University of Southern California Dissertations and Theses

Investigation of the role of ICOS in the regulation of ILC2 dependent airway hyperreactivity

(USC Thesis Other)

Investigation of the role of ICOS in the regulation of ILC2 dependent airway hyperreactivity

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content Copyright 2022 Marshall Hin Sing Fung

Investigation of the Role of ICOS in the Regulation of ILC2 Dependent Airway Hyperreactivity
by
Marshall Hin Sing Fung

A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

August 2022
ii

Acknowledgements
I would like to thank my Mentor and Chair of my M.S. Thesis committee, Dr. Omid Akbari,
for all his support and sharing his expertise with me over the past two years. He has been very
welcoming and dependable during these unprecedented times of the COVID-19 pandemic. I
greatly appreciate my committee members, Dr. Joseph R. Landolph, Jr., and Dr. Kiego Machida,
and I thank them for their time and knowledge that they have passed onto me during my enrollment
in the M.S. program in Molecular Microbiology and Immunology.
I would like to thank the postdoctoral fellows in Dr. Omid Akbari’s lab oratory, Drs.
Georges Helou and Benjamin Hurrell, for their guidance and support for me. They have taught me
valuable lessons that pertain to both inside and outside of the laboratory. I would also like to thank
the graduate students in Dr. Akbari’s labora tory, Emily Howard, Jacob Painter, Christine Quach,
Stephen Shen, and lab manager Pedram Jahani for their assistance on helping me conduct
experiments.
iii

Table of Contents

Acknowledgements ........................................................................................................................................ ii
List of Figures .............................................................................................................................................. iv
Abbreviations ................................................................................................................................................ v
Abstract ........................................................................................................................................................ vi
Chapter 1: Introduction of Asthma ........................................................................................................... 1
1.1 General Overview ............................................................................................................................... 1
1.2 Immunopathogenesis of Type 2 Asthma ............................................................................................ 3

Chapter 2: ILC2s ........................................................................................................................................ 5
2.1 General Overview of ILC2s ................................................................................................................ 5
2.2 Stimulation of ILC2 ............................................................................................................................ 6
2.3 Functions of ILC2 ............................................................................................................................... 6
2.4 Regulation of ILC2 Immune Receptors .............................................................................................. 8
2.5 Intracellular Domain and Signaling of ICOS ...................................................................................... 9
2.6 The Role of ICOS in ILC2s .............................................................................................................. 11

Chapter 3: Materials and Methods ......................................................................................................... 13
3.1 Mice .................................................................................................................................................. 13
3.2 Lung processing for flow cytometry ................................................................................................. 13
3.3 ILC2 Processing and Sorting ............................................................................................................ 14
3.4 RNA sequencing and data analysis ................................................................................................... 14
3.5 Supernatant cytokine measurement .................................................................................................. 15
3.6 Glucose uptake assay and measurement of glycolytic function ........................................................ 15
3.7 Fatty acid uptake assay and measurement of fatty acid oxidation function ...................................... 16
3.8 Agilent Seahorse ............................................................................................................................... 16
3.9 Statistical analysis ............................................................................................................................. 16

Chapter 4: Results ..................................................................................................................................... 18
4.1 Objectives of the study ...................................................................................................................... 18
4.2 Lack of ICOS affects the transcriptomic profile of aILC2s .............................................................. 18
4.3 Lack of ICOS affects IL-10 regulator gene expression and increases IL-10 cytokine production ... 21
4.4 Lack of ICOS improves the IL-4-induced IL-10 production in aILC2s ........................................... 22
4.5 Bhlhe40 participates in the regulation of IL-10 dependently from ICOS ......................................... 25
4.6 Lack of ICOS induces a metabolic change from fatty acid oxidation to glycolysis in aILC2s ........ 27

Chapter 5: Discussion and Future Directions ........................................................................................ 31

References ................................................................................................................................................... 36

iv

List of Figures

Fig. 1 Type 2 immune response to inhaled allergens ..................................................................... 5
Fig. 2 ILC2s and ILC210s cytokine secretion ................................................................................. 8
Fig. 3 ICOS:ICOS-L interaction promotes ILC2 pro-inflammatory cytokine secretion and
increases AHR and eosinophilic inflammation............................................................................... 9

Fig. 4 ICOS:ICOS-L interaction and signaling pathway in Treg cells ........................................... 11
Fig. 5 Lack of ICOS affects the transcriptomic profile of aILC2s ............................................... 21
Fig. 6 Lack of ICOS affects IL-10 regulator gene expression and IL-10 cytokine production .... 22
Fig. 7 Lack of ICOS improves the IL-4 induced IL-10 production in aILC2s ............................. 25
Fig. 8 Bhlhe40 participates in the regulation of IL-10 dependently from ICOS .......................... 26
Fig. 9 Lack of ICOS induces a metabolic change from fatty acid oxidation to glycolysis in
aILC2s ........................................................................................................................................... 30

v

Abbreviations
AHR Airway hyperreactivity
APC Antigen presenting cell
AREG Amphiregulin
DAMPs Damage-associated molecular patterns
DCs Dendritic cells
ECAR Extracellular acidification rate
ECM Extracellular matrix
FAO Fatty acid oxidation
i.n. Intranasal
IBD Inflammatory bowel disease
ICOS Inducible T-cell co-stimulator
ICOS KO Icos
-/-

ICOS-L ICOS-Ligand
IFN-γ interferon-γ
IL Interleukin
ILC Innate lymphoid cell
ILC2 Group 2 innate lymphoid cell
LAIR-1 Leukocyte-associated immunoglobulin-like
receptor 1
MHC Major histocompatibility complex
OCR Oxygen consumption rate
PAMPs Pathogen-associated molecular patterns
PD-1 Programmed cell death protein 1
PRRs Pattern recognition receptors
TH1 T helper 1
TH17 T helper 17
TH2 T helper 2
TLRs Toll-like receptor
TN Naïve T
TNFRSF Tumor necrosis factor receptor superfamily
TNF-α Tumor necrosis factor-α
TSLP Thymic stromal lymphopoietin

vi

Abstract
Allergic asthma is a growing heterogenous disease that is characterized by chronic lung
inflammation and airway hyperreactivity (AHR). Group 2 innate lymphoid cells (ILC2s) are from
a subset of immune cells that contribute to the development of lung inflammation and AHR by
secreting type 2 cytokines. Because inducible co-stimulatory molecule (ICOS) has been shown to
play a role in ILC2 effector functions and homeostasis, we hypothesize that ICOS plays a role
in regulating ILC2-mediated IL-10 production and a metabolic shift in allergic asthma.
Therefore, to test this hypothesis, we investigated the role of ICOS in ILC2s during IL-33-induced
airway inflammation. We found that ICOS regulated the production of IL-10 with and without the
presence of IL-4 via the following IL-10 gene regulators: Nfil3, Bhlhe40, cMaf, and Prdm1.
Moreover, ICOS regulated pro-inflammatory IL-5 and IL-13 cytokines production in ILC2s. ICOS
deficiency induced a metabolic change from fatty acid oxidation (FAO) to glycolysis, while the
presence of IL-4 rescued partial dependency on FAO. Additionally, this study highlights a possible
immunoregulatory role of ICOS in allergic asthma and presents ICOS as a potential therapeutic
target.
1

Chapter 1: Introduction of Asthma
1.1 General Overview
Asthma is a heterogenous group of diseases characterized by chronic inflammation and
remodeling of the airways. It is considered to be one of the most common major non-
communicable diseases, affecting more than 300 million people worldwide
1
. The prevalence of
asthma follows a rural to urban gradient, with up to 10% of developed western countries affected
with the disease
2
. However, developing countries are displaying rapid increases of asthma
prevalence as they have started adopting westernized lifestyles, increasing the burden on
healthcare systems, and negatively impacting societies and patient outcomes. Asthma prevalence
is also correlated with other immune-mediated diseases such as type 1 diabetes mellitus and atopic
dermatitis
2
. The cause of increased asthma prevalence is currently unknown. However, it is likely
to involve external and genetic factors.
The combination of environmental exposures and individuals’ genetic variability may
contribute to the heterogeneity of asthma
3
. Environmental exposures depend on the geography
surrounding the affected individuals, while genetic variability may contribute to the individuals’
increased susceptibility to react to certain allergens or to develop certain allergic diseases
3
.
Therefore, the interactions between the environment and the individuals’ genetics may lead to
affected individuals displaying varying pathophysiological mechanisms at different disease stages.
Severe asthma is the exception, as it is characterized by early childhood onset and multi-allergen
sensitization throughout the patient’s life
4
.
Multiple hypotheses have been proposed to determine the cause of asthma. First, it was
thought that increased exposure to indoor allergens combined with plush furniture and carpets may
have contributed to the increase of asthma. Second, the “hygiene hypothesis” propo ses that the
2

decreased exposure to unhygienic environments during early life led to the development of
asthma
5
. Third, the “microbial diversity” hypothesis suggests that the lack of immunogen contact ,
such as non-pathogenic microbes and commensal organisms, contributes to affected priming and
regulation of the immune system, leading to increased prevalence of asthma
6
. Ultimately, it is
thought that genetics, environmental factors, and host factors influence the development of asthma.
Genetic factors may include immunological factors, age, and sex, as 80% of children diagnosed
with asthma by the age of 6 years are atopic or genetically predisposed to allergic
hypersensitivity
2,7
. Moreover, asthma is more common in boys in early childhood compared to
girls, while girls tend to acquire asthma during puberty and early adulthood. Environmental factors
include air pollution, pollen, and weather, while host factors include obesity, cigarette smoking,
and allergic sensitization
1
.
Current treatment for asthma includes both non-pharmacological and stepwise
pharmacological treatment through inhalation of corticosteroids and short and long-acting β 2
agonists. Patients are encouraged to seek treatment for any present co-morbidities that may
augment the severity of asthma, such as avoidance of cigarette smoking and weight loss.
Minimizing exposure to environmental allergens is suggested, as it lowers allergic sensitization to
allergens. However, this strategy has not worked, as it does not reduce asthma significantly. Self-
management education and inhaler training are offered, as they allow the patient to recognize the
signs and symptoms of asthma and to respond accordingly with proper inhaler training. Stepwise
pharmacological treatment can also be used if non-pharmacological methods do not significantly
reduce asthma. A combination of short-acting, long-acting β 2 agonists and inhaled corticosteroids
is used with each treatment
8
.
3

