THE EFFECTS OF VITAMIN A DEFICIENCY ON HOST DEFENSE …

The Pennsylvania State University

The Graduate School

Department of Veterinary and Biomedical Sciences

THE EFFECTS OF VITAMIN A DEFICIENCY ON HOST DEFENSE

DURING CITROBACTER RODENTIUM INFECTION

A Dissertation in

Pathobiology

by

Kaitlin L. McDaniel

© 2015 Kaitlin L. McDaniel

Submitted in Partial Fulfillment

of the Requirements for the Degree of

Doctor of Philosophy

December 2015

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The dissertation of Kaitlin L. McDaniel was reviewed and approved* by the following:

Margherita T. Cantorna Distinguished Professor of Nutrition and Immunology Dissertation Advisor Co-Chair of Committee

A. Catharine Ross Huck Chair and Professor of Nutrition Co-Chair of Committee

Na Xiong Associate Professor of Veterinary Science and Biomedical Sciences

Mary K. Kennett Professor of Veterinary Science and Biomedical Sciences

Connie J. Rogers Assistant Professor of Nutritional Science Anthony Schmitt Associate Professor of Molecular Virology Director, Pathobiology Graduate Program

*Signatures are on file in the Graduate School

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ABSTRACT

Vitamin A deficiency is still a prevalent problem in resource limited countries, affecting

more that 250 million preschool aged children. Vitamin A deficiency is associated with

increased severity of respiratory and diarrheal diseases, a major cause of childhood

mortality. Vitamin A supplementation is associated with increased vitamin A status and

decreased disease severity, indicating a link between vitamin A status and immune

function. Using the murine enteric pathogen, Citrobacter rodentium, I explored the

relationship between host vitamin A status, intestinal infection and immune response.

The first objective was to determine the effect of vitamin A deficiency on C. rodentium

infection. My study showed that vitamin A deficient (A-) hosts did not clear the infection

and 40% of the A- animals developed a lethal infection. Additionally, retinoic acid

treatment of A- hosts promoted infection clearance and survival. The findings from this

study suggest that A- hosts may harbor enteric pathogens but retinoic acid treatment was

effective during C. rodentium infection. The second objective was to determine how

retinoic acid promotes C. rodentium clearance in the A- host. My data shows that 1 dose

of retinoic acid in an A- host was effective at clearing infection. Two doses of retinoic

acid treatment promoted il17a expression in the colon of A- mice yet, retinoic acid

treatment of splenocytes in vitro inhibited IL-17 secretion. Vitamin A host status was

found to have no effect on the microbiota and B cell numbers in the gut, indicating that

other acquired immune cells may be targets of RA. To understand the effects of retinoid

signaling on T cells, T-dnRAR mice were generated. T-dnRAR mice had significantly

more CD8+ thymocytes but significantly fewer CD8+ T cells in the spleen and

mesenteric lymph node. Together, the work presented in this dissertation indicates that

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vitamin A deficient hosts may be asymptomatic carriers of enteric infections and that

vitamin A treatment during infection promotes a local IL-17a response that is associated

with clearance of C. rodentium.

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TABLE OF CONTENTS

List of Figures……………………………………………………………………….

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List of Tables……………………………………………………………………..… Abbreviations……………………………………………………………………….

viii xi

Acknowledgements………………………………………………………………….

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Chapter 1: Introduction……………………………………………………………. Chapter 1 References……………………………………………………………….

1 17

Chapter 2: Vitamin A deficient hosts become non-symptomatic reservoirs of Escherichia coli-like enteric infections……………………………………………. Chapter 2 References……………………………………………………………….

27 45

Chapter 3: Short term retinoic acid treatment promote mucosal IL-17 expression leading to clearance of enteric infection……………………………… Chapter 3 References……………………………………………………………….

57 76

Chapter 4: Summary and conclusions …………………………………………… Chapter 4 References………………………………………………………………

91 97

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LIST OF FIGURES Figure 1-1: Vitamin A metabolism………………………………………………

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Figure 2-1: A- mice are more susceptible to C. rodentium infection than A+ mice…………………………………………………………………………

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Figure 2-2: Quantification of whole body imaging during C. rodentium infection…………………………………………………………………………..

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Figure 2-3: T cells in the IEL of the colon from A+ and A- mice before and after infection…………………………………………………………………….

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Figure 2-4: Colon histology from C. rodentium infected animals……………...

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Figure 2-5: RA treatment of A- mice eliminates C. rodentium infection……..

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Figure 2-6: Systemic C. rodentium in tissues…………………………………..

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Figure 2-7: Systemic spread of C. rodentium following GI infection…………

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Figure 2-8: The effect of vitamin A status on LPS response and i.v challenge with C. rodentium…………………………………………………………………

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Figure 2-9: Natural transmission of C. rodentium is not affected by the host vitamin A status. ………………………………………………………………..

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Figure 3-1: RA treatment starting at d7 results in similar clearance when RA is given at d0………………………………………………………………………

79

Figure 3-2: Short term RA treatment results in increased il17a mRNA…….. 80

Figure 3-3: The effects of RA treatment on mRNA expression in duodenum, ileum and colon at d11 post-infection and 2 doses of RA ……………………

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Figure 3-4: The microbiota is not affected by host VA status……………….. 83

Figure 3-5: RA inhibition of IFN-γ and IL-17 in A- whole splenocytes and CD4+ T cell in vitro………………………………………………………………..

84

Figure 3-6: Serum antibody titers and GI B cell numbers are not affected by host vitamin A status …………………………………………………………...

85

Figure 3-7: RA treatment in vitro does not affect T-dnRAR splenocytes response to stimulation ………………………………………………………...

86

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Figure 3-8: RA signaling inhibition in T cells leads to increased CD8+ T cells in the thymus and decreased CD8+ T cells in the periphery…………………...

88

Figure 3-9: Gating strategy for flow cytometry analysis ………………….…... 89

Figure 5-1: Retinoic acid treatment during enteric infection promotes protective mucosal immune responses in the vitamin A deficient host……....

95

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LIST OF TABLES Table 2-1: Serum cytokines in A+ and A- mice………………………………...

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Table 3-1: RT-PCR primer sequences…………………………………………..

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Table 3-2: RA signaling inhibition in T cells leads to immune cell population imbalance in the periphery…………………………………………...…………

83

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ABBREVIATIONS

A-: vitamin A deficient hosts

A+: vitamin A sufficient hosts

CCL: chemokine ligand

CCR: CC-chemokine receptor

CD: cluster of differentiation

CFU: colony forming units

CTLA-4: cytotoxic T lymphocyte antigen 4

DBD: DNA binding domain

DC: dendritic cell

DN: double negative

dn-RAR: dominant negative retinoic acid receptor

DP: double positive

EtOH: ethanol

FOXP3: Forkhead box P3

GALT: gut associated lymphoid tissues

GI: gastrointestinal tract

IEL: intraepithelial lymphocytes

IFN: interferon

Ig: immunoglobulin

IL: interleukin

ip: intra-peritoneal

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iv: intra-venous

KO: knock out

LBD: ligand binding domain

LCK: lymphocyte specific protein tyrosine kinase

LPL: lamina propria lymphocytes

LPS: lipopolysaccharide

MAdCAM-1: mucosal addressin cell adhesion molecule-1

MLN: mesenteric lymph nodes

NK: natural killer

NKT: natural killer T

NTD: NH2 terminal domain

PP: Peyer’s patches

RA: retinoic acid

RAR: retinoic acid receptor

RARE: retinoic acid response elements

RBP: retinol binding protein

ROR-γt: RAR orphan receptor γt

RXR: retinoid X receptor

SI: small intestine

STRA6: stimulated by retinoic acid gene 6

TCR: T cell receptor

T-dnRAR: T cell specific dn-RAR

TFG: transforming growth factor

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Th: T helper

TNF: tumor necrosis factor

Treg: regulatory T cell

TTR: transthyretin

VA: vitamin A

VAD: vitamin A deficiency

WT: wild type

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ACKNOWLEDGMENTS

First, I would like to thank my advisor, Dr. Margherita Cantorna for her help, guidance

and patience during my time at Penn State. Dr. Cantorna has given me invaluable advice

and life lessons that I will carry with me throughout my career. I am grateful for my

committee members: Dr. Catharine Ross, Dr. Na Xiong, Dr. Mary Kennett and Dr.

Connie Rogers for sharing their knowledge and their valuable support through my

studies. A special thanks to Dr. Rogers for her constant encouragement and mentoring

throughout my graduate school experience.

I would like to thank my lab members, Jamaal James and Yang-ding Lin for their support

and advice. Additionally, I have been fortunate enough to have three wonderful friends

in the lab. Veronika Weaver, Stephanie Bora and Lindsay Snyder have helped me with

countless experiments and have been friends in and out of lab. They truly made coming

to work in the lab enjoyable and helped me keep my sanity. I don’t know how I would

have survived graduate school without them. I would like to thank the past and present

members of the Cantorna lab, the Ross lab, the Veterinary and Biomedical Science

program, the members of the Flow Cytometry Core and the office staff for all of their

help during my time at Penn State. A special thanks to Dr. Ruth Nissly for her

encouragement and for letting me know that everything will work out in the end.

My family and friends deserved the largest and deepest thanks. I have been lucky

enough to have three families on my graduate school journey: my family in Wyoming,

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my farm family on Rocky Top, and my CrossFit Nittany family. Not many people are

lucky enough to have experienced the love and fun that I have being so far from home.

My Wyoming family (Dad, Mom, and sister) always supported me no matter what, even

my insane decision to move across the country. They instilled qualities in me that have

helped me persevere to the end of graduate school. They have made me who I am today.

My Rocky Top family made me feel at home in a new place and I am forever grateful for

that. My CrossFit family gave me a place to be myself and learn and grow in ways I

didn’t know possible. I am a stronger, better person because of CrossFit Nittany and they

have saved my life. From the bottom of my heart, thank you to everybody who helped

me get to this point.

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Chapter 1

Introduction

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Vitamin A: Sources and metabolism

Vitamin A (VA) is a fat-soluble essential micronutrient that humans obtain

through their diet. VA is absorbed from the diet as previtamin A retinyl esters present

in dairy products and liver and provitamin A carotenoids (from leafy green vegetables

and bright colored fruits) (1). The most common form of dietary vitamin A is β-

carotene which is found in vegetables and fruits such as sweet potatoes, kale, carrots

and cantaloupe. Proper vitamin A intake is associated with eye health, cell

development and immune system health, making it an important nutrient.

Dietary vitamin A needs to be converted into an active form so that the body

can use it. The metabolism of vitamin A has been summarized in Figure 1-1.

Briefly, once vitamin A has been absorbed into intestinal epithelial cells, the

associated proteins and fatty acids are removed. This frees the retinyl esters and

carotenoids so they can be processed by enzymes in the epithelial cells which convert

retinyl esters and carotenoids to retinol (2). Retinol becomes esterified into retinyl

palmitate and incorporated into chylomicrons where it enters the lymphatic system

and travels to the liver (3). The liver is the major storage organ of vitamin A and also

produces retinol binding protein (RBP) (4). Once the chylomicron enters the liver,

the retinyl palimitate is hydrolyzed into retinol where it is stored in the liver stellate

cells (5, 6). Retinol can associate with RBP which binds with transthyretin (TTR)

forming a traveling complex in the serum that can be transported to extrahepatic

tissues (1, 7). The RBP:TTR complex delivers retinol to the plasma membrane

receptor, STRA6 (stimulated by retinoic acid gene 6), on target cells. The binding to

the transport complex to STRA6 releases retinol from its binding proteins and retinol

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enters the cell where it is converted to retinoic acid (RA) (Fig. 1-1A and 1-1B).

Dietary vitamin A must be converted into RA for the use of cells.

RA is the active metabolite of vitamin A. The effects of RA are exerted when

RA binds the retinoic acid receptor (RAR), which is found inside the nucleus (8).

The RAR is a member of the superfamily of nuclear receptors and has three isoforms,

RAR α, β and γ (8, 9). RARs are expressed in every cell of the body, but RARα is the

most common form (10). The RARs are divided into three domains that are

necessary for its function, including the COOH-terminal ligand binding domain

(LBD) which binds RA, the central DNA binding domain (DBD) which binds to

DNA sequences and the NH2 terminal domain (NTD) whose mechanism of action is

yet to be determined (11-13). The RAR heterodimerizes with the retinoid X receptor

(RXR) to form a complex that, in the presence of ligand, binds to retinoic acid

response elements (RARE) in the promoter regions of RA-inducible genes (11) (Fig.

1-1B). All-trans retinoic acid binds to the RAR, inducing conformational changes in

the ligand-binding domain which releases co-repressor proteins and recruits activator

proteins. The combination of RA, RAR:RXR heterodimer and activator proteins

make up a complex that induces gene transcription (11, 12). RAR signaling can also

inhibit gene transcription, but the mechanisms of inhibition are largely unknown. RA

largely regulates gene expression through nuclear receptor binding and co-activator

protein recruitment to gene promoter sites.

Vitamin A Deficiency

Vitamin A deficiency (14) remains a public health issue in resource-limited

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countries, such as India and most of Africa (15). VAD is a critical concern in young

children and pregnant women (16, 17). It is estimated that over 20% of preschool

aged children are clinically vitamin A deficient (15, 17). Vitamin A deficiency in

infants occurs during breastfeeding because of the vitamin A deficient status of the

mothers. VAD causes xerophthalmia (night blindness) and is associated with

increased severity of infectious disease (18, 19). Vitamin A supplementation resulted

in a 26-30% reduction of mortality from respiratory and diarrheal diseases by

reducing disease severity in populations studied in Africa and India (18, 20). The

World Health Organization (WHO) reports vitamin A supplementation reduced

measles related mortality by 50% and diarrheal related mortality by 33% (21).

Additionally, studies showed that high dose vitamin A supplementation reduced

childhood mortality and morbidity in children without VAD (14, 22). Based on these

studies the WHO recommends that preschool aged children in counties where VAD is

common receive vitamin A supplementation (11, 15). The collective reports indicate

that there is a strong link between vitamin A status and resistance to infectious

disease.

