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The role of endogenous neuregulin-4 during immune-mediated intestinal injury

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The role of endogenous neuregulin-4 during immune-mediated intestinal injury

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The Role of Endogenous Neuregulin-4 During Immune-Mediated Intestinal Injury

by

Jessica Kathleen Bernard

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
(CRANIOFACIAL BIOLOGY)

August 2022

Copyright 2022 Jessica Kathleen Bernard
ii

“Adopt the pace of nature: her secret is patience”
-Ralph Waldo Emerson

iii

Acknowledgements

First, I want to thank my husband, Byron, for the constant support that he has given me during this
process. He helped to motivate me to achieve something that, at times, I believed was unattainable.
He constantly pushed me to press forward and is my constant cheerleader, even before this process
began.

Secondly, I would also like to thank Dr. Mike Schumacher and Dr. Cambrian Liu, without which
the development of this project may not have been possible. Their collaboration and support during
this time was essential for research planning, analysis, and general scientific understanding. They
are always eager to help in any way they can.

Next, I’d like to thank my friends and family. To my family, though halfway across the US, were
always eager to listen to my struggles and offer whatever support they could. And to my friends,
Dr. Soula Danopolous who was always there for me, especially during difficult times. Her support
really helped get to where I am today. Nandini Girish, who was always supportive in my decisions
and even helped babysit when I needed to study for tests.

Lastly, I’d like to thank my mentor Dr. Mark Frey. He took a chance on me as a young research
technician with little lab experience. The experience I gained from working with him at Vanderbilt
University and at Children’s Hospital Los Angeles helped to drive me to further my knowledge

iv
and career in research. His support during, and even before, this process has been important in the
development of my project and career.

v
Table of Contents
Epigraph………………………………………………………………………………….......ii
Acknowledgements………………………………………………………………………….iii
List of Tables………………………………………………………………………………..vii
List of Figures.……………………………………………………………………………. viii
Abstract……………………………………………………………………………………...ix
Introduction…………………………………………………………………………………..1
1. The Gastrointestinal Tract……………………………………………………………….. ..1
1.1 Overview of Intestinal Anatomy and Physiology……………………………… ..1
1.2 Inflammatory Bowel Disease (IBD)…………………………………………… ..2
1.3 Growth Factors in the Intestinal Tract…………………………………………. ..4
1.4 Epidermal Growth Factor (EGF) Family………………………………………. ..7
1.5ErbB4……………………………………………………………………………...8
1.6 Neuregulin-4 (NRG4)………………………………………………………….. 10
2. Means of Intestinal Regulation………………………………………………………….. 12
2.1 Intestinal Epithelial Barrier…………………………………………………….. 12
2.2 Mucosal Immunology………………………………………………………….. 16

Chapter 1: The loss of endogenous NRG4 impairs intestinal recovery after
LPS-injury…………………………………………………………………………………. 21
Abstract……………………………………………………………………………. 21
Introduction………………………………………………………………………... 22
Methods……………………………………………………………………………. 25
Results……………………………………………………………………………… 28
Discussion………………………………………………………………………….. 32

Chapter 2: Endogenous Neuregulin-4 regulates lymphocyte recruitment by colonic
epithelial St3gal4 during IL-10R neutralization colitis……………………………………. 43
Abstract…………………………………………………………………………….. 44
Introduction………………………………………………………………………… 45
Methods……………………………………………………………………………. 47
Results……………………………………………………………………………… 54
Discussion………………………………………………………………………….. 58
Conclusion…………………………………………………………………………………. 71
References………………………………………………………………………………….. 73

vi

List of Tables

Table 1. Overview of Intestinal Growth Factors…………………………………………...6
Table 2. Tight Junction (TJ) expression in CD and UC………………………………….. 15
Table 3. Chapter 1 qPCR Assays………………………………………………………….41
Table 4. Gsea Pathways Analysis from Bulk RNA Sequencing………………………….42
Table 5. Chapter 2 qPCR Assays………………………………………………………….69
Table 6. Cell Cytometry (CyTof) Mouse Immune Cell Profile…………………………..70

vii
List of Figures
Figure 1. General overview of homeostatic (Normal) intestinal regulation
vs. IBD affected intestinal regulation………………………………………………………...4

Figure 2. Epidermal growth factor receptor activation and signaling……………………….8

Figure 3. A simplified overview of lymphocyte subsets required for
numerous biological responses…………………………………………………………….. 19
Figure 4. Nrg4 and ErbB4 are inversely expressed along the mouse
intestinal tract………………………………………………………………………………. 34

Figure 5. NRG4 deletion promotes intestinal permeability and
enterocyte apoptosis at 24h after LPS challenge…………………………………………... 35

Figure 6. NRG4
-/-
mice demonstrate a decreased
inflammatory cytokine expression at baseline, but not after LPS challenge………………. 36

Figure 7. ZO-1 redistribution is delayed with the loss of NRG4,
after LPS challenge………………………………………………………………………… 37

Figure 8. NRG4 deletion shows a trend towards a decrease
in baseline Shh signaling…………………………………………………………………... 38

Figure 9. Baseline Muc2 mRNA expression is altered in NRG4
-/-
mice…………………... 39

Figure 10. ER stress and autophagy markers are
reduced with the loss of NRG4…………………………………………………………….. 40

Figure 11. Whole body NRG4 deletion leads to reduced IL-10R
neutralization- induced inflammation in the colon………………………………………… 63

Figure 12. NRG4 deletion reduces inflammatory cytokines and CD8
+
T cell
infiltration in IL-10R neutralization colitis………………………………………………… 64

Figure 13. NRG4 deletion limits splenic CD8
+
T cells……………………………………. 65

Figure 14. RNA-sequencing analysis indicates significant loss of St3gal4
in mice with NRG4 deletion……………………………………………………………….. 66

Figure 15. NRG4 deletion leads to the loss of colonic epithelial ST3GAL4……………… 67

Figure 16. The loss of NRG4 may shift microbial composition, promoting
an increase in known protective bacterial species…………………………………………. 68

viii
Abstract

Inflammatory Bowel Disease (IBD), a chronic inflammatory condition of the intestinal tract, is
comprised of two diseases: Crohn’s Disease (CD) and Ulcerative Colitis (UC). Although the direct
cause of IBD is unknown, it is likely a result from epithelial barrier dysfunction, overactive
immune responses, and microbial dysregulation. The ErbB family of receptor tyrosine kinases are
essential for the regulation of colonic epithelial cell proliferation, differentiation, survival, and
wound healing. We previously showed that ErbB4 is induced as a compensatory response by
intestinal inflammation; treatment with exogenous NRG4 (an ErbB4 ligand) protects colonocytes
against cytokine-induced apoptosis and ameliorates murine experimental colitis. Exogenous
NRG4 can further alleviate intestinal inflammation by suppressing the activity of pro-
inflammatory macrophages. However, the role of endogenous NRG4 in the intestine has not been
described. Here we tested the function of endogenous NRG4 in the intestine by two immune-
mediated injury models: acute bacterial LPS and IL-10R neutralization colitis. NRG4
-/-
mice
demonstrated a significant increase in intestinal permeability and apoptosis 24h after acute
bacterial LPS injury. Tight junctional component ZO-1 also showed a dysregulated localization
pattern in NRG4
-/-
mice at 24h, compared to WT cage mates, suggesting an impaired epithelial
barrier recovery. The loss of endogenous NRG4 reduced baseline ileal Muc2, Chop, Sqstm1, and
Map1lc3, known factors that aid in barrier protection and/or epithelial repair. However, NRG4
-/-

mice displayed a reduced inflammatory response, indicated by significantly reduced lipocalin-2,
total histology damage, myeloperoxidase, spleen weight, and CD8
+
T cell numbers, during IL-10R
neutralization colitis. Baseline CD8
+
T cells were not altered in the colon, but were significantly
reduced in the spleen. Bulk RNA sequencing of colonic homogenates determined a significant loss
of ST3GAL4 in NRG4
-/-
mice when compared to WT cage mates. This loss of ST3GAL4 was

ix
verified by qPCR analysis from colonic homogenates and colonic organoid cultures, while
localization was determined by in situ hybridization. ST3GAL4 levels were partially rescued with
exogenous NRG4 treatment in vivo. Taken together, these results suggest that NRG4 promotes
CD8
+
T cell recruitment through the interaction with ST3GAL4. Furthermore, endogenous NRG4
may contribute to the intestinal epithelial integrity and recovery during the innate immune
response, while suppress deleterious over production of T cells during adaptive immune mediated
injury.

1
Introduction

1. The Gastrointestinal Tract

1.1 Overview of Intestinal Anatomy and Physiology

The gastrointestinal (GI) tract is a cohesive assembly of organs that starts at the mouth and ends
at the anus. The components of the GI tract consist of the mouth, esophagus, stomach, small
intestine, colon, rectum, and anus. The GI tract performs a variety of essential functions including
digestion, absorption, excretion, and host protection. The stomach is responsible for the physical
digestion of food (retropulsion in the stomach) (1). The small intestine (regionally separated into
the duodenum, jejunum, and ileum) processes proteins, carbohydrates, and fats facilitated by
chemical digestion via bile and digestive enzymes that facilitate nutrient absorption (1). Other
organs that aid in digestion are salivary glands, liver, pancreas, and gallbladder. After the small
intestine, the contents reach the colon (also known as the large intestine), where they are desiccated
and compacted then expelled from the rectum through the anus.

Anatomy: The intestinal tract is composed of 4 different layers that include the serosa, muscularis,
submucosa, and mucosa. The inner layer (mucosa) of the small and large intestine is composed of
absorptive and secretory epithelial cells, connective tissue (lamina propria), and smooth muscle
(muscularis mucosa) (1). The submucosa includes a network of nerves, lymphatics, and connective
tissue (1). This region is surrounded by a layer of smooth muscle (longitudinal and circular) and
encased in an outer serosal layer. Together, this network of cells maintains the dynamic and rapid
repair response to injury.

2
1.2 Inflammatory Bowel Disease (IBD)
Inflammatory Bowel Disease (IBD) is comprised of two diseases: Crohn’s Disease (CD) and
ulcerative colitis (UC). IBD is generally characterized as the chronic inflammation of the intestinal
tract likely resulting from impaired epithelial barrier function, overactive immune responses,
and/or dysregulated microflora. IBD afflicts about 3 million Americans (2). The onset of IBD often
occurs between the ages of 15-30 but can be diagnosed at any age (2). Although IBD is diagnosed
worldwide, increased incidence of IBD occurs in northern climates from urban cities or towns in
developed countries (2).

CD and UC present as two distinct diseases with altered prognosis and treatment options, and the
pathophysiology greatly varies between the two diseases (3). CD can affect tissue throughout the
entire gastrointestinal tract (4), commonly with elevated disease at the ileum and the proximal
colon. Intestinal inflammation occurs in “patchy” patterns, surrounded by unaffected areas, and
afflicts the entire thickness of the bowel wall. UC on the other hand is primarily a disease of the
colon and affects the mucosal layer of the distal colon and rectum (3).

Common risk factors for IBD include environment, stress, diet, genetic susceptibility, immune
dysregulation, and intestinal microbial imbalance. 1.5-28% of patients diagnosed with CD have a
first degree relative with IBD (4) while 1.6-30% of UC patients have a first degree relative with
IBD (3). More than 200 susceptibility genes have been identified in either and/or both CD and UC
by genome-wide association studies (2). Some of these include immune responsive genes in CD
(e.g., IL-10R), while UC susceptibility genes are generally targeted to intestinal epithelial barrier
(e.g IL-13) and the T cell response (e.g., IL-36) (3, 4). Mucosal inflammatory responses are

3
characteristic of both CD and UC. However, CD predominantly elicits a T helper response (TH1
or TH17 cells), where cytokine regulation by TH17 and TH2 cells are characteristic of UC (3, 4).

Although UC and CD have different characteristics, the colonization and balance of intestinal
microbial communities contribute to the pathogenesis of each. The intestinal microflora consists
of approximately 10
11
bacteria, 10
6
fungi, 10
8
archaea, and 10
8
viruses in a single gram of stool
(5). Among its many roles, the microbiota is essential in the regulation of metabolism and
immunity. In fact, dysregulation in microbial diversity and function underlies many intestinal
diseases, such as IBD (6). IBD patients are known to have decreased levels of Bacteroides,
Firmicutes, Clostridia, Ruminococcaceae, Bifidobacterium, Lactobacillus, and Faecalibacterium
prausnitzii, and increased levels of Gammaproteobacteria, Escherichia coli, and Fusobacterium
species (7). Furthermore, the loss of protective effects from short chain fatty acids (a microbial
nutrient source) and the increase in pro-inflammatory factors (such as oxidative stress, toxin
secretion, and lipopolysaccharide structure) contribute to the pathogenesis of IBD (8).