1.2 Immunopathogenesis of Type 2 Asthma
At the immune level, asthma can be characterized into two main subtypes: type 2 asthma
and non-type 2 asthma
9
. As the most common form of asthma, type 2 asthma is found in 70% of
patients affected with asthma
10
. Type 2 asthma is identified by the presence of type 2 inflammation
and encompasses allergic and eosinophilic asthma
11
.
Type 2 asthma may be caused by the inhalation of and sensitization to generally non-
offensive exogenous allergens found in the environment. These allergens include house dust mites,
pollutants, pollen, and cigarette smoke particulates and are inhaled into the bronchioles
2
. The
mucosal epithelium releases damage-associated molecular patterns (DAMPs) in the form of
nonspecific alarmins after allergens cause damage to the epithelium (Fig. 1)
12
. These epithelial-
derived nonspecific alarmins include IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) and
induce dendritic cell (DC) maturation
2,13
. The mature DCs then take in the inhaled allergens,
process allergens, present them onto major histocompatibility complex (MHC) class II, and release
chemokines that recruit TH2 cells and group 2 innate lymphoid cells (ILC2s)
14
. The secretion of
IL-25, IL-33, and TSLP from the damaged epithelium can also directly recruit TH2 cells and
ILC2s
14
.
After the recruitment of TH2 cells and ILC2s, activation of transcription factor GATA3 in
both cells types induces the release of type 2 cytokines: IL-4, IL-5, IL-9, and IL-13
14
. IL-4 and IL-
13 bind to IL-4 receptor subunit-α (IL -4Rα) to induce nitric oxide release from th e epithelium,
goblet cell metaplasia, and airway fibrosis
14
. IL-5 binds to IL-5Rα and induces tissue eosinophilia
by driving eosinophil differentiation near the bronchial mucosal epithelium where the release of
eosinophil granules induces tissue damage
14–16
. IL-9 drives mast cell survival and growth near the
mucosal epithelium and leads to the release of histamine, a pro-inflammatory molecule, after mast
4

cell degranulation
17
. All of these events may lead to remodeling of the airways by increasing
airway smooth muscles, increasing mucus production, and developing airway hyperreactivity
(AHR), a cardinal feature of allergic asthma
14
.
Emerging therapeutics have tried to target cytokines, chemokines, and receptors to treat
type 2 asthma. As TSLP regulates DC maturation and downstream type 2 inflammation, TSLP is
a potential target for therapeutic treatments. Tezepelumab is a blocking antibody that binds to
TSLP and was able to attenuate early and late asthmatic responses in allergic asthma
18
.
Additionally, Tezepelumab was able to reduce blood and sputum eosinophils in patients with mild
asthma
14
. Lebrikizumab and tralokinumab are IL-13 blocking antibodies that have been made to
inhibit nitric oxide production and goblet cell hyperplasia
14
. However, these treatments were
ineffective and showed little improvement in alleviating symptoms and reducing eosinophilic
exacerbations
14
.
Blockade of IL-4 and IL-13 receptor (IL-4Rα) using dupilumab has shown greater efficacy
than anti-IL-4 and anti-IL-13 antibodies. Dupilumab was found to significantly reduce
exacerbations of symptoms and improve lung function in patients experiencing moderate to severe
asthma
19
. Mepolizumab and reslizumab are IL-5 blocking antibodies that show moderate efficacy
in affected patients under specific asthmatic conditions as patients displaying moderate to severe
asthma often showed no improvements in lung function or symptoms
14
. Antagonists for
chemokines such as CCR3 and CCR4 have been developed with the hopes of inhibiting
eosinophilic and TH2 cell activity, respectively. However, most chemokine antagonists failed to
display any clinical relevance as they had toxicological problems or had no significant reduction
of asthmatic responses
14
. GATA3 has also been identified as a potential target, as this transcription
factor regulates both TH2 cells and ILC2 type 2 cytokine expression and subsequent type 2
5

immunity. Patients with mild asthma have shown reduced immune responses after administration
of ST010, a DNAzyme that inactivates GATA3 RNA, although eosinophil levels were not
drastically reduced
14
.

Fig. 1 Type 2 immune response to inhaled allergens (Adopted from Barnes, 2018, Nature
Immunology)

Chapter 2: ILC2s

2.1 General Overview of ILC2s
ILC2s are a subset of ILCs that were first discovered 14 years ago and were the latest
subpopulation of lymphoid cells to be identified after the discovery of T and B lymphocytes
20
.
Both human and mice ILC2s are identified as lineage negative and CD127 (IL-7Ra), CD45, and
6

ST2 (IL-33R) positive, which allows epithelial-derived IL-33 alarmin to bind to and activate
ILC2s
21
. ILC2s share similar functions to TH2 cells as they secrete IL-4, IL-5, IL-9 and IL-13 after
activation of GATA3 transcription factor
22,23
. Therefore, ILC2s are highly implicated in allergic
asthma as they contribute to type 2 immune response by secreting type 2 cytokines
24–26
.

2.2 Stimulation of ILC2
ILC2 activation relies on alarmins secreted by mucosal epithelial cells that have been
activated by allergens or by chemokines released by activated mature epithelial DCs
2
. These
alarmins include IL-25, TSLP, and IL-33, whose receptor can be found in ILC2s as ST2 (IL-33R).
Moreover, ILC2s require IL-2 and IL-7 for survival and development, hence the expression of the
CD127 (IL-7Ra) marker
27–29
. Other types of molecules that regulate ILC2 stimulation include
cytokines, hormones, neuropeptides, lipid mediators, co-stimulatory and co-inhibitory molecules,
and adhesion molecules
21
. For this study, only alarmins and cytokines will be further mentioned.

2.3 Functions of ILC2
The functions of ILC2s are dependent on tissue type as ILC2s may reside in the lungs,
adipose tissue, skin, gut, and brain
30
. Specifically in the lung and in the context of allergic asthma,
ILC2s can have an inflammatory phenotype through the secretion of IL-4, IL-5, IL-9, and IL-13
while an immunoregulatory phenotype is induced through IL-10 and amphiregulin (AREG)
secretion (Fig. 2)
31
.
IL-4 induces an IgE isotype switch that upregulates mast cell activation and promotes CD4
+

TH2 cell differentiation
32,33
. Activated mast cells then release histamine into the airways and
increase lung inflammation and airway obstruction. IL-5 recruits eosinophils to the mucosal
7

epithelium and contributes to airway remodeling through the release of toxic granules and IL-4,
further driving lung inflammation
24,34
. IL-9 contributes to the survival and proliferation of ILC2s
and induces IL-5 and IL-13 production through autocrine signaling
35
. IL-13 contributes to mucosal
epithelium leakiness by dysregulating tight junctions in the epithelium, allowing allergens to enter
the ECM and activate downstream TH2 effector functions via mature DCs
36
.
IL-10 has been shown to downregulate pro-inflammatory cytokines and lung inflammation
by suppressing by the recruitment of eosinophils
37
. Important genes that positively regulate IL-10
production include Nfil3, cMaf, and Prdm1 while Bhlhe40 negatively regulates IL-10 production
38
.
Nfil3 has been described to be an essential gene for ILC2s development and a contributor to the
regulation of IL-10 in CD4+ T cells. cMaf and Prdm1 have been reported to be important genes
in regulating IL-10 expression in ILC210s
39
. Bhlhe40 has been described to repressive IL-10
expression in T cells during Mycobacterium tuberculosis infection
40
. Characterized as a subset of
ILC2s that produce IL-10, ILC210s contribute to the suppression of ILC2 proinflammatory
functions and reduction of AHR and lung inflammation
39
. AREG was first described as an
epidermal growth factor responsible for tissue healing and is secreted by epithelial cells upon
damage and inflammation
41
. Later studies have shown that ILC2s are able to secrete AREG upon
IL-33 signaling and are able to downregulate lung inflammation by promoting repair of damaged
mucosal epithelium
42
.
8

Fig. 2 ILC2s and ILC210s cytokine secretion (Adapted from Bartemes, 2021, The Journal of
Allergy and Clinical Immunology)

2.4 Regulation of ILC2 Immune Receptors
Several immune receptors have been identified on ILC2s that work to mediate ILC2
survival, homeostasis, proliferation, and effector functions
21
. These receptors include programmed
cell death protein-1 (PD-1), leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1),
tumor necrosis factor receptor superfamily (TNFRSF), and inducible T-cell co-stimulator
(ICOS)
21
.
PD-1 has been shown to be an inhibitory receptor found on all T cells and is responsible
for regulating T cell effector function and tolerance towards self-tissue
43
. PD-1 can also be found
on ILC2s as it reduces AHR and lung inflammation by downregulating ILC2 viability and effector
functions such as the secretion of pro-inflammatory cytokines IL-5, IL-9, and IL-13
44
. LAIR-1 is
an inhibitory receptor that is expressed on most leukocytes and can inhibit B and T cells, NK cells,
and DCs
45
. The receptor has been shown to be inducible in lung ILC2s and can reduce AHR
9

through the downregulation of cytokine secretion and effector function
46
. Several receptors of the
TNF superfamily and their ligands have been observed to be implicated with ILC2s
21
. TNFRSF25
can induce activation of ILC2 while contributing to ILC2 survival and proliferation; TNFRSF18
promotes IL-5 and IL-13 secretion through autocrine signaling of IL-9, leading to increased lung
inflammation
47,48
.