Retinoic acid regulation of peripheral immune responses

The immune system is a network of cells, tissues and systems that protect the

host from disease. This defense system is composed of innate immune defenses

(barrier defenses and mechanisms that distinguish self from non-self) and adaptive

immune defenses (mechanisms that have immunological memory). The innate

immune response occurs quickly and initiates the adaptive immune response which

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takes over once the initial innate response ends. Fully functional innate and adaptive

immunity are required for the host to fight infections.

Retinoic acid is a potent immune cell regulator. It has been shown to affect

both the innate and adaptive branches of the immune system. The effects of RA on

the innate immune system have been reviewed in detail elsewhere (23). The focus of

this dissertation is to elaborate on the effects of RA on the adaptive immune system,

mainly T cells.

T cells are a major part of the adaptive immune system. These cells mature in

the thymus and express T cell receptors, CD3 and either CD4 or CD8 on their

surface. CD4+ T cells are T helper (Th) cells that mediate immune responses

primarily though secretion of cytokines which regulate other immune cells such as B

cells and macrophages (24). Th cells are able to respond to a wide variety of

pathogens, regulate immune responses to control the magnitude of the immune

system’s response and protect against autoimmunity (24). Th cells are further

categorized based on the cytokines they secrete. Th1 cells are regulated by the

transcription factor, T-bet, and are potent cytokine producers, particularly interferon

(IFN) -γ that aids in the response to intracellular pathogens (25). Th2 cells secrete

interleukin (IL) -4, IL-5 and IL-10 and are regulated by the transcription factor,

GATA-3 (26). Th2 cells are important for host responses to helminthes and promote

B cell antibody production, the other half of the adaptive immune system (25). IFN-γ

secreted from Th1 cells inhibits Th2 cell function and IL-4 produced from Th2 cells

inhibit Th1 functions, indicating that Th1 and Th2 cells antagonize each other (27).

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Another type of CD4+ T cells are Th17 cells. Th17 cells produce IL-17A,

IL17-F, IL-21 and IL-22 to control bacterial infections (28). Th17 cells are induced

in the presence of transforming growth factor (TFG)-β, IL-6 and IL-23 (29) and are

important for mucosal barrier function (24, 26, 28, 30). These cells are regulated by

the transcription factor, ROR-γt. The opposing cell lineage is regulatory T cells

(Tregs). Tregs express the transcription factor, FoxP3, and suppress the functions of

target T cells to inhibit immune responses (27). Naïve CD4+ T cells express both

FOXP3 and ROR-γt and when RA, TFG-β and low concentrations of IL-6 are

present, FOXP3 will inhibit Th17 cell development by inhibiting ROR-γt expression

(31, 32). Humans and mice with mutations in the foxp3 gene have severe immune

impairments indicating that Treg cells are vital to maintaining the balance between

inflammatory and anti-inflammatory responses (24, 25). Treg cells secrete anti-

inflammatory cytokines such as IL-10 and TGF-β to inhibit effector cell function

(27). They also express the surface marker CTLA-4 (cytotoxic T lymphocyte antigen

4) which competes for co-stimulatory receptors on effector cells (25). When CTLA-4

binds to effector cells, an inhibitory signal is sent to the effector cell and

inflammatory responses recede (25) . Additionally, IFN-γ and IL-17 has been

implicated in autoimmune disease pathology while IL-4 and IL-10 have been

associated with inflammation resolution (22). Maintaining the balance between

Th1/Th17 and Th2/Treg cells is required for inflammatory homeostasis in the host.

The balance between inflammatory and regulatory responses is mediated in

part by RA. RA promoted Th2 responses (IL-4, IL-5, IL-10) and inhibited Th1

responses (IFN- γ) in vitro and in vivo (33, 34). T cells treated in vitro with RA

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produced less IFN-γ (34) while T cells from A- mice overproduced IFN-γ (35) and

under-produced regulatory cytokines (IL-4, IL-10) (36). Additionally, RA treatment

of A- T cells resulted in decreased IFN-γ transcription (36). However, a recent study

showed that RA signaling was needed for T-bet expression in memory CD4+ T cells

(37). Expression of a dominant negative RAR (dnRAR) in T cells (i.e. inhibition of

RA signaling in T cells) caused T cells to express high levels of IL-17 and express

both IL-17 and IFN-γ (37). Additionally, when dnRAR T cells were transferred into

Rag knockout (Rag KO) mice (mice that do not have T or B cells), the RagKO mice

that received the dnRAR T cells developed more severe colitis due to the dnRAR T

cells overproducing IL-17 and IL-17/IFN-γ (37). This new report is consistent with

RA inhibiting IL-17 and IFN-γ. Overall, the literature suggests a regulatory role for

RA in maintaining a balance between inflammatory and regulatory cytokine

production.

In addition to maintaining cytokine balance, RA can regulate the balance

between Th17 and Treg cells. RA, in combination with TGF-β, inhibited Th17 cell

induction from naïve CD4+ T cells in vitro and in vivo while promoting FoxP3

expression and IL-10 production from Treg cells (38, 39). Furthermore, RA inhibited

IL-6 mediated Th17 conversion, further promoting FoxP3+ Treg cell differentiation

(40). RA regulation of FoxP3 expression is largely mediated through RAR/RXR

binding at the FoxP3 promoter region (41). Maintaining balance between Th17 and

Treg cell responses is critical for ensuring that the immune system responds correctly

to stimuli and RA is a mediator of that balance (42-44). The literature suggests that

RA is an important mediator of immune response balance.

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The mucosal immune system

The intestine is a complex environment that is constantly exposed to dietary

and bacterial antigens. The local immune system must distinguish between

commensals and pathogens by reacting effectively against pathogens but benignly to

commensals. The gut epithelial cells, microbiota and innate and adaptive immune

systems must work in conjunction to protect the host from breaches in the

gastrointestinal barrier.

The physical barrier created in the gut is the absolute first line of host defense

in the intestine. A thick layer of mucus secreted by goblet cells (specialized epithelial

cells) separates the microbiota and gut lumen from the host epithelial cells. Some

epithelial cells produce anti-microbial peptides, further bolstering the physical

defense between host and microbe. Furthermore, innate and innate-like immune cells

secrete cytokines that promote gut epithelial cell production of anti-microbial

peptides. Maintaining the physical barrier between host and intestinal lumen is vital

for host survival.

The immune compartments associated with intestinal immunity have been

termed gut associated lymphoid tissue (GALT). T cells in the gut reside in the

mesenteric lymph nodes (MLN), Peyers patches (45), and within the intraepithelial

lymphocyte (IEL) and lamina propria lymphocyte (LPL) compartments of the small

intestine (SI) and colon (46). Additionally, other immune cells such as dendritic

cells reside in the GALT. Dendritic cells (DCs) in the MLN, PP and LPL bridge the

gap between innate and adaptive immunity by initiating T cell responses (47). The

DC presents antigen to the T cell (47). In addition, the DC also provides co-

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stimulation and cytokine signals that regulate T cell development into the effector T

cell types (47). The relationship between T cells and DCs is an important mediator of

immunity in the gut because these cells within the GALT are the first immune cells to

interact with pathogens that breach the host through the breaching the intestinal

epithelial barrier (48). The adaptive immune cells contained within these GALT

compartments are essential for maintaining a healthy host.

Retinoic acid and immune cell trafficking

One of the major functions of RA is the promotion of immune cell trafficking

to the mucosa. In a pivotal study, Iwata and colleagues determined that RA treatment

of T cells induced expression of the gut homing receptors, CCR9 and α4β7, on CD4+

T cells (44). CCR9 interacts with CCL25 on intestinal epithelial cells while α4β7

mediates cell trafficking to the intestinal endothelium though interactions with

mucosal addressin cell adhesion molecule-1 (MAdCAM-1) (11). Additionally, RA

induced CCR9 and α4β7 on other immune cells (49, 50). In support of the

observation that RA is important for immune homing to the gut, multiple groups have

reported that A- animals had fewer effector T cells in the gut and decreased gut

homing receptor expression (49, 51-53). Surprisingly, RA signaling was not needed

for homing since dnRAR T cells homed effectively to the gut of RagKO mice (37).

RA is an important metabolite for host defense.

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Retinoic acid and barrier function

RA affects gut barrier function by supporting intestinal barrier integrity. VAD

in humans was associated with increased gut permeability (54) and vitamin A

supplementation was associated with improved gut integrity (55-57). Additionally,

studies done in animals models have shown that A- status resulted in increased gut

permeability and decreased goblet cell function (58-63). RA is important for

maintaining epithelial barrier in the GI tract.

When the epithelial barrier is breached, RA can promote epithelial repair.

During colitis, RA was bound to RAR/RXR and the IL-22 promoter inducing IL-22

production in innate lymphoid cells and γδ T cells (64). IL-22 is an important

cytokine for barrier function that largely targets epithelial cell to promotes tissue

regeneration (65) and is required for survival during enteric infection (66). These

studies suggest that RA plays a role in gut epithelial repair.

Once the epithelial barrier has been breached, immune cells respond. GALT

DCs are one of the first responders. These cells express CD103+ and have been

shown to express RA producing enzymes unlike their splenic counterparts (51, 67).

The RA produced by these DCs induced expression of the gut homing receptors,

CCR9 and α4β7, on T cells (51, 68, 69). Local production of RA, in conjunction with

the presence of TFG-β resulted in an environment that promoted Treg cell

development and IL-10 secretion (67, 70). Furthermore, DCs isolated from the

GALT of A- mice promoted the differentiation of Th17 cells in vitro (71). The SI has

a relatively high concentration of RA as determined by liquid chromatography–mass

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spectrometry (11, 72). These findings support a role for RA in the promotion of

barrier functions.

Retinoic acid and the balance of mucosal Th17 and Treg cells

The balance between Th17 and Treg cells is highly important in the gut

because both are needed for host defense. If the Th17 response dominates over the

Treg response, excess inflammation and damage to the host can occur. However if

there is too much inhibition of inflammation, then pathogens can escape the gut and

become systemic, leading to sepsis. RA plays a role in maintaining the balance

between Th17 and Treg cells.

The effects of RA on Th17 cells are still not completely understood. Many in

vitro studies have shown that RA inhibited Th17 cell induction from naïve CD4+ T

cells (reviewed in (8)). Concentrations used in cell culture studies range from 10nM

to 1uM and result in suppressed Th17 cell induction and reduced IFN-γ and IL-17

secretion in vitro (11, 73, 74). Paradoxically, multiple groups have shown that A-

mice have almost no Th17 cells (73, 75, 76). In support of this observation, two

studies reported that 1-30nM RA in vitro is needed for IL-17 production from T cells

(73, 74) and upregulation of CCR9 and α4β7 expression on IL-17 producing T cells

(73), indicating that these concentrations of RA may promote Th17 cell induction.

Additionally, species of the gut microbiota can induce Th17 cell generation,

indicating that the intestinal environment may be a factor regarding the induction of

Th17 cells (77, 78). While there is no evidence that shows that RA directly affects

the microbiota (as bacteria do not have RARs or RXRs), the combination of local RA

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in low concentrations (<30nM) and the presence of the microbiota may result is

different regulation of effector T cell differentiation in vivo. These data indicate that

the local environment and the concentration of RA may have different effects on

Th17 cells in vitro and in vivo.

Genetic mouse models have led to insight as to how RA signaling regulates

Th17 and Treg cells. Reports showed that Th17 cell differentiation is impaired in

RARα-deficient and A- CD4+ T cells, indicating that RA is needed for the

development of Th17 cells (75, 76). Conversely, when T cells expressed the dnRAR

(inhibited RA signaling in only T cells), they had a propensity to secrete more IL-17

than IFN-γ (37). dnRAR CD4+ T cells trafficked to the gut and caused more

intestinal inflammation when transferred into RagKO mice compared to WT cells

(37). More work is needed to fully understand how RA-RAR signaling affect Th17

cells in vivo.

T reg cell are critical to dampen the immune response so that the host is not

damaged by overactive inflammation. Treg cells have suppressive function, such as

inhibition of effector cell proliferation, secretion of regulatory cytokines and the

ability to traffic to the gut through CCR9 and α4β7 (79). CD103+ DCs can promote

the conversion of naïve CD4+ T cells to Treg cells in the gut (67). RA induced Tregs

are more suppressive during antigen specific CD8+ T cell driven acute intestinal

inflammation than Tregs induced without RA (80). However suppression following

CD4+ T cell transfer was no different between Treg cells induced with or without RA

in vitro (80). Interestingly, FoxP3+ T cell frequencies were not different in WT or

dnRAR T cells (37). Overall, RA promotes Treg cell induction in vitro however, the

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role of RA in the promotion of Treg cell differentiation in vivo is less clear.

Currently, the effects of RA and RA signaling on Th17 and Treg cells are not

completely understood.

Vitamin A and disease outcomes

Due to its immune-regulatory functions, vitamin A is sometimes termed “the

anti-infective vitamin” (81, 82). In humans, VA supplementation was associated with

improved recovery from a wide variety of diseases, including malaria and measles

(83). Many studies looking at the interaction between VAD and human infections

have shown that VAD was associated with exacerbated disease (84). In HIV-1

infected humans, vitamin A supplementation decreased vertical transmission of HIV-

1 from mother to child (83, 85, 86) and decreased HIV-1 associated mortality in

children (83) (87). High dose vitamin A supplementation promoted recovery from

measles and decreased severity of malaria infection in young children (83, 88).

However, the effects of vitamin A supplementation in respiratory diseases were

conflicting as to whether vitamin A supplementation was beneficial, detrimental or

not different (83). Laboratory models of vitamin A deficiency indicated that multiple

immune responses were impaired in A- hosts. A- rodents had impaired antibody

responses and more Th1 cells and fewer Th2 cells (89-92). Conversely, A- mice had

decreased Th1 and Th17 responses following infection with Toxoplasma gondii (75).

RA treatment in vivo restored IFN-y production in A- T. gondii infected mice (75).

RA treatment in vitro was able to inhibit effector and memory T cell cytokine

production (93). The data suggest a role for vitamin A in host resistance to infection.

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Vitamin A and gastrointestinal inflammation

Gastrointestinal infections are also regulated by vitamin A. A- mice

developed a more severe enterohemorrhagic E. coli infection compared to A+ mice

(94). Furthermore, RA treatment reduced colonic inflammation caused by dextran

sodium sulfate (DSS) and Citrobacter rodentium infection (64). RA treatment during

chemical and bacterial inflammation inhibited production of IL-17 and IFN-γ (64)

(76). In addition to inhibiting inflammatory cytokine production, RA treatment

increased anti-microbial peptide production and IL-22 production which

corresponded with decreased inflammation (64, 76). During C. rodentium infection,

A+ mice cleared the infection more quickly than A- mice (95, 96). VA

supplementation shortened the duration of enterotoxigenic E. coli infection in

children (97). These data suggest that vitamin A is a critical factor in limiting

intestinal inflammation.