Currently the most commonly used effective treatments of IBD target the immune system, leading
to lower rates of relapse, risk of surgical intervention, as well as increased quality of life. Over the
past few decades, this has been provided by anti-inflammatory treatments, such as anti-tumor
necrosis factor (TNF) monoclonal antibody therapeutics (4). However, there is a risk for
developing immune intolerance to these and similar therapies and lost efficacy among patients is
common. No therapeutics currently target the mucosal barrier. Therefore, advances in our
understanding of IBD are required to achieve more effective long-term treatments or even a
potential cure.

4

1.3 Growth Factors in the Intestinal Tract
Growth factors are generally defined as polypeptides that bind to surface membrane receptors and
promote various cellular responses that include growth, proliferation, or differentiation (9). There
are numerous growth factors (Table 1) that are expressed in a variety of tissue and cell types, such
as the epithelium (10, 11), sub-epithelium (12), exogenous sources (i.e. breast milk) (13-16),
immune cells (17), and various source tissue types (18) within in the GI tract (9). Although their
roles vary, they can include growth, maintenance, repair, restitution, immune cell regulation, and
may be involved in either the advancement or suppression of cancer (9).

Figure 1. General overview of homeostatic (Normal) intestinal regulation vs
IBD affected intestinal regulation. Characteristics of IBD include: a loss of the
mucus layer, increase in epithelial permeability and bacterial translocation,
expansion of the Lamina propria, and dysregulation and overactivation of
immune cells, inflammatory cytokines, and chemokines. Created with
BioRender.com

5
Sequential growth factor signaling generally begins with the release or presentation of the ligand,
followed by the binding to a receptor, leading to receptor activation (primarily through kinase
activity). Growth factor signaling can occur by autocrine/juxtacrine (acting upon a receptor from
the same cell or neighboring cell), paracrine (acting upon nearby cells or tissues such as growth
factors from the mesenchyme acting upon the epithelium), endocrine (growth factors acting at a
distance, usually carried in the circulation to target cells), or exocrine (carried by a duct to target
cells) activity. Pharmacological treatment with exogenous growth factors can also be used to
activate endogenous receptors and signaling networks. Growth factor receptors receptors often
have multiple ligands that bind to one receptor, potentially driving numerous cellular responses
(9).

6

Growth Factor Ligand(s) Receptor(s) Target
Epidermal growth factor EGF, TGF-⍺,
NRG1-4,
HB-EGF,
amphiregulin,
betacellulin,
epiregulin,
epigen
EGFR/ErbB1,
ErbB2,
ErbB3, ErbB4
Endothelium
Epithelium
Immune
Fibroblast growth factor FGF1, 2, 4,
7, 10, 15/19,
18, 20, 21, 23
FGF1-4
⍺/β-klotho
coreceptors
Epithelium
Mesenchyme
Hedgehog Shh, Ihh, Dhh Ptch1-2 Epithelium
Immune
Hepatocyte growth factor HGF c-Met
CD44 coreceptor
Endothelium
Epithelium
Mesenchyme
Immune
Insulin-like growth factor IGF1-2 IGFR1-2 Endothelium
Epithelium
Transforming growth factor-β TGF-β,
BMP2-7,
activin,
inhibin,
nodal
TβR-1-2 Endothelium
Epithelium
Immune
Trefoil factor TFF1-3 CXCR4 Epithelium
Immune

Table 1. Overview of Intestinal Growth Factors
Adapted from Schumacher et al. 2018

7
1.4 Epidermal Growth Factor (EGF) Family
The EGF family of receptor tyrosine kinases (EGFR, ErbB2, ErbB3, ErbB4), expressed in
epithelial, endothelial, and immune cells (9), recognizes numerous growth factor ligands that
include EGF, TGF-⍺, heparin-binding EGF-like growth factor (HB-EGF), amphiregulin,
betacellulin, epigen, and heregulin (HRG)/neuregulin (NRG) (Figure 2). These growth factor
ligands are homologous but can vary in cellular activity. The activation of the transmembrane
growth factor receptor occurs by the binding of these ligands at the cell surface, followed by homo-
and hetero-dimerization of these receptors. Although ErbB family members share substantial
homology, each contains distinct properties and molecular docking sites that contribute to various
downstream signaling cascades and cellular processes. For example in the case of ErbB4, once
dimerization occurs, the metalloproteinase ADAM 17 (a.k.a. TACE) cleaves the extracellular
domain, while 𝛾-secretase cleaves the cytoplasmic domain, leading to the translocation of an
intracellular domain to the nucleus (19).

8

1.5 ErbB4
ErbB4, the least well-understood ErbB family member, was first cloned from a human mammary
carcinoma cell line (19). It contains a cysteine rich ecto domain and cytoplasmic domain composed
of a juxtamembrane region, a tyrosine kinase domain, and a carboxyterminal domain (19). ErbB4
has 4 functionally different isoforms, JM-a, JM-b, CYT-1, and CYT-2. JM-a and JM-b influence
shedding of the ectodomain from the cell surface, while CYT-1 influences the phosphotiylinositol-
Figure 2. Epidermal growth factor receptor family member activation and
signaling. Created with BioRender.com

9
3 kinase (PI-3 kinase) docking to the cytoplasmic domain, promoting cell proliferation and
migration, which is deleted in CYT-2 (19).

ErbB4 activation, similar to most ErbB family members, is initiated by ligand binding of growth
factors. However, ErbB4 recognizes a wider range of these growth factors than other ErbB
members (Neuregulin (NRG)1-4, Betacellulin, Epiregulin, and Heparin binding-Epidermal
Growth Factor (HB-EGF)) (19). Neuregulin growth factors (NRGs) undergo extensive alternative
splicing, which leads to a greater diversity of protein expression and cellular functions (20). This
neuregulin class of ligands interacts with the heterodimers EGFR/ErbB4, ErbB2/ErbB4, and
ErbB3/ErbB4 (21). NRGs 1 and 2 are recognized with higher affinity to ErbB3, while NRGs 3 and
4 interact with only ErbB4 (22). However, it has been shown that NRGs 1 and 2 have a higher
potency than NRGs 3 and 4 in activating ErbB4 (16, 20).

After activation and dimerization, ErbB4 can, like other ErbBs, undergo slow internalization
through clathrin-coated pits or through endosomal trafficking, depending on type of ligand binding
(19). Additionally, ErbB4 is subject to a two-step proteolytic cleavage mechanisms reminiscent of
Notch processing. The ErbB4 ectodomain is cleaved by TACE (tumor necrosis factor-alpha
converting enzyme), an ADAM matrix metalloprotease designated ADAM17 (23). Cleavage of
the ectodomain leaves behind an 80kDa membrane-bound fragment, which is then proteolytically
released to the cytoplasm by γ-secretase (16, 19). This 80kDa intracellular domain is then directly
translocated into the nucleus, activating many downstream signaling pathways such as JAK,
STAT, Shc, p85 subunit of PI-3 kinase, GrbB2, GrbB7 (19).

10
ErbB4 expression is found in both at low levels in human adult and fetal tissues, including the
gastrointestinal tract (16, 20). Our lab has previously reported that ErbB4 is up-regulated during
inflammation in both the adult human and mouse colon (24). We have also demonstrated that
ErbB4 promotes mouse colon epithelial cell survival, but not migration, through the
phosphorylation of PI-3 kinase/AKT pathway (25). The deletion of intestinal epithelial ErbB4
demonstrated exacerbated colitis and a slower recovery after DSS colitis injury (26). Furthermore,
the overexpression of ErbB4 in cultured colonic epithelial cells has been shown to be protective
against cytokine-induced apoptosis, while the knockdown of ErbB4 in a cultured colorectal cancer
cell line led to an increase in apoptosis (16). Together, these results suggest that ErbB4 is expressed
as a compensatory response in order to preserve the epithelium, rather than as a driver of the
pathogenesis of intestinal disease.

1.6 Neuregulin-4 (NRG4)
NRG4, an ErbB4-specific ligand, is expressed at low levels in all mammalian tissues except the
nervous system (20, 27). There are at least 5 different splice variants of NRG4 (NRG4A1,
NRG4A2, NRG4B1, NRG4B2, and NRG4B3), with 2 (NRG4A1 and NRG4A2) containing
transmembrane regions and transported to the cell membrane (20). NRG4A variants are comprised
of a transmembrane domain, containing an 8kDa Epidermal growth factor (EGF) motif, but vary
at the C-terminal domain (i.e., NRG4A2 has a PDZ binding motif) (21). NRG4B variants differ
from NRG4A variants by the loss of a transmembrane domain and a truncated EGF domain (only
the first four cysteine residues) (20). All variants are present in the small intestine, but only the
A1/A2 are weakly expressed within the colon (20).

11
In mouse colitis models, including dextran sodium sulfate (DSS) and IL10
-/-
, intestinal tissues have
high levels of epithelial apoptosis, cytokine expression, and inflammatory cell infiltration. During
these colitic conditions there is an upregulation of ErbB4 and a downregulation of NRG4 in the
colon (17, 25). Exogenous treatment with NRG4 promoted colonocyte survival during cytokine-
induced apoptosis, as determined by cleaved caspase-3 and Apoptag-ISOL apoptotic markers, in
vivo and in vitro (25). Similarly, exogenous NRG4 alleviates inflammation during a murine DSS
colitis model, demonstrated by a reduced histology score, elevated weight percentage over time,
and increased colon length, compared to PBS control mice. Exogenous NRG4 treatment of ex-
vivo bone marrow derived macrophages also led to increased apoptosis, while the loss of myeloid
ErbB4 or NRG4 deletion led to up-regulation of pro-inflammatory cytokines (LPS/INF𝛾) (17, 28).
These results suggest that NRG4 promotes clearance of pro-inflammatory macrophages during
inflammation. Furthermore, both UC and CD showed a significant reduction of NRG4 relative
mRNA levels, when compared to normal tissue (25).

In the intestinal epithelium, NRG4-ErbB4 signaling is also important in the maintenance of Paneth
cells (cells that support the stem cell niche by the secretion of growth factors and antimicrobial
peptides) and the intestinal epithelium, as determined by intestinal organoid culture (16). Intestinal
organoids from mice with a deletion of intestinal epithelial ErbB4 showed reduced lysozyme (a
Paneth cell marker) and abnormal budding, with a sensitivity to TNF treatment, when compared
to control organoids (16). Furthermore, exogenous treatment with NRG4 is protective against
Paneth cell ablation in a necrotizing enterocolitis mouse model (known to reduce Paneth cells) and
blocks bacterially induced apoptosis in cultured intestinal epithelial cell apoptosis through a Src

12
dependent pathway (16). This suggests that NRG4 is important for the prevention of injury/disease
and recovery of the intestinal epithelium.

2. Means of Intestinal Regulation
2.1 Intestinal Epithelial Barrier
The intestinal epithelial barrier is a physical barrier formed by a monolayer of columnar
epithelial cells, separating the intestinal lumen from the mucosa. Intestinal epithelial cells form
interepithelial junctional complexes (tight, gap, and adherens junctions) that polarize the cell to
apical or basolateral regions (or fence function) and regulate the passage of molecules into the
body (or gate function) (29). These activities depend on cytoplasmic scaffolding proteins Zonula
Occludins (ZO) and Cingulin; tight junction proteins occludin, tricellulin, and marvelD3;
Claudins, and others (30) (Table 2). This barrier serves as the first line of defense, as it protects
from the infiltration of pathogenic material and serves as a sentinel that ensures rapid epithelial
repair by either molecular mechanisms or recruitment of immune cells. Other factors such as mucin
(goblet cells), Paneth cells (that secrete antimicrobial peptides), and intestinal epithelial
lymphocytes contribute to the maintenance of the intestinal barrier. The disruption of the intestinal
barrier causes increased intestinal permeability, which may lead to disease (as seen in IBD and
NEC).

Although it is unclear if barrier dysfunction occurs prior to disease or following inflammatory
cytokine insult, a study by Zeissig et al. looking at CD remission clinical samples determined that
TJ protein expression and epithelial apoptosis were similar to healthy controls (31). Here, the
expression of Claudins 1, 3, 4, 5, 7 and 8 were present in CD samples, while Claudin 2, 11, 12, 14

13
were not detectable (31). In vitro intestinal epithelial cell monolayers also demonstrated that TNF
treatment led to increased epithelial apoptosis and decreased tight junction strand numbers,
suggesting a secondary barrier defect (32). Furthermore, increased intestinal permeability is
apparent in quiescent IBD and first-degree relatives with CD (33, 34).