Fig. 3 ICOS:ICOS-L interaction promotes ILC2 pro-inflammatory cytokine secretion and
increases AHR and eosinophilic inflammation (Maazi, 2015, Immunity)

2.5 Intracellular Domain and Signaling of ICOS

Expressed as a disulfide bond-linked homodimer, ICOS is the third member of the CD28
superfamily expressed on T cells identified as a type 1 transmembrane glycoprotein
49
. With its N
terminus located outside of the cell, ICOS bears a single immunoglobulin (Ig) variable-like
domain that is anchored to the transmembrane with 23 amino acids, and ends with a cytoplasmic
10

tail of 35 amino acids
49
. However, ICOS does not include the MYPPY motif that CD28 uses to
bind to its receptor, B7-1. This suggests that ICOS binds to a different receptor that was later
identified as ICOS-L
49,50
.
Although signaling pathways involving ICOS have not yet been explored specifically in
ILC2s, it is predicted to be similar to signaling pathways found in other ICOS
+
cells such as Treg
cells. A partial analysis of ICOS signaling pathways can be described through the downstream
PI3K signaling of ICOS as it has been thoroughly studied in Treg cells
51
. Upon binding of ICOS-
L to ICOS receptor, the YMFM motif found on the ICOS cytoplasmic tail is phosphorylated and
binds to the SH2 domain of p50 , p85, and p110 , which are subunits of class IA
phosphatidylinositol 3-kinase (Fig. 3) (PI3K)
51
. The initial recruitment of p50  and p85  to the
YMFM motif induces NFAT to bind to FOXP3, a gene responsible for the regulation of anti-
inflammatory IL-10 cytokine production. Meanwhile, the binding of p50 , p85, and p110 
causes PIP2 to be converted into PIP3, which later phosphorylates protein kinase B (PKB), also
known as Akt
51
. Akt is a protein kinase that plays a major role in regulating cellular processes as
the phosphorylation of Akt can activate or inhibit multiple downstream signaling pathways
responsible for regulating metabolism, survival, and effector functions.
For regulating metabolism, the PI3K/mTORC2/PTEN pathway is responsible for glucose
metabolism while Treg cell cholesterol and lipid metabolisms are regulated through the
PI3K/mTORC1/Raptor pathway
51
. Interestingly, ICOS expression can be upregulated through
the PI3K/mTORC1/Raptor pathway, suggesting that ICOS may have a connection to the
regulation of cholesterol metabolism in Treg cells
52
. Meanwhile, phosphorylation of Akt in
addition to the presence of IL-2 has been shown to upregulate anti-apoptotic molecule Bcl-2,
promoting Treg cell survival
51
. Additionally, phosphorylated Akt can also upregulate T-bet
11

expression, increasing CXCR3 levels and, subsequently, enhancing Treg cells’ inhibitory
properties
51
.

Fig. 4 ICOS:ICOS-L interaction and signaling pathway in Treg cells (Adapted from Li et al.,
Frontiers in Immunology, 2020)

2.6 The Role of ICOS in ILC2s
ICOS was first identified as part of the CD28 superfamily expressed on T cells
53
. Studies
have shown that ICOS regulates T cell activation, proliferation, and differentiation in conjunction
with its ligand (ICOS-L) expressed on B cells and antigen presenting cells
54
. ICOS and ICOS-L
were later discovered to be expressed on ILC2s and contribute to ILC2 effector function and
homeostasis
55
. The lack of ICOS and the disruption of ICOS:ICOS-L interaction exhibited an
impairment of pSTAT5 and BCL-2, leading to a reduction of pro-inflammatory IL-13 cytokine
secretion (Fig. 4)
56
. STAT5 contributes to IL-13 production in mast cells and in T cells
51
.
Additionally, ICOS regulates the survivability of ILC2s as the lack of ICOS was shown to increase
cell death without affecting ILC2 proliferation
56
. Consequently, the reduction in ILC2 survival
12

exhibited lower IL-5 production
56
. Taking these results together, the absence of ICOS and the
disruption of ICOS:ICOS-L interaction were shown to reduce AHR, lung inflammation, and
eosinophilia
56
.
Interestingly, ICOS is highly implicated with IL-10, as ICOS deficiency in Treg cells has
been shown to decrease IL-10 production
58
. While studying Toxoplasma gondii parasite infection
of the central nervous system, previous studies have shown that blocking IL-10 receptor (IL-10R)
in the brain contributed to an increase of APCs, CD4+ T cells, and neutrophils while blocking
ICOS-L increased CD8+ and CD4+ T cell levels without affecting IL-10 production
59
. These
findings suggest that ICOS and IL-10 differentially regulate T cells during parasite infection of the
brain
59
.
Other studies suggest that ICOS and IL-10 appear to synergize and reduce gut
inflammation in the development of inflammatory bowel disease (IBD)
60
. The lack of ICOS-L was
shown to be linked with an increase of IL-10 producing CD4+ T cells along with a decrease of
IgG antibodies in the gut, reducing gut inflammation
60
. In addition, the absence of both IL-10
producing cells and ICOS-L resulted in colitis, suggesting that ICOS-L and IL-10 share an
immunoregulatory role in the gut
60
. Despite the brain and gut showing different immunoregulatory
relationships between ICOS and IL-10, the interplay between pulmonary ILC2s and IL-10 should
still be investigated, as it may be tissue-dependent and has yet to be fully explored in the context
of allergic asthma.

13

Chapter 3: Materials and Methods
3.1 Mice
ICOS-deficient mice (C.129S4-Icostm1Shr/J) on the BALB/c background mice were
obtained from Dr. Arlene Sharpe (Harvard Medical School, Boston, Massachusetts). Wild-type
(WT) BALB/cByJ mice were purchased from the Jackson Laboratory (Bar Harbor, ME). All mice
were bred in the animal facility of the Keck School of Medicine, University of Southern California
(USC) in Los Angeles, California. Mice were maintained at a macroenvironmental temperature of
21–22 °C, humidity (48–52%), in a conventional 12:12 light/dark cycle with lights on at 6:00 a.m.
and off at 6:00 p.m. Four to eight-week-old aged and sexed-matched mice were used in the study.
All experimentation protocols were approved by the USC Institutional Animal Care and Use
Committee (IACUC).

3.2 Lung processing for flow cytometry
Mice were sensitized intranasally for 3 days with carrier-free recombinant mouse (rm)-IL-33
(0.5 µg per mouse in 50µL, BioLegend) to induce AHR. Lung tissue was cut into small pieces and
incubated in type IV collagenase (1.6 mg ml
-1
; Worthington Biochemicals) at 37°C for 60 min,
Single-cell suspensions were obtained by passing the lung tissue digest through a 70-µm cell
strainer and incubated with red blood cell lysis buffer (RBC Lysis Buffer 1x, BioLegend). The
lungs were then stained with antibodies to identify ILC2s (ILC2s). ILC2s were gated as lineage-
negative (CD3e–, CD5–, CD45R–, Gr-1–, CD11c–, CD11b–, Ter119–, TCR  –, TCR –, and
FCεRI –), CD45
+
, ST2
+
, CD127
+
cells. Fluorescent live/dead fixable stains (ThermoFisher
Scientific) were used to exclude dead cells, according to manufacturer’s instructions.
14

CountBright Absolute Count Beads were used to count lung immune cells (Invitrogen).
Antibodies were added at a 1:200 dilution, when no recommendations were provided by the
manufacturer. Acquisition was performed on a BD FACSCanto II (BD Biosciences) using the BD
FACSDiva software v8.0.1. Data were analyzed with FlowJo software (TreeStar) version 10.

3.3 ILC2 Processing and Sorting

Murine ILC2s were sorted on a FACSARIA III system (Becton Dickinson) based on the
lack of expression of classical lineage markers (CD3e–, CD5–, CD45R–, Gr-1–, CD11c–,
CD11b–, Ter119–, TCR  –, TCR –, and FCεRI –), CD45
+
, ST2
+
, CD127
+
cells. Sorted ILC2s
were cultured at 37 °C, in the presence of rm-IL-2 (10 ng mL
-1
) and rm-IL-7 (10 ng mL
-1
) with
and without rm-IL-4 (10 ng mL
-1
) in RPMI (Gibco) supplemented with 10% heat-inactivated fetal
bovine serum (FBS) and 100 units per mL penicillin–streptomycin. For knockdown studies, 2.5
M Nfil3 in vivo morpholinos 5’ -GCTGCATCAGAAAGGACCTCCTCGT-3’, Bhlhe40 5’-
ATCCGTTCCATGATGCGGCGAGC-3’, Prdm1 5’-ACATCTGAGATAAGCCTCTCTCATG-
3’, cMaf 5’-GTTCATTGCCAGTTCTGAAGC CATC-3’ or random control oligo (Gene Tools,
LLC, Philomath, Ore) were added as free uptake oligos 24 hours before the addition of rm-IL-4
(10 ng/mL).

3.4 RNA sequencing and data analysis
Freshly sorted ILC2s after three intranasal administrations of rm-IL-33 (0.5 μg per mouse),
defined in this study as activated ILC2s (aILC2s), were incubated (5 × 10
4
per mL) with rm-IL-2
(10 ng mL
-1
) and rm-IL-7 (10 ng mL
-1
) for 24 hours or with rm-IL-2 (10 ng mL
-1
) and rm-IL-7 (10
ng mL
-1
) and rm-IL-4 (10 ng mL
-1
) for 48 hours. Total RNA was isolated using MicroRNAeasy
15

(Qiagen). In total, 10 ng of input RNA was used to produce cDNA for downstream library
preparation. Samples were sequenced on a NextSeq 500 (Illumina) system. Raw reads were
aligned, normalized, and further analyzed using Partek Genomics Suite software, version 7.0
Copyright; Partek Inc. Normalized read counts were tested for differential expression using
Partek’s gene -specific analysis (GSA) algorithm.

3.5 Supernatant cytokine measurement
aILC2s were cultured (5 × 10
4
per mL) for 24 hours in the presence of rm-IL-2 (10 ng mL
-1
)
and rm-IL-7 (10 ng mL
-1
) or for 48 hours in the presence of rm-IL-2 (10 ng mL
-1
) and rm-IL-7 (10
ng mL
-1
) and rm-IL-4 (10 ng mL
-1
). The levels of IL-5, IL-9, IL-10, and IL-13 were measured in
supernatants using LEGENDplex multiplex kits (BioLegend) and data were analyzed via the
LEGENDplex data analysis software v8.0.