Citrobacter rodentium: disease and pathogenesis

C. rodentium is a mouse pathogen that models human infections with

enteropathogenic Escherichia coli by causing attaching and effacing lesions of the

cecum and colon in mice (98). C. rodentium is transmitted via the fecal-oral route (as

mice are known to be coprophagic) and causes inflammation of the colon. The

severity of C. rodentium infection can range from a self-limiting colitis to severe

inflammation and death, usually due to dehydration (99-102). This mouse model of

infection is widely used to study host-pathogen interactions in the gastrointestinal

tract.

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C. rodentium infection with laboratory grown bacteria is a highly reproducible

infection model. Following colonization, C. rodentium becomes more virulent,

adapting to the host and becoming more infectious to naïve animals (102, 103). This

adaptation allows C. rodentium to colonize the colon and rectum. By day 3 post

infection, the bacteria moves to the colon (100). Fecal shedding of C. rodentium

peaks between day 7 and 14 post-infection. Crypt hyperplasia develops and effector

immune cell infiltration occurs during this time period as well. During the peak of

infection, mice can exhibit weight loss and diarrhea (98, 100). Resistant mouse

strains clear the infection by day 30 post infection, while susceptible strains succumb

to persistent infections without clearing the pathogen (98). C. rodentium is a useful

model for studying host/pathogen interactions and mucosal immunity.

Immunity to Citrobacter rodentium

The innate immune system is required for early protection as demonstrated in

RagKO mice lacking T and B cells; these mice were unable to clear the bacteria and

eventually succumbed to the infection (104). Macrophages and innate lymphoid cells

produce IL-22 early during infection which promoted epithelial repair (100, 105,

106). IL-22 KO mice developed fatal infections within the first two weeks of C.

rodentium infection (66). Furthermore, IL-22 production induced an essential

adaptive Th17 responses, leading to clearance of C. rodentium (100, 105).

T cells and B cells are essential for resolution of C. rodentium infection since

mice without T cells (CD4 KO) or B cells (Igμ KO) developed fatal infections (100).

Furthermore, T cell dependent antibody, Th1 and Th17 responses were required for

16

bacterial clearance (99) (107). IFN-γ KO and IL-17 KO mice succumbed to lethal C.

rodentium infections (38, 108, 109). Together, the innate and adaptive immune

responses are required for protection from C. rodentium.

Objectives

In this dissertation, the effects of host vitamin A status and RA

supplementation on host defense to a bacterial infection with C. rodentium was

investigated. Some reports have shown that RA inhibits IFN-γ and IL-17 production

in vitro and in vivo from T cells however, other studies indicate that RA is critical for

Th1 and Th17 cell responses in vivo. It was unknown how the vitamin A deficient

host would respond to a C. rodentium infection, which requires Th1/Th17 immune

responses for clearance. Based on experiments that used A- mice and RA dosing in

models of gut inflammation, we hypothesized that the A- mice might be resistant to

C. rodentium infection. In Chapter 2, we tested the susceptibility of A- mice to C.

rodentium infection. In Chapter 3, we determined some of the RA targets in the gut

mucosa that would mediate host resistance to C. rodentium infection. The last

Chapter summarizes the major findings and puts the work in the context of the

growing literature on the role of vitamin A in regulating mucosal T cells in the gut.

17

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108. Geddes, K., S. J. Rubino, J. G. Magalhaes, C. Streutker, L. Le Bourhis, J. H. Cho, S. J. Robertson, C. J. Kim, R. Kaul, D. J. Philpott, and S. E. Girardin. 2011. Identification of an innate T helper type 17 response to intestinal bacterial pathogens. Nat Med 17: 837-844.

109. Simmons, C. P., N. S. Goncalves, M. Ghaem-Maghami, M. Bajaj-Elliott, S. Clare, B. Neves, G. Frankel, G. Dougan, and T. T. MacDonald. 2002. Impaired resistance and enhanced pathology during infection with a noninvasive, attaching-effacing enteric bacterial pathogen, Citrobacter rodentium, in mice lacking IL-12 or IFN-gamma. J Immunol 168: 1804-1812.

25

Figure 1-1

Figure 1-1. Vitamin A metabolism. A) Vitamin A is absorbed from the diet and converted into a storable, stable form of vitamin A, retinol. Retinol is transported to and stored in the liver. It can be mobilized and released into the bloodstream for cell utilization. Adapted from Perusek and Maeda. Nutrients 2013, 5, 2646-2666. B) When retinol enters the target cell, it is converted into the active form, retinoic acid (RA). RA translocates to the nucleus where it binds the retinoid receptors which regulate gene transcription. Adapted from Maden, M. Nat Rev Neurosci 2002. 3: 843-853.

27

Chapter 2

Vitamin A deficient hosts become non-symptomatic reservoirs of Escherichia coli-like enteric infections.

Chapter adapted from the manuscript entitled:

“Vitamin A deficient hosts become non-symptomatic reservoirs of Escherichia coli-like

enteric infections.”

Authors: Kaitlin L. McDaniel, Katherine H. Restori, Jeffery W. Dodds, Mary J.

Kennett1, A. Catharine Ross and Margherita T. Cantorna

Infect Immun. 2015 Jul;83(7):2984-91

28

ABSTACT

Vitamin A deficiency (A-) remains a public health concern in developing

countries and is associated with increased susceptibility to infection. Citrobacter

rodentium was used to model human Escherichia coli infections. A- mice developed a

severe and lethal (40%) infection. Vitamin A sufficient (A+) mice survived and cleared

the infection by d25. Retinoic acid treatment of A- mice at the peak of the infection

eliminated C. rodentium within 16d. Inflammation was not different in A+ and A- colons

although the A- mice were still infected at d37. Increased mortality of A- mice was not

due to systemic cytokine production, an inability to clear systemic C. rodentium or

increased virulence. Instead A- mice developed a severe gut infection with most of the A-

mice surviving and resolving inflammation but not eliminating the infection.

Improvements in vitamin A status might decrease susceptibility to enteric pathogens and

eliminate potential carriers from spreading infection to susceptible populations.

29

INTRODUCTION

Vitamin A deficiency is a significant problem in developing countries where

inadequate micronutrient intake remains a public health concern (1). The World Health

Organization estimates that over 20% of preschool aged children are clinically vitamin A

deficient (1, 2). Vitamin A deficiency contributes to the higher prevalence of respiratory

and diarrheal diseases as well as increased childhood mortality. Conversely, vitamin A

supplementation is practiced as a means to reduce mortality in preschool-aged children

by reducing severity of infectious diseases (3).

Vitamin A and its active metabolite, retinoic acid (RA) are important regulators of

T cell responses. RA inhibited IFN-γ production from T cells in vitro (4). In addition, T

cells from vitamin A deficient (A-) mice overproduced IFN-γ (5). RA also inhibited

Th17 cells in vitro and in vivo (6). RA increased the expression of the gut homing

receptors, α4β7 and CCR9, on T cells (7), which recruits T cells to the gut mucosa. In

vitro, RA inhibited IL-17 and induced expression of the transcription factor FoxP3,

associated with regulatory T cells, and IL-10 production (8). Vitamin A and RA are key

regulators of T cell cytokine production and gut homing.

Several lines of experimental evidence support a beneficial effect of vitamin A

and RA on the host response to infection (9, 10). In the gut, the mechanisms that account

for the anti-infective effects of vitamin A include support of B cell function and T cell-

dependent B-cell antibody responses (11). Gut infection of A- mice with Trichinella

spiralis resulted in T cells that produced IFN-γ but not IL-4, and as a result, reduced the

rate of parasite clearance (9). Furthermore, RA treatment reduced colonic inflammation

caused by dextran sodium sulfate and infection (12). The reduction in gastrointestinal

30

(GI) inflammation with RA treatment was attributed to the inhibition of IL-17 and IFN-γ

(12, 13). These data suggest that vitamin A and RA regulate T cell function to limit

inflammation following chemical and infectious injury in the gut.

Citrobacter rodentium is a mouse pathogen that models human infections with

enteropathogenic Escherichia coli and causes attaching and effacing lesions of the cecum

and colon in mice (14). The natural route of C. rodentium transmission is fecal-oral.

Resistant mouse strains including C57BL/6 mice are infected transiently with C.

rodentium and clear the infection within 2-3 weeks (14). The acquired immune system

was required for early protection from C. rodentium, as demonstrated in recombination-

activating gene (Rag) knockout (KO) mice lacking T and B cells that were unable to clear

the infection (15). Robust IL-22 production from innate lymphoid cells and macrophages

has been shown to induce protective Th17 responses that resulted in clearance (16, 17).

T cells and B cells were essential for resolution of C. rodentium infection since mice

without T cells (CD4 KO) or B cells (Igμ KO) developed fatal infections (17). Host

resistance to C. rodentium depends on IL-22 production from innate cells, T cells, B cells

and Th17 cells.

Here, we determined the effect of vitamin A deficiency on host resistance to C.

rodentium infection in C57BL/6 mice; a normally resistant mouse strain. Because of the

well-demonstrated inhibitory effects of RA on the differentiation of Th1 and Th17 cells,

we predicted that a bacterial infection that required Th17 cell responses for resistance

might be less severe in A- mice, and exacerbated in RA-treated mice. Interestingly and

contrary to expectations, A- mice developed a chronic infection with C. rodentium. The

31

C. rodentium infection was lethal for 40% of A- mice while none of the vitamin A

sufficient (A+) or RA treated A- mice died prematurely from infection.

32

MATERIALS AND METHODS

Mice. C57BL6 mice were originally from Jackson Laboratories (Bar Harbor, MN) and

bred at the Pennsylvania State University (University Park, PA) for experiments. Vitamin

A deficient (A-) and vitamin A sufficient (A+) mice generated as previously described (5,

18). Briefly, mice were fed a purified diet that did not contain any vitamin A (A-) or that

contained 25 µg of retinyl acetate (vitamin A) per day (A+). At weaning, mice were

continuously fed the A- or A+ diet until the end of the study. Serum retinol status was

determined by ultra pressure liquid chromatography at 6-7 weeks of age in pooled

samples. For some experiments, A- mice were treated with 37.5 µg of all-trans RA

(Sigma Aldrich, St Louis, MO) administered orally in 10 μl corn oil three times per week

(19) . For some experiments, mice were injected i.p. with E. coli O111:B4 LPS (6

mg/kg, Sigma-Aldrich). Experimental procedures were approved by the Office of

Research Protection Institutional Animal Care and Use Committee of the Pennsylvania

State University, University Park, PA.

C. rodentium infection. C. rodentium strain ICC169 (nalidixic acid resistant) and

bioluminescent strain ICC180 (kanamycin resistant) were kind gifts of Gad Frankel

(London School of Medicine and Dentistry, London UK). C. rodentium ICC169 was

cultured in Luria-Bertani (LB, EMD Chemicals, Inc., Gibbstown, NJ) broth containing 50

μg/ml nalidixic acid (Sigma-Aldrich) while, C. rodentium ICC180 was cultured in LB

broth containing 100 μg/ml kanamycin (Sigma-Aldrich). Mice 8-10 weeks of age were

infected by oral gavage with 5×109 CFU in 200 μl of C. rodentium strain unless

otherwise noted. For studies looking at in vivo infection kinetic, 5×109 CFU C.

rodentium ICC180 was used. Animals were imaged every other day using the IVIS50

33

small animal imaging system (Xenogen Corp., Alameda, CA, USA). Images were

analyzed by Living Image software (PerkinElmer, Waltham, MA). Additional groups of

mice were injected i.v. with 104-108 CFU of C. rodentium strain ICC169. Feces and

other tissues were collected, homogenized and plated in serial dilutions on LB agar plates

containing naladixic acid.

For most experiments mice were housed 1 per cage from the time of infection to prevent

transmission from mouse to mouse. Natural transmission experiments were performed as

previously described (20). Briefly, A-, RA d0, and A+ mice were infected via oral

gavage with 5×109 CFU. Three days post-infection, each infected “seed” mouse was co-

housed with two naive A+ mice.

Histology: Distal colon sections were fixed in 10% formalin, sectioned and stained with

hematoxylin and eosin (Pennsylvania State University Animal Diagnostic Laboratory,

University Park, PA). Specimens were coded and evaluated in a blinded fashion by a

board certified laboratory animal veterinarian with training in pathology. Crypt

measurements were taken at 100X using the cellSens software (Olympus Corp., Center

Valley, PA, USA). Sections were scored on a scale from 0 to 4 as follows: severity of

inflammation (0 = none, 1= minimal, 2= mild, 3= moderate, 4= extensive); epithelial

sloughing (0 = none, 1 = minimal, 2 = mild, 3= moderate, 4= extensive); distention of

muscularis (0 = none, 1 = minimal, 2 = mild, 3 = moderate, 4 = extensive); edema (0 =

none, 1 = minimal, 2= mild, 3 = moderate, 4 = extensive). Total histology scores were

generated by adding the scores for each category together, generating a value from 0-16

for each sample.

Flow cytometry: Colonic IELs were isolated as previously described and stained for

34

flow cytometry (21). Cells were counted and stained with PE-Cy5 TCRβ (BD

Pharmingen, San Jose, CA USA), FITC CD8β (eBioscience San Diego, CA, USA), PE-

Cy7 CD8α (BioLegend, San Diego, CA, USA) or PE Texas Red CD4 (Invitrogen,

Carlsbad, CA, USA). Cells were analyzed on a FC500 Bench top cytometer (Beckman

Coulter, Brea, CA, USA) and data was analyzed using FlowJo 7.6.1 software (Tree Star,

Ashland, OR, USA).

ELISA: Cytokine production in the serum was measured for TNF-α, IL-1β and IFN-γ

levels by ELISA and following the manufacturer’s instructions (BD Biosciences,

Minneapolis, MN).

