Investigating the Role of Innate Inflammatory Pathways During Infection with Murine Coronavirus

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Publicly Accessible Penn Dissertations

2015

Investigating the Role of Innate Inflammatory Pathways During Infection With Murine Coronavirus

Zachary Bestor Zalinger University of Pennsylvania, [email protected]

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Recommended Citation Zalinger, Zachary Bestor, "Investigating the Role of Innate Inflammatory Pathways During Infection With Murine Coronavirus" (2015). Publicly Accessible Penn Dissertations. 2123. https://repository.upenn.edu/edissertations/2123

This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2123 For more information, please contact [email protected]. Investigating the Role of Innate Inflammatory Pathways During Infection With Murine Coronavirus

Abstract Multicellular organisms are constantly exposed to microorganisms, such as viruses and bacteria, many of which are infectious pathogens. The immune system evolved to provide protection against these organisms, and includes numerous components to defend against a diverse, rapidly evolving pool of pathogens. The immune system is classically divided into two arms, the innate and adaptive systems. The time and metabolic cost of activating the T and B cell responses are considerable, and can also have deleterious side effects if the response is not properly controlled. Therefore, the adaptive immune system is carefully regulated so as to be activated only when necessary. The innate immune system serves to control most pathogens in a more rapid, less energetically expensive manner than the adaptive immune system, and to help regulate subsequent immune responses, guiding and controlling them in order to most efficiently clear pathogens with a minimum of pathological side effects. Among the first elements of the innate system that pathogens encounter are pattern recognition receptors, invariant receptors that detect conserved pathogen associated molecular patterns. Upon recognizing its binding ligand, a pattern recognition receptor will activate a signaling cascade that often triggers production and/or release of one or more pro- inflammatory cytokines. These in turn modulate additional elements of both the innate and adaptive immune responses, and are often critical lynchpins of host defense. Due to this central role in the immune system we investigated the roles of two distinct innate inflammatory pathways, the inflammasome and the MDA5-dependent portion of the type 1 interferon response, during infection with the murine coronavirus mouse hepatitis virus (MHV). Utilizing transgenic mice deficient in Caspase-1 and -11, which catalyze all inflammasome processing, the IL-1 receptor, the IL-18 receptor, or MDA5, we characterize the disease course and subsequent immune response following infection with MHV, a model murine coronavirus. We find that inflammasome signaling is otectivpr e during infection, and that much or all of this protection is mediated by IL-18 signaling driving production of interferon gamma by T cells. We also demonstrate that MDA5 signaling controls viral tropism and replication, and that in its absence the host immune response becomes over active, likely leading to lethal pathology. Overall, our studies help define how innate inflammatory pathways can have diverse influences on pathogenesis and the immune response.

Degree Type Dissertation

Degree Name Doctor of Philosophy (PhD)

Graduate Group Cell & Molecular Biology

First Advisor Susan R. Weiss

Subject Categories Allergy and Immunology | Immunology and Infectious Disease | Medical Immunology