3.6 Glucose uptake assay and measurement of glycolytic function
aILC2s were incubated (5 × 10
4
per mL) with rm-IL-2 (10 ng mL
-1
) and rm-IL-7 (10 ng mL
-1
)
for 24 hours with and without rm-IL-4 (10 ng mL
-1
) for 48 hours. To assess glucose uptake, 50 μg
mL
−1
of 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]-amino)−2-deoxyglucose (2-NBDG) from
ThermoFisher Scientific were added to the culture for 20 min. Acquisition was performed using a
BD FACSCanto II (BD Biosciences). The expression of the glucose transporter 1 (Glut-1) was
assessed using a PE-Cy7 anti-Glut-1 (polyclonal, Novus Biologicals).

16

3.7 Fatty acid uptake assay and measurement of fatty acid oxidation function
aILC2s were incubated (5 × 10
4
per mL) with rm-IL-2 (10 ng mL
-1
) and rm-IL-7 (10 ng mL
-1
)
for 24 hours with and without rm-IL-4 (10 ng mL
-1
) for 48 hours in RPMI 1640 medium without
glucose or glutamine (BI Biological Industries, Beit-Haemek, Israel) but supplemented with 10%
heat-inactivated FBS (Omega Scientific), 100 units/mL of penicillin and 100 mg/mL of
streptomycin (GenClone). For lipid droplet quantification, cells were incubated in media
containing 1000 ng/mL of Bodipy FL C16 (4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-
Indacene-3-Hexadecanoic Acid) or Bodipy 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-
3a,4a-Diaza-s-Indacene) (Thermo Fisher Scientific) at 37 C for 30 minutes. Acquisition was
performed using a BD FACSCanto II (BD Biosciences).

3.8 Agilent Seahorse
The real-time extracellular acidification rate (ECAR) and oxygen consumption rate (OCR)
were measured using a Seahorse XF HS Mini Analyzer (Seahorse Agilent). Briefly, 5 × 10
4
aILC2s
were plated with and without rm-IL-4 (10 ng mL
-1
) in Seahorse media supplemented with 1mM
pyruvate, 2mM glutamine, and 10mM glucose. Mito stress test kit (Agilent, San Diego CA), using
2 μM oligomycin, 2 μM FCCP, and 1 μM rotenone/ antimycin A, was performed according to the
manufacturer’s instructions.

3.9 Statistical analysis
The experiments were repeated 2 times (n=2), and the data are shown as the representative of
more than 2 independent experiments. A two-tailed Student’s t -test for unpaired data was applied
for comparisons between 2 groups. Data were analyzed with Prism Software (GraphPad Software
17

Inc.). Error bars represent standard error of the mean. p value < 0.05 was considered to denote
statistical significance

18

Chapter 4: Results
4.1 Objectives of the study
Type 2 asthma is characterized by the presence of type 2 inflammation and airway
hyperreactivity (AHR) that is induced by pro-inflammatory cytokines such as IL-5 and IL-13.
These cytokines have been shown to be secreted by ILC2s, which are identified as major players
in type 2 asthma. Additionally, ICOS has been identified as a co-stimulatory molecule on ILC2s
that regulates IL-5 and IL-13 production and ILC2 viability and effector functions, suggesting that
ICOS may contribute to the development of AHR. Moreover, ICOS is implicated in IL-10
production in studies focusing on parasite infection in the brain and on the developing of IBD in
the gut. As IL-10 displays anti-inflammatory properties through the downregulation of pro-
inflammatory cytokine production and suppression of eosinophil recruitment, we decided to
examine the role of ICOS in IL-10 production in pulmonary ILC2s during the development of
allergic asthma.

4.2 Lack of ICOS affects the transcriptomic profile of aILC2s

We first established an asthmatic mice model with wild-type (WT) BALB/cByJ mice and
ICOS knock-out (KO) mice as described by a previous study performed in our lab
56
. Mice were
intranasally (i.n.) challenged with rm-IL-33 (1 µg in 50 µL per mouse) on 3 consecutive days. On
day 4, the mice were euthanized, and their lungs were harvested and processed for flow cytometry
(Fig. 5A). Activated ILC2s (aILC2s) harvested from the IL-33-challenged mice were identified as
CD45
+
lineage ST2
+
CD127
+
(Fig. 5B). We compared the transcriptional profile of FACS-sorted
aILC2s from WT and ICOS KO mice using RNA-sequencing analysis. The absence of ICOS
resulted in 866 significantly modulated genes in aILC2s: 381 genes were upregulated while 485
19

genes were downregulated (Fig. 5C). To identify the role of ICOS in regulating aILC2 effector
functions, we compared the cytokine gene expressions including TH2 cytokines in WT and ICOS
KO aILC2s. Interestingly, we saw IL-4 and a set of cytokines (IL-10, IL-22, IL-24) belonging to
the IL-10 superfamily that were highly upregulated in the absence of ICOS (Fig. 5D)
61
. We then
identified and examined the expression of the genes responsible for IL-10 superfamily cytokine
production with an emphasis on IL-10 expression (Fig. 5E). Il4, cMaf, Nfil3, and Prdm1 were
observed to be highly upregulated while negative regulators such as Bhlhe40 were highly
downregulated. These genes are known to regulate IL-10 and have been previously described as
essential genes responsible for the regulation of IL-10 production
38
. Altogether, these results
suggest that the lack of ICOS in aILC2s upregulates the expression of genes responsible for IL-10
production.
20

3
2
1
0
1
2
0 10
3
10
3
10
4
10
5
0
10
3
10
3
10
4
10
5
0 10
3
10
4
10
5
0
10
4
10
4
10
5
CD45 APCCy
Lineage FITC
ST 2 PerCP
eFluor 10
CD12 P Cy
IL 33
Day 1 2 3
uthanasia
T
ICOS O
4
A.
.
C. D.
.
Sorted aILC2s
Culture 24h
R A se uencing
LIS A
Functional assay
10 5 0 5 10
0
5
10
15
Log
2
(fold change)
log
10
(p value)
4 2 0 2 4
0
5
10
15
Log
2
(fold change )
log
10
(p value)
Downregulated genes pregulated genes
Log
2
(fold change )
IL 10
IL 24
IL 22
IL 4
4 5 down
regulated
genes
3 1 up
regulated
genes
21

Fig. 5 Lack of ICOS affects the transcriptomic profile of aILC2s. a-e ILC2s were sorted from WT
and ICOS KO mice after three intranasal challenges with 1 µg of rm-IL-33. Sorted cells were
incubated with rm-IL-2 (10 ng mL
-1
) and rm-IL-7 (10 ng mL
-1
) for 24 hours. a WT and ICOS KO
mice were intranasally challenged for 3 consecutive days with 1 μg rm -IL-33. On day 4, mice were
euthanized. b Representative flow cytometry plots of ILC2s gated as CD127+ ST-2+ from CD45+
lineage- lung cells. c Volcano plot of total differentially regulated genes. Gene-specific analysis
(GSA) algorithm was used to test for differential expression of genes (p-value < 0.05, n=3). d
Volcano plot of IL-10 cytokine family genes (p-value < 0.05, n=3). e Fold change comparison of
positive and negative IL-10 regulator genes. Solid plot represents statistical significance (p-value
< 0.05). Dotted plot represents statistical insignificance (p-value > 0.05). GSA algorithm was used
to test for differential expression of genes (p-value < 0.05, n=3)

4.3 Lack of ICOS affects IL-10 regulator gene expression and increases IL-10 cytokine
production

To validate the findings that we observed in the comparison of the transcriptional profile,
we decided to measure TH2 cytokine levels such as IL-5 and IL-13, IL-10. IL-33-activated ILC2s
from WT and ICOS KO mice were cultured with rm-IL-2 and rm-IL-7 for 24 hours. The culture
supernatant was collected, and cytokines were quantified using LEGENDplex multiplex kits from
BioLegend. As expected, ICOS KO aILC2s confirmed previous reports of lower expression levels
of IL-5 and IL-13 compared to WT aILC2s (Fig. 6A)
56
. ICOS KO aILC2s produced significantly
more IL-10 cytokines than WT aILC2s, confirming the high IL-10 gene expression observed in
the RNA-sequencing (Fig. 6B). Expression of intracellular IL-10 levels was also higher in ICOS
KO aILC2s when compared to WT aILC2s (Fig. 6C). We then examined genes that coded for
transcription factors that are highly implicated with IL-10 secretion, Nfil3 and cMAF. cMaf
expression significantly increased in ICOS KO aILC2s with a similar but statistically insignificant
trend of Nfil3 expression in ICOS KO aILC2s (Fig. 6D). These results suggest that the absence of
ICOS is associated with lower expression of TH2 cytokines with higher expression of IL-10 that is
reflected in increases in cMaf and Nfil3 expression.
22

Fig. 6 Lack of ICOS affects IL-10 regulator gene expression and IL-10 cytokine production. a-d
ILC2s were sorted from T and ICOS O mice after three intranasal challenges with 1 μg of rm -
IL-33. Sorted cells were incubated (10
4
cells/200 μL) with rm-IL-2 (10 ng mL
-1
) and rm-IL-7 (10
ng mL
-1
) for 24 hours. a the level of IL-5 and IL-13 and b IL-10 produced by purified lung ILC2
as measured by ELISA. c Quantification of intracellular IL-10 and d intranuclear NFIL3 and
cMAF presented as MFI in WT and ICOS KO aILC2s. Data are representative of at least 3
independent experiments and shown as mean ± SEM (two-tailed Student’s t -test).