Statistical Analysis: Statistical analyses were performed using GraphPad Prism

software (Graphpad, La Jolla, CA, USA). Two-tailed Student’s t-tests were used for

serum retinol analysis. One-way ANOVA with Tukey’s post-hoc test were used to

compare the systemic C. rodentium loads, bioluminescence quantification, and cell

population analysis. Two-way ANOVA with Bonferonni’s post-hoc test were used to

compare CFU, histology scores and crypt lengths. Log-rank tests were used for the

survival curves and ratios of serum cytokine producers. For all analyses, P<0.05 was

used as the limit for significance.

35

RESULTS

A- mice are more susceptible to C. rodentium infection than A+ mice. A+ and A-

mice were generated as previously described (5, 18). As expected (22), 6-7 week old A-

mice had significantly lower serum retinol than A+ mice (Fig. 2-1A). Bacterial fecal

shedding in A+ mice peaked around d10 and cleared within 25 days post-infection (Fig.

2-1B). The infection in A- mice followed the same kinetics as A+ mice until d14. A+

and A- mice had similar numbers of C. rodentium in the cecum and feces at d10 post-

infection (cecum data not shown and Fig. 2-1A). After d14 the A- mice continued to

shed high numbers of C. rodentium in their feces, whereas the A+ mice began to clear the

infection (Fig. 2-1B). All of the infected mice showed a small amount of weight loss (5%

of starting weight) within the first 2-4 days of infection but all of the A+ mice recovered

and no A+ mice died following C. rodentium infection (Fig. 1C). Conversely, some of

the A- mice failed to recover and instead lost significantly more of their initial body

weight (10-20%), which resulted in the premature lethality of 40% of the A- mice (Fig. 2-

1C). The A- mice that survived did not lose any more weight than the A+ mice, had

normal exploratory behaviors (data not shown) and otherwise were undistinguishable

from their A+ infected counterparts. A subset of A- mice die following weight loss and

the remaining mice resume normal behaviors although they have not cleared C.

rodentium.

To visualize the intestinal passage of C. rodentium in vivo, we made use of a

bioluminescent C. rodentium strain, and live animal imaging. Between d2 and d6 post-

infection, low levels of C. rodentium bioluminescence were detected in the upper parts of

the gastrointestinal (GI) tract of A+ mice (Fig. 2-1D). Just before the peak in fecal

36

shedding (Fig. 2-1B) the intensity of bioluminescence increased and moved into the

lower GI tract of A+ mice (Fig. 2-1D). After d10 of infection, the intensity decreased in

A+ mice and by d14, when fecal shedding of C. rodentium was on the decline in A+

mice, less bioluminescence was detected in the intact A+ animals (Fig. 2-1D). The

kinetics of C. rodentium transit in the A+ mice was similar to that reported previously in

wild-type mice (23). The bioluminescence in the A- mice matched that of the A+ mice at

d2 (Fig. 2-1D). However, as early as d4, the A- mice showed bioluminescence

throughout the GI tract, which persisted in intensity through d14 (Fig. 2-1D). Unlike the

A+ mice, the intensity of bioluminescence in A- mice between d4 and d14 was high

throughout the GI tract and significantly higher than in A+ mice (Fig. 2-1D and Fig. 2-2).

Thus, the transit of C. rodentium through the GI tract occurred more rapidly and persisted

for longer in A- than A+ mice and reflected the fecal shedding results.

T cell frequencies are lower in the vitamin A deficient gut. To determine if vitamin A

status causes differences in gut mucosal immune cell populations, the intraepithelial

lymphocyte (IEL) populations of the colon were characterized. IELs are in direct contact

with the intestinal epithelium and a source of T cells from the colon (24). In addition, the

T cells in the IEL are in close contact with the microbiota and enteric pathogens in the

colon. Fewer total IEL cells were isolated from the colons of uninfected A- versus A+

mice (Fig. 2-3A). C. rodentium infection resulted in a significant increase in the number

of cells in the colon of both A- and A+ mice (Fig. 2-3A). Interestingly, although the A+

mice resolved the infection by d37, as shown in Fig. 2-1B, the numbers of T cells

(TCRβ+) did not return to baseline by d37 pi (Fig. 2-3A and 2-3B). The total number of

IELs and TCRβ+ T cells in the colon of A- mice increased following infection but never

37

reached the levels present in infected A+ mice (Fig. 2-3A). The numbers of TCRβ+,

TCRβ+/CD8α+, and TCRβ+/CD8αβ+ T cells in the colon IEL compartment were lower

in A- than in A+ mice, both before and after infection (Fig. 2-3A-E). Therefore, although

the increases in total cell numbers and T cell subpopulations were similar in A+ and A-

mice, the A- mice consistently had fewer T cells in the colonic IEL compartment

compared to A+ mice. Differences in T cell numbers and populations between A+ and

A- mice were not associated with clearance of C. rodentium in A+ mice and persistence

of C. rodentium in A- mice.

Exacerbated inflammation and epithelial hyperplasia in A- mice following C.

rodentium infection. Histopathology sections of the colon were evaluated before and

after infection for signs of inflammation, tissue damage and hyperplasia, including

measurements of crypt length. Before infection, A+ and A- mice had low histopathology

scores that did not differ with vitamin A status (Fig. 2-3F and Fig. 2-4). Crypt lengths

also did not differ between uninfected A+ and A- mice (Fig. 2-3G). After infection, the

histopathology scores of A+ colons did not change significantly, either at peak infection

(d10) or after resolution of infection (d37) (Fig. 2-3F and 2-4). In A- mice,

histopathology scores were significantly higher at d10 post-infection, both compared with

baseline A- scores and scores for A+ mice at d10 (Fig. 2-3F and 2-4). By d37, the

histopathology scores of A+ and A- mice were the same in spite of the fact that A+ mice

had cleared the infection, while A- mice had not (Fig. 2-3F and 2-4). Crypt length in the

A+ mice was not affected by infection (Fig. 2-3G). Crypt length in the A- mice increased

significantly after infection and were significantly longer on both d10 and d37 than at

baseline and as compared to the A+ mice at all time points (Fig. 2-3G). Overall, although

38

A- mice exhibited more inflammation than A+ mice at d10 post-infection the effect was

not present at d37 even though A- mice continued to harbor C. rodentium and A+ mice

did not.

RA treatment of A- mice results in clearance of C. rodentium. To determine if RA

could rescue the severe infection in A- mice, A- mice were orally dosed with RA. RA

dosing began either 2 wks before infection (RA -d14), or on the day of infection (RA d0)

(Fig. 3). Once started, RA dosing continued 3 times weekly throughout the remainder of

the experiment. Treatment of A- mice with RA, either 2 wks before (RA -d14) or on the

day of infection (RA d0), resulted in fecal shedding of C. rodentium similar to that of A+

mice (Fig. 1B and Fig. 2-5). In a separate experiment, the RA d0 mice were also infected

with bioluminescent C. rodentium. The bioluminescence in the RA d0 mice was of a

high intensity and resembled that in A- rather than A+ mice (Fig. 2-1D and 2-2).

Interestingly, by d14 the intensity of bioluminescence in the RA d0 group was lower and

resembled the A+ rather than the A- mice (Fig. 2-1D and 2-2).

To determine whether RA could be used therapeutically in A- mice with an established

C. rodentium infection, the RA treatment of A- mice was started on d14 post-infection

(RA +d14). Day 14 of infection occurs just after the peak of fecal shedding and before

A+ mice show a rapid decline in C. rodentium shedding (Fig. 2-1B). Treatment of A-

mice with RA, three times weekly, beginning on d14 resulted in a gradual decline in fecal

shedding of C. rodentium (Fig. 2-5), which was significant as early as 4 days following

the start of RA treatment, as compared to untreated A- mice (d18 post-infection, Fig. 2-

5). As observed in earlier experiments, untreated A- mice maintained a persistent

infection while the RA +d14 treated mice cleared the infection 18d after the start of the

39

RA treatment (d32 post-infection, Fig. 2-5). Thus, RA treatment of A- mice cleared the

C. rodentium infection, which otherwise was persistent.

Increased C. rodentium CFU in the liver and spleen of A- mice. At d14 post-infection

mice that had been infected with the bioluminescence strain of C. rodentium were

euthanized to determine which organs harbored C. rodentium. In the A+ group,

bioluminescence was detected in the colon but not in other parts of the GI tract (Fig. 2-

6A). In addition, A+ mice did not have visible bioluminescence in their spleen or liver at

d14 post-infection (Fig. 2-6B). A- mice had high levels of bioluminescence in the upper

portions of the GI tract that were significantly higher than in A+ mice (Fig. 2-6C). A-

mice also had visible bioluminescence at d14 in both the spleen and liver (Fig. 2-7A and

Fig. 2-6B). The bioluminescence in the spleen and liver of RA d0 mice was more similar

to A+ mice than to A- mice at d14 post-infection (Fig. 2-7A and Fig. 2-6B).

Quantification of the bioluminescence showed that A- mice had higher bioluminescence

in the spleen, liver and SI than either the A+ or the RA d0 mice (Fig. 2-7A and Fig. 2-

6B).

Next we determined whether viable C. rodentium was detectable in the spleen and liver

of our mice following gastric infection with C. rodentium. Cultures of the spleen and

liver at d10 and d14 post-infection showed that A+ mice had detectable C. rodentium in

their internal organs (Fig. 2-7B, and d14 data not shown). A- mice had significantly

more C. rodentium in the spleen and liver than A+ mice (Fig. 2-7B), while the spleen and

liver of RA d0 mice did not differ from those of A+ mice but were significantly different

than those of A- mice (Fig. 2-7B). At d37 of infection the A- mice no longer had C.

40

rodentium present in the spleen and liver even though they continued to shed C.

rodentium in the feces (data not shown).

Mortality of A- mice following infection with C. rodentium is not due to over-

production of systemic cytokines. To determine whether A- mice died due to cytokine

over-production, serum cytokine levels were determined at before (d0) and at the peak

(d10) of infection in A+ and A- mice (Table 2-1). None of the A+ mice had detectable

levels of any cytokines before infection. At d0, several of the A- mice had detectable

levels of TNF-α, IL-1β or IFN-γ in their serum (Table 2-1). Infection resulted in

detectable TNF-α, IL-1β and IFN-γ in the serum of both A+ and A- mice. There was

significantly more A- mice with IL-1β in the serum (10 of 19) than A+ (1 of 13) mice at

d10 post-infection (Table 2-1). Serum IFN-γ was higher but not significantly different in

A- mice compared to A+ mice at d10 post-infection (P=0.0817). To measure the

capacity for cytokine production, LPS was injected ip into A- and A+ mice to measure

serum cytokine response. A- mice produced significantly more TNF-α than A+ mice

following LPS injection, while the IL-1β response was the same in A+ and A- mice (Fig.

2-8A and IL-1β data not shown). Overall A- mice were more likely to have IL-1β

detectable in the serum after C. rodentium infection and produced more TNF-α after LPS

than A+ mice.

We hypothesized that A- mice were dying following oral challenge with C. rodentium

because of systemic spread. We attempted to determine the LD50 of i.v. injected C.

rodentium in A+ mice. All of the A+ mice survived an i.v. dose of 108 C. rodentium and

readily cleared the bacteria. A- mice also survived an i.v. injection of 108 C. rodentium.

A+ mice had the highest number of organisms in the spleen and liver 1d post-i.v.

41

infection with 108 CFU, which declined at d3 and again at d5 and was completely

cleared by d10 post-infection (Fig. 2-8B and 2-8C). The group x time interaction showed

no differences in the clearance of i.v. injected C. rodentium between A+ and A- mice

(Fig. 2-8B and 2-8C). In addition, i.v. injection was not lethal and did not induce a

systemic cytokine response in either A+ or A- mice (data not shown).

Host vitamin A status does not alter C. rodentium infectivity. Previous reports have

demonstrated that natural infection of C. rodentium to co-housed uninfected mice occurs

with several fewer log units of bacteria than with laboratory grown C. rodentium (20).

We hypothesized that vitamin A and RA were attenuating the infectivity (virulence) of

C. rodentium and that C. rodentium that has been passaged through A- mice might be

more virulent. A+, RA d0, and A- seed mice were infected by gavage using laboratory

grown C. rodentium and 3d later each was co-housed with naïve A+ mice, housed in

groups of 2 with the seed mice. As described previously (20), all seed mice, despite their

vitamin A status, infected all co-housed naïve A+ mice within 3 days of exposure (data

not shown). Culturing of the feces, spleen and liver from the cage mates of A+, A- and

RA d0 seed mice showed that there were no differences in the CFU of C. rodentium

recovered in the spleen, liver and feces via natural transmission from either A+, A- or RA

d0 seed mice (Fig. 2-9). Thus the data do not show an effect of vitamin A status and or

RA treatment on C. rodentium infectivity.

42

DISCUSSION

A- mice were found to be significantly more susceptible to C. rodentium infection

compared to A+ mice. The increased susceptibility of A- mice included lethality of 40%

of the A- mice by d14. The kinetics of shedding of C. rodentium in A+ and A- mice were

not different before d14, after which the A+ mice proceeded to clear the infection while

the A- mice either died or developed a persistent infection. The data suggest that early

innate immune responses are adequate in A- mice during the initial phase of the infection,

when fecal bacterial counts are increasing, and instead suggest that A- mice failed to

generate an acquired immune response necessary to clear the infection. Our results

unexpectedly showed that A- mice became chronic carriers of C. rodentium, while no

longer showing symptoms (inflammation of the colon, diarrhea etc.). Correcting vitamin

A status or treating A- mice with RA effectively eliminated the infection and improved

survival of A- mice, suggesting an added benefit not previously recognized for vitamin A

and/or RA interventions.

The increased mortality of A- mice following GI infection was not due to the systemic

spread of C. rodentium, increased systemic cytokine production or the virulence of C.

rodentium. Others have reported mortality following gavage of C. rodentium in mice and

have attributed that mortality to systemic spread and poly-microbial sepsis (15, 25, 26).

