The lysis media was 20mM
Tris HCl pH8 (J.T.Baker 4103-02), 140mM NaCl, 1% IGEPAL® CA-630 (Sigma I8896),
with 1mM EDTA. It was sterile filtered and protease phosphatase inhibitor was added
before use. Using a cell scraper the cells were gathered and pulverized with a cell
pulverizer. After spinning down the samples at 100 rcf for 5 minutes at 4 °C, the
supernatant was collected. The protein G beads were washed with the lysis buffer 3 times
before usage with the antibodies and samples. The samples were precleared with mouse
IgG and lysis buffer washed Protein G beads, before being incubated overnight with rabbit
anti-mouse NOX2 antibody overnight in at 4 °C on a tube rotator. Next day, the Protein G
beads were added to the sample and NOX2 antibody mixture and incubated for 1 hour at
4 °C on a tube rotator. After the incubation, the beads were washed and FLIM imaged
with 2mM concentration NADH using the the Leica SP8 DIVE FALCON inverted
69
microscope using the 63x water immersion objective (1.2 NA). At least 7 images were
taken of LPS treated cells (LPS+), cell media treated cells (LPS-) and NADH only.
The rest of the samples were used for Western Blotting for NOX2 using a goat anti
mouse gp91phox antibody. The samples were reduced with reducing dye and bmercaptoethanol (6x) at 95°C for 5 min. Each sample in sample buffer were loaded onto
precast 10% Tris-Glycine gels and resolved by SDS-PAGE at 120 V at 4°C for 2 hr.
Proteins on gels were transferred to nitrocellulose membranes using an iBlot™ 2 gel
transfer machine. After blocking for 1 hr with blocking buffer at room temperature while
shaking. Membranes were washed 3 times for 5 min with TBS-T, then incubated with goat
anti-mouse gp91phox antibody in blocking buffer at 4°C overnight. After 3 washes, 5 min
each with TBS-T, membranes were incubated with donkey anti-Goat IR 680 (1:4000),
respectively, in blocking buffer at room temperature for 1 hr. After another 3 washes for 5
min each with TBS-T, membranes were imaged with the LiCor Odyssey Fc machine.
Signal quantification was done with Image Studio Version 5.2. Controls included blots
processed without exposure to primary antibody.
4.2.4 Metabolic Imaging Protocol for old and young LG
LG were removed from C57BL/6J mice (n = 3 each age) at young (11-12 wks) and old
(93-96 weeks) ages, they were kept in -20°C dry ice. 10x Tris-buffered Saline (TBS)
consisting of 200 mM Tris, 1.5 M NaCl and pH 7.5 was combined with de-ionized water
and 40% acrylamide to produce 15% acrylamide and 1x TBS. 10% ammonium-persulfate and tetramethylethylenediamine were added to the mixture and poured onto a
small cell culture dish. The LG was dipped multiple times into the liquid as to produce
70
maximum exposure and minimum bubbles. After polymerization of the acrylamide gel
around the LG, it was cut in a trapezoidal shape to accommodate optimal cutting with the
Leica vibratome. Sections were cut at 100 µm thickness and the 25% 50% and 75% depth
cuts were used for FLIM imaging. These sections were put on a slide with a coverslip and
were imaged on the Leica SP8 DIVE FALCON inverted microscope with a 25x objective
(0.95 Numerical Aperture) water immersion lens. The multiphoton laser line used was at
740nm wavelength and the z-stack image of the whole LG section was taken. The FLIM
decay measurements from each pixel were transformed using FAST-Fourier
transformation resulting in cosine and sine terms plotted in a FLIM phasor. This analytical
tool can quantify changes in metabolic states through observed changes in pixel densities
and positions in the phasor. The images were analyzed with Leica LAS-X program.