4.4 Lack of ICOS improves the IL-4-induced IL-10 production in aILC2s
Given that ICOS KO aILC2s produce more IL-10 than WT aILC2s, we decided to explore
the transcriptional profile of FACS-sorted ICOS KO aILC2s with and without rm-IL-4 using RNA-
sequencing analysis as IL-4 stimulation has been shown to induce cMaf and IL-10 expression in
ILC2s
39
. IL-33-activated ILC2s were isolated from WT and ICOS KO mice and cultured for 48
hours with rm-IL-2, rm-IL-7, and with and without rm-IL-4 (10 ng mL
-1
) (Fig. 7A). The presence
of rm-IL-4 resulted in 809 significantly modulated genes in aILC2s: 472 genes were upregulated
while 337 genes were downregulated (Fig. 7B). We analyzed TH2 cytokine gene expressions to
1
2
3
T ICOS O
125
130
135
140
145
150
155
A.
.
C. D.
IL 5 (x10
1
pg mL
1
)
IL 10 (x10
1
pg mL
1
)
IL 10 MFI (x10
1
)
FIL 3 MFI (x10
1
)
cMAF MFI (x10
1
)
IL 13 (x10
1
pg mL
1
)
0
0
1
2
3
4
25
30
35
40
45
50
5
0
5
0
5
50
55
0
5
p 0.0433 p 0.02 5
p 0.0001
p 0.04 0
p 0.0110 p 0.02 5
23

identify the role of IL-4 in regulating ICOS KO aILC2 IL-10 secretion. Interestingly, the IL-10
superfamily cytokines including IL-10 and IL-24 were highly upregulated while pro-inflammatory
TH2 cytokines such as IL-5 and IL-13 were downregulated (Fig. 7C). We then examined the
previously mentioned set of genes that coded for IL-10 transcription factors and observed they
exhibited similar trends of expression as seen earlier. Il4, cMaf, Nfil3, and Prdm1 were highly
upregulated while Bhlhe40 was highly downregulated (Fig. 7D). Next, these findings were
confirmed at the protein level. The culture supernatant was collected, and cytokines were
quantified after culturing WT and ICOS KO aILC2s with rm-IL-4 for 48 hours. ICOS KO aILC2s
cultured with rm-IL-4 exhibited lower levels of IL-5 and IL-13, following the observation seen at
the transcription level (Fig. 7E). Additionally, we observed higher levels of secreted IL-10 from
ICOS KO aILC2s cultured with rm-IL-4 (Fig. 7F). Interestingly, rm-IL-4-cultured ICOS KO
aILC2s secreted lower intracellular IL-10 levels while showing no significant differences in Nfil3
and cMaf expression levels (Fig. 7G, H). Taken together, the addition of rm-IL-4 in conjunction
with the lack of ICOS further increases IL-10 production in aILC2s independently from Nfil3 and
cMaf.
24

2
0
2
4

IL 33
Day 1 2 3
uthanasia
ICOS O
4
A.
. C.
D.
Log
2
(fold change )
Sorted aILC2s
Culture 4 h
IL 4
R A se uencing
LIS A
Functional assay
10 5 0 5 10
0
5
10
15
20
log
2
(fold change)
log
10
(pvalue)
4 2 0 2 4
0
5
10
15
20
log
2
(fold change)
log
10
(pvalue)
Downregulated genes pregulated genes
IL 10
IL 24
IL 5
IL 13
0
1
2
3
4
50
55
0
5
0
IL 4 IL 4
IL 4
22
24
2
2
IL 4
IL 4 IL 4
T ICOS O
.
F.
. G.
IL 5 (x10
2
pg mL
1
)
IL 13 (x10
1
pg mL
1
)
IL 10 (x10
1
pg mL
1
)
IL 10 MFI (x10
1
)
FIL3 MFI (x10
1
)
cMAF MFI (x10
1
)

10
11
12
13
14
50
55
0
5
0
5
0
1
2
3
4
5
33 down
regulated
genes
4 2 up
regulated
genes
p 0.005
p 0.04
p 0.0 5 p 0.10 2
p 0.0 15 p 0.5435
25

4.5 Bhlhe40 participates in the regulation of IL-10 dependently from ICOS

We decided to further investigate the roles of genes coding for IL-10 transcription factors
in the regulation of IL-4 stimulated IL-10 production in a functional assay. We isolated aILC2s
from rm-IL-33-challenged WT and ICOS KO and cultured the aILC2s with Nfil3, Bhlhe40, cMaf,
and Prdm1 morpholino sequences, antisense oligomers, for 24 hours before culturing with and
without IL-4 for an additional 48 hours (Fig. 8A). These morpholino sequences were used to knock
down their respective IL-10-encoding genes with the goal of decreasing IL-10 secretion through
Nfil3, cMaf, and Prdm1 knockdown and increasing IL-10 secretion through Bhlhe40 knockdown.
We subsequently measured IL-10 cytokine levels using LEGENDplex multiplex kids from
BioLegend. In line with earlier results, we observed higher IL-10 expression levels from ICOS
KO aILC2s cultured with and without IL-4 when compared to their respective WT counterparts
(Fig. 8B). However, the addition of cMaf and Prdm1 morpholino sequences to ICOS KO aILC2s
cultured with rm-IL-4 showed an increase of IL-10 production. This result goes against the trend
of decreasing IL-10 expression level that we had expected to see (Fig. 8B). Multiple reasons may
be given to explain this anomaly in IL-10 expression. Most of which can be attributed to technical
Fig. 7 Lack of ICOS improves the IL-4 induced IL-10 production in aILC2s. a-c ILC2s were sorted
from ICOS KO mice after three intranasal challenges with 1 µg of rm-IL-33. Sorted cells were
incubated with rm-IL-2 (10 ng mL
-1
), rm-IL-7 (10 ng mL
-1
), and rm-IL-4 (10 ng mL
-1
) for 48 h. a
ICOS KO mice were intranasally challenged for 3 consecutive days with 1 μg rm -IL-33. On day
4, mice were euthanized and aILC2s were cultured with and without rm-IL-4. b Volcano plot of
total differentially regulated genes. Gene-specific analysis (GSA) algorithm was used to test for
differential expression of genes (p-value < 0.05, n=3). c Volcano plot of IL-10 cytokine family
genes (p-value < 0.05, n=3). d Fold change comparison of positive and negative IL-10 regulator
genes. Solid plot represents statistical significance (p-value < 0.05). Dotted plot represents
statistical insignificance (p-value < 0.05). GSA algorithm was used to test for differential
expression of genes (p-value < 0.05, n=3) e The level of IL-5 and IL-13 and f IL-10 produced by
purified lung aILC2 as measured by ELISA. g Quantification of intracellular IL-10 and h
intranuclear NFIL3 and cMAF presented as MFI in WT and ICOS KO aILC2s. Data are
representative of at least 3 independent experiments and shown as mean ± SEM (two-tailed
Student’s t -test).

26

problems within the design of the functional assay. The addition of Bhlhe40 morpholino sequence
showed a significant increase of IL-10 expression from ICOS KO aILC2s cultured with rm-IL-4
(Fig. 8B). As Bhlhe40 is a gene whose expression is responsible for the negative regulation of IL-
10 production, its knockdown should result in higher expression of IL-10, which was observed
during this assay.

Fig. 8 Bhlhe40 participates in the regulation of IL-10 dependently from ICOS. a-b ILC2s were
sorted from WT and ICOS KO mice after three intranasal challenges with 1 µg of rm-IL-33. Sorted
cells were incubated with rm-IL-2 (10 ng mL
-1
), rm-IL-7 (10 ng mL
-1
), and with or without rm-
IL-4 (10 ng mL
-1
) for 72 h. a WT and ICOS KO mice were intranasally challenged for 3
consecutive days with 1 μg rm -IL-33. On day 4, mice were euthanized. aILC2s were incubated
with rm-IL-2, rm-IL-7, with or without rm-IL-4 and appropriate MO. b The levels of IL-5, IL-13,
and IL-10 produced by aILC2s cultured with or without MO (2 µM) and rm-IL-4 (10 ng mL
-1
) for
72 h. Data are representative of at least 3 independent experiments and shown as mean ± SEM
(two-tailed Student’s t -test).

T ICOS O
A.
IL 33
Day 1 2 3
uthanasia
T
ICOS O
4
Sorted aILC2s
Culture 24h
Morpholino
Culture 4 h
IL 4
LIS A
T T IL4 ICOS O ICOS O IL4
.
IL 10 (x10
1
pg mL
1
)
0
5
10
15
20
0
5
10
15
20
25
0
5
10
15
20
0
5
10
15
20
25
p 0.11 0
p 0.0042 p 0.02 0 p 0.031
27

4.6 Lack of ICOS induces a metabolic change from fatty acid oxidation to glycolysis in
aILC2s

ILC2s found in the gut primarily use fatty acid oxidation (FAO) as their main source of
energy
62
. However, upon the generation of ILC210s from ILC2s, this subset of cells exhibited a
possible preference of glycolysis over FAO for IL-10 production while downregulating lung
inflammation, supporting the notion that a change in metabolism can alter ILC2 effector functions
and differentiation
39
. By extending this line of rationale to ICOS KO aILC2s, we decided to
investigate the impact of ICOS on aILC2 metabolism and to identify metabolic changes appear if
any.
Although expression of the glucose transporter 1, Glut-1, did not show significant
differences between ICOS KO and WT aILC2s, we saw a slight trend of increased Glut-1
expression in ICOS KO aILC2s (Fig. 9A). To explore this trend, we used 2-NBDG, a fluorescent
glucose analog, and flow cytometry to measure glucose uptake. We observed that ICOS KO
aILC2s had significantly higher 2-NBDG uptake, suggesting that the lack of ICOS in aILC2s
increased glucose uptake and metabolism (Fig. 9B). To determine whether fatty acid metabolism
was affected, we used Bodipy-C16 and Bodipy-493 fluorescent fatty acid analogs to measure fatty
acid uptake and utilization, respectively. We saw that ICOS KO aILC2s exhibited less Bodipy-
C16 and more Bodipy-493 fluorescence, suggesting that ICOS KO aILC2s may have lower fatty
acid uptake and less utilization when compared to WT aILC2s as the Bodipy-493 were being
accumulated within ICOS KO ILC2s (Fig. 9C). The same glucose and fatty acid uptake and
utilization tests were performed on WT and ICOS KO aILC2s cultured in the presence of rm-IL-
4 for 48 hours. Although statistically insignificant, we saw a similar trend in rm-IL-4-cultured
aILC2s as ICOS KO aILC2s exhibited higher glucose uptake and lower fatty acid uptake; however,
we observed a trend of higher lipid utilization in rm-IL-4 cultured ICOS KO aILC2s than in rm-
28