On the other hand, barrier defects may also predispose the intestinal epithelium to a greater
inflammatory response. UC patients demonstrated an upregulation of IL-13, leading to increased
epithelial permeability and apoptosis, compared to CD and normal controls (35). The upregulation
of IL-13 led to increased expression of Claudin 2, a pore-forming tight junction protein (35). Mice
null for Myosin Light Chain Kinase (MLCK)—a scaffolding protein that directly interacts with
actinomyosin ring—showed increased phosphorylation of MLC, barrier defects, and immune
activation (36). These mice did not develop disease until immune-mediated injury, but then had
accelerated colitis associated disease (36). MLCK deleted mice also showed a mild increase in
TNF and IFN-γ (indicating at least mild baseline inflammation), with no microbial translocation
detected (37). Similarly, JAM-A (which normally contributes to barrier function and the migration
of inflammatory cells) deleted mice showed barrier dysfunction but disease was only evident after
DSS injury (38). Taken together, this suggests that an increased risk for disease incidence with
barrier dysfunction and chronic inflammatory conditions.

Although it is unclear if IBD barrier dysfunction occurs from a primary or secondary defect,
therapeutic targets of the barrier architecture could alleviate these barrier defects. An example of
this outcome was demonstrated by the inhibition of MLCK to reverse the phosphorylation of MLC
and prevent TJ reorganization in CACO-2 monolayers (39). Similarly, Sulfasalazine, a protective

14
agent in the disruption of TJs, prevented MLCK upregulation during the treatment of inflammatory
cytokine TNF/IFN-γ monolayers (37). Therefore, targeting the proteins that regulate the structure
of the epithelial barrier might be therapeutic in preventing further clinical disease, whether patients
are predisposed or not.

15

TJ proteins Expression in CD Expression in UC Function
Claudin 1 Increase Increase Tightens the
epithelium; formation
of TJ strands
Claudin 2 Increase Increase Pore-forming;
formation of TJ
strands; decreases of
CLDN1 and 4
Claudin 4 Decrease
Active
Inflammation:
Increase
Decrease
Active
Inflammation:
Increase
Tightens the
epithelium; decreases
paracellular
conductance by
reduced sodium
permeability
Claudin 5 Decrease Tightens the epithelium
Claudin 8 Decrease Tightens the epithelium
Claudin 12 Increase Tightens the epithelium
Claudin 18 Increase undetermined
Occludin Decrease Decrease Binds ZO-1; cell
adhesion; regulates
paracellular
permeability
ZO-1 Decrease Decrease Protein-Protein
interactions; link to
actin cytoskeleton
“anchoring”

Table 2. Tight Junction (TJ) expression in CD and UC. Adapted from Landy et al.
2016.

16
2.2 Mucosal Immunology
Immune surveillance and recruitment to the intestinal epithelium is critical for homeostatic
epithelial maintenance and repair. However, during IBD both the innate and adaptive immune
responses can potentiate inflammation through the infiltration of the intestinal mucosa. Thus,
immune cell overactivation or impairment of immune mediated tolerance is a deleterious
contributor to the pathogenesis of IBD.

The innate immune response is an inherent, rapidly activated (minutes to four days) collection of
immune cells and immune regulatory substances (40). Physical or chemical barriers involved in
this response mainly consist of neutrophils, monocytes, macrophages, dendritic cells, natural killer
cells, mucus, epithelial barrier, inflammatory cytokines, and antimicrobial/bactericidal substances
(41). After infiltration of the epithelial barrier, pathogens enter the body and pathogen-associated
molecular patterns (PAMPs) or damage associated molecular patterns (DAMPs) are recognized
by residential immune mediators through different cell surface and intracellular receptors (41).
Once activated, these immune cells elicit numerous cellular responses in order to clear pathogen(s)
and/or repair the site of injury. Phagocytes (monocytes, macrophages, neutrophils, and dendritic
cells) are the first to respond to injury or pathogen infiltration and contain pattern recognition
receptors (PRR) that bind to PAMPs or DAMPs to promote the engulfment/degradation of various
pathogens and/or the recruitment of other immune cells by cytokine or chemokine production (41).
Dendritic cells also aid in the activation of the adaptive immune response by migrating to the
lymph nodes and presenting intact or degraded pathogens to T cells through the Major
Histocompatibility Complex (MHC), ultimately activating and recruiting necessary T cells. Thus,

17
the innate immune response is critical for the activation, maturation, differentiation, and
recruitment of the adaptive immune response, through their role in antigen presentation (42).

T cells are specialized lymphocytes that are important for adaptive immunity, providing
immunological memory and self-tolerance (43). T cells are derived from the bone marrow and
migrate to the thymus, where they undergo maturation and selection. These naïve T cells are
carried through the blood to secondary lymphoid organs (i.e., lymph nodes, spleen, Peyer’s
patches, and mucosal tissues), providing additional maturation, stimulation, and proliferation (43).
The spleen is critical for antibody production, lymphocyte activation, and storage of mononuclear
phagocytes (44), allowing for the redistribution of CD4
+
and CD8
+
T cells to nonlymphoid tissues
after antigen presentation (45). Inflammatory cytokines are a critical part of the broad spectrum of
responses to antigens by the immune system. These cytokines, which are mainly produced by
CD4+ helper T cells and CD8+ cytotoxic T cells during the adaptive immune response, enhance
the initiation and proliferation of other cells (mainly antibody producing B cells, phagocytic cells,
anti-microbial, etc.) (41).

CD4 T helper cells are classified as two subgroups (Th1 and Th2) that produce different cytokines
by distinct T cell clones (41). The first subgroup, Th1, is defined as more of a pro-inflammatory
response and secretes IL-2, IFN-γ, and TNF cytokines (41). Activation of this subgroup may then
promote the initiation of cytotoxic T cells and macrophages in order to protect against parasitic
infection, enhance T cell activation, perpetuate autoimmunity, and many other functions (41). The
Th2 response secretes IL-4, IL-5, IL-6, IL-13, and IL-10 cytokines and is responsible for the
regulation B cells (41). Activation of the Th2 subgroup is seen in response to allergies and

18
protective against pathogens (41). It has been suggested that Th2 is necessary to counteract an
excess of Th1 (41).

Although previous IBD studies have focused on the contribution of CD4
+
T cells, there are
conflicting data on the role of CD8
+
T cells during disease due to the lack of understanding of each
subset of CD8
+
T cells. Previous studies clearly show that cytolytic CD8
+
T cells produce IFN-γ
and TNF, known mediators of intestinal inflammation. The dysregulation in the recognition of
intestinal enterocyte-specific antigens by CD8
+
T cells can lead to the destruction of epithelial
cells by increased reactive CD8
+
T cells and the loss of barrier integrity (46). Thus, both the
destruction of the epithelium and the increase in luminal anitgens can promote overactivation and
recuitment of immune cells, promoting inflammation. However, in 2005 Brimnes and colleagues
reported that CD8
+
regulatory T cells (known to promote immune suppresssion during
homeostasis) were reduced during IBD and that isolated CD8
+
T cells from the lamina propria
could not suppress immune responses. Furthermore, highly heterogenic CD8
+
T cell populations
have been identified in IBD patients, with variable functions (47, 48). Taken together, these results
demonstrate the complexity and individual differences in patients with IBD.

19

Within IBD, the Th1 response is commonly associated with Crohn’s disease (CD), whereas
ulcerative colitis (UC) has a greater Th2 tone (49). However, both UC and CD have been reported
to have high levels of TNF, IFN-γ, and IL-6, common Th1 cytokines (50). Although spontaneous
apoptosis is a normal homeostatic function of the intestinal epithelium, TNF has been shown to
increase apoptosis as well as decrease tight junction (TJ) strand assembly and/or stability, leading
to increased intestinal permeability (32). Intestinal barrier defects can ultimately cause increased
antigen leakage, activation of mucosal immunity, and disease. In fact, transepithelial electrical
resistance (a measure of permeability) was reduced by ~50% in biopsies from CD and UC patients
Figure 3. A simplified overview of lymphocyte subsets required for numerous
biological responses. Created with BioRender.com

20
versus healthy control tissue, though the difference in UC was more pronounced (31). This
difference may be because CD, with its elevated Th1 cytokine levels, displays decreased epithelial
restitution rates, leading to reduced epithelial focal lesions, or TJ Claudin 2 (a pore forming
protein) expression (31).

21
Chapter 1

The loss of endogenous NRG4 impairs intestinal barrier recovery after acute
LPS injury

Jessica K. Bernard
1,2
, Michael A. Schumacher
1,3
, Esteban Fernadez
1
, and *Mark R. Frey
1,3,4

1. The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, CA 90027
2. Craniofacial Biology Program, Herman Ostrow School of Dentistry, University of Southern
California, Los Angeles, CA 90089
3. Department of Pediatrics, University of Southern California Keck School of Medicine, Los
Angeles, CA 90089
4. Department of Biochemistry and Molecular Medicine, University of Southern California Keck
School of Medicine, Los Angeles, CA 90089

Abstract

Inflammatory Bowel Disease (IBD) is a chronic, incurable inflammatory condition that involves
the loss of intestinal epithelial barrier function (epithelial damage, cell-cell junction permeability,
bacterial translocation, and impaired restitution). Activation of ErbB receptor tyrosine kinases can
ameliorate damage and reduce disease activity in both animal IBD models and human patients,
though the molecular mechanisms of this protection are not entirely clear. ErbB4 activation with
exogenous NRG4, a growth factor that only binds to this receptor, promotes intestinal epithelial
cell survival and reduces experimental intestinal inflammation. However, it is not known what role
endogenous NRG4 plays in maintaining the epithelial barrier, or what its primary intracellular

22
mechanisms of action are. Here, we demonstrate that loss of endogenous NRG4 compromises the
ability to repair the intestinal barrier, as shown by increased permeability, apoptosis, and
redistribution of tight junction protein ZO-1 after bacterial antigen challenge. A potential
mechanism for these effects is suggested by our observation that NRG4 deleted mice exhibit
decreased intestinal epithelial mRNA expression of Shh, while having altered markers for Muc2,
ER stress, and autophagy. A link between hedgehog signaling and barrier function in the gut has
not been explored. However, Shh signaling facilitates the recovery from injury in lung, heart, and
brain (where it stabilizes the blood brain barrier through regulation of ZO-1). This suggests that
endogenous NRG4 promotes intestinal barrier maintenance and recovery by altering secretory and
mitochondrial functions, possibly through Shh signaling.

Introduction

Inflammatory Bowel Disease (IBD) is a condition of chronic inflammation within the
gastrointestinal tract, which afflicts >3 million Americans (2). Many patients do not respond to
current therapies, or if they do, eventually relapse. Therefore, better therapeutic approaches, or a
combination of approaches, may be necessary to sufficiently treat IBD. Although the cause of this
disease is multifactorial and etiology can differ between patients, a significant component of IBD
pathobiology is the destruction of the intestinal epithelial layer (or epithelial barrier), leading to
increased permeability, microbial migration, and diminished restitution/repair (51, 52). Available
treatments for IBD may alleviate symptoms, but as yet do not directly target the destruction and/or
repair of this barrier. This gap represents an opportunity if key mechanisms of epithelial
maintenance and repair can be defined.

23
The intestinal barrier is a tightly regulated network of multiple layers that include: a microbial
layer, an extracellular layer (or mucus layer), physical barrier (or epithelial barrier), and
immunological layer (53). The physical tissue barrier, comprised of a single layer of columnar
epithelial cells and interepithelial tight junctions, remains the first line of defense against
potentially harmful pathogens, toxins, and antigens from the intestinal lumen (29). This barrier
undergoes transient damage and repair during homeostasis, resulting in a limited acute
inflammatory response to prevent translocation of the intestinal contents. Therefore, an innate
mucosal defense is vital to reduce invasion of pathogens and quickly repair a compromised
epithelial monolayer, facilitated by stem and progenitor cells that allow for rapid epithelial
turnover.

Growth factor receptors and their ligands promote intestinal epithelial cell survival and restitution
(54-57) and regulate important cellular processes that support intestinal epithelial barrier function
(58). Previous studies indicate that growth factor receptors can protect the integrity of this barrier,
preventing paracellular permeability and tight junction disruption in injury models such as
hydrogen peroxide, early weaning, and ethanol (59-63). Furthermore, both growth factor receptors
and junctional components stimulate similar signal transduction pathways (i.e. PI3K and Src) (16,
25), making them potential therapeutic targets for improved epithelial maintenance and repair.