4.2.5 F4/80, Acridine orange nuclear labeling of live LG sections.
LG from 3 old mice were polymerized and cut in the same way (25%, 50%, and
75%) as aforementioned. These sections were then submerged in 1:20 F4/80-AF564 and
1:200 acridine orange in TBS for 1 hour in an incubator at 37°C with gentle shaking. Then
the sections were washed with TBS 3 times, for 10 minutes in the incubator with gentle
shaking. These sections were then put on a slide with a coverslip and were imaged on
the Leica SP8 DIVE FALCON inverted microscope using the 63x water immersion
objective (1.2 NA). Images were taken using the 740nm wavelength and the images were
taken using z-stack regions of the LG enriched in the F4/80 stain. At least 5 to 7 images
were taken of each section. Then the images were then analyzed with the Leica LAS-X
program.
71
4.2.6 Phospho-p47 and p47 Immunofluorescence in mice LG and macrophage cells
After fixing in 4% paraformaldehyde and 4% sucrose in PBS for 3 hr at room
temperature, LG from female young and old C57BL/6J mice were incubated overnight in
a 30% sucrose PBS solution at 4°C. LG were embedded in O.C.T. the following day,
frozen on dry ice, and stored at –80 °C. Frozen O.C.T. blocks were sectioned at 5 µm
thickness and mounted on superfrost plus microscope slides. While submerged in sterile
phosphate-buffered saline (PBS), the slides were photobleached with a LED desk lamp
(Rotary LED Light Model Q1) in a 4 °C cold room for 7 days to get rid of the
autofluorescence from lipofuscin. These slides were quenched with ammonium chloride
in PBS for 15 min at room temperature with shaking between each step. The slides were
then blocked with 5% BSA in 0.3% Triton X-100 for 3 hr at room temperature. The tissue
was then incubated with primary antibodies as follows: rabbit anti-mouse to p47 (1:100),
or goat anti-mouse phospho-p47 (1:100), and rat anti-mouse F/480 (1:100) in blocking
buffer overnight at 4°C. On the second day, slides were washed with PBS 3 times, for 15
min each at room temperature with gentle shaking. The tissue was then incubated with
secondary antibodies as follows: donkey anti-rat Alexafluor (AF) 488 (1:200, for F4/80),
and donkey anti-rabbit AF 594 (1:200, for p47) or donkey anti-goat AF 568 (1:200 for
pP47). DAPI and Phalloidin AF 647 (1:200) were added concurrently with secondary
antibodies and incubated for 1 hr at 37°C. After incubation with secondary antibodies,
slides were washed again with PBS 3 times, then mounted with ProLong antifade
mounting medium and a glass coverslip and were left to dry overnight. Images were
acquired with either a Zeiss LSM 800 with Airyscan processing using a 63x oil objective,
72
NA 1.4. All the images were subjected to equivalent image processing with brightness
and contrast of the proteins of interest being conserved on the QuPath program version
0.4.2.
RAW 264.7 cells (mouse macrophage cells) were obtained from Sigma and cultured
in DMEM/F-12 complete media with 10% FBS in a 12 well plate with 20mm cover glass.
The cells were exposed to 100ng/mL LPS or just cell media for 24 hours. Then the cover
glasses were fixed and permeabilized with -20 °C acetone and methanol mixture (1:1) for
20 min. After the cells were blocked with 1% BSA in PBS, they were incubated with the
aforementioned antibodies and imaged accordingly.
4.3. Results
4.3.1 NOX 2 from RAW 264.7 cells show different characteristics in FLIM image
RAW 264.7 cells that were either stimulated with LPS or non-stimulated were
harvested and lysed. Using a NOX2 (gp91phox) antibody, immunoprecipitation was
conducted and confirmed through Western blotting using a NOX2 antibody with another
host animal (Fig 2C). A lot of background was observed due to a combination of high IgG
background suspected at 60kDa and an incomplete stripping of antibodies from a prior
blot with the same antibody used in the immunoprecipitation. The first 4 lanes being the
solution from the washes of the beads after preclearing, show a clear signal in the bands
at 70kDa, which is not observed in the last two lanes on the left membrane. Compared to
the secondary antibody only treated control on the right membrane, the signal seems
stronger on the last two lanes of the left membrane. This may be due to the remnants of
73
mouse IgG (heavy chain size between75 to 50kDa) that was used in the preclearing step
of the immunoprecipitation and the overlap with the NOX2 signal (60kDa). The
quantification of these signals is not feasible as the samples’ protein concentration was
not able to be measured through the Bradford Assay and nanodrop, due to the
interference of the lysis buffer. However, we are able to confirm the existence of the NOX2
signal.