IL-4-cultured WT aILC2s (Fig. 9D, E). To confirm that the lack of ICOS KO induces a shift from
fatty acid metabolism to anaerobic glycolysis, we measured oxygen consumption rate (OCR) and
extra cellular acidification rate (ECAR) of WT and ICOS KO aILC2s cultured with and without
rm-IL-4. OCR is an indicator of mitochondrial respiration by measuring oxygen consumption
while ECAR is an indicator of anaerobic glycolysis by measuring proton production levels. ICOS
KO aILC2s did not exhibit any significant differences in the measurement of OCR but exhibited
greater ECAR than WT aILC2s, suggesting that ICOS KO aILC2s uses more anaerobic glycolysis
as higher protons levels are detected (Fig. 9F). Interestingly, ICOS KO aILC2s cultured with rm-
IL-4 exhibit a trend of high levels of OCR, suggesting that there is increased mitochondrial
respiration reflected in higher oxygen consumption (Fig. 9F). This finding is in line with our earlier
observation of rm-IL-4-cultured ICOS KO aILC2s exhibiting higher lipid utilization. The
inclusion of IL-4 may play a role in maintaining a partial reliance on FAO as fatty acid utilization
is reflected in higher levels of OCR in rm-IL-4-cultured ICOS KO aILC2s. Moreover, we observed
higher ECAR levels in rm-IL-4-cultured ICOS KO ILC2s when compared to their WT
counterparts, suggesting that there is partial dependency on glycolytic metabolism (Fig. 8G).
Taken together, these results suggest that the lack of ICOS in aILC2s drives a metabolic shift from
FAO with a partial reliance on FAO towards glycolytic metabolism when stimulated with IL-4.
29

p 0.02 5
15
20
25
30
T ICOS O
0
5
10
15
20
IL 4
0 20 40 0 0
0
5
10
15
20
0 20 40 0 0
0
5
10
15
20
0 20 40 0 0
0
20
40
0
0
0 20 40 0 0
0
20
40
0
0
2 DG
0 10
3
10
3
10
4
10
5
Cell count
5
0
5
0
0 10
3
10
3
10
4
10
5
GL T1
Cell count
T IL 4 ICOS O IL 4
0 10
3
10
3
10
4
10
5
2 DG
Cell count
1
1
20
22
24
2
IL 4
0
20
40
0
0
IL 4
Oligomycin FCCP Rot/AA Oligomycin FCCP Rot/AA
Oligomycin FCCP Rot/AA Oligomycin FCCP Rot/AA
A.
C.
D. .
F.
G.
.
Glut 1 MF I (x10
1
)
2 D G MFI (x10
2
)
odipy C1 MF I (x10
3
)
odipy 4 3 MF I (x10
2
)
2 D G MFI (x10
2
)
odipy C1 MFI (x10
2
)
odipy 4 3 MFI (x10
3
)
OCR (pmol/min)
CAR (mp /min)
OCR (pmol/min)
CAR (mp /min)
Time (minutes)
Time (minutes)
Time (minutes)
Time (minutes)
25
30
35
40
45

10
p 0.55
p 0.0002
p 0.0321
p 0.0133
p 0. p 0.110
p 0.0 12
T ICOS O
30

Fig. 9 Lack of ICOS induces a metabolic change from fatty acid oxidation to glycolysis in aILC2s.
a-g ILC2s were sorted from WT and ICOS KO mice after three intranasal challenges with 1 μg of
rm-IL-33. a-c, f Sorted cells were incubated with rm-IL-2 (10 ng mL
-1
), rm-IL-7 (10 ng mL
-1
) for
24 hours. d-f, g Sorted cells were incubated with rm-IL-2 (10 ng mL
-1
), rm-IL-7 (10 ng mL
-1
), rm-
IL-4 (10 ng mL
-1
) for 48 hours. a Glut-1 quantification (left) and corresponding representative
histogram (right) of Glut-1 expression presented as MFI in WT and ICOS KO ILC2s; n=3. b
Quantification (left) and corresponding representative histogram (right) of 2-NBDG uptake
presented as MFI in FACS-sorted WT and ICOS KO aILC2s; n=3. c Bodipy-C16 and Bodipy-493
quantification presented as MFI in FACS-sorted WT and ICOS KO aILC2s. d Quantification (left)
and corresponding representative histogram (right) of 2-NBDG uptake presented as MFI in FACS-
sorted WT and ICOS KO aILC2s cultured with rm-IL-4. e Bodipy-C16 and Bodipy-493
quantification presented as MFI in FACS-sorted WT and ICOS KO aILC2s cultured with rm-IL-
4. f Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)
under basal conditions and in response to indicated drugs. g Measure of OCR and ECAR of aILC2s
cultured with rm-IL-4 under basal conditions and in response to indicated drugs

31

Chapter 5: Discussion and Future Directions
Overall, we delineated the implication of ICOS in regulating IL-10 production and
inducing a metabolic shift in IL-33-induced ILC2s. We have shown that the lack of ICOS
contributes to decreased expression levels of pro-inflammatory TH2 cytokines such as IL-5 and
IL-13 and increased expression levels of IL-10. Moreover, this pattern of cytokine expression
levels is reflected in aILC2s stimulated with IL-4 as well. Knockdown of IL-10 regulator gene,
Bhlhe40, exhibited the upregulation of IL-10 production in ICOS KO aILC2s. Additionally, the
lack of ICOS mediates a metabolic switch from FAO to glycolysis.
Both human and mice models of allergic asthma are characterized by type 2 immune
responses that include the secretion pro-inflammatory cytokines: IL-4, IL-5, and IL-13
14
. Recent
studies have proposed that TH2-secreted IL-4 has immunoregulatory functions as IL-4 was
observed to induce a subset of IL-10-producing ILC2s (ILC210s) that can inhibit ILC2 activation
and effector functions and diminish the development of AHR
39
. In this study, we showed that
ICOS has a role in regulating IL-10 production as the lack of ICOS showed increased IL-10
production level and cMaf expression, a critical IL-10 regulator gene
39
. The addition of IL-4 further
increased IL-10 production especially in the absence of ICOS, suggesting that IL-4 and ICOS may
have some interplay in the production and regulation of IL-10. Previous studies described
immunoregulatory properties of Treg-secreted IL-10 in the context of asthma
63
. Although this study
focuses on ILC2-secreted IL-10, this source of IL-10 may share some immunoregulatory functions
found in Treg-secreted IL-10 and should be further explored. If ILC2-secreted IL-10 does exhibit
an immunoregulatory phenotype, then it is possible that ICOS blockade contributes to ILC2s’
immunoregulatory function when taking the possible interplay between ICOS and IL-4-stimulated
IL-10 production into consideration.
32

We then turned towards IL-10-encoding genes to study their role in aILC2s that lack ICOS.
Previous studies have shown that cMaf and Prdm1 are critical genes that code for IL-10 production
in immune cells including ILC210s
39
. We generated morpholino knockdown sequences for IL-10
gene regulators: Nfil3, Bhlhe40, cMaf, and Prdm1. Using these morpholino sequences, we
attempted to recreate the morpholino functional assay described by Howard et al using WT and
ICOS KO aILC2s cultured with and without IL-4 and with the appropriate morpholino
sequences
39
. We were able to show that Bhlhe40 knockdown upregulated IL-10 expression and
suggest that Bhlhe40 contributes to IL-10 expression in ILC2s. However, we were unable to see
any significant differences in IL-10 and TH2 cytokines levels after Nfil3, cMaf, and Prdm1
knockdown. It is not to say that these transcription factors are not responsible for IL-10 production
but rather, technical problems may have contributed to the lack of significant differences. Different
conditions and techniques might be necessary to explore the role of these IL-10 transcription
factors in the regulation of IL-10 production in ICOS KO aILC2s. Techniques may include
combinational use of morpholino sequences or the use of flow cytometry to check expression of
IL-10 regulator genes to determine if the morpholino sequences succeeded in knocking down
expression. Despite not fulfilling its intended purpose, the morpholino functional assay confirmed
earlier results of increased IL-10 production of ICOS KO aILC2s cultured with and without IL-4.
Further optimization is needed to obtain significant results from the functional assay.
Building on previous reports of an interplay between metabolic changes and immune
response shifts, we decided to look at the impact that the absence of ICOS has on ILC2 metabolism.
Traditional ILC2s use FAO as the primary metabolic pathway use for proliferation and effector
functions
62
. Recently, Howard et al have shown that the generation of ILC210s does not rely on
FAO but, instead, exhibits a shift towards glycolysis dependency
39
. Extending this finding to other
33

phenotypes, we examined ICOS O aILC2s’ glucose and fatty acid metabolism through
fluorescent tags and Seahorse Agilent technology. We have shown that ICOS KO aILC2s has a
higher preference for glycolysis than FAO and is reflected in higher ECAR levels. Moreover, the
addition of IL-4 shows a similar preference for glycolysis in rm-IL-4-cultured ICOS KO aILC2s
but, interestingly, shows an increase in lipid utilization. The increase in lipid utilization is
supported by higher OCR levels produced by rm-IL-4-cultured ICOS KO aILC2s, suggesting that
there is a connection between IL-4 stimulated IL-10 production and FAO. This connection is
similar to Treg cells’ dependency on FAO for IL-10 production in the gut microbiome
64
. However,
further studies are needed to strengthen the link between increased IL-10 production caused by a
shifting dependency onto glycolysis in ICOS KO aILC2s.
This study did not include results describing ICOS intracellular signaling via ICOS-L.
However, we may be able to speculate ICOS signaling pathway mechanisms related to cytokine
production and metabolism in ILC2s using results from other studies. As described earlier, ICOS
requires IL-2 and IL-7 to maintain homeostasis and to regulate production of both pro-
inflammatory cytokines and anti-inflammatory cytokines
65
. Previous studies have described that
ICOS:ICOS-L interaction and the binding of IL-2 to its receptor promote production of pro-
inflammatory IL-5 and IL-13 cytokines through STAT5 signaling
65
. Moreover, the binding of
IL-2 has been shown to activate the PI3K/Akt pathway through the recruitment of JAK1 to the
IL-2 receptor intracellular domain
66
. As PI3K is implicated in both mTORC2/PTEN and
mTORC1/Raptor pathways, it is possible that ICOS:ICOS-L interaction in the presence of IL-2
contributes to the regulation of glucose and lipid metabolism. This association may be a potential
mechanism that can explain the shift towards glucose metabolism as ICOS induces a preference
towards the mTORC2/PTEN pathway. Further studies analyzing mTORC2/PTEN and
34