Susceptible strains of mice (C3H/HeJ, C3H/HeOuJ and C3H/HeN) develop a severe

infection with 100% lethality due to bacterial translocation from the gut, cytokines in the

serum and crypt cell apoptosis (25). By comparison, C57BL/6 mice are relatively

resistant to C. rodentium (15, 17, 25). For the first time, our data clearly show that C.

rodentium does not grow following i.v. injection of large numbers of bacteria in either

43

A+ or A- mice, indicating that vitamin A deficiency does not affect the ability to survive

a systemic infection with C. rodentium. However, A- mice were more susceptible to oral

infection with C, rodentium. We necropsied one recently deceased and one moribund A-

mouse. Very little food was found in the stomach of either mouse, suggesting that the

mice had stopped eating. The necropsy of A- mice failed to identify evidence of systemic

infection (data not shown). It has been shown that LPS-induced proinflammatory

cytokines such as TNF-α and IL-1β can result in anorexia (27). Our data are consistent

with TNF-α and IL-1β induced anorexia, since the A- mice that died had lost significant

amounts of weight (10-20% of original body weight). In addition, A- mice had higher

TNF-α levels after LPS challenge and were more likely to have detectable IL-1β in their

serum after infection than A+ mice. Surviving A- mice may have been just below the

threshold of the response and therefore survived the infection-induced anorexia. Based

on the inability of C. rodentium to grow following an iv injection and the necropsies of

moribund and dead A- mice, we concluded that the mortality in our A- mice was not due

to sepsis and/or the systemic spread of the infection.

A- mice had reduced numbers of total colonic IELs and all IEL T cell subsets in the colon

before infection. Infection induced homing and expansion of T cells in the gut of both

A+ and A- mice. Although the A- mice were persistently infected with C. rodentium,

colonic inflammation as determined by histological score resolved and was not different

at d37 from A+ mice. This same phenomenon occurred in germfree mice mono-

associated with C. rodentium (28). In germfree mice C. rodentium infection was not

cleared but the inflammatory response in the colon was nevertheless resolved (28). It

therefore seems that vitamin A is not required to resolve inflammation in the colon and

44

that in the absence of vitamin A, T cells are able to arrive and respond to the infection.

However, the acquired immune response in the A- mice was ineffective at eliminating the

infection. The data suggest a requirement for vitamin A/RA to mount effective protective

immunity after the initial infection. Since RA treatments effectively reduced C.

rodentium numbers in already infected A- mice within 4 days of treatment, it will be of

interest to identify the targets of RA in the GI tract for C. rodentium clearance.

In our study, A- mice developed persistent and sometimes fatal enteric infections. The

cause of the premature lethality of A- mice was not due to sepsis and/or systemic spread

of C. rodentium. Instead it seems that a high load of C. rodentium in the gut of A- mice

results in infection-induced anorexia in 40% of the mice. When given early, vitamin A

and/or RA interventions protected A- mice from the lethality following C. rodentium

infection and when administered later RA cleared the persistent infections that occurred

in surviving A- mice. Our work has important implications for the developing world

where vitamin A deficiency is prevalent. In particular, vitamin A-deficient humans and

animals could be reservoirs for E. coli-like enteric pathogens. Vitamin A and RA

treatments might be useful interventions to decrease morbidity and mortality from enteric

infections. We show here a novel and unappreciated role for vitamin A/RA for

eliminating persistent enteric infections and show added benefits to improving vitamin A

status in the developing world.

45

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and women of reproductive age. J Nutr 132: 2857S-2866S. 3. Fawzi, E. V. a. W. W. 2000. Vitamin A supplementation: implications for

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4. Cantorna, M. T., F. E. Nashold, and C. E. Hayes. 1994. In vitamin A deficiency multiple mechanisms establish a regulatory T helper cell imbalance with excess Th1 and insufficient Th2 function. J Immunol 152: 1515-1522.

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9. Carman, J. A., L. Pond, F. Nashold, D. L. Wassom, and C. E. Hayes. 1992. Immunity to Trichinella spiralis infection in vitamin A-deficient mice. J Exp Med 175: 111-120.

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13. Spencer, S. P., C. Wilhelm, Q. Yang, J. A. Hall, N. Bouladoux, A. Boyd, T. B. Nutman, J. F. Urban, Jr., J. Wang, T. R. Ramalingam, A. Bhandoola, T. A. Wynn, and Y. Belkaid. 2014. Adaptation of innate lymphoid cells to a micronutrient deficiency promotes type 2 barrier immunity. Science 343: 432-437.

14. Collins, J. W., K. M. Keeney, V. F. Crepin, V. A. Rathinam, K. A. Fitzgerald, B. B. Finlay, and G. Frankel. 2014. Citrobacter rodentium: infection, inflammation and the microbiota. Nat Rev Microbiol 12: 612-623.

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15. Bry, L., and M. B. Brenner. 2004. Critical role of T cell-dependent serum antibody, but not the gut-associated lymphoid tissue, for surviving acute mucosal infection with Citrobacter rodentium, an attaching and effacing pathogen. J Immunol 172: 433-441.

16. Sonnenberg, G. F., and D. Artis. 2012. Innate lymphoid cell interactions with microbiota: implications for intestinal health and disease. Immunity 37: 601-610.

17. Mundy, R., T. T. MacDonald, G. Dougan, G. Frankel, and S. Wiles. 2005. Citrobacter rodentium of mice and man. Cell Microbiol 7: 1697-1706.

18. Smith, S. M., N. S. Levy, and C. E. Hayes. 1987. Impaired immunity in vitamin A-deficient mice. J Nutr 117: 857-865.

19. Ma, Y., Q. Chen, and A. C. Ross. 2005. Retinoic acid and polyriboinosinic:polyribocytidylic acid stimulate robust anti-tetanus antibody production while differentially regulating type 1/type 2 cytokines and lymphocyte populations. J Immunol 174: 7961-7969.

20. Wiles, S., G. Dougan, and G. Frankel. 2005. Emergence of a 'hyperinfectious' bacterial state after passage of Citrobacter rodentium through the host gastrointestinal tract. Cell Microbiol 7: 1163-1172.

21. Lefrancois, L., and N. Lycke. 2001. Isolation of mouse small intestinal intraepithelial lymphocytes, Peyer's patch, and lamina propria cells. Curr Protoc Immunol Chapter 3: Unit 3 19.

22. Restori, K. H., K. L. McDaniel, A. E. Wray, M. T. Cantorna, and A. C. Ross. 2014. Streptococcus pneumoniae-induced pneumonia and Citrobacter rodentium-induced gut infection differentially alter vitamin A concentrations in the lung and liver of mice. J Nutr 144: 392-398.

23. Basu, R., D. B. O'Quinn, D. J. Silberger, T. R. Schoeb, L. Fouser, W. Ouyang, R. D. Hatton, and C. T. Weaver. 2012. Th22 cells are an important source of IL-22 for host protection against enteropathogenic bacteria. Immunity 37: 1061-1075.

24. Meresse, B., G. Malamut, and N. Cerf-Bensussan. 2012. Celiac disease: an immunological jigsaw. Immunity 36: 907-919.

25. Vallance, B. A., W. Deng, K. Jacobson, and B. B. Finlay. 2003. Host susceptibility to the attaching and effacing bacterial pathogen Citrobacter rodentium. Infect Immun 71: 3443-3453.

26. Ghosh, S., C. Dai, K. Brown, E. Rajendiran, S. Makarenko, J. Baker, C. Ma, S. Halder, M. Montero, V. A. Ionescu, A. Klegeris, B. A. Vallance, and D. L. Gibson. 2011. Colonic microbiota alters host susceptibility to infectious colitis by modulating inflammation, redox status, and ion transporter gene expression. Am J Physiol Gastrointest Liver Physiol 301: G39-49.

27. Sarraf, P., R. C. Frederich, E. M. Turner, G. Ma, N. T. Jaskowiak, D. J. Rivet, 3rd, J. S. Flier, B. B. Lowell, D. L. Fraker, and H. R. Alexander. 1997. Multiple cytokines and acute inflammation raise mouse leptin levels: potential role in inflammatory anorexia. J Exp Med 185: 171-175.

28. Kamada, N., Y. G. Kim, H. P. Sham, B. A. Vallance, J. L. Puente, E. C. Martens, and G. Nunez. 2012. Regulated virulence controls the ability of a pathogen to compete with the gut microbiota. Science 336: 1325-1329.

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Figure 2-1

Figure 2-1. A- mice are more susceptible to C. rodentium infection than A+ mice. A) Serum retinol analysis from groups of 6-7 week old A+ and A- mice. Each data point represents the serum retinol values in pooled samples from a different litter of mice (n= 10-13 liters). B) C. rodentium CFU in the feces. Data is shown as mean + SEM and 1 representative of 5 independent experiments with n=3-5/group. C) Survival of A- and A+ mice during C. rodentium infection with data combined from 5 experiments n=9-15/group. D) Whole body imaging of A+, A-, and RA d0 treated mice infected with bioluminescent C. rodentium and imaged. The image shows 1 mouse from a total of 3-4 mice/treatment group. Means of the bioluminescence from all the mice are shown in SFig.1. Two-tailed Student t-test (A), two way ANOVA with Bonferroni post-hoc tests (B), log-rank test (C), *P<0.05, **P<0.01, ***P<0.001

48

Figure 2-2 Figure 2-2: Quantification of whole body imaging during C. rodentium infection. Quantification of bioluminescence from whole body imaging of A+ A-, and RA d0 imaged at d2-14 post-infection. Data is representative from one set of images with n=3-4/treatment group. Two way ANOVA with Bonferroni post-hoc tests, data marked by “a” is significantly different from data marked by “b” with a P values of P<0.001.

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Figure 2-3

Figure 2-3: T cells in the IEL of the colon from A+ and A- mice before and after infection. A) Immune cell numbers in the colon IEL. B) TCRβ+ T cell numbers in the colon IEL. C) CD8α+ T cell numbers in the colon IEL. D) CD8αβ+ T cell numbers in the colon IEL. E) CD4+ T cells numbers in the colon IEL. Values are the mean + SEM combined data from 2-3 independent experiments with n=6-15 /group. Two way ANOVA with Bonferroni post-hoc tests. Asterisks indicate significant differences between groups at specific days by post test, *P<0.05, **P<0.01, ***P<0.001. F) Histology scores and G) crypt length of colons during infection. Values with different letters are significantly different from each other, P<0.05. A) Two way ANOVA with Bonferroni post-hoc tests, *P<0.05, ***P<0.001.

50

Figure 2-4

Figure 2-4: Colon histology from C. rodentium infected animals. Representative H&E stained distal colon sections from A + mice (A-D) and A- mice (E-H). A+ mice: A) d0, score of 4; B) d10, score of 5; C) Magnification of d10; D) d37, score of 3; A- mice: E) d0, score of 5; F) d10,score of 9; G) Magnification of d10; H) d37 (score of 3). Lines for scale = 200um for A, B, D-F, and H. Lines for scale = 100um for C and G. Photographs were taken with an Olympus BX61 camera.

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Figure 2-5

Figure 2-5: RA treatment of A- mice eliminates C. rodentium infection. A) CFU in the feces of A+ mice A- mice, A- mice treated with RA for 2wks before infection (RA –d14), A- mice treated with RA on the day of infection (RA d0) and A- mice treated with RA starting on d14 of infection (RA d+14). Values are the mean + SEM of one representative of 2 independent experiments and n=3-5/group. Two-way ANOVA with Bonferroni post-hoc tests. Values with different letters are significantly different from each other, P<0.01-0.0001.

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Figure 2-6

Figure 2-6: Systemic C. rodentium in tissues. Images of (A) GI tract and (B) spleen and liver. (C) Quantification of bioluminscence from the small intestine. One way ANOVA with Tukey post-hoc tests, * P<0.05. n.s. - not significant.

53

Figure 2-7

Figure 2-7: Systemic spread of C. rodentium following GI infection. Mice were sacrificed at d14 post-infection with the bioluminescent strain of C. rodentium and the A) GI tract and B) spleen and liver were imaged and quantitated. An example image is shown in Fig. 2-6. Values are from the images of n=3-4 mice/treatment group. CFU of C. rodentium recovered in the B) spleen and liver of d10 infected mice. Values in B) are the mean + SEM combined from three independent experiments with n=9-15/group. One-way ANOVA with Tukey post-tests.

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Table 2-1

55

Figure 2-8

Figure 2-8: The effect of vitamin A status on LPS response and i.v challenge with C. rodentium. A) Serum TNF-α in A- and A+ mice treated with LPS. CFU in the B) spleen and C) liver of mice following i.v. injection of C. rodentium. Values are the mean + SEM combined from two-three independent experiments with n=8-9/group/time-point. Two-tailed Student t-test (A), Two-way ANOVA with Bonferroni post-hoc tests (B and C). Asterisks indicate significant differences between groups at specific days by post test.; *P<0.05.

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Figure 2-9

Figure 2-9: Natural transmission of C. rodentium is not affected by the host vitamin A status. CFU in the feces A), spleen B), and liver C) of A+ cage mates of A-, RA d0 and A+ laboratory inoculated mice. N=4-6/group. Values are the mean + SEM combined for two independent experiments. One-way ANOVA with Tukey post-tests.

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Chapter 3

Short term retinoic acid treatment promotes mucosal IL-17 expression leading to clearance of enteric infection

58

ABSTRACT

Retinoic acid (RA), the active metabolite of vitamin A, is an important factor in

Citrobacter rodentium clearance. One dose of RA at d7 post infection was effective at

clearing C. rodentium infection in vitamin A deficient (A-) mice. The early effects of RA

included induction of il17a mRNA expression in the colon and regIIIy expression in the

GI tract that occurred after 4 days or 2 doses of RA treatment. One important source of

IL-17a in the gut are CD4+ T cells. Paradoxically, in vitro RA treatment of A- CD4+ T

cells inhibited IL-17a secretion. To study the specific effects of RA on T cells, mice

expressing a dominant negative form of the retinoic acid receptor (dnRAR) were crossed

with Lck-cre mice to generate T-dnRAR mice. T-dnRAR mice had significantly more

CD8+ single positive thymocytes and significantly fewer CD8+ T cells in the spleen and

mesenteric lymph nodes than WT mice. The targets of RA include T cells, IL-17a

production and CD8 T cell development and/or differentiation. In the gut induction of

mRNA for IL-17A was associated with the ability of RA treated mice to clear C.

rodentium.

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INTRODUCTION

Vitamin A deficiency remains a significant problem in the human population,

particularly in resource limited countries where nutritional intake is a public health

concern (1). Vitamin A deficiency is suggested to contribute to higher prevalence of

diarrheal and respiratory diseases which leads to increased childhood mortality and

vitamin A supplementation effectively decreased mortality in young children by reducing

infection severity (2). Vitamin A deficient (A-) mice treated with retinoic acid (RA), the

active form of vitamin A, promoted clearance of an enteric bacterial infection (3).