In the phasor diagrams in Fig 2A, both NOX2 immunoprecipitated beads of RAW
264.7 cell lysates that were non-stimulated (LPS -) or LPS stimulated (LPS +), were
imaged at least 7 times per condition. Yellow and cyan circles represent the most
abundant pixel positions of LPS + and LPS -. As can be observed in Fig 2B, it is evident
that there is more cyan signal present on the beads in the non-stimulated samples, while
there is more yellow in the LPS stimulated bead samples. As activated NOX2 will bind
more to NADH, the signal emitted by this enzyme would be more towards the top left of
the phasor. The yellow circle is situated more to the top left versus the cyan circle,
indicating that the NOX2 has more bound NADH. This experiment set the standard for
the future phasor analysis in keeping an activated and non-activated NOX2 signal.
74
LPS + LPS -
A
B
50
75
50
75
C Beads
LPS - + - + - + - + - + - +
Secondary Control
LPS- LPS+
Wash
10µm
10µm
10µm
10µm
10µm
10µm
75
Figure 2. Immunoprecipitated NOX2 from LPS stimulated RAW 264.7 cells FLIM
image and phasor. A. Phasors are shown of beads containing NOX2 from
immunoprecipitation with RAW 264.7 cells with LPS- (LPS non-treated, cell media only),
LPS+ (LPS treated) on the top left and right respectively. Yellow colored ring was of the
LPS+ signal and the cyan colored ring is of the LPS-. The exact placement of these rings
are consistent throughout. B. Representative FLIM images of LPS- and LPS + RAW 264.7
cell NOX2. C. Western blotting of NOX2. First 10 lanes are solvents that came from
cleaning the beads, where the first and second are the first washes of LPS- and LPS+
beads and the next washes every two rows. The last two lanes are the beads themselves
with LPS- and LPS+ samples. The left membrane was exposed to the goat anti-mouse
NOX2 antibody plus the anti-goat IR680 antibody, while the right membrane was only
exposed to the anti-goat IR680 antibody.
4.3.2 Old LG show less oxidative phosphorylation and more lipofuscin
I imaged LG by FLIM from young (11 week old) versus old (93 week old) mice,
which measures the cells’ free/bound NADH+ redox states 159-162. FLIM signatures were
measured but the intense signal arising from lipofuscin granules overpowered the subtle
differences in NADH signal, as lipofuscin has an overlapping emission spectrum with
NADH. By thresholding out the brightest pixels corresponding to lipofuscin, we highlighted
differences in NADH FLIM signals. The remaining fluorescent signal was relatively
photon-limited and therefore needed de-noising and amplification of signal-to-noise ratio
(SNR) by applying Leica’s Complex Wavelet Filter.164 Using this approach, the FLIM
phasor distribution of young LGs was distinct from that of old LGs.
First, we found tissue-wide metabolic differences in LGs of 11-week mice versus
93-week mice. LGs of young mice had greater OxPhos FLIM signature than LGs of old
mice (Fig 3A). Younger mice have more even distributions of metabolic states where the
older mice have regions that have metabolism similar to young mice. With the same
rainbow scale bar applied to both phasors, we observed a higher distribution of the higher
OxPhos signal (red colored) on the young versus the old (Fig 3B top and bottom). It is
76
interesting that the higher OxPhos area in the young LG are distributed in the outer edges
throughout the 3 mice samples, while the old LG have the higher OxPhos areas in the
interior regions. The distribution of the lower OxPhos (blue colored) are evident in the old
(Fig 3B) in both the whole LG and magnified (top and bottom row respectively) images.