mTORC1/Raptor expression levels in ICOS KO aILC2s are needed to be examined if ICOS are
implicated in either pathway.
Additionally, ICOS was observed to regulate IL-10 production in Treg cells through the
recruitment of p50  and p85  to the YMFM motif. p50  and p85  subunits can regulate NFAT
and FOXP3 activity and induce IL-10 production. This regulatory pathway may be a potential
mechanism to explain how IL-10 is produced in ICOS
+
ILC2s but has yet to be be fully explored.
As results show higher levels of IL-10 in ICOS KO aILC2s than in WT aILC2s, it is possible that
ICOS KO ILC2s may not be able to use the recruitment of p50  and p85  for IL-10 production.
Instead, ICOS KO ILC2s may prefer to use a different pathway as suggested by an increase of
cMaf expression
67
. Again, further studies are needed to examine the mechanisms that are involved
in ILC2-derived IL-10 production such as analyzing p50  and p85  expression levels and other
proteins implicated with cMaf expression.
This study highlights new roles of ICOS that involve regulation of IL-10 production and
metabolic modulation. Our results suggest that ICOS may mediate ILC2s’ immunoregulatory
functions as the lack of ICOS with and without IL-4 increases IL-10 production and decreases IL-
5 and IL-13 expression levels. Additionally, ICOS may play a role in modulating ILC2 glucose
and fatty acid metabolism as ILC2s that lack ICOS prefer glycolysis but still retain partial
dependency on FAO in the presence of IL-4 stimulated IL-10. We believe that the findings in this
study may provide potential therapeutic treatments focusing on increasing ILC2s’ capacity on
producing immunosuppressive IL-10.
Novel therapeutic treatments may include an ICOS antagonist that is capable of modulating
Bhlhe40 expression coupled with an administration of IL-4 cytokine. The ICOS antagonist would
be able to block the ICOS:ICOS-L interaction, which has been shown previously to downregulate
35

IL-5 and IL-13 secretion, reducing AHR and eosinophilia
68
. Moreover, based on the findings
presented in this study, an ICOS antagonist would be able induce ILC2s to produce IL-10, an
immunoregulatory cytokine that may suppress secretion of inflammatory cytokines and
development of AHR. Although IL-4 contributes to type 2 asthma, the administration of IL-4 may
increase the efficacy of the ICOS antagonist by inducing the secretion of additional IL-10, thereby
overcoming the negative effects of administering exogenous IL-4. As the interplay among ILC2s,
IL-10, and ICOS has not yet been fully explored, this study provides novel and exciting findings
that may provide a new perspective on development of type 2 asthma and offer a breakthrough in
developing effective therapeutic treatments for allergies and asthma.

36

References

1. Dharmage, S. C., Perret, J. L. & Custovic, A. Epidemiology of Asthma in Children and
Adults. Front. Pediatr. 7, (2019).

2. Holgate, S. T. et al. Asthma. Nat. Rev. Dis. Primer 1, 1–22 (2015).

3. Carr, T. F. & Bleecker, E. Asthma heterogeneity and severity. World Allergy Organ. J. 9,
41 (2016).

4. Tai, A. et al. Outcomes of childhood asthma to the age of 50 years. J. Allergy Clin.
Immunol. 133, 1572-1578.e3 (2014).

5. Strachan, D. P. Hay fever, hygiene, and household size. BMJ 299, 1259–1260 (1989).

6. Rook, G. A. W., Martinelli, R. & Brunet, L. R. Innate immune responses to mycobacteria
and the downregulation of atopic responses. Curr. Opin. Allergy Clin. Immunol. 3, 337–
342 (2003).

7. Yunginger, J. W. et al. A Community-based Study of the Epidemiology of Asthma:
Incidence Rates, 1964–1983. Am. Rev. Respir. Dis. 146, 888–894 (1992).

8. Parsons, J. P. et al. An Official American Thoracic Society Clinical Practice Guideline:
Exercise-induced Bronchoconstriction. Am. J. Respir. Crit. Care Med. 187, 1016–1027
(2013).

9. Moore, W. C. et al. Identification of Asthma Phenotypes Using Cluster Analysis in the
Severe Asthma Research Program. Am. J. Respir. Crit. Care Med. 181, 315–323 (2010).

10. When Asthma Is Not Just Asthma: Type 2 Inflammation | Allergy & Asthma Network.

11. Type 2 Inflammation and the Evolving Profile of Uncontrolled Persistent Asthma.
European Medical Journal (2018).

12. Neill, D. R. et al. Nuocytes represent a new innate effector leukocyte that mediates type-2
immunity. Nature 464, 1367–1370 (2010).

13. Holgate, S. T. Innate and adaptive immune responses in asthma. Nat. Med. 18, 673–683
(2012).

14. Barnes, P. J. Targeting cytokines to treat asthma and chronic obstructive pulmonary
disease. Nat. Rev. Immunol. 18, 454–466 (2018).

15. Lambrecht, B. N., Hammad, H. & Fahy, J. V. The Cytokines of Asthma. Immunity 50,
975–991 (2019).
37

16. Mukherjee, M., Sehmi, R. & Nair, P. Anti-IL5 therapy for asthma and beyond. World
Allergy Organ. J. 7, 32 (2014).

17. Koch, S., Sopel, N. & Finotto, S. Th9 and other IL-9-producing cells in allergic asthma.
Semin. Immunopathol. 39, 55–68 (2017).

18. Gauvreau, G. M. et al. Effects of an anti-TSLP antibody on allergen-induced asthmatic
responses. N. Engl. J. Med. 370, 2102–2110 (2014).

19. Wenzel, S. et al. Dupilumab efficacy and safety in adults with uncontrolled persistent
asthma despite use of medium-to-high-dose inhaled corticosteroids plus a long-acting β2
agonist: a randomised double-blind placebo-controlled pivotal phase 2b dose-ranging
trial. Lancet Lond. Engl. 388, 31–44 (2016).

20. Vivier, E. The discovery of innate lymphoid cells. Nat. Rev. Immunol. 21, 616–616
(2021).

21. Frontiers | Social Networking of Group Two Innate Lymphoid Cells in Allergy and
Asthma | Immunology.

22. Spits, H. et al. Innate lymphoid cells — a proposal for uniform nomenclature. Nat. Rev.
Immunol. 13, 145–149 (2013).

23. Hoyler, T. et al. The transcription factor GATA-3 controls cell fate and maintenance of
type 2 innate lymphoid cells. Immunity 37, 634–648 (2012).

24. Klose, C. S. N. & Artis, D. Innate lymphoid cells control signaling circuits to regulate
tissue-specific immunity. Cell Res. 30, 475–491 (2020).

25. Spellberg, B. & Edwards, J. E. Type 1/Type 2 immunity in infectious diseases. Clin.
Infect. Dis. Off. Publ. Infect. Dis. Soc. Am. 32, 76–102 (2001).

26. Zhu, J. Mysterious ILC2 tissue adaptation. Nat. Immunol. 19, 1042–1044 (2018).

27. Spits, H. & Cupedo, T. Innate lymphoid cells: emerging insights in development, lineage
relationships, and function. Annu. Rev. Immunol. 30, 647–675 (2012).

28. Wilhelm, C. et al. An IL-9 fate reporter demonstrates the induction of an innate IL-9
response in lung inflammation. Nat. Immunol. 12, 1071–1077 (2011).

29. van Rijt, L., von Richthofen, H. & van Ree, R. Type 2 innate lymphoid cells: at the cross-
roads in allergic asthma. Semin. Immunopathol. 38, 483–496 (2016).

30. Ricardo-Gonzalez, R. R. et al. Tissue signals imprint ILC2 identity with anticipatory
function. Nat. Immunol. 19, 1093–1099 (2018).
38

31. Bartemes, K. R. & Kita, H. Roles of innate lymphoid cells (ILCs) in allergic diseases:
The 10-year anniversary for ILC2s. J. Allergy Clin. Immunol. 147, 1531–1547 (2021).

32. Steinke, J. W. & Borish, L. Th2 cytokines and asthma — Interleukin-4: its role in the
pathogenesis of asthma, and targeting it for asthma treatment with interleukin-4 receptor
antagonists. Respir. Res. 2, 66–70 (2001).

33. Karta, M. R., Broide, D. H. & Doherty, T. A. Insights into Group 2 Innate Lymphoid
Cells in Human Airway Disease. Curr. Allergy Asthma Rep. 16, 8 (2016).

34. Liu, L.-Y. et al. Human airway and peripheral blood eosinophils enhance Th1 and Th2
cytokine secretion. Allergy 61, 589–597 (2006).

35. Wilhelm, C., Turner, J.-E., Van Snick, J. & Stockinger, B. The many lives of IL-9: a
question of survival? Nat. Immunol. 13, 637–641 (2012).

36. Sugita, K. et al. Type 2 innate lymphoid cells disrupt bronchial epithelial barrier integrity
by targeting tight junctions through IL-13 in asthmatic patients. J. Allergy Clin. Immunol.
141, 300-310.e11 (2018).

37. Seehus, C. R. et al. Alternative activation generates IL-10 producing type 2 innate
lymphoid cells. Nat. Commun. 8, 1900 (2017).

38. Zhang, H. et al. An IL-27-Driven Transcriptional Network Identifies Regulators of IL-10
Expression across T Helper Cell Subsets. Cell Rep. 33, 108433 (2020).

39. Howard, E. et al. IL-10 production by ILC2s requires Blimp-1 and cMaf, modulates
cellular metabolism, and ameliorates airway hyperreactivity. J. Allergy Clin. Immunol.
147, 1281-1295.e5 (2021).