Vitamin A is important for protection against infection but the mechanisms of protection

are unclear.

Vitamin A regulates immune cell development, phenotype, function and homing.

RA has a wide variety of effects on the immune system including upregulating gut

homing receptors, α4β7 and CCR9, on T and B cells (4-6) and balancing inflammatory

and regulatory immune responses (7). The balance between inflammatory and regulatory

responses are particularly important in the gut because the mucosal immune system has

to constantly discriminate pathogen from commensal and self from non-self. Dendritic

cells in the gut associated lymphoid tissue (GALT) have the capability to produce RA

(4). RA, in conjunction with transforming growth factor-β (TGF-β), promoted FoxP3+

expression in naïve CD4+ T cells (8, 9). Conversely, RA suppressed-6 and TGF-β

mediated Th17 cell induction (10, 11). These studies suggest that RA is an important

mediator of Th17 and Treg immune responses in vitro. Interestingly, in vivo A- mice had

fewer total T cells in the gut and decreased Th17 responses and increased Th1 responses

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(12-16). How RA regulates T cells differently in vitro compared to in vivo is not

completely understood.

C. rodentium is a murine pathogen that models human enteropathogenic E. coli

infection and colonizes the intestinal epithelium of the colon through attaching and

effacing lesions (17). C. rodentium must compete with the host microbiota to first

establish colonization (17). Following colonization with C. rodentium, a protective

immune response is mounted, which requires IL-22 production from innate lymphoid

cells, macrophages and γδ T cells to promote intestinal epithelial barrier protection and

induce an adaptive Th17 cell response (18-22). Additionally, T cells and B cells are

required for clearance C. rodentium as mice deficient in T cells, B cells, or both develop

fatal infections within 20 days of inoculation (21, 23).

We have previously reported the effects of host vitamin A status on C. rodentium

infection. A+ mice cleared the bacteria but A- mice failed to clear the infection and

~40% of the A- mice died between d7 and d14 post-infection (3). RA treatment before,

at the time of infection and during infection promoted C. rodentium clearance in the A-

host (3). There was no difference in fecal shedding in A+, A-, or RA dosed animals

before day 10, which suggested that early innate immunity in the A- mice was adequate.

A- mice had significantly fewer T cells in the gut before infection, and although the

numbers of T cells increased with infection, A- mice had fewer T cells in the gut

throughout the course of infection (3). Other studies have shown that animals with

acquired immune defects succumb to C. rodentium infection between d7 and d14 post-

infection (23). Our studies indicate that A- mice are more susceptible to C. rodentium

61

infection, possibly due to an inability to mount an adequate mucosal acquired immune

response.

Our previous study suggested that inadequate acquired T cell responses in A-

mice might be the cause of increased susceptibility. Experiments were done to determine

the targets of RA that promote clearance of C. rodentium. RA treatment starting as early

as d7 post-infection was effective for clearance of C. rodentium. One dose, two doses

and three doses of RA starting at d7 post-infection induced bacterial clearance as well.

RA induced il17a mRNA expression in the colon within 4 days of treatment that was

associated with the successful clearance of C. rodentium. Two doses of RA also induced

regIIIy expression in the GI tract. Interestingly, RA treatment in vitro inhibited IFN-γ

and IL-17a secretion from A- CD4+ T cells. Expression of the dominant negative RAR

(dnRAR) in T cells using an Lck promoter resulted in increased CD8+ single positive

cells in the thymus but decreased CD8+ cells in the spleen and mesenteric lymph node

(MLN). Other possible targets of RA were B cells and the microbiota however no

differences in B cell function or microbiota were detected as a function of vitamin A

status and/or RA treatments. Our data indicates that in vivo RA treatment promoted IL-

17a production in the colon and RA signaling was important in CD8+ T cell development

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MATERIALS AND METHODS

Mice: C57BL6 mice from Jackson Laboratories (Bar Harbor, MN) were bred at the

Pennsylvania State University (University Park, PA) for experiments. Vitamin A

deficient (A-) and vitamin A sufficient (A+) mice generated as previously described (15,

24). Briefly, mice were fed a purified diet that did not contain any vitamin A (A-) or that

contained 25 µg of retinyl acetate (vitamin A) per day (A+). At weaning, mice were

continuously fed the A- or A+ diet until the end of the study. Vitamin A status was

determined by using ultra pressure liquid chromatography to examine pooled serum

retinol levels in 6-7 week old mice. For some experiments, A- mice were treated with

37.5 µg of all-trans RA (Sigma Aldrich, St Louis, MO) administered orally in 10 μl corn

oil (25). Wild type (WT) C57Bl6 mice fed standard chow diets were used as controls for

microbiota studies.

T-dnRAR mice were generated by crossing Lck-cre mice from Jackson Laboratories (Bar

Harbor, MI) and dominant negative RARα mice, a kind gift from Dr. Randolph Noelle

(Dartmouth University, Lebanon, NH). Experimental procedures were approved by the

Office of Research Protection Institutional Animal Care and Use Committee of the

Pennsylvania State University, University Park, PA.

C. rodentium infection: C. rodentium strain ICC169 (nalidixic acid resistant) was a kind

gifts of Gad Frankel (London School of Medicine and Dentistry, London UK). C.

rodentium ICC169 was cultured in Luria-Bertani (LB, EMD Chemicals, Inc., Gibbstown,

NJ) broth containing 50 μg/ml nalidixic acid (Sigma-Aldrich) and mice 6-8 weeks of age

were infected by oral gavage with 5×109 CFU in 200 μl. Feces and other tissues were

63

collected, homogenized and plated in serial dilutions on LB agar plates containing

naladixic acid.

Cell isolation and culture: Spleens, thymus and MLNs were homogenized and lysed

with red blood cell lysis buffer to obtain single-cell suspensions. SI LPLs and colonic

LPLs were isolated at previously described and stained for flow cytometry (26). For

some experiments, CD4+ T cells from the spleen were purified using mouse CD4 cell

recovery column kits following the manufacturer’s instructions (Cedarlane Laboratories

Ltd, Burlington, NC). The purity of the CD4+ cells after enrichment was between 75-

82% CD4+ cells as determined by flow cytometry.

Cultures included the following concentrations of antibodies: 0.5 μg/ml anti-CD3 alone

or 0.5 μg/ml anti-CD3 with 5 μg/ml anti-CD28 (BD Pharmingen, San Diego, CA) in

RPMI 1640-C containing 10% FBS (Equitech-Bio, Inc, Kerrville, TX), 2 mM L-

glutamine, 5 mM β-mercaptoethanol (Invitrogen, Carlsbad, CA), and 10 μg/ml

gentamycin (Teknova, Hollister, CA). Some cultures contained 10nM of all-trans RA.

ELISAs: Supernatants were collected for cytokine detection and production of IFN-γ, IL-

4, IL-10 and IL-17 (BD Biosciences) were measured by ELISAs following the

manufacturer’s instructions. Serum was collected for antibody titer determination. Total

IgA production was measured by the Mouse IgA ELISA Quantitation Set (Bethyl

Laboratories, Inc, Montgomery, TX). C. rodentium specific antibody titers were

determined by coating ELISAs plates with 30ng/ml sonicated C. rodentium. Horseradish

peroxidase conjugated anti-mouse IgG1 and IgG2 (BD Pharmagen) were used as

detection antibodies and serially diluted pooled serum samples were used as standards to

determine relative values.

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Flow cytometry: SI LPLs, colonic LPLs, splenocytes and thymocytes were collected and

processed according to standard protocol. Cells were counted and stained with FITC

B220, PE CD4, PE-CD19, PE-Cy5 TCRβ (BD Pharmingen, San Jose, CA USA) and

PE-Cy7 CD8α (BioLegend, San Diego, CA, USA). Cells were analyzed on a FC500

Bench top cytometer (Beckman Coulter, Brea, CA, USA) and data was analyzed using

FlowJo 7.6.1 software (Tree Star, Ashland, OR, USA).

RT-PCR: Whole tissues were snap frozen and RNA was isolated with TRiZOL

(Invitrogen). cDNA was synthesized using TaqMan reverse transcription reagents kit

(Applied Biosystems, Carlsbad, CA). RT- PCR was performed using SYBR green mix

(BioRad) and MyiQ Single-Color Real-Time PCR machine (BioRad). Serially diluted

standards for each gene of interest were run to determine relative copy number.

Expression levels were normalized to the expression of the average of HPRT and

GAPDH and values from A+ uninfected tissue samples were set at 1.

For bacterial DNA isolation, feces were collected from A- cagemates or A+ cagemates,

or from A- cagemates before and after two weeks of RA treatment (37.5 ug/10ul orally

three times a week). DNA was isolated from fecal samples using QIAamp DNA stool

minikit (Qiagen, Valcencia, CA). 10 ng/sample was amplified using SYBR green mix

(BioRad) and MyiQ Single-Color Real-Time PCR machine (BioRad). Data was analyzed

using the ΔΔCT method using 16S as the reference gene and expressed as relative

expression of specific bacterial phyla (27) compared to bacterial DNA isolated from mice

on standards chow diet set as 1. Primers for all RT-PCRs are in Table 3-1.

Statistical Analysis: Statistical analyses were performed using GraphPad Prism

software (Graphpad, La Jolla, CA, USA). Two-way ANOVA with Bonferonni’s post-

65

tests were used to compare CFUs, systemic bacterial loads and tissue RT-PCR across

different dietary groups. Student’s t-tests or nonparametric Mann Whitney tests were

used to compare bacterial DNA analysis, cytokine production and immune cell

populations between dietary groups or genotypes. For all analyses, P<0.05 was used as

the limit for significance.

66

RESULTS

RA treatment of A- mice at d7 promoted clearance.

A- mice failed to clear a C. rodentium infection (Figure 3-1A) and previously published

(3). RA treatment of A- mice at the time of infection (RA d0) resulted in bacterial

clearance (Figure 3-1A) (3). To determine whether RA treatment at the peak of infection

was as effective as at the day of infection, A- mice were treated continuously beginning

at day 7 post-infection (RA d7). RA d7 treatment was as effective as RA d0 treatment for

eliminating C. rodentium (Figure 3-1A). To determine how many RA doses were needed

to protect A- mice from C. rodentium, mice were infected for 7 days and received one

dose of RA (RA 1x) at d7, two doses of RA (RA 2x) at d7 and d9, or three doses of RA

(RA 3x) on d7, d9 and d11 post-infection. All three dosing schemes resulted in clearance

of C. rodentium from the A- host (Figure 3-1a and 31B). In one of 2 experiments, the

RA 3x treated A- mice cleared the infection by d23 and the 3X RA treatment was as

effective as continuous dosing with RA beginning at d0 or d7 (Figure 3-1A and 3-1B). In

the second experiment, the RA 3x group was not cleared more quickly than the other

groups (Figure 3-1C) and therefore the 1X, 2X and 3X RA treatment groups were equally

effective at clearing C. rodentium in A- mice. The average day of clearance from the

combined experiments was not significantly different between the RA 3x and the RA 1x

groups (d22 +1 vs. d26 +2, P=0.0892 by Mann-Whitney test). However, both

experiments showed that 1 dose of RA given at d7 post-infection effectively cleared C.

rodentium in the A- host.

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RA treatment induced il17a expression in the colon.

To determine the short term effects of RA during C. rodentium infection, A- mice were

infected with C. rodentium for 7 days. At d7, A- mice received one oral dose of RA or

were left untreated. Half of the mice were sacrificed at d9 post-infection while the

remaining were dosed a second time with RA (RA 2X) or remained untreated and then

sacrificed at d11 post-infection (Figure 3-2A). The C. rodentium CFU in the spleen and

liver were not different at d9 or d11 post-infection in the RA 1x, RA 2x and A- mice

(Figure 3-2A). Short term RA treatment had no effect on the CFU in the spleen and liver.

To identify potential targets of RA in the colon, mRNA analysis of colon tissues were

performed. There were no differences in mRNA for il17a, il22, regIIIy, foxp3 or rorc in

the d9 and RA 1x treated mice compared to the A- d9 mRNA values (Figure 3-2B).

There were no differences in mRNA for il22, regIIIy, foxp3 or rorc mRNA expression in

the colons of d11 post-infection A- and 2x RA mice (Figure 3-2A). il17a mRNA

expression was significantly increased in the colon of RA 2x dosed mice compared to A-

mice at the same time point (P<0.01) (Figure 3-2A). Two doses of RA induced

expression of il-17a in the colon of A- mice. In addition, there was a significant

interaction effect on il17a between the VA groups and time (P=0.0105), suggesting that

the group effect differed over time. There was a significant time effect on il22 expression

(P=0.0344), indicating that IL-22 production increased during C. rodentium infection but

was not induced by oral RA treatment.

mRNA levels were measured in the duodenum and ileum of the small intestine (SI) and

colon after RA 2x and at d11 post C. rodentium infection. The duodenum contains the

highest relative concentration of host produced RA in the GI tract (28) and the ileum is

68

still colonized with C. rodentium at d10 post-infection (3). Expression of rorc, foxp3, il6,

il17a, il22 and regIIIy were significantly different based on location in the gut

(P<0.0001). We observed a significant increase in il17a expression in the colon of RA

treated mice (P<0.0286) and a significant interaction effect (P=0.0044) suggesting that

the group effect differs over time. Average expression was highest in the ileum and

lowest in the duodenum for all studied genes, indicating that gene expression can differ in

the GI tract based on location (Figure 3-3). A group effect was observed for regIIIy

(P=0.0169), indicating that RA treatment induced regIIIy expression throughout the GI

tract (Figure 3-3).

Vitamin A and the gut microbiota.

Previous reports have suggested that the microbiota regulated the ability to produce IL-17

(29). Additionally, one report showed that A- mice had fewer gut microbes in the SI and

feces as well as fewer mucosal Th17 cells (16). If the microbiota was different between

A+ and A-mice, this could explain the differences in IL-17 production and the inability of

the A- mice to clear C. rodentium. There were no differences in the bacterial 16S

ribosomal content of the feces from the mice as a consequence of vitamin A status

(Figure 3-4A). In addition, the feces from A+ and A- mice contained similar ratios of

Bacteriodies, Firmicutes, Actinobacter and γ-Proteobacter phyla DNA (Figure 3-4B).