The ErbB4 receptor tyrosine kinase protects colon epithelial cells in the presence of inflammatory
cytokines such as tumor necrosis factor (TNF) and is important in the recovery and repair of the
epithelium after injury (16, 24). ErbB4 is highly expressed in human IBD, while the ErbB4 specific
ligand NRG4 is paradoxically found at low levels in disease (25). Loss of this ligand appears to be

24
part of the disease process, as exogenous NRG4 prevents cytokine-induced apoptosis in cultured
colon epithelial cells and in vivo, by AKT and Src signaling pathways (16, 25). However, it is
unknown if NRG4-ErbB4 signaling regulates the structural components of this barrier in order to
regulate barrier integrity either at homeostasis or during injury and disease.

Similar to NRG4-ErbB4, Hedgehog signaling ligands promote cell survival and proliferation in a
wide variety of tissue types (64, 65). Sonic Hedgehog (Shh), a growth factor ligand that promotes
routine epithelial turnover (64), contributes to intestinal development by epithelial-mesenchyme
interactions through epithelial secretion of ligand, promoting mesenchymal differentiation (66). In
adults, Shh is expressed in endoderm-derived tissues, with greatest expression in the ileum and
proximal colon of the GI tract (67). Shh and its receptor, Ptch1, are expressed along the human
intestinal epithelium, localized to the base of villus or crypt during homeostasis (66). During
intestinal inflammatory conditions, such as CD, a similar expression pattern of Shh and Ptch1 was
demonstrated in the intestinal epithelium, along with increased CD4
+
T cell staining (66).
Furthermore, the loss of intestinal epithelial Shh led to alterations in secretory cell and metabolic
signaling targets (67). Although the direct relationship between Shh signaling and the intestinal
epithelial barrier is unknown, a previous study suggested that Shh is necessary to promote repair
and maintenance of tight junctions in the blood brain barrier (68).

Here we investigated the role of endogenous NRG4 in intestinal barrier function by subjecting
NRG4
-/-
and wildtype (WT) cage mates to an acute lipopolysaccharide (LPS) injection model. Our
results show that NRG4
-/-
mice had worsened inflammation and epithelial permeability, consistent
with a protective role for intestinal NRG4 as previously found in other models of acute injury and

25
inflammation (16, 25, 26). Our data suggest that NRG4 promotes intestinal epithelial barrier
integrity and recovery by limiting intestinal permeability, epithelial apoptosis, and ZO-1
redistribution following LPS-stimulated inflammation. Baseline analysis of barrier components in
NRG4
-/-
mice showed alterations in inflammatory cytokines, Shh signaling, and metabolic
signaling target levels. Together, these findings are potentially indicative of an impaired recovery
response. In summary, this work shows that NRG4 regulates a complex network that promotes
intestinal barrier stabilization, and furthermore that resulting alterations in cellular signaling
mechanisms in NRG4
-/-
mice impair intestinal recovery.

Methods
Animal Study Approval
The use of all animals and animal experiments were approved and monitored by the Children’s
Hospital Los Angeles Animal Care and Use Committee (Animal Welfare Assurance #A3276-01).
All experiments complied with appropriate ethical animal testing and research regulations.

Animal Experiments
NRG4
-/-
mice were purchased from the Mutant Mouse Regional Resource Center (MMRRC) at
University of California, Davis and then bred and maintained at Children’s Hospital Los Angeles.
Males and females from either NRG4
+/-
and/or NRG4
-/-
breeding cages were generated from mice
backcrossed to a C57Bl/6J background for at least 8 generations. Cage mates were housed together
for at least 2 weeks prior to experiments to negate potential microbiota-mediated cage effects.

26
11-12 week-old male and female NRG4
-/-
and WT cage mates, or mice harboring a LoxP-flanked
ErbB4 allele (ErbB4 FF) and intestinal specific ErbB4-deleted mice generated by crossing these
animals to a Villin-Cre driver line (LVC/ErbB4FF), were challenged with 4 kDa (22 mg/ml)
fluorescein isothiocyanate (FITC)-dextran gavage and 10 mg/kg LPS by IP injection. 6-12 week-
old male and female NRG4
-/-
and WT cage mates were used for all other baseline experiments.
Full thickness or intestinal scrapings from the ileum or distal colon were collected for mRNA
isolation and analysis.

Real-Time Quantitative PCR
mRNA from full thickness tissue or colonic organoids was extracted using an on-column total
RNA isolation kit (OMEGA Biotek, R6834-02), and cDNA was generated with a high-capacity
cDNA reverse transcriptase kit (Applied Biosystems, 4368814). Quantitative gene expression
analysis using TaqMan assays (Thermofisher, Table 3) was performed using an Applied
Biosystems StepOne thermocycler. The average fold change, calculated by the 2
−ΔΔCt
method,
represents the gene expression relative to WT control groups with Hprt as the reference gene.

Immunofluorescence Staining
Ileal sections (5 µm) from 4% formaldehyde fixed, paraffin embedded tissue were dewaxed and
blocked with 5% goat serum for 1 h at room temperature. This was followed by an incubation with
primary antibody against ZO-1 (Invitrogen, #61-7300, 1:50) and E-cadherin (BD Biosciences,
#610181, 1:500) overnight at 4 °C. Cells were washed and incubated with secondary anti-mouse
Alexa Fluor-488 (Life Technologies, #A11029, 1:200) or anti-rabbit Alexa Fluor-546 (Life
Technologies, #A11035, 1:200) for 1 h at room temperature. After washing the cells, DAPI

27
(Invitrogen, #D3571, 1:500) was incubated for 15 min, followed by 2 PBS washes, and mounted
with Vectashield Hardset mounting media (Vector Labs, #H-1400).

To assess apoptosis, TUNEL stain (Roche, #11684795910) was performed per manufacturer
instructions on ileal sections from baseline and LPS-treated mice.

RNA Sequencing and Analysis
RNA extracted from NRG4
-/-
and WT distal colon homogenates were barcoded using Illumina
index primers and sequenced on a NextSeq machine to obtain single-ended reads of 75 bp length
to a depth of ~10 M reads/sample by SeqMatic LLC (San Francisco, CA). Kallisto (69) was used
to pseudoalign the FASTQ files to the mouse transcriptome, for estimated transcript per million
abundances. For a pathway enrichment comparison, transcript abundances were summed to obtain
a per-gene expression value, then input into Gene Set Enrichment Analysis (GSEA) (70, 71).

Statistics
Statistical plots and analyses were created using Prism software (GraphPad Inc., La Jolla, CA).
Each dot represents one biological replicate and is portrayed as the mean ± SEM in dot and bar
graphs. Statistical testing includes Student’s two-sided t test or one-way ANOVA, with Tukey’s
post hoc test, as appropriate.

28

Results

NRG4 and ErbB4 are inversely expressed throughout the mouse intestinal
tract
ErbB4 is generally expressed at low levels in all mammalian epithelial tissues (Srinivasan et al.
1998) but is induced by challenge (Frey et al. 2009). However, NRG4 expression patterns along
the GI tract are unknown. To determine regional ErbB4 and Nrg4 levels, we analyzed full thickness
biopsy homogenates from the stomach, duodenum, jejunum, ileum, proximal colon, and distal
colon by qPCR, in adult C57Bl/6J littermates. We observed highest levels of Nrg4 expression in
the duodenum, decreasing distally along the GI tract (Figure 4). Interestingly, these results were
inverse to ErbB4 expression patterns, which (other than high expression in the stomach) increased
distally along the GI tract (Figure 4).

The loss of NRG4 impairs recovery from LPS induced injury
Growth factors receptors, such as ErbB4, are known to promote intestinal epithelial
restitution/wound healing in colonocytes (24). We previously showed that exogenous treatment
with NRG4, an ErbB4-specific ligand, stimulated colonocyte survival in vitro and in vivo (25).
However, the role of endogenous NRG4 in maintenance and repair the intestinal epithelial barrier
is unclear. To test this, we used an LPS-induced injury model to determine barrier integrity and
recovery at 4h and 24h. Although NRG4
-/-
mice showed a significant increase in FITC-dextran
permeability only at 24h after LPS injury (p= 0.03) (Figure 5A), a similar trend was observed at
both baseline and 4h after LPS injury (Figure 5A), when compared to WT cage mates.
Interestingly, the loss of epithelial ErbB4 showed a trend towards decreased intestinal permeability

29
after 24h, compared to their cage mate controls (Figure 5A). Further investigation of this response
by the loss of ligand and loss of receptor may provide insight into the location and tissue specific
signaling mediating these outcomes and intestinal homeostasis.

Increased intestinal permeability and loss of epithelial integrity frequently result from increased
epithelial apoptosis rates. To determine intestinal epithelial recovery after LPS challenge, we
examined apoptosis over time using Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP
nick end labeling (TUNEL) staining. NRG4
-/-
mice displayed a significant increase in TUNEL
positive cells at 24h post-LPS compared to WT (p= 0.033) (Figure 5B), with no difference at either
baseline or 4h. These results are consistent with our permeability results observed at 24h,
indicating a compromised epithelial barrier.

Although LPS-induced injury showed an increase in permeability and epithelial apoptosis at 24h,
there was no difference in inflammatory cytokine levels at either 4 or 24h after injection. However,
interestingly we observed a decrease in baseline levels of Ifn𝛾 (p= 0.0139), Il-1β (p=0.053), and
Il-10 (p=0.048), with a similar trend in Tnf and Il-6, in NRG4
-/-
mice compared to WT cage mates
(Figure 6). Although the LPS injury model is widely used for barrier function analysis, the exact
mode of injury is unclear and likely involves innate immune activation. Thus, we hypothesize that
the baseline inflammatory cytokine defect demonstrated in NRG4
-/-
mice may be overcome after
LPS injections, due to the significant inflammatory response. However, intestinal immune cell
profiles by flow cytometry or ELISA, at different time points, may provide a more comprehensive
analysis.

30
NRG4 deletion caused a delay in ZO-1 redistribution after LPS-induced
injury
The tight junctional protein ZO-1 has previously been shown to be critical for mucosal repair after
injury, with a decrease in expression during IBD (72). Therefore, we wanted to determine ZO-1
expression patterns during intestinal recovery in NRG4
-/-
and WT cage mates after LPS-induced
injury. LPS induced a redistribution from the cell membranes to the cytoplasm by 4h in all mice
(Figure 7A). Localization was partly recovered in WT mice by 24h, but not in NRG4
-/-
cage mates.
Although Tjp1 (codes for ZO-1) RNA expression was not altered at baseline, NRG4
-/-
mice showed
reduced mRNA level relative to WT at 4h (Figure 7B) (p=0.017) after injury, but was not sustained
to 24h. Together, these results indicate that endogenous NRG4 may play a role in the intestinal
epithelial distribution and recovery of ZO-1 after injury.

The loss of NRG4 leads to alterations associated with Sonic Hedgehog (Shh)
signaling during homeostasis
Shh contributes to intestinal development and epithelial repair (66). GSEA Hallmark pathways
comparison of bulk RNA sequencing transcript abundances in NRG4
-/-
and WT colonic
homogenates indicated alterations in Hedgehog signaling (Table 4). Therefore, we investigated
ileal expression of Shh signaling targets during homeostasis. Shh, Gli1 (a downstream transcription
factor and readout of Shh signaling), and Ptch1 (Shh receptor) mRNA levels showed a trend
towards decreased expression in ileal scrapings from NRG4
-/-
mice (Figure 8) versus WT, with no
change in Ihh, another hedgehog ligand implicated in intestinal homeostasis and the intestinal
inflammatory response (73). Further analysis of Shh and/or its downstream signaling molecules
are necessary to determine the interaction of Shh with NRG4.

31

Previously, intestinal epithelial deletion of Shh demonstrated alterations in intestinal secretory
production (67). Intestinal mucus layers are integral for separation and protection of the epithelial
barrier from potentially invasive bacteria. While the small intestine contains a single mucus layer,
the colon has an inner and outer mucus layer, due to intestinal function and bacterial load (74, 75).
Depleted mucus levels are observed during IBD, allowing for significant bacterial translocation to
occur (76, 77). Since intestinal mucus is mainly composed of the secreted glycoprotein Muc2, we
wanted to determine the baseline Muc2 expression levels in NRG4
-/-
mice compared to WT mice
in both the ileum and colon. Small intestinal scrapings showed a significant loss of Muc2 (p=0.002)
in NRG4
-/-
mice compared to WT cage mates, while colonic scrapings demonstrated a trend
towards an increase (Figure 9). Although these data suggest there are differential expression
patterns in the ileum vs the colon, these variable patterns in fact may be due to a compensatory
transcriptional expression associated with Muc2 expression.