40. Huynh, J. P. et al. Bhlhe40 is an essential repressor of IL-10 during Mycobacterium
tuberculosis infection. J. Exp. Med. 215, 1823–1838 (2018).

41. Zaiss, D. M. W., Gause, W. C., Osborne, L. C. & Artis, D. Emerging functions of
amphiregulin in orchestrating immunity, inflammation and tissue repair. Immunity 42,
216–226 (2015).

42. Salimi, M. et al. A role for IL-25 and IL-33–driven type-2 innate lymphoid cells in atopic
dermatitis. J. Exp. Med. 210, 2939–2950 (2013).

43. PD-1 and Its Ligands in Tolerance and Immunity | Annual Review of Immunology.

44. Helou, D. G. et al. PD-1 pathway regulates ILC2 metabolism and PD-1 agonist treatment
ameliorates airway hyperreactivity. Nat. Commun. 11, 3998 (2020).

39

45. Meyaard, L. et al. LAIR-1, a Novel Inhibitory Receptor Expressed on Human
Mononuclear Leukocytes. Immunity 7, 283–290 (1997).

46. Helou, D. G. et al. LAIR-1 acts as an immune checkpoint on activated ILC2s and
regulates the induction of airway hyperreactivity. J. Allergy Clin. Immunol. 149, 223-
236.e6 (2022).

47. Yu, X. et al. TNF superfamily member TL1A elicits type 2 innate lymphoid cells at
mucosal barriers. Mucosal Immunol. 7, 730–740 (2014).

48. Nagashima, H. et al. GITR cosignal in ILC2s controls allergic lung inflammation. J.
Allergy Clin. Immunol. 141, 1939-1943.e8 (2018).

49. Hutloff, A. et al. ICOS is an inducible T-cell co-stimulator structurally and functionally
related to CD28. Nature 397, 263–266 (1999).

50. Wikenheiser, D. J. & Stumhofer, J. S. ICOS Co-Stimulation: Friend or Foe? Front.
Immunol. 7, (2016).

51. Li, D.-Y. & Xiong, X.-Z. ICOS+ Tregs: A Functional Subset of Tregs in Immune
Diseases. Front. Immunol. 11, (2020).

52. Zeng, H. et al. mTORC1 couples immune signals and metabolic programming to
establish T(reg)-cell function. Nature 499, 485–490 (2013).

53. Hutloff, A. et al. ICOS is an inducible T-cell co-stimulator structurally and functionally
related to CD28. Nature 397, 263–266 (1999).

54. Simpson, T. R., Quezada, S. A. & Allison, J. P. Regulation of CD4 T cell activation and
effector function by inducible costimulator (ICOS). Curr. Opin. Immunol. 22, 326–332
(2010).

55. Maazi, H. & Akbari, O. ICOS regulates ILC2s in asthma. Oncotarget 6, 24584–24585
(2015).

56. Maazi, H. et al. ICOS:ICOS-Ligand Interaction Is Required for Type 2 Innate Lymphoid
Cell Function, Homeostasis, and Induction of Airway Hyperreactivity. Immunity 42, 538–
551 (2015).

57. Zhu, J., Cote-Sierra, J., Guo, L. & Paul, W. E. Stat5 activation plays a critical role in Th2
differentiation. Immunity 19, 739–748 (2003).

58. Redpath, S. A. et al. ICOS controls Foxp3+ regulatory T-cell expansion, maintenance
and IL-10 production during helminth infection. Eur. J. Immunol. 43, 705–715 (2013).
40

59. O’ rien, C. A., atista, S. J., Still, . M. & arris, T. . IL -10 and ICOS Differentially
Regulate T Cell Responses in the Brain during Chronic Toxoplasma gondii Infection. J.
Immunol. 202, 1755–1766 (2019).

60. Landuyt, A. E. et al. ICOS ligand and IL-10 synergize to promote host–microbiota
mutualism. Proc. Natl. Acad. Sci. 118, e2018278118 (2021).

61. Commins, S., Steinke, J. W. & Borish, L. The extended IL-10 superfamily: IL-10, IL-19,
IL-20, IL-22, IL-24, IL-26, IL-28, and IL-29. J. Allergy Clin. Immunol. 121, 1108–1111
(2008).

62. Wilhelm, C. et al. Critical role of fatty acid metabolism in ILC2-mediated barrier
protection during malnutrition and helminth infection. J. Exp. Med. 213, 1409–1418
(2016).

63. Rigas, D. et al. Type 2 innate lymphoid cell suppression by regulatory T cells attenuates
airway hyperreactivity and requires inducible T-cell costimulator-inducible T-cell
costimulator ligand interaction. J. Allergy Clin. Immunol. 139, 1468-1477.e2 (2017).

64. Cai, F., Jin, S. & Chen, G. The Effect of Lipid Metabolism on CD4+ T Cells. Mediators
Inflamm. 2021, e6634532 (2021).

65. Maazi, H. et al. ICOS:ICOS-Ligand Interaction Is Required for Type 2 Innate Lymphoid
Cell Function, Homeostasis, and Induction of Airway Hyperreactivity. Immunity 42, 538–
551 (2015).

66. The role of Stat5a and Stat5b in signaling by IL-2 family cytokines | Oncogene.

67. Howard, E. et al. IL-10 production by ILC2s requires Blimp-1 and cMaf, modulates
cellular metabolism, and ameliorates airway hyperreactivity. J. Allergy Clin. Immunol.
147, 1281-1295.e5 (2021).

68. Maazi, H. et al. ICOS:ICOS-Ligand Interaction Is Required for Type 2 Innate Lymphoid
Cell Function, Homeostasis, and Induction of Airway Hyperreactivity. Immunity 42, 538–
551 (2015).

Abstract (if available)

Abstract Allergic asthma is a growing heterogenous disease that is characterized by chronic lung inflammation and airway hyperreactivity (AHR). Group 2 innate lymphoid cells (ILC2s) are from a subset of immune cells that contribute to the development of lung inflammation and AHR by secreting type 2 cytokines. Because inducible co-stimulatory molecule (ICOS) has been shown to play a role in ILC2 effector functions and homeostasis, we hypothesize that ICOS plays a role in regulating ILC2-mediated IL-10 production and a metabolic shift in allergic asthma. Therefore, to test this hypothesis, we investigated the role of ICOS in ILC2s during IL-33-induced airway inflammation. We found that ICOS regulated the production of IL-10 with and without the presence of IL-4 via the following IL-10 gene regulators: Nfil3, Bhlhe40, cMaf, and Prdm1. Moreover, ICOS regulated pro-inflammatory IL-5 and IL-13 cytokines production in ILC2s. ICOS deficiency induced a metabolic change from fatty acid oxidation (FAO) to glycolysis, while the presence of IL-4 rescued partial dependency on FAO. Additionally, this study highlights a possible immunoregulatory role of ICOS in allergic asthma and presents ICOS as a potential therapeutic target.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Immune regulation of ILC2 dependent asthma by sodium butyrate

PDF

IFN-α treatment inhibits induction of type-2 innate lymphoid cell dependent airway hyperresponsiveness

PDF

FoxP3⁺ regulatory T cells inhibit ILC2 dependent AHR

PDF

PD-1 is a regulator of ILC2-mediated airway hyperresponsiveness and inflammation

PDF

Investigating the modulatory mechanisms of ILC2s as a therapeutic strategy in the context of health and disease

PDF

A novel therapeutic approach in asthma: depleting CD52-expressing leukocytes suppresses airway hyperreactivity and ameliorates lung inflammation

PDF

Impact of cannabinoid receptor 2 stimulation on adipose tissue-resident ILC2s and its potential in alleviating obesity-induced type 2 diabetes mellitus

PDF

BIA-ALCL cells have characteristics of ILC2 and Th2 cell phenotypes

PDF

The essential role of histone H2A deubiquitinase MYSM1 in natural killer cell maturation and HSC homeostasis

PDF

Role of a novel transmembrane protein, MTTS1 in mitochondrial regulation and tumor suppression

PDF

Yeast-based nanobody identification for MCEMP1: mast-cell expressed membrane protein 1

PDF

Adaptive metabolic strategies of Mycobacterium tuberculosis to combat stress from antibiotics and ROS

PDF

Regulation of inflammation and angiogenesis by Kaposi’s sarcoma-associated herpesvirus

PDF

Airway inflammation and respiratory health in the Southern California children's health study

PDF

Mechanism of ethanol-mediated increase of SARS-COV-2 entry through GRP78 induction

PDF

Placental growth factor mediated transcriptional and post-transcriptional regulation of hemeoxygenase-1

PDF

The histone methyltransferase SMYD5 plays a role in regulating plasma-cell antibody production and macrophage inflammatory cytokine secretion in vivo

PDF

A truncating mutation in the autophagy gene UVRAG drives inflammation and tumorigenesis in mice

PDF

Hepatitis B virus X protein regulation of β-catenin and NANOG and co-regulatory role with YAP1 in HCC malignancy

PDF

WAVE1 is a novel mediator of HPV trafficking to the Golgi apparatus

Asset Metadata

Creator Fung, Marshall (author)

Core Title Investigation of the role of ICOS in the regulation of ILC2 dependent airway hyperreactivity

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Degree Conferral Date 2022-08

Publication Date 07/18/2022

Defense Date 06/06/2022

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

Tag airway hyperreactivity,allergic asthma,Bhlhe40,cMaf,fatty acid oxidation,glycolysis,ICOS,IL-10,IL-13,IL-33,IL-4,ILC2,Nfil3,OAI-PMH Harvest,Prdm1: IL-5

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Akbari, Omid (committee chair), Landolph, Joseph (committee member), Machida, Keigo (committee member)

Creator Email marshalf@usc.edu,marshallfung@berkeley.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC111373210

Unique identifier UC111373210

Legacy Identifier etd-FungMarsha-10852

Document Type Thesis

Format application/pdf (imt)

Rights Fung, Marshall

Type texts

Source 20220719-usctheses-batch-955 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions 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. The original signature page accompanying the original submission of the work to the USC Libraries is retained by the USC Libraries and a copy of it may be obtained by authorized requesters contacting the repository e-mail address given.

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Repository Email cisadmin@lib.usc.edu