Our results indicate that vitamin A status did not affect total bacteria in the feces or the

representation of 4 of the common phyla of bacteria found in mice.

69

Serum antibody levels and B cell numbers were not affected by vitamin A

deficiency.

Vitamin A regulates B cell responses (5, 7, 25). We hypothesized that A- mice failed to

mount a robust C. rodentium specific antibody response that might explain the inability to

clear an infection. There was no difference in total serum IgA or C. rodentium IgA levels

between A- and A+ mice but there was a significant interaction effect (P=0.0400) in total

IgA levels, indicating that the group effect differed over time (Figure 3-6A). C.

rodentium specific IgG1 and IgG2 titers were not affected by host vitamin A status

(Figure 3-6B). There was an observed time effect for C. rodentium specific IgA

(P=0.0005) and IgG2 (P=0.0025), indicating that antigen specific antibody levels

increased during infection. Additionally, there was no difference in B cell numbers in the

SI LPL (Figure 3-6C) or colonic LPL (Figure 3-6 D). B cell numbers and C. rodentium

specific antibody responses were not affected by vitamin A.

In vitro RA treatment inhibited Th1/Th17 cytokine production from T cells.

As previously published, 10nM RA inhibited IFN-γ secretion from A- splenocytes

(Figure 3-6A) (30). IL-17a production from A- and RA treated splenocytes was below

the limit of detection. RA treatment of enriched CD4+ T cells from mice significantly

inhibited IFN-γ and IL-17a production (Figure 3-6B). IL-4 and IL-10 levels were

undetectable in splenocyte and CD4+ T cell cultures (data not shown). Our data confirms

data in the literature demonstrating an inhibitory effect of RA on IFN-γ and IL-17

production from T cells in vitro.

70

Thymic development was impaired in mice that express a dnRAR receptor in T

cells.

T-dnRAR mice were generated to study the effects of retinoid signaling in T cells. To

confirm that the dnRAR was functioning in T cells, splenocytes were stimulated with

anti-CD3 antibody and treated with 10nM RA or ethanol as a control. RA treatment

significantly inhibited IFN-γ production in WT cells (Figure 3-7). RA treatment had no

effect on IFN-γ secretion from anti-CD3 stimulated T-dnRAR splenocytes (Figure 3-7).

There was no effect of RA on IL-17a in either the WT or T-dnRAR cultures for these

assays (Figure 3-7). These data indicated that T cells from the T-dnRAR mice were

unable to respond to RA treatment in vitro.

In the thymus, T cells develop through a double negative (DN) to a double positive (DP)

stage and then to single positive either CD4 or CD8 T cells. Frequencies of DN, DP,

CD4+ and CD8+ cells from the WT littermates were consistent with other reports (31).

WT mice had significantly more total thymocytess than T-dnRAR littermates (Table 3-2,

Figure 3-8 A). There were more Tcrβ+ T cells in the WT thymus compared to T-dnRAR

mice (Table 3-2). WT mice had more DP and less CD8 single positive thymocytes than

the T-dnRAR mice (Table 3-2, Figure 3-8A). In the periphery, WT mice had

significantly increased Tcrβ+ cells and CD8+ T cells in the spleen (Table 3-2, Figure 3-

8B). WT mice had more total cells and fewer CD8+ T cells in MLN than the T-dnRAR

mice (Table 3-2, Figure 3-8C). There were not differences in B cells (B220+) in the

spleen and MLN between WT and T-dnRAR mice (data not shown). Overall, the T-

dnRAR mice had fewer T cells in the thymus as well as in the periphery than their WT

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counterparts, specifically decreased CD8+ T cells in the spleen and MLN. These data

indicate that RA signaling is important for CD8+ T cell development.

72

DISCUSSION

Here, for the first time, we show that short term treatment with RA during enteric

infection promoted bacterial clearance. Additionally, RA treatment of A- mice starting at

d7 post-infection was as effective as at the time of infection for clearance of C.

rodentium. Only 1 dose of RA was required for A- mice to clear a C. rodentium

infection. Induction of IL-17a mRNA expression in the colon of RA treated mice

corresponded with the elimination of C. rodentium in the RA treated A- mice. IL-17a

was induced in the colon following RA treatment. RA treatments of A- hosts could be

effective for eliminating carriers of Escherichia coli-like infections in the A- host.

The effect of RA in vitro and the effects of vitamin A in vivo on IL-17 and Th17 cells is

paradoxical. Our data confirms that RA treatment in vitro inhibited IL-17a production

from A- CD4+ T cells in vitro. Previously published in vitro studies have shown that RA

inhibited Th17 induction when IL-6 was absent (7, 32). Other in vitro experiments also

showed that 10nM to 1uM RA can inhibited Th17 cell induction and reduced IFN-γ and

IL-17a secretion (12, 33, 34). Two independent studies have shown that in vitro

treatment of naive T cells with 1nM RA was needed for IL-17a production (12, 34). All

referenced in vitro assays used naïve CD4+ T cells but used a variety of mouse strains

(C57BL/6 and BALB/c (12), C57BL6 OT-II (34), B6.SJL (13), or AKR/J (12)),

indicating that different mouse strains may respond to RA or metabolize vitamin A

differently (35). Our data show that RA in vivo induced IL-17a expression in the colon

of C57BL/6 mice. At this time, we do not know the source of the IL-17a mRNA in the

colon of RA treated mice. Several different populations of cells produce IL-17a

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including T cells, innate lymphoid cells and monocytes (36). The effects of vitamin A in

vivo are not completely understood.

One explanation for the increased IL-17a expression as a result of RA treatment may be

the recruitment of Th17 cells from the periphery to the colon. We have observed that A-

mice had fewer colonic T cells than A+ mice (3) and it is known that RA upregulates gut

homing receptors on T cells in vitro (4). A- mice and retinoic acid receptor (RAR)α

deficient mice had very few Th17 cells in the gut (12-14, 16) . The decreased Th17 cells

in the gut of A- mice was also observed in a genetic mouse model of Crohn’s disease

(SAMP1/YP mouse model) however this seemed to have no effect on the model (37).

IL-17+ T cells generated in vitro in the presence of 1nM RA expressed high levels of

CCR9 and α4β7 (12). Furthermore, when these IL-17+ T cells were transferred into

RagKO mice, they trafficked to the gut and caused inflammation (12). If RA was

required to get Th17 cells to home to the gut during infection, this could account for the

observed increased expression of IL-17a in the RA treated animals compared to the

untreated mice.

To further determine possible targets of vitamin A in host resistance to C. rodentium, the

microbiota and B cells were examined. Because C. rodentium competes with the

commensal microbiota for colonization of the host, we measured 16S DNA and bacteria

phyla in the feces of A+ and A- mice. We found no differences in total bacterial DNA.

Additionally, we found no effect of vitamin A status on Bacteriodetes, Firmicutes,

Actinobacteria or γ-Proteobacteria. Others have reported that A- mice have fewer gut

bacteria in the ileum and feces by RT-PCR and bacterial cultures (16). A wide range of

factors affect gut microbiota composition (i.e. diet, animal facility, vendors) and these

74

factors along with experimental differences could account for the different results (38-

40). In addition, we found no differences in serum IgA, antigen specific IgA or antigen

specific IgG1 and IgG2 antibody levels in A- and A+ mice throughout infection or in B

cell numbers in the LP of the GI tract. Currently, our data does not support an effect of

vitamin A on the microbiota or B cells in the gut, however the relationship between RA

and gut microbiota or RA and mucosal B cells has not yet been thoroughly studied.

These data indicate that other acquired immune system components, like T cells, may be

a main target of RA during C. rodentium infection.

To examine the effects of RA on T cells, we generated T-dnRAR mice. Others have used

the dnRAR mouse to inhibit RA signaling in T cells using a CD4-cre mouse (31, 41).

Reports using the CD4 cre-dnRAR mice showed that, like in our study, the CD8+ single

positive thymocytes frequencies were increased compared to WT littermates (31).

However, there were no differences in the CD8+ T cell populations in the periphery

compared to WT mice in the CD4 cre-dnRAR mice and we showed that there were fewer

CD8+ in the periphery using the Lckcre-dnRAR mouse (31). Memory CD4+ T cell

numbers were not different between WT and CD4 cre-dnRAR mice although the CD4cre

dnRAR T cells produced more IL-17 and less IFN-γ than the WT T cells (41). By using

the Lck cre mouse, we have inhibited RA signaling at the DN stage of development while

the groups that use the CD4 cre mouse inhibited retinoid signaling at the DP stage (31,

41). Our data showed that when RA signaling is inhibited in T cells, double positive

(CD8+CD4+) thymocyte numbers were significantly decreased but CD8+ thymocyte

numbers were significantly increased. The Lck cre dnRAR CD8+ T cells may have a

defect in survival, maturation or proliferation. Defects in CD8+ T cells could result in

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the inability of host to survive viral or intracellular bacterial infections. Thymic organ

culture and flow cytometry showed that in vitro RA treatment of human thymocytes

promoted CD4+ thymocyte development and inhibited CD8+ thymocyte development

from the DP stage, indicating that RA may affect CD8+ T cell development (42). The

data suggest that RA signaling is important for CD8+ T cell development and for

maintaining CD8+ T cells periphery.

From this study, we determined that RA treatment of A- mice at d7 post infection was

just as effective as RA treatment starting at d0 in clearing C. rodentium infection.

Additionally, we found that one dose of RA at d7 post infection eliminated C. rodentium

infection from the A- host. One target of RA in the colon that corresponded with C.

rodentium clearance was IL-17a mRNA levels. However, we do not know what cells

were making IL-17a. Determining which cells were producing IL-17 would be critical to

understanding the mechanisms underlying the effects of RA on host resistance in A-

mice.. The work shows that RA is a critical regulator of T cell development and the local

IL-17 response during C. rodentium infection.

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Figure 3-1 Figure 3-1: RA treatment starting at d7 results in similar clearance when RA is given at d0. A) CFU in the feces of A- mice, A- mice treated with RA starting at d7 post infection (RA d7) and A- mice treated with RA on the day of infection (RA d0). Values are the mean + SEM of one experiment and n=4-5/group. B and C) CFU in the feces of A- mice given one dose of RA at d7 post-infection (RA 1x), one dose of RA at d7 and 9 post-infection (RA 2x), or one dose of RA on d7, 9 and 11 post-infection (RA 3x). Values are the mean + SEM of one experiment and n=3-4/group. Two-way ANOVA with Bonferroni post-hoc tests. (A)Values with different letters are significantly different from each other, P<0.01-0.0001.

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Figure 3-2

Figure 3-2: Short term RA treatment results in increased il17a mRNA. A) Experimental timeline B )Spleen and liver CFU and C) RT-PCR of whole colon after RA 1x or RA 2x in A- mice. The RA treatments were started at d7 post-infection. Mice were sacrificed at d9 and d11 post infection. Values are the mean + SEM of two combined experiments and n=8-10/group. Two-way ANOVA with Bonferroni post-hoc tests

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Table 3-1: RT-PCR primer sequences HPRT: F: 3’-GTAATGTTATCGAGAAGTCAGAC-5’

R: 3’-CACCTTCTATATTAACTGTGACC-5’

GAPDH: F: 3’-TAGCTTCCACCTTCTACACCAAA;-5’ R: 3’-GATGTCGTTGTCCCACCACC-5’

FOXP3: F: 3’-GACGTATCGAGGGTCGAAGA-5’ R: 3’-CGAGAACGACGTAGCATCG-5’

RORC: F: 3’-AGGAGGGCACTTTTCTCCA-5’ R: 3’-GTTACACCCTCTACACCCT-5’

IL-17A: F: 3’-TGTGTCTACTTCGAGAGGGAC-5’ R: 3’-TAGTAGGGAGTTTCGACTCG-5’

IL-22: IL-6: REGIIIy

F: 3’-AGACCTACAAGACCAGCAGT-5’ R: 3’-CACCTCTCTAGTTCCGCTAA-5’ F: 3’-AGGACTAATATAGGTCAAACCCATCG-5’ R: 3’-AAGACCTCATGGTATCGATGGACC-5’ F: 3’-AAAACTAGTACCTCCTGTCCTT-5’ R: 3’-ACTTGGGTTGTCTCCACCTAC-5’

16S: F: 3’-AAACTCAAAKGAATTGACGG-5’ R: 3’-CTCACRRCACGAGCTGAC-5’

Bacteriodetes: F: 3’-CRAACAGGATTAGATACCCT-5’ R: 3’-GGTAAGGTTCCTCGCGTAT-5’

Firmicutes: F: 3’-TGAAACTYAAAGGAATTGACG-5’ R: 3’-ACCATGACCACCTGTC-5’

Actinobacteria: F: 3’-TACGGCCGCAAGGCTA-5’ R: 3’-TCRTCCCACCTTCCTCCG-5’

y-Proteobacteria F: 3’-TCGTCAGCTCGTGTYGTGA-5’ R: 3’-CGTAAGGGCCATGATG-5’

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Figure 3-3 Figure 3-3: The effects of RA treatment on mRNA expression in duodenum, ileum and colon at d11 post-infection and 2 doses of RA. Whole tissue mRNA was collected from the duodenum, ileum and colon after two RA doses begun at d7 post-infection. Mice were scarified at d11 post-infection. Data is log transformed to account for distribution and combined from two independent experiments with n=5-9/group. Two-way ANOVA with Bonferroni post-hoc tests.

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Figure 3-4

Figure 3-4: The microbiota is not affected by host VA status. RT-PCR analysis of A) fecal 16S bacterial DNA from A+ and A- mice and (B) bacterial phyla from A+ and A- mice. Values are the mean + SEM of three experiments and n=8-9group. Mann Whitney test.

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Figure 3-5

Figure 3-5: RA inhibition of IFN-γ and IL-17a in A- whole splenocytes and CD4+ T cells in vitro A) Anti-CD3 stimulated IFN-γ production from A+ and A- splenocytes after 72 hours. A- + RA samples were treated with 10nM RA. B) Anti-CD3/CD28 CD3 stimulated IFN-γ and IL-17a production from A+ and A- CD4+ T cells after 72 hours. A- + RA samples were treated with 10nM RA. Values are the mean + SEM of two combined experiments and n=5-7/group. Student’s t test or Mann Whitney test.