77
100µm
Young Old
Phasor Metabolism
500µm 500µm
100µm
Magnified
A B
78
Figure 3. Metabolism FLIM imaging of young and old LG. (A) Phasor diagram shows
the lifetime distribution of young LG (left) and old LG (old). The rainbow color scheme is
consistent between the two phasors. N = 3 mice, 3 layers (25%, 50%, and 75%) each
mouse LG, thus 9 image composite per phasor. (B) Left young LG shows a more Oxphos signal on the outer side of the acini, while the old LG shows less Ox-phos signal. 5
times magnified image of each highlighted area (white box) is shown below. Scale bar:
500 µm and 100 µm respectively.
Second, in the same phasor from Figure 3A, the signal of the LPS+ (cyan) and
LPS- (magenta) NOX 2 circles and lipofuscin (dark blue) were superimposed. While a
green circle shows the tail that is resident in both phasors but stronger on the old LG
phasor. When the images were magnified for both, the presence of lipofuscin is markedly
increased in the old LG. Also, there is an increase in the cyan and magenta signal in the
old LG, especially around the lipofuscin (blue) signal. While the phasor for young do not
show as much accumulation of lipofuscin, it is present in the whole LG image and the
magnified images. Through the FLIM images, we can see that the old LG has less
OxPhos state, a unique apparent NOX2 signal, and increased accumulation of lipofuscin
versus the young LG.
79
Young Old
Phasor Pathological Signal Magnified
A
B
500µm 500µm
100µm 100µm
80
Figure 4. Pathological Signal FLIM imaging of young and old LG. (A) Phasor diagram
with NOX2 (activated and non-activated are yellow and cyan respectively), Lipofuscin
(blue) and a tail signal from the main metabolic signal (red) are observed. The positions
of the circles are consistent between the two LG phasors. N = 3 mice, 3 layers (25%, 50%,
and 75%) each mouse LG, thus 9 image composite per phasor. (B) FLIM imaging with
same color coding from young LG on the left and old LG on the right. Scale bar: 500 µm
and 100 µm respectively.
Third, following the discovery of the yellow circle and cyan circle in the magnified
images(Fig 4A), we were interested in which cell population that these signals may be
contributing from. As we had known the increase in multinucleate macrophages in the old
LG, as discussed in Chapter 3, we pursued a higher magnification image with F4/80
staining of the LG in the acrylamide.
The higher magnification images showed that the apparent NOX2 signal is
localized to these macrophages (Fig 5A). When the image is transposed with the same
four circles from the whole LG image phasor, we see that the NOX whether it be the
activated or non-activated is present in the multinucleated macrophages. Also, these cells
co-localized with the lipofuscin signal (Fig 5B). The red circle which was a tail prominent
in the old lg phasor (Fig 4A), can be seen throughout the acini and other cells but not in
the multinucleated macrophage. Also, this red signal can be seen in the old LG areas that
overlap with the blood vessel.
81
Figure 5. Higher magnification FLIM Image of Old LG shows NOX2 and lipofuscin
signal in multinucleated macrophages. A. F4/80 and acridine orange stained old LG
were divided in 3 different layers (25, 50, and 75%) and FLIM images with 63x lens were
taken on F4/80 (macrophage marker) stained areas. NOX2 (yellow and cyan) and
LPS- NOX LPS+ NOX Nuclei F4/80
LPS-NOX LPS+ NOX Red Circle Lipofuscin F4/80
A
B
20µm
20µm 20µm 20µm 20µm
82
lipofuscin (blue) overlaps with F4/80 (green) stained areas. The macrophage appears to
have multiple nuclei (white). (phasors were of n=3 mice) B. The same image but
highlighted in the same phasor circles as prior whole LG FLIM image phasor is shown.