Cellular stress responses, such as ER stress and autophagy, are observed with the loss of Muc2
(78) and intestinal epithelial Shh (67). IBD susceptibility is also associated with alterations in
metabolic cellular responses (79, 80). Since appropriate responses to ER stress and functional
autophagy are vital for barrier repair, we examined the expression of key targets for ER stress
(Chop and Ern1) and autophagy (Map1lc3 and Sqstm1), in homeostatic ileal scrapings. NRG4
-/-
mice had significantly reduced Chop (p<0.0001), Map1lc3 (p< 0.001) and Sqstm1 (p= 0.003), with
a similar trend in Ern1 (Figure 10). These deficiencies may contribute to impairments in epithelial
recovery, tissue barrier function, or maintenance of the epithelial barrier. However, further work
will be necessary to determine whether these connections are direct or secondary. Also, it is

32
necessary to further investigate protein expression of these identified ER stress and autophagy
readouts by immunostaining and/or western blots to verify mRNA expression results.

Discussion
The ErbB4 receptor tyrosine kinase is induced during inflammation (24, 25), while its specific
ligand NRG4 is expressed at low levels (25). Herein we report that baseline ErbB4 and Nrg4
expression varies along the intestinal tract (Figure 4), with greatest expression in the distal colon
and duodenum, respectively. Previous literature established a critical role for ErbB4 in the
maintenance of the stem cell compartment (by Paneth cell stabilization) and epithelial structure
(16). This was determined by the observation that intestinal epithelial specific knockout enteroids
have significantly reduced lysozyme, dysmorphic phenotype, and loss of architecture when
challenged with TNF, compared to WT enteroids (16). Furthermore, exogenous NRG4 protects
against the ablation of Paneth cells, when challenged with dithizone/Klebsiella pneumonia
(necrotizing enterocolitis model) (16). It remains unclear if this interaction of NRG4-ErbB4 with
stem cell and/or Paneth cells contributes to intestinal regeneration and/or to the mechanics of
barrier function.

Here we demonstrate that the deletion of endogenous NRG4 impairs intestinal epithelial recovery
from acute LPS induced injury (Figure 5), determined by increased intestinal permeability and
apoptosis (TUNEL) at 24 hours. Epithelial barrier recovery was also determined by localization
and expression of the junctional scaffolding component Tjp1/ZO-1 at 4 hours and 24 hours, with
significant reduction of Tjp1 expression at 4 hours and ZO-1 epithelial redistribution at 24 hours
(Figure 6). The loss of ZO-1 at tight junctions is associated with increased permeability, WNT/β-

33
catenin (a key regulator of the stem cell compartment and involved in epithelial recovery)
dysregulation, and disease (IBD) (72). Similarly, Hedgehog signaling mechanisms can promote
epithelial cell survival and proliferation during intestinal homeostasis and repair (64). We
demonstrate the hedgehog ligand, Shh, and its signaling targets trend towards a decrease in NRG4
null mice (Figure 8). These data are consistent with previous studies, suggesting that ErbB family
members and their ligands are important for response and repair of the intestinal epithelium during
inflammation (25, 27, 54, 57). Together, these results suggest that endogenous NRG4 promotes
intestinal barrier function, possibly through the interaction with Shh signaling.

Although there is little known about the contribution of Shh to homeostatic intestinal barrier
regulation, there is evidence from other systems to suggest a role. Administration of recombinant
Shh after permanent middle cerebral artery occlusion significantly reduced brain edema by the
upregulation of tight junctional proteins ZO-1 and occludin, while blocking Shh signaling reversed
this effect (68). Also, Shh disruption in gastric epithelium caused re-localization of ZO-1 and
breakdown of tight junctions (81), and Shh protects against endotoxin-mediated lung injury (82).
Furthermore, the intestinal epithelial deletion of Shh led to homeostatic dysregulation of goblet
and Paneth cell numbers, alterations in ER stress signaling targets, and decreased autophagy (67).
Similarly, the loss of NRG4 led to baseline alterations in Muc2 expression (Figure 9), ER stress
signaling targets (Figure 10), and reduced autophagy (Figure 10), compared to WT cage mates.
Although further verification of baseline and inflammatory protein expression are necessary for a
definitive assessment of NRG4-Shh barrier alterations, these observations provide a possible
mechanism for the delay in epithelial barrier recovery during acute intestinal injury, as seen by
LPS-injection

34

35

36

37

38

39

40

41
Gene Life Technologies Taqman
Gene Expression Assay

Hprt Mm03024075_m1
ErbB4 Mm01256793_m1
Nrg4 Mm00446254_m1
Tnf Mm00443258_m1
Ifn𝛾 Mm01168134_m1
Il-6 Mm00446190_m1
Il-1β Mm00434228_m1
Il-10 Mm01288386_m1
Tjp1/Zo-1 Mm01320638_m1
Ihh Mm00439613_m1
Shh Mm00436528_m1
Gli1 Mm00494654_m1
Ptch1 Mm00436026_m1
Muc2 Mm01276696_m1
Ddit3/Chop Mm01135937_g1
Ern1 Mm00470233_m1
Map1lc3 Mm00782868_sH
Sqstm1 Mm00436026_m1

Figure 3. Chapter 1 qPCR Assays
Table 3. Chapter 1 qPCR Assays

42

Table 3. qPCR Assays
Table 4. Gsea Pathways Analysis from Bulk RNA Sequencing

43
Chapter 2
Endogenous Neuregulin-4 regulates lymphocyte recruitment by colonic
epithelial St3gal4 during IL-10R neutralizing colitis

Jessica K. Bernard
1,2
, Michael A. Schumacher
1,3
, Cambrian Y. Liu
1,4
, Kay Katada
1
, Mary K.
Washington
5
, and *Mark R. Frey
1,3,6

5. The Saban Research Institute, Children’s Hospital Los Angeles, Los Angeles, CA 90027
6. Craniofacial Biology Program, Herman Ostrow School of Dentistry, University of Southern
California, Los Angeles, CA 90089
7. Department of Pediatrics, University of Southern California Keck School of Medicine, Los
Angeles, CA 90089
8. Department of Medicine, The University of Chicago, Chicago, IL 60637
9. Department of Pathology, Microbiology, and Immunology, Vanderbilt Ingram Cancer
Center, Vanderbilt University Medical Center, Nashville, TN
10. Department of Biochemistry and Molecular Medicine, University of Southern California
Keck School of Medicine, Los Angeles, CA 90089

*Corresponding author
Email: mfrey@chla.usc.edu
Address: Children’s Hospital Los Angeles, 4650 Sunset Blvd. MS#137, Los Angeles, CA 90027
Phone: 323-361-7204

44
Disclosures: Dr. Frey and Dr. Schumacher are inventors on a patent held by Children's Hospital
Los Angeles on the possible therapeutic use of NRG4 in intestinal inflammation. No other
conflicts.

Abstract
Growth factors are essential for maintenance of intestinal health at homeostasis and during
inflammation. We have previously shown that exogenous treatment with the growth factor
neuregulin-4 (NRG4) promotes colonocyte survival during cytokine challenge and is protective in
acute models of intestinal inflammation. However, the role of endogenously expressed NRG4 in
colitis is not well understood. Using NRG4
-/-
mice, we tested the effect of endogenous NRG4 on
colitis. NRG4
-/-
and wild type cage-mate mice were subjected to an IL-10R neutralization model
of chronic colitis. NRG4
-/-
mice displayed reduced inflammatory cytokine expression in the colon
and a reduction in tissue CD8
+
T cells at 5 weeks post induction of colitis. In unchallenged
NRG4
-/-
mice, baseline numbers of colonic CD8
+
T cell numbers were unchanged. However, there
was a significant decrease in splenic CD8
+
T cells. RNA sequencing from colonic homogenates
showed a loss of St3gal4, a sialyltransferase involved in immune cell trafficking, in the NRG4-
null animals. This loss was verified in both tissue and epithelium. The regulation of St3gal4 by
NRG4 was confirmed with ex vivo epithelial colon organoid cultures from NRG4
-/-
mice and by
induction of St3gal4 in vivo following NRG4 treatment. Together our data suggest that NRG4
regulates colonic epithelial ST3GAL4 and thus the recruitment of CD8
+
T cells, but also has
systemic effects on T cell maturation. These effects may combine to stimulate immune mediated
inflammation during IL-10R neutralization colitis.

45
Introduction

Neuregulin-4 (NRG4) is a member of the neuregulin subfamily of EGF-like growth factors that
activate ErbB receptor tyrosine kinases. In particular, NRG4 is a selective ligand for ErbB4 with
negligible reported binding to other ErbB family receptors (27). A variety of tissue responses have
been described for this ligand, including context-dependent regulation of cellular apoptosis (17,
25), control of cytokine expression (28, 83) tuning of energy metabolism and insulin susceptibility
(84), and others. We previously showed that NRG4 levels in the colon are reduced in mouse
models of colitis and in inflammatory bowel disease (IBD) (25), and that the inflammatory
cytokine tumor necrosis factor (TNF) downregulates its expression.

Treatment with exogenous NRG4 protects against intestinal inflammation in several experimental
models (16, 17, 25). Furthermore, deletion of the NRG4 receptor ErbB4 in either macrophages
(17) or the intestinal epithelium (26) worsens chemically-induced experimental colitis. However,
the role of endogenously expressed NRG4 in regulating colonic epithelial function, gene
expression, and response to insult is unknown, as is the role of NRG4 signaling in adaptive immune
response-driven colitis.

IBD is a multifaceted disease that broadly involves genetic susceptibility, aberrant immune and
epithelial responses, and dysregulation of the intestinal microbiome. One specific example of the
complexity of the pathogenesis of IBD is with the alteration of interleukin (IL)-10 signaling. IL-
10 is essential to dampen both innate and adaptive immune responses, and genetic mutations in
IL-10 signaling are associated with early onset IBD in children (85-87). In mice, experimental

46
disruption of IL-10 signaling drives colitis and can promote a dysregulated, colitogenic microbiota
(88-91).

ST3GAL4 is a β-galactoside α2,3-sialyltransferase that regulates production of 𝛼2,3 sialyllactose
(3SL) by mediating the addition of the sialic acid N-acetylneuaminic acid to lactose (92). 3SL can
in turn stimulate Th1/Th17 proinflammatory responses (93), linking this pathway to immunity and
inflammation. ST3GAL4 is also involved in the formation of selectin ligands (94) necessary for
immune cell function, and thus may regulate the development of localized immune responses.
Both E-selectin and Sialyl Lewis X, one known selectin binding partner, are expressed during
active IBD (95, 96). Furthermore, ST3GAL4 deficient mice show reduced disease in acute dextran
sodium sulfate and IL-10
-/-
colitis mouse models (92, 93).

Here we tested role of endogenous NRG4 in colitis by subjecting NRG4
-/-
mice to an immune-
mediated (IL-10R neutralization) model of colitis (97). In contrast to the protective role of NRG4
and ErbB4 signaling in other colitis models (16, 25, 26), NRG4 signaling appeared to enhance
disease in the IL-10R neutralization colitis model. NRG4
-/-
mice exhibit reduced levels of
ST3GAL4, reduced T cell infiltration to the colon, reduced inflammatory cytokine levels, and a
shift in intestinal microbial species that may favor an anti-inflammatory profile. Overall, our
results suggest that while NRG4-ErbB4 signaling reduces innate immunity-driven inflammation
(17, 28) and protects the epithelium from injury (16, 25), it also participates in T cell recruitment
during inflammation driven by disruption of IL-10 signaling. Thus, NRG4 participates in both
protective and pathogenic signaling in colonic inflammation and understanding the balance

47
between these will be key for future therapeutic targeting of this axis in IBD and other
inflammatory disorders.

Methods
Animal Study Approval
The use of all animals and animal experiments were approved and monitored by the Children’s
Hospital Los Angeles Animal Care and Use Committee (Animal Welfare Assurance #A3276-01).
All experiments complied with appropriate ethical animal testing and research regulations.

Animal Experiments
NRG4
-/-
mice were purchased from the Mutant Mouse Regional Resource Center (MMRRC) at
University of California, Davis and then bred and maintained at Children’s Hospital Los Angeles.
Males and females from either NRG4
+/-
and/or NRG4
-/-
breeding cages were generated from mice
backcrossed to a C57Bl/6 background for at least 8 generations. Male and Female cage mates were
housed together for at least 2 weeks prior to experiments.

5 week-old NRG4
-/-
and WT controls were used for colonic baseline flow cytometry analysis, while
6-12 week-old mice were used for all other baseline experiments. For NRG4 rescue experiments,
6 week-old NRG4
-/-
mice were i.p. injected with either 100 µg/kg NRG4 (Reprokine, Q8WWG1)
or PBS for 24 hours. Full thickness distal colon was collected, and flash frozen for later mRNA
isolation and analysis.