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Figure 3-6

Figure 3-6: Serum antibody titers and GI B cell numbers are not affected by host vitamin A status: A) Total and C. rodentium specific IgA titers and B) C. rodentium specific IgG1 and IgG2 titers in the serum of A+ and A- mice during infection. Samples were combined from 1-3 experiments, n=4-12/time point/VA group. Two way ANOVA. C) Total SI LPL and B cell number and D) total colonic LPL and B cell numbers. Data are combined from two independent experiments, n=8-10/group. Student’s t-test.

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Figure 3-7

Figure 3-7: RA treatment in vitro does not affect T-dnRAR splenocyte response to stimulation. Anti-CD3 stimulated IFN-γ and IL-17a production from WT and T-dnRAR mice after 72 hours with or without 10nM RA treatment. Values are the mean + SEM from one experiment and n=3-4/group. Mann Whitney test.

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Table 3-2: RA signaling inhibition in T cells leads to immune cell population imbalance in the periphery.

Frequency (%)

Cell Number x (106)

Cell Type WT T-dnRAR WT T-dnRAR Thymus CD4+ 8 + 1 11 +2 56 + 1 4.2 + 0.7 CD8+ 3 + 0.5 12 + 2 *** 2 + 0.5 4.6 + 0.8 ** DN (CD8-CD4-) 4 + 0.5 5 + 0.5 23 + 0.5 1 + 0.2 DP (CD8+CD4+) 83 + 2 67 + 3 ** 60 + 8 26 + 3 ** Tcrβ+ 8 + 1 90 +1 * 62 + 8 34 + 4 ** Total cell number 73 + 10 31+ 4 ** Spleen Tcrβ+ 40 + 1 31 + 12 ** 12 + 2 7.8 + 0.9 * CD4+ 52 + 3 63 + 2 ** 6.5 + 1 4.9 + 0.5 CD8+ 41 + 1 33 + 2 ** 5.1 + 0.7 2.6 + 0.3 ** Total cell number 30 + 6 25 + 3 Mesenteric Lymph Node Tcrβ+ 84 + 2 77 + 1 * h 2.5 + 0.6 1.1 + 0.2 * CD4+ 51 + 3 65 +2 ** 1.3 + 0.3 0.7 + 0.2 CD8+ 48 + 3 35 +1 ** 1.2 + 0.3 0.4 + 0.07 ** Total cell number 3.2 + 0.7 1.5 + 0.3 *

Values are the frequencies as percentiles. Frequency and cell numbers are expressed as means + SEM for 5-10 mice/group. Data is combined from two independent experiments. CD4 and CD8 frequencies are expressed as percentages of Tcrβ+ populations. Gating strategy can be found in Figure 3-9. * P<0.05, ** P<0.01, *** P<0.001 by Student’s t-test or Mann Whitney test.

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Figure 3-8

Figure 3-8: RA signaling inhibition in T cells leads to increased CD8+ T cells in the thymus and decreased CD8+ T cells in the periphery. Graphical data from Table 3-2. A) Total thymocyte cell number, DP cell number and CD8+ T cell numbers from the thymus. B) Total cell and CD8+ T cell numbers from the spleen. C) Total cell and CD8+ T cell numbers from the MLN.

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Figure 3-9

Figure 3-9: Gating strategy for flow cytometry analysis. A) shows gating strategy for analysis of thymocytes. B) shows gating strategy for analysis of spleen and MLN.

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Chapter 4

Summary and conclusions

92

The role of vitamin A in enteric infection was investigated in this thesis. In

Chapter 2, the effect of host vitamin A status during Citrobacter rodentium infection was

evaluated. While it has been known that vitamin A deficiency is associated with

respiratory and diarrheal diseases in children (1, 2), the effects of vitamin A during C.

rodentium infection were unknown. I show here that A- mice failed to clear C.

rodentium infection. T cell numbers were decreased in the colon of A- mice compared to

A+ mice. Additionally, my work showed that RA treatment before infection, at the time

of infection and during infection eliminated the pathogen from the host. I observed 40%

mortality in A- mice following C. rodentium infection. Death following C. rodentium

infection had been attributed to sepsis (3-5), however I did not find elevated serum

cytokines in the A- mice compared to A+ mice. Furthermore, necropsies of moribund

mice did not show signs of sepsis but did find indicators of severe gut damage. Together

the data suggests that the C. rodentium related mortality in the A- host was not caused by

sepsis. However, this manuscript did not determine why the A- mice did not clear C.

rodentium or how RA treatment promoted infection clearance in A- mice. The major

conclusions from the first manuscript were that A- mice are susceptible to C. rodentium

infection and RA can be given before, at the time of or during infection to promote

survival and clearance of the infection. The work demonstrated a new role for vitamin A

for eliminating possible reservoirs of enteric bacteria in A- hosts (6).

The timing of RA treatment and the mechanisms by which RA regulated host

resistance to enteric infection were probed in Chapter 3. RA treatment starting at d7

post- infection was as effective as at the time of infection for C. rodentium clearance.

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Furthermore, it was determined that one 37.5 ug dose of RA starting at d7 post infection

was enough to clear C. rodentium from the A- host. The data showed that RA treatment

during infection induced IL-17a production which was associated with clearance of C.

rodentium. Other reports indicated that A- mice have inadequate Th17 immune

responses and fewer Th17 cells in the gut (7) and our data showed that A- mice were

unable to clear an infection. Our previous study also showed that A- mice had fewer total

T cells in the colon compared to A+ mice (6) (7-10). However, in vitro RA inhibited IL-

17a production from A- CD4+ T cells. In addition to CD4+ T cells, IL-17a is produced

by many other cell types including CD8+ T cells, γδ T cells, innate lymphoid cells,

natural killer (NK) T cells, NK cells and neutrophils (11, 12). There are at least three

possibilities that could explain the induction of RA mRNA in the colon of A- mice

infected with C. rodentium and directly inhibiting IL-17a from CD4+ T cells: 1) RA

treatment promoted IL-17a production from other (CD8, γδT, ILC etc.) cells in the gut 2)

RA treatment promoted IL-17 producing immune cells to home to the gut or 3) RA

indirectly resulted in CD4+ T cells making more IL-17a in the gut via innate cell

production of IL-22. These three possibilities are not mutually exclusive. Further work

needs to be done to determine which of the mechanisms account for increased IL-17a in

the colon following RA treatment of C. rodentium infected mice.

Because the early shedding kinetics (before day 10-11) of C. rodentium were not

different in A-, A+ and RA treated mice, we hypothesized that the adaptive immune

response and not the early innate immune responses was the target of vitamin A in the

gut. In addition, it has been shown that both T cell and B cell responses are required for

the host to clear a C. rodentium infection (3). Our data showed that vitamin A status had

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no effect on antibody responses during C. rodentium infection or B cell numbers in the

lamina propria of the SI and colon. T cells are well-described vitamin A targets. To

study the role of vitamin A in T cells we generated T-dnRAR mice. These mice

expressed the dnRAR in T cells under the Lck promoter and were therefore non-

responsive to RA treatment. Preliminary characterizations of the immune cell

populations in the T-dnRAR mice indicated that RA signaling was important for T cell

development and in particular the CD8+ T cells from the T-dnRAR mice were lower in

the periphery. The role of CD8+ T cells in host resistance is unclear since CD8 KO mice

survived and presumably cleared C. rodentium infection (3). It would be interesting to

see if IL-17a production from T-dnRAR CD8+ T cells is impaired compared to WT

CD8+ T cells.

The findings from this dissertation have implications in the treatment of enteric

pathogens, particularly in vitamin A deficient hosts. The work presented here highlights

a potential role for vitamin A supplements to eliminate reservoirs of enteric pathogens.

Vitamin A deficiency has been implicated as a factor important in the severity of

diarrheal disease and as a contributor to childhood mortality. I show that

supplementation with RA, the active form of vitamin A, reduced infection severity and

promoted survival of A- mice, indicating that vitamin A supplementation during enteric

infections in humans may be beneficial. Additionally, my work showed that RA

treatment may regulate IL-17a which is needed to clear C. rodentium (Figure 5-1).

Further experiments are needed to determine other targets of RA during C. rodentium

infection such as intestinal epithelial cells and other cytokines. It remains to be

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determined whether vitamin A deficient humans become asymptomatic carriers of E. coli

or other enteric bacteria that have the potential to cause disease. A vitamin A supplement

that could clear bacterial infections and decrease carriers would be an effective way to

limit mortality and disease from enteric pathogens.

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Figure 5-1

Figure 5-1. Retinoic acid treatment during enteric infection promotes protective mucosal immune responses in the vitamin A deficient host. A) In a vitamin A sufficient or RA supplemented host, immune cells home to the gut and during C. rodentium infection produce adequate amounts of IL-17A. B) In a vitamin A deficient host, immune cells fail to home to the gut and contain C. rodentium in the gut. IL-17A production is reduced and the immune response is not robust enough to clear the infection.

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REFERENCES

1. WHO. 2009. Global prevalence of vitamin A deficiency in populations at risk 1995–2005: WHO global database on

vitamin A deficiency. In World Health Organization, Geneva. 2. West, K. P., Jr., G. R. Howard, and A. Sommer. 1989. Vitamin A and infection:

public health implications. Annu Rev Nutr 9: 63-86. 3. Bry, L., and M. B. Brenner. 2004. Critical role of T cell-dependent serum

antibody, but not the gut-associated lymphoid tissue, for surviving acute mucosal infection with Citrobacter rodentium, an attaching and effacing pathogen. J Immunol 172: 433-441.

4. Vallance, B. A., W. Deng, K. Jacobson, and B. B. Finlay. 2003. Host susceptibility to the attaching and effacing bacterial pathogen Citrobacter rodentium. Infect Immun 71: 3443-3453.

5. Ghosh, S., C. Dai, K. Brown, E. Rajendiran, S. Makarenko, J. Baker, C. Ma, S. Halder, M. Montero, V. A. Ionescu, A. Klegeris, B. A. Vallance, and D. L. Gibson. 2011. Colonic microbiota alters host susceptibility to infectious colitis by modulating inflammation, redox status, and ion transporter gene expression. Am J Physiol Gastrointest Liver Physiol 301: G39-49.

6. McDaniel, K. L., K. H. Restori, J. W. Dodds, M. J. Kennett, A. C. Ross, and M. T. Cantorna. 2015. Vitamin A-Deficient Hosts Become Nonsymptomatic Reservoirs of Escherichia coli-Like Enteric Infections. Infect Immun 83: 2984-2991.

7. Cha, H. R., S. Y. Chang, J. H. Chang, J. O. Kim, J. Y. Yang, C. H. Kim, and M. N. Kweon. 2010. Downregulation of Th17 cells in the small intestine by disruption of gut flora in the absence of retinoic acid. J Immunol 184: 6799-6806.

8. Wang, C., S. G. Kang, H. HogenEsch, P. E. Love, and C. H. Kim. 2010. Retinoic acid determines the precise tissue tropism of inflammatory Th17 cells in the intestine. J Immunol 184: 5519-5526.

9. Hall, J. A., J. L. Cannons, J. R. Grainger, L. M. Dos Santos, T. W. Hand, S. Naik, E. A. Wohlfert, D. B. Chou, G. Oldenhove, M. Robinson, M. E. Grigg, R. Kastenmayer, P. L. Schwartzberg, and Y. Belkaid. 2011. Essential role for retinoic acid in the promotion of CD4(+) T cell effector responses via retinoic acid receptor alpha. Immunity 34: 435-447.

10. Spencer, S. P., C. Wilhelm, Q. Yang, J. A. Hall, N. Bouladoux, A. Boyd, T. B. Nutman, J. F. Urban, Jr., J. Wang, T. R. Ramalingam, A. Bhandoola, T. A. Wynn, and Y. Belkaid. 2014. Adaptation of innate lymphoid cells to a micronutrient deficiency promotes type 2 barrier immunity. Science 343: 432-437.

11. Mills, K. H. 2008. Induction, function and regulation of IL-17-producing T cells. Eur J Immunol 38: 2636-2649.

12. Jin, W., and C. Dong. 2013. IL-17 cytokines in immunity and inflammation. Emerg Microbes Infect 2: e60.

Vita Kaitlin L. McDaniel

Education 2010-present The Pennsylvania State University

Ph.D. in Pathobiology Advised by Dr. Margherita Cantorna, GPA: 3.8/4.0

2008-2010 University of Wyoming B.S. in Microbiology/B.S. in Molecular Biology Advised by Dr. E. Lee Belden, GPA 3.7/4.0

2006-2008 Northwest College A.S. in Biology, GPA 3.8/4.0

Publications

• McDaniel, K.L., K.H. Restori, J. Dodds, M.J. Kennett, A.C. Ross and M.T. Cantorna. 2015. Vitamin A deficient hosts become non-symptomatic reservoirs of Escherichia coli-like enteric infections. Infect Immun. 83:2984-91.

• Cantorna, M.T., K.L. McDaniel, S. Bora, J. Chen and J. James. 2014. Vitamin D, immune regulation, the microbiota and inflammatory bowel disease. Exp Biol Med (Maywood). 239: 1524-30.

• Ooi, J.H., K.L. McDaniel, V. Weaver and M.T. Cantorna. 2014. Murine CD8+ T cells but not macrophages express the vitamin D 1α-hydroxylase. J Nutr Biochem. 25: 58-65.

Professional Presentations

• Vitamin A deficient causes resistant Citrobacter rodentium intestinal infection and retinoic acid treatment mediates the local immune response. American Association of Immunologist Annual Meeting. New Orleans, LA. 2015. Poster

• Vitamin A deficient mice have a decreased ability to clear the enteric pathogen, Citrobacter rodentium and increased mortality during infection. American Association of Immunologist Annual Meeting. Pittsburgh, PA. 2014. Oral presentation and poster.

• Retinoic acid supplementation of vitamin A deficient mice promotes clearance of an infection with Citrobacter rodentium. 16th Annual Upstate New York Immunology Conference. Albany, NY. 2013. Poster

Awards and Honors

American Association of Immunologists Trainee Abstract Award 2014 PSU College of Agriculture Graduate Student Grant 2014 NIH Training Grant: Animal Models of Inflammation 2013 PSU College of Agriculture Mr. and Mrs. E. Bowsworth Grier Scholarship 2013 Pennsylvania State University Graduate School FEGR Scholarship Award 2010