The green circle shows the overlap with acini and other cells but not in the multinucleated
macrophages.
4.3.2 Old LG have more activated p47phox in the macrophages
After the photobleaching of the LG with LED desk lamps, autofluorescence from
the lipofuscin was minimal. We observed the presence of a population of multinucleated
macrophages that were labeled with F4/80 in the old (Fig 6A, B). These multinucleated
macrophages contained the p47phox (p47) and the phosphorylated p47phox (pP47),
whereas the single nucleated macrophages in the young LG did not label as strongly for
the pP47. This suggests the activated transmembrane NOX2, which should be bound to
the pP47, is mostly present on the multinucleated macrophages in the old LG.
83
Figure 6. Old LG shows increased phosphor-p47phox in macrophages. (A) Young
LG have less macrophages (magenta F4/80 stain) while in the old LG p47phox is present
in the macrophage cells. Scale bar: 20 µm (B) Young LG have macrophages but not
phosphorylated p47phox signal (pP47). Old LG have more F4/80-stained cells that have
pP47 stain. Scale bar: 500 µm (n=3 mice) Each column labeled Mouse 1, Mouse 2, and DAPI Actin P47 F4/80 DAPI Actin pP47 F4/80Young LG Old LG
A
B
Young LG Old LG
Mice 1 Mice 2 Mice 3
84
Mouse 3 are images from different mice for different age groups. Mouse 1, 2, and 3 are
not necessarily the same across different panels or figures.
4.3.3 M1 type macrophages have higher activated NOX2
After the exposure of 100ng/mL LPS for 24 hours, the RAW 264.7 cells (mouse
macrophages) in vitro, should have turned into the classically activated M1
phenotype.165,166 This can be confirmed with Fig. 7 A,B., in which there was a marked
increase in both the P47 and pP47 signal in the in the LPS stimulated macrophages. The
staining on the LPS stimulated macrophages show the punctate formation of both P47
and pP47 on the membrane which is in accordance with the NOX2 activation mechanism
and the mice LG in Fig 7. These stimulated macrophages also show an enlarged
morphology with increased pseudopodia. Also, there are some multinuclear
macrophages present in the cells which have been observed in a prior publication.167
85
Figure 6. LPS stimulated RAW 264.7 cells have higher p47phox and phosphorylated
p47phox. (A) Mice macrophage cells have p47phox on the membrane. LPS stimulated
cells have higher signal intensity and punctae formation on the membrane. Scale bar: 20
µm (B) While there are resident phosphorylated p47phox signal in both non-stimulated
and LPS-stimulated macrophages, there was higher signal in the LPS stimulated
macrophages. Green arrows indicate the punctae formation. Scale bar: 20 µm LPS Stimulated Non-stimulated DAPI Actin pP47phox
A
B
LPS Stimulated Non-stimulated
DAPI Actin P47phox
86
4.4. Discussion
In the comparison between the FLIM phasors of young and old LGs, we were able
to observe the young LG having a higher OxPhos characteristic versus the old LG,
suggesting that the old LG are less metabolically active or perhaps less healthy. This is
evident with the buildup of lipofuscin in the LG which was clearly present in the phasor as
a long tail (Fig 4A blue circle), and is in accordance with prior publications indicating the
increase in lipofuscin accumulation as early as 8 months.19 As it is known that oxidative
stress increase is correlated with aging lacrimal gland in both humans and mice, 23,168,169
and the concurrent increase of lipofuscin granules suggest its’ role for oxidative stress.