48
For 16S analysis, stool was collected at both 5 weeks and 9 weeks from 6 WT and 6 NRG4
-/-
cage
mates, along with WT and NRG4
-/-
dam stool that was collected at weaning. Colonic scrapings
were collected on week 9 at euthanasia. All stool and scrapings were stored at -20 degrees until
16s processing and analysis.

For IL-10R neutralization, 5 week-old NRG4
-/-
and WT controls were i.p. injected with 1 mg/kg
InVivoMAb anti-mouse IL-10R (CD210) antibody (BioXcell, BE0050) weekly, for 5 weeks (97).
All mice were euthanized on week 5 (one week after the last injection). Stool was collected weekly
for Lipocalin-2 analysis. Post-mortem plasma from blood collected by cardiac puncture was used
to analyze myeloperoxidase. Full thickness distal colon was collected, and flash frozen for RNA
analysis. Swiss rolls of each colon were formaldehyde fixed and paraffin embedded for histology
scoring similar to (98). Histology scores (0-3) established from the amount and depth of
inflammation, the percentage of inflamed crypts, crypt damage, and the percentage of crypts
involved in crypt damage were determined by an experimentally blinded pathologist. The total
histology score is the sum of each category score.

ELISAS
Fecal lipocalin-2 was analyzed from homogenized fecal contents using Mouse Lipocalin-2 DuoSet
ELISA (R&D systems, DY1857), per manufacturer instructions. Myeloperoxidase levels were
analyzed by using 10% of plasma, in PBS, from post-mortem cardiac puncture by Mouse
Myeloperoxidase DuoSet ELISA (R&D systems, DY3667), per manufacturer instructions.

49
Real-Time Quantitative PCR
mRNA from full thickness tissue or colonic organoids was extracted using an on-column total
RNA isolation kit (OMEGA Biotek, R6834-02), and cDNA was generated with a high-capacity
cDNA reverse transcriptase kit (Applied Biosystems, 4368814). Quantitative gene expression
analysis using TaqMan assays (Thermo-Fisher, Table 5) was performed using an Applied
Biosystems StepOne thermocycler. The average fold change, calculated by the 2
−ΔΔCt
method,
represents the gene expression relative to WT or PBS control groups as appropriate, with Hprt as
the reference gene.

Microbial 16S rRNA Community Profiling
Cage mate mice of wildtype or NRG4
-/-
genotypes were weaned and co-housed. Fresh fecal pellets
were obtained from mice at 5 and 9 weeks of age. Mice were euthanized and the colonic mucosa
scraped with a razor blade to obtain a representation of the mucosal adherent microbiome at 9
weeks old. Fecal and tissue samples were flash frozen and stored at -80°C prior to profiling.

Sequencing of the V4 16S rRNA bacterial variable region using PCR primers targeted to 515/806
was performed by MR DNA (Shallowater, TX, USA). After 30 cycles of PCR amplification,
samples were multiplexed and pooled for generation of an Illumina DNA library. Sequencing was
performed on the MiSeq platform following the manufacturer’s (Illumina) guidelines. Sequence
data were processed by MR DNA. Sequences that were <150 bp or containing ambiguous bases
were filtered out; the remaining sequences were quality filtered using a maximum expected error
threshold of 1.0, dereplicated, denoised, and filtered to remove chimeras and point errors. The
resulting zOTUs were taxonomically classified using BLASTn against a closed-reference, curated

50
database derived from NCBI (www.ncbi.nlm.nih.gov) to obtain species-level nearest-neighbor
annotations, when possible.

Raw and percentage-normalized abundance matrices were loaded into R for analysis and principal
component visualization. Overall taxonomic diversity was quantified by generating rarefaction
curves using the vegan 2.5-7 package. The PERMANOVA routine was used to perform statistical
evaluations of genotype-based and other metadata-based associations of microbial community
structure. The randomforest 4.7-1 package was used to identify individual taxa that facilitate
discrimination of wildtype and NRG4
-/-
stool microbiomes.

RNAScope In Situ Hybridization
Sections from paraffin embedded, colonic swiss rolls were probed using an RNAScope Mm-
ST3GAL4 probe (Advanced Cells Diagnostics, 543061), and processed using the RNAScope 2.5
HD Detection (Advanced Cell Diagnostics, 322310), per manufacturer’s protocol.

Colonic Organoids
Colonic organoids (“colonoids”) were produced and passaged from NRG4
-/-
and WT mouse
colons, based on previously published protocols (99-101). Briefly, colonic crypts were extracted
by an incubation in chelation buffer (2 mM EDTA and 43.4 mM sucrose in D-PBS), followed by
trituration in a shaking buffer (43.8 mM sucrose and 54.9 mM sorbitol in PBS). Isolated crypts
were then embedded in Matrigel (BD Biosciences) and overlaid with Intesticult (StemCell
Technologies). For experiments, cultures were starved in DMEM and collected after 24 hours.

51
RNA Sequencing and Analysis
RNA extracted from NRG4
-/-
and WT distal colon homogenates were barcoded using Illumina
index primers and sequenced on a NextSeq machine to obtain single-ended reads of 75 bp length
to a depth of ~10 M reads/sample by SeqMatic LLC (San Francisco, CA). Kallisto (69) was used
to pseudoalign the FASTQ files to the mouse transcriptome, for estimated transcript per million
abundances. For a pathway enrichment comparison, transcript abundances were summed to obtain
a per-gene expression value, then input into enrichr (102-104). Immune cell population estimates
were determined by CIBERSORT (http://cibersort.stanford.edu), using the LM22 signature per
100 permutations (105). These results were normalized as the relative abundance to the total
predicted cells.

Single Cell Mass Cytometry (CyTof) Processing and Analysis
For CyTof preparation, colonic mucosal peelings from 4 WT and 5 NRG4
-/-
cage mates were
extracted under a dissection microscope on week 5 of IL-10R neutralization colitis. Separated
peelings were segmented and incubated in a digestion solution (0.2 Wunsch units/ml Liberase TM
+ 200 Kuntz units/ml DNase I in DMEM/12 +15mM HEPES) at 37 degrees for 30 min, with
continuous shaking at 175 rpm. A neutralizing solution (DMEM/F12/HEPES + 10%FBS) was
added to the digested tissue, and the tissue was triturated and passed through a 70-μm-pore cell
strainer. The strainer was then washed with neutralization solution and isolated cells/solution were
centrifuged at 300 x g for 8 min. After washing the pellet in a FACS buffer (HEPES-buffered
saline + 1% BSA) and centrifuged, an RBC lysis solution (Biolegend, 420301) was used to
resuspend the cells for 5 min at room temperature. This was followed by two more FACS buffer
washes and centrifugation steps. The pellet was then passed through a 40-μm-pore cell strainer,

52
and dead cells were removed using EasySep Dead Cell Removal Kit (StemCell Technologies), per
manufacturer’s instructions. Live cells were resuspended in PROT1 fixative (Smart Tube Inc.)
prior to processing by the Children’s Hospital Los Angeles Single Cell, Sequencing, and CyTof
Core using the Fluidigm Maxpar staining protocols of a mouse immune cell panel (Table 6).
Stained cells were run on a Fluidigm Helios operating system.

Flow cytometry
Colons and spleens from 6 NRG
-/-
and 6 WT cage mates were extracted and processed similar to
previously reported methods (106). For colons, dissected tissues were segmented into 2mm pieces
and placed in digestion solution (0.2 Wunsch units/ml Liberase TM + 200 Kuntz units/ml DNase
I in DMEM/12 +15mM HEPES) for 30 min at 37°C with continuous 180 x g agitation. Segmented
tissue was then triturated, passed through a 70-μm-pore cell strainer, washed with DMEM:F12 +
10% FBS, and with HEPES-buffered saline (107) with 0.5% BSA.

Cells were isolated from spleens using a method similar to StemCell Technologies T cell isolation
extraction method (www.stemcell.com). Extracted spleens were washed in EasySep Buffer
(StemCell Technologies, 20144), minced on a 70-μm-pore cell strainer, and washed with EasySep
Buffer. Cells were centrifuged at 300 x g at 4°C for 10 min. This was followed by a 5-10 min RBC
lysis and wash with PBS. After centrifugation, cells from the spleens and colons were processed
at the same time.

53
Cells were blocked for 15 min at 4°C with 5% mouse/rat serum + Fc block (anti-CD16/32
antibody, Biolegend “truStain fx”), and probed with a mixture of preconjugated antibodies for
30 min at 4°C. Antibodies (working dilutions) were targeted to CD45 (Biolegend 103107; 1:250),
CD8 (Biolegend 100751; 1:100), CD4 (Biolegend 100451; 1:200), and CD3 (BD Biosciences
563565; 1:200), followed by washes. Propidium iodide (Life Technologies, R37108) was added
to cells before analysis on a BD LSR II. Compensation was adjusted using references obtained by
the analysis of single antibodies to unstained cells.

Flow cytometry analysis was performed by FlowJo (FlowJo, LLC). Gating was based on the
proportion live cells per CD45+ cells.

Statistics
Statistical plots and analyses were created using Prism software (GraphPad Ince., La Jolla, CA).
Each dot represents one biological replicate and is portrayed as the mean ± SEM in dot and bar
graphs. Statistical testing includes Student’s two-sided t test, one-way ANOVA, and two-way
ANOVA, with Tukey’s post hoc test or Kruskal-Wallis test, as appropriate.

54
Results
Whole body NRG4 deletion protects against IL-10R neutralization-mediated
chronic colitis.
Colonic neuregulin-4 (NRG4) expression is reduced in IBD and in experimental inflammation
models (e.g., DSS colitis, TNF exposure). We previously showed that administration of
exogenous NRG4 reduces injury and inflammation in DSS colitis (25). However, the role of
endogenously expressed NRG4 in the response to colonic injury and inflammation, and NRG4’s
role in a more T cell-dependent disease, are unknown. To test the role of endogenous NRG4 in a
T cell driven chronic colitis model, we injected NRG4
-/-
mice and wildtype cage mates with IL-
10R neutralizing antibody (97) weekly for 4 weeks. While both genotypes developed colitis
within one week as shown by fecal lipocalin-2 levels (Fig 11A), the response was significantly
attenuated in NRG4
-/-
animals compared to wildtypes, especially early in disease development
(Figure 11D). After 5 weeks, NRG4
-/-
mice continued to show reduced markers of ongoing
inflammation, including plasma levels of the neutrophil-produced factor myeloperoxidase
(p=0.0005) and spleen weight (p=0.001), compared to wildtype cage-mates (Figure 12D-E). This
correlated with reduced tissue pathology scores (measuring epithelial damage and immune cell
influx) in NRG4
-/-
mice (p= 0.03) (Figure 12B-C).

55
Inflammatory cytokines and CD8+ T cell infiltration are reduced in chronic
colitis in NRG4
-/-
mice.
The chronic colitis mediated by IL-10R neutralization involves substantial recruitment of both
innate and adaptive immune cells that produce inflammatory cytokines including TNF, IFN-g, IL-
1b, and IL-6 (97). To test whether the reduced severity of colitis is associated with altered immune
cell responses, we assessed levels of these cytokines in colonic homogenates at baseline and after
IL-10R treatments. Baseline relative mRNA analysis indicated no difference in cytokine
production (Figure 12A), but after IL-10R neutralization colitis IFN-g (p=0.0068) and IL-1b
(p=0.033) levels were significantly lower in NRG4
-/-
colons versus wildtype (Figure 12B). As both
IFN-g and IL-1b are T cell produced cytokines, we next asked whether this change in cytokine
level represented an altered immune cell profile. Using CyTOF analysis of whole colon
homogenates 5 weeks after IL-10R injury, we found that NRG4
-/-
colons contained significantly
reduced CD8
+
T cell populations (p=0.018), with a trend in reduced numbers of CD45
+
immune
cells overall (Figure 12C).

NRG4 deletion limits splenic CD8
+
T cells but does not alter intestinal T cell
populations at homeostasis.
To test if baseline immune population shifts might contribute to the altered T cell immune profile
in colitic NRG4
-/-
mice, we quantified T cell populations in colon and spleen of 5 week-old animals
by flow cytometry. This analysis found no difference in basal intestinal T cell numbers (Figure
13B) in the colons of NRG4
-/-
mice versus wildtype cage mates, consistent with no change in
baseline colonic cytokine levels (Figure 2B). This correlated with colonic immune cell profiles
predicted by CIBERSORT (Figure 3C), a computational analysis of bulk RNA sequencing data to

56
detect immune cell signatures. In contrast to the findings in colon, however, we saw a significant
reduction in splenic CD8
+
T cell numbers in NRG4
-/-
mice (p=0.02) (Figure 13A). These results
suggest an altered systemic T cell repertoire in the NRG4
-/-
mice, which may contribute to reduced
overall inflammation during IL-10R neutralization colitis.

NRG4 promotes ST3GAL4 expression in the colonic epithelium.
To identify candidate mediators of the differential susceptibility to colitis in NRG4
-/-
and WT cage
mates, we investigated baseline transcriptomic changes in an unbiased manner by bulk RNA
sequencing of colonic tissue. We analyzed the most significant changes by volcano plot as shown
in Figure 4B. Highly decreased transcripts in NGR4
-/-
mice included St3gal4, Nxpe4, and Hsp8,
with a significant increase in Bco2 (Figure 14A-B). Several of these genes are generally altered
during inflammation (St3gal4: (108); Bco2:(109)) or have been previously identified to change
expression level in IBD (Nxpe4:(110); Hspa8:(111)). The most significant alteration was the loss
of St3gal4 (p=0.0008). Furthermore, a BioPlanet (112) 2019 pathways catalogue, using an EnrichR
algorithm, of NRG4
-/-
downregulated genes indicated a significant loss in membrane trafficking,
immune system signaling, and O-glycan biosynthesis, known functions of ST3GAL4 and/or other
sialyltransferases (Figure 14C) (113, 114).

As St3gal4 was the largest and most significant change, we next wanted to determine if a direct
causal regulatory relationship exists between NRG4 and St3gal4. First, we verified and extended
our RNA sequencing results. St3gal4 localization by in situ hybridization showed a striking
absence of expression in the colonic epithelial crypts of NRG4
-/-
mice (Figure 15A), also
quantitated by qPCR analysis of colonic tissue and colonic organoids. We found a significant

57
decrease in St3gal4 expression in NRG4
-/-
mice versus wildtype in both whole tissue (p=0.002)
and isolated colonic epithelium (p= 0.0007) (Figure 15B-C). Furthermore, the loss of tissue
St3gal4 was also seen after IL-10R colitis in NRG4
-/-
mice compared to wildtype (p<0.0001)
(Figure 15D).

Next, we wanted to determine if exogenous treatment with NRG4 could rescue St3gal4 loss in
NRG4
-/-
colons, and thus show a direct interaction. We injected NRG4
-/-
mice i.p. with 100 µg/kg
NRG4 or PBS. After 24 hours, this treatment resulted in a partial rescue of St3gal4 expression (p=
0.0011) in colonic homogenates (Figure 15E). These results show a direct regulation of epithelial
St3gal4 by NRG4 induced signaling.

NRG4 may limit microbial diversity.
Dysbiosis of the intestinal microbiota is observed in IBD. Since ST3GAL4 and 3SL are known to
impact early-life bacterial colonization and microbial composition (92, 108), we tested whether
microbial taxonomic composition was altered in NRG4
-/-
mice compared to WT cage mates. We
sampled the 16S rRNA community sequences of the stool microbiome at 5 wks-old and at 9 wks-
old, and of the mucosa-associated microbiome at 9 wks-old. We found a trend towards increased
taxonomic diversity in both stool at 5 wks-old (p= 0.09) and 9 wks-old (p= 0.06) in NRG4
-/-
mice
compared to WT cage mates (Figure 16A). At 5 wks-old and 9 wks-old, the stool microbiomes
appeared globally similar on a principal component plot (Figure 16B). The composition of the
mucosa-associated microbiome (intestinal scrapings) at 9 wks-old also appeared globally similar.
Random forest analysis of individual species (Figure 16C-D) showed that NRG4
-/-
mice had
modest elevations in some bacteria that are known to be protective in colitis, such as Lactobacillus

58
spp. (115), Butyriciccus pullicaecorum (116), and Barnesiella spp (117). Taken together, these
data suggest that NRG4 loss is associated with subtle changes in the stool microbiome consistent
with an anti-inflammatory profile marked by slight elevations in bacterial diversity and
abundances of bacterial symbionts.

Discussion
Growth factors play a multitude of roles in maintaining intestinal homeostasis and promoting
appropriate responses to injury and inflammation. NRG4 is widely expressed in various tissues of
the body and multiple cell types in the intestine. We have previously shown that intestinal NRG4
levels are reduced during colitis, and that administration of exogenous NRG4 limits disease (16,
25) by preventing epithelial apoptosis (25) and suppressing inflammatory macrophage activity (17,
28). Here, we used both unbiased and targeted approaches to understand the role of endogenously
expressed NRG4 in colonic homeostasis. Furthermore, we describe how whole-body deletion of
NRG4 affects chronic, adaptive immunity-driven colitis.

Our data demonstrate that NRG4 loss leads to reduced colonic CD8+ T cell recruitment in IL-10R
neutralization colitis. NRG4
-/-
mice also showed significantly lower expression of inflammatory
cytokines IL-1β and IFN𝛾 (cytokines produced by T cells) during disease. These parameters were
not different in unchallenged NRG4
-/-
versus WT cage-mate mice, suggesting that NRG4 does not
directly alter homeostatic colonic immune populations. Interestingly, while colonic T cell
populations were unchanged in unchallenged mice, splenic CD8
+
T cell numbers were
significantly decreased in NRG4
-/-
mice at baseline. As the spleen is an important site of T cell
development (45), we can speculate that NRG4 regulation of the micro-environment in this organ

59
can alter the readiness of these cells for recruitment to the colon in response to a pro-inflammatory
signal.

Given previous work from our lab and others showing that both exogenous NRG4 and its receptor
ErbB4 are protective in experimental colitis (17, 25, 26, 28), it was somewhat surprising to see
protection against IL-10R neutralization colitis. However, most growth factor and cytokine
signaling pathways have pleiotropic effects that can be context, concentration, or localization
dependent. It may be that the level of signal (endogenous versus pharmacological), specific
immune context (primarily innate immunity and ulceration versus adaptive immune-driven
inflammation), or timing (acute versus sustained/chronic), or some combination of these, is critical
for defining outcomes. Alternatively, given our findings in spleen (Figure 13), it is possible that
NRG4 plays a long-term developmental role in the immune system. Future studies to distinguish
between these possibilities will require additional tools such as tissue-specific and inducible
deletion models.

Multiple T cell subtypes (e.g., cytotoxic CD8+ T cells, memory CD8+ T cells) participate in the
colonic immune response (118). Although our results clearly show systemic NRG4’s effect on
CD8+ cells (Figures 12 & 13), future work will be necessary to understand the precise mechanism
of action since we did not detect the NRG4 receptor ErbB4 on T cells (17). Cytotoxic CD8+ T
cells participate in IBD pathogenesis (119-121), whereas tissue resident memory CD8 T cells may
either be pro- or anti-inflammatory (118, 122). Future work to clarify the effect of NRG4 on these
sub-types, and whether this response is directly occurring in peripheral and lymphoid tissues, will
be important.

60
We found that whole body NRG4 deletion leads to changes in the colonic gene expression profile.
Strikingly, NRG4
-/-
colons showed a dramatic loss of epithelial St3gal4, which was partially
rescued by exogenous NRG4 treatment (Figure 15). This was accompanied by reduced T cell
recruitment, as discussed above. Impaired leukocyte infiltration and reduced colitis was previously
reported in murine models after deletion of St3gal4. In this setting, colitis sensitivity was rescued
by treatment with the ST3GAL4 activity product ⍺2,3-sialyllactose (3SL) (92, 93). Kurakevich et
al. also determined that 3SL upregulated TGF-β (a known T cell regulator and an inhibitor of P-
selectin (123), and activated dendritic cells isolated from the mesenteric lymph nodes.
Furthermore, functional human endothelial ST3GAL4 studies showed that ST3GAL4 is the
primary sialyltransferase that regulates the synthesis of E-, P-, and L-selectin ligands (124). Taken
together, these results suggest that the loss of epithelial ST3GAL4 after NRG4 deletion could
inhibit immune cell recruitment and infiltration during IL-10R neutralization mediated injury. T
cell recruitment could be pathogenic or protective depending on context. Thus, future studies will
assess the effects of endogenous NRG4 in other models.

Interaction between NRG4 and ST3GAL4 in the intestine has not previously been identified. Both
NRG4 and ST3GAL4 are produced and regulated by many cells and tissues (125, 126). We
previously showed that NRG4 is an important factor that is produced in breast milk that promotes
early life intestinal health (16). Similarly, ST3GAL4 and 3SL are critical factors in breast milk,
components of milk oligosaccharide signaling mechanisms, that produce a plethora of systemic
effects including the regulation of the immune response (126).

61
As ST3GAL4 plays a role in mucin glycosylation (127), and therefore may alter the function of
mucus and its effects on the microbiota, we performed analysis of fecal and adherent microbiota
in NRG4
-/-
mice. Previous work by Fuhrer et al. demonstrated a significant decrease in
Ruminococcaceae at baseline in ST3GAL4
-/-
mice, as determined both by the cross-fostering of
WT and ST3GAL4
-/-
litters and the microbial reconstitution of germ-free mice. Ruminococcaceae
are abundantly found in IBD patients (128-130) compared to normal controls. Though NRG4
-/-

mice did not display major shifts in bacterial species, a few previously identified protective species
showed a trend towards elevation with NRG4 deletion, as determined by random forest plot
(Figure 16). NRG4-null mice also demonstrated an increase in related diversity in stool at 5 weeks
(p=0.09) and 9 weeks (p=0.06). Together, this suggests a potentially healthier result to intestinal
injury. Furthermore, we demonstrated that epithelial ST3GAL4 is expressed within proximity to
both mucus and bacteria, and is lost in NRG4
-/-
mice (Figure 15). Together, these data suggest that
NRG4 could promote selective bacterial colonization and diversity possibly through epithelial
ST3GAL4. Thus, NRG4 may support specific systemic elements of immune-mediated
inflammation, such as IL-10R neutralizing colitis.

In this study, we have shown that NRG4 regulates intestinal epithelial ST3GAL4 expression and
may play a role in splenic T cell maturation and recruitment of CD8+ T cells to the intestine during
inflammation. Through the loss of these factors, NRG4-null mice show a reduction in overall
inflammation in IL-10R neutralization colitis. These findings provide important insight into the
tissue specific effects of NRG4. Though our previous work showed that NRG4 directly promotes
survival of intestinal epithelial cells and death of inflammatory macrophages in the colon, our
current results suggest that developmental extra-intestinal effects of this growth factor, such as in

62
T cell development in lymphoid tissue, add a layer of complexity to how this growth factor can
regulate the body’s immune responses.

63

WT
A B
C
D
NRG4
-/-
E
IL-10R neutralization-induced colitis
Figure 11. Whole body NRG4 deletion leads to reduced IL-10R neutralization-
induced inflammation in the colon. 5 week-old NRG
-/-
and WT cage mates were injected
with IL-10R neutralizing antibody (1mg/kg) for 4 weeks. A. Hematoxylin and Eosin-
stained sections from paraffin embedded colonic swiss rolls collected on experimental
week 5 B. Total colonic damage score; n=7 WT and n=12 NRG4
-/-
. Analyzed by Kruskal-
Wallis test. C. Fecal Lipocalin-2 ELISA (****p<0.0001, **p<0.001); n=10 WT and n=19
NRG4
-/-
analyzed by two-way ANOVA. D. Post-mortem plasma MPO concentration
levels by ELISA (** p=0.0005); n=5 WT and n=8 NRG4
-/-
. E. Total spleen weight
(**p=0.001). Data are presented as the mean ± SEM. Statistical testing by Student’s two-
sided t-tests.

64

A
B
% total cells
% total cells
% total cells
% total cells
% total cells
Figure 12. NRG4 deletion reduces inflammatory cytokines and CD8+ T cell infiltration
in IL-10R neutralization colitis. A. Relative mRNA levels (qPCR) of Tnf, Ifng, Il1b, and Il6
from full thickness colonic homogenates after 5 weeks of IL-10R neutralization colitis,
compared with baseline colon homogenates; n=10 baseline WT, n= 10 baseline NRG4
-/-
, n=8
colitic WT, and n=12 colitic NRG4
-/-
, analyzed by one-way ANOVA. The following outliers
were identified and removed by ROUT testing (Q=1.0%): Tnf (1 from the WT control group),
Ifng (1 from the NRG4
-/-
control group), Il1b (1 from the WT control and 2 from the NRG4
-/-

colitis groups), and Il6 (1 from the NRG4
-/-
control and 1 from NRG4
-/-
colitis groups); IL-1.
B. CyTof analysis of colonic cells isolated after IL-10R induced injury, and gating by FlowJo
software (*p=0.018). Data are presented as the mean ± SEM. Statistical testing used Student’s
two-sided t-tests.