As lipofuscin is not degradable by lysosomal enzymes, the accumulation rate is
inversely related to the longevity in cells, and this is similarly reflected in the rainbow
images of Fig 3A. While we and others have shown accumulation of lipofuscin in the aged
LG, 19,112,123 lipofuscin is also known to be accumulated in both the macrophages and T
lymphocytes in aged mice.170 Perhaps this is not surprising as macrophages are
abundant in lysosomes, which is the primary site for lipofuscin. This is particularly evident
in Fig 5B with the F4/80-stained multinucleated macrophages outside of the acini
showing a lipofuscin signal in the cytosol. The multinucleated macrophages seen in the
old LG have been published to contain lipid metabolizing enzymes123 but the phenotype
(M1 versus M2) of these macrophages has not been characterized. Both in
atherosclerosis and non-alcoholic fatty liver disease, diseases concerning high lipid
content, the M1 type macrophage seem to exacerbate the disease.171-174 Likewise, in the
old LG, we observe the NOX2 activated macrophages, suggesting the phenotype to be
87
M1 pro-inflammatory type. Although we have presented the whole LG FLIM imaging
showing an increase in a NOX2 activated macrophages, a flow cytometry-based
approach may be definitive in the quantification of the different phenotypes.
Here we present a characterization of the old LG having a less OxPhos metabolic
profile with a discrete lipofuscin content. Building up on the prior study of multinucleate
macrophages that contain lipid metabolizing enzymes and cholesterol transporters, we
have identified these cells to have an activated NOX2 profile, which suggests their
classically activated, pro-inflammatory phenotype. Further studies that build up on the
distinct macrophages and possible modulation of these macrophages may elucidate a
possible mechanism in treating or preventing age related dry eye disease.
88
Conclusion
First, I demonstrate in this thesis that the modulation of the p38, PP2A, TTP, and CTSS
pathway in the male NOD mice may be a possible drug target in ameliorating the dry eye
disease symptoms. Through the cell experiments involving a P38 inhibitor and PP2A
activator, I showed that controlling the p38 phosphorylation status may be linked to a
decrease in the CTSS mRNA. Unfortunately, this was not able to be replicated in the male
NOD Mice studies that involved systemic injection of the p38 inhibitor, SB202190. A
temporal relationship of the p38 inhibitor and the phospho-p38 to p38 ratio suggests a
more systemic pharmacodynamic analysis and local delivery to the LG may be necessary.
Second, by comparing the old and young LG of female C57BL/6 mice, I was able to
discover and characterize a novel multinucleate macrophage population that increased
with age. These cells had cholesterol transporters NPC1, NPC2, and a lipid metabolizing
enzyme Cathepsin L in the cells. I was also able to present a reproducible photobleaching
method that was used for immunofluorescence in the lipofuscin rich aged LG. The old LG
showed accumulation of the lipid droplets intra and extra of the acinar cells and which
may indicate a role for the multinucleate macrophages in the lipid rich environment.
Finally, I showed through FLIM imaging that the old LG had a unique metabolic profile in
the phasor with lower OxPhos state than the young LG. There was a unique metabolic
contribution by the novel multinucleate macrophages in the aged LG having an activated
NOX2 profile that suggests the macrophages to be in a pro-inflammatory M1 type
macrophage state.
89
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Creator
Choi, Minchang
(author)
Core Title
Characterization of changes in metabolism and inflammation in the lacrimal gland in dry eye disorders
School
School of Pharmacy
Degree
Doctor of Philosophy
Degree Program
Pharmaceutical Sciences
Degree Conferral Date
2024-08
Publication Date
07/12/2024
Defense Date
06/21/2024
Publisher
Los Angeles, California
(original),
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
aging,cathepsin S,dry eye disease,lacrimal gland,macrophage,multinucleated macrophage,OAI-PMH Harvest,p38,tristetraproline
Format
theses
(aat)
Language
English
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Electronically uploaded by the author
(provenance)
Advisor
Hamm-Alvarez, Sarah (
committee chair
), MacKay, Andrew (
committee member
), Stiles, Bangyan (
committee member
)
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minchanc@usc.edu,minchoipharmdphd@gmail.com
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theses (aat)
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Choi, Minchang
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texts
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20240712-usctheses-batch-1180
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The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright.
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Tags
cathepsin S
dry eye disease
lacrimal gland
macrophage
multinucleated macrophage
p38
tristetraproline