65

A
B
C
WT
NRG4
-/-
Figure 13. NRG4 deletion limits splenic CD8
+
T cells. A-B. Splenic and colonic
cells were isolated from 5 week-old NRG4
-/-
mice and WT cage mates. Flow
cytometry was gated and analyzed by FlowJo software, based on the proportion of
live cells per positive CD45 cells; n=4 WT and n=4 NRG4
-/-
. C. Baseline RNA
sequencing from colonic biopsies in adult NRG4
-/-
and WT cage mates. The predicted
immune cell abundances were analyzed by CIBERSORT; n=6 WT and n=5 NRG4
-/-
. Data are presented as the mean ± SEM. Analyzed by Student’s two-sided t test.

66

-/- WT -/- WT
A B
C
Figure 14. RNA-sequencing analysis indicates significant loss of St3gal4 in mice
with NRG4 deletion. RNA-sequencing of full thickness colonic biopsies from n=5
NRG4
-/-
and n=6 WT cage mates aged 11-12 weeks. A. Heat map of expression levels
of altered genes. Red indicates an increase in relative expression and blue indicates a
decrease in relative expression, as shown in the figure legend. B. A volcano plot of
altered genes identified by RNA-sequencing; St3gal4 p=0.0008. C. Pathway analysis of
downregulated genes that are above 2 -log10 p-value by BioPlanet_2019 in NRG4
-/-
mice
compared to WT cage mates.

67

WT NRG4
-/-
A
B
C D
Figure 15. NRG4 deletion leads to the loss of colonic epithelial ST3GAL4. A. St3gal4
in situ hybridization from adult NRG4
-/-
and WT colonic swiss rolls sections at
homeostasis. B. Relative St3gal4 mRNA expression from NRG4
-/-
and WT colonic
organoids after starvation (***p=0.0007), analyzed by Student’s two-sided t-test. C.
Relative St3gal4 mRNA expression from full thickness NRG4
-/-
and WT colonic
homogenates at baseline and after IL-10R neutralization colitis. Statistical analysis by one-
way ANOVA, with 1 outlier from the NRG4
-/-
control group was identified and removed
by ROUT testing (Q=1.0%). D. NRG4
-/-
mice were i.p. injected with NRG4 (100 µg/kg)
or PBS for 24 hours. Relative St3gal4 mRNA expression from n=4 NRG4
-/-
and n=4 WT
full thickness colonic biopsies (**p=0.0011). Statistical testing used Student’s two-sided
t-tests.

68

A
B
C
C
D
Figure 16. The loss of NRG4 may shift microbial composition, promoting an increase in
known protective bacterial species. 6 WT mice (WT breeding cage) and 6 NRG4
-/-
mice
(NRG4
-/-
breeding cage) were weaned together at approximately 3 weeks of age, in 3 different
cages. Stool was collected at 5 weeks and 9 weeks of age. Stool from WT and NRG4
-/-
dams
were collected at litter weaning. Colonic scrapings were collected at 9 weeks. A. 16s analysis
of microbial diversity for each age and genotype visualized from rarefaction. Statistical
analysis using Student’s two-sided t-test; Stool 5 weeks (p= 0.09) and 9 weeks (p= 0.06) B.
Principal component analysis (PCA) of microbial abundances for each age and genotype, as
compared with each dam. C. Random Forest techniques identify discriminating taxa as those
whose elimination reduces the accuracy of the classifier for the tested NRG4
-/-
and WT
genotypes. D. The abundance, as determined by the percentage of biomass, from
discriminating species predicted by the random forest methods in WT and NRG4
-/-
cage
mates. Statistical testing by Student’s two-sided t-test.

69

Gene Life Technologies Taqman Gene Expression
Assay
Hprt Mm03024075_m1
St3gal4 Mm00501503_m1
Tnf Mm00443258_m1
Il-1β Mm00434228_m1
Il-6 Mm00446190_m1
Ifn! Mm01168134_m1
Table 5. qPCR Assays
Table 5. Chapter 2 qPCR Assays

70

Table 6. Cell Cytometry (CyTof) Mouse Immune Cell Panel
Reactivity Label Vendor Catalog
CD45 89Y Fluidigm 3089005B
CD11b 115/113In Biolegend 101249
GD2 139La Biolegend 357302
CD25 141Pr Biolegend 102002
Ly6G 142Nd Biolegend 127602
CD8a 143Nd Biolegend 100702
CD19 144Nd Biolegend 115502
CD69 145Nd Biolegend 104502
T-bet 147Sm Biolegend 644802
CD140a (PDGFα) 148Nd
Biolegend 135902
CD4 149Sm Biolegend 100506
CD366 (TIM-3) 150Nd Biolegend 119702
CD206 151Eu Biolegend 141702
CD279 152Sm Biolegend 135202
Ly6C 153Eu Biolegend 128002
CD62L 154Sm Biolegend 104402
FSP1 155Gd Abcam ab220213
CD223 (LAG-3) 156Gd Biolegend 125204
CD31 158Gd Biolegend 102402
CD274 (PD-L1) 159Tb Biolegend 124302
CD317 160Gd
Biolegend 127002
FAP 161Dy Sigma MABC1145
CD170 (SiglecF) 163Dy
R&D Systems 750620
Eomes 164Dy
R&D Systems MAB8889
FoxP3 165Ho Biolegend 320002
CD326 (EpCAM) 166Er Biolegend
118202
TGFβ (LAP) 167Er Biolegend 141402
CD161 (NK1.1) 168Er
Biolegend 108743
CD102 169Tm Biolegend 105602
CD140b (PDGFβ) 170Er
Biolegend 136002
Phox2b 171Yb Santa Cruz sc-376997
CD3ε 172Yb Biolegend 100302
F4/80 173Yb Biolegend 123102
CD44 174Yb
Biolegend 103002
Granzyme B 175Lu Biolegend 372202
I-A/I-E (MHC) 176Yb Biolegend 107602
CD11c 209Bi
Fluidigm 3209005B

71
Conclusion

The regulation and repair of intestinal barriers (i.e., microbial, mucus, epithelial, and immune) are
critical for homeostasis, where dysregulation leads to intestinal diseases, such as IBD. Growth
factor signaling, such as NRG4-ErbB4 and Shh, promotes barrier integrity by epithelial cell
restitution and immune cell regulation. Herein, we demonstrate that NRG4
-/-
mice displayed
increased intestinal permeability and apoptosis 24h after acute bacterial LPS challenge. Also,
NRG4
-/-
mice showed altered distribution of ZO-1 at 24h compared to WT cage mates, who
exhibited ZO-1 localization similar to baseline. These data suggest that endogenous NRG4
promotes intestinal barrier recovery after acute LPS injury. Furthermore, an analysis of baseline
ileal scrapings from NRG4
-/-
and WT mice determined decreased Muc2 levels and reduced ER
stress and autophagy signaling targets. Taken together, baseline alterations in both a major mucosal
barrier component and cell stress response mechanisms may impair the intestinal barrier during
recovery, as seen with the loss of NRG4.

Although the response to acute intestinal injury led to an impaired intestinal recovery, NRG4
-/-

mice showed reduced inflammation (indicated by fecal Lipocalin ELISA, total histology score,
tissue myeloperoxidase ELISA, spleen weight, and inflammatory cytokine levels) after IL-10R
neutralization colitis, compared to WT cage mates. A CyTof profile analysis of mouse immune
cells, from colonic cells after injury, determined a similar decrease in inflammation by the
reduction of total T cell numbers, with a significant decrease in CD8
+
T cells. Although a baseline
flow cytometry analysis of CD8
+
T cell numbers did not detect a difference in the colon, there was
however a significant decrease in the spleen. This suggests that NRG4 may promote CD8
+
T cell

72
recruitment during adaptive immune-mediated injury in part by regulating maturation in the
spleen.

Further analysis of baseline colonic alterations by bulk RNA sequencing indicated a significant
reduction in St3gal4, verified by qPCR analysis from colonic homogenates, colonic organoids
cultures, and in situ hybridization. This loss of ST3GAL4 was partially rescued by the treatment
of exogenous NRG4. Considering ST3GAL4 plays a role in immune cell trafficking, this loss is
also likely to promote a reduced inflammatory recruitment or injury.

In summary, these results demonstrate the complexity of the intestinal inflammatory response. We
have determined that NRG4 contributes to mechanisms that drive both immune cell regulation and
epithelial barrier recovery, necessary for intestinal health. It is plausible that NRG4 plays various
roles during the innate (LPS) v adaptive (IL-10R neutralization) immune response, or acute versus
chronic injury. Considering that NRG4 contributes to numerous tissues in a variety of ways and
that our mouse model is a whole body NRG4 deletion, this indicates that NRG4 is required in
response to diverse actions necessary in a range of tissues. However, further investigation is
necessary to determine the specific tissue responses, and the immune cells that regulate them, by
using tissue specific deletions in mouse models, such as NRG4 LVC or ST3GAL4 LVC. It is also
critical to assess the expression patterns of many of these altered genes (i.e., ST3GAL4) in human
intestinal disease, thus leading to a possible therapeutic target of NRG4 and/or ST3GAL4.

73
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Abstract (if available)

Abstract Inflammatory Bowel Disease (IBD), a chronic inflammatory condition of the intestinal tract, is comprised of two diseases: Crohn’s Disease (CD) and Ulcerative Colitis (UC). Although the direct cause of IBD is unknown, it is likely a result from epithelial barrier dysfunction, overactive immune responses, and microbial dysregulation. The ErbB family of receptor tyrosine kinases are essential for the regulation of colonic epithelial cell proliferation, differentiation, survival, and wound healing. We previously showed that ErbB4 is induced as a compensatory response by intestinal inflammation; treatment with exogenous NRG4 (an ErbB4 ligand) protects colonocytes against cytokine-induced apoptosis and ameliorates murine experimental colitis. Exogenous NRG4 can further alleviate intestinal inflammation by suppressing the activity of pro-inflammatory macrophages. However, the role of endogenous NRG4 in the intestine has not been described. Here we tested the function of endogenous NRG4 in the intestine by two immune-mediated injury models: acute bacterial LPS and IL-10R neutralization colitis. NRG4-/- mice demonstrated a significant increase in intestinal permeability and apoptosis 24h after acute bacterial LPS injury. Tight junctional component ZO-1 also showed a dysregulated localization pattern in NRG4-/- mice at 24h, compared to WT cage mates, suggesting an impaired epithelial barrier recovery. The loss of endogenous NRG4 reduced baseline ileal Muc2, Chop, Sqstm1, and Map1lc3, known factors that aid in barrier protection and/or epithelial repair. However, NRG4-/- mice displayed a reduced inflammatory response, indicated by significantly reduced lipocalin-2, total histology damage, myeloperoxidase, spleen weight, and CD8+ T cell numbers, during IL-10R neutralization colitis. Baseline CD8+ T cells were not altered in the colon, but were significantly reduced in the spleen. Bulk RNA sequencing of colonic homogenates determined a significant loss of ST3GAL4 in NRG4-/- mice when compared to WT cage mates. This loss of ST3GAL4 was verified by qPCR analysis from colonic homogenates and colonic organoid cultures, while localization was determined by in situ hybridization. ST3GAL4 levels were partially rescued with exogenous NRG4 treatment in vivo. Taken together, these results suggest that NRG4 promotes CD8+ T cell recruitment through the interaction with ST3GAL4. Furthermore, endogenous NRG4 may contribute to the intestinal epithelial integrity and recovery during the innate immune response, while suppress deleterious over production of T cells during adaptive immune mediated injury.

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Asset Metadata

Creator Bernard, Jessica Kathleen (author)

Core Title The role of endogenous neuregulin-4 during immune-mediated intestinal injury

School School of Dentistry

Degree Doctor of Philosophy

Degree Program Craniofacial Biology

Degree Conferral Date 2022-08

Publication Date 01/29/2023

Defense Date 07/29/2022

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

Tag ErbB receptor tyrosine kinases,experimental colitis,neuregulin growth factors,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Frey, Mark (committee chair), Merrell-Brugger, Amy (committee member), Paine, Michael (committee member), Warburton, David (committee member)

Creator Email jess.bernard@gmail.com,jkbernar@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC111375832

Unique identifier UC111375832

Legacy Identifier etd-BernardJes-11047

Document Type Dissertation

Format application/pdf (imt)

Rights Bernard, Jessica Kathleen

Type texts

Source 20220801-usctheses-batch-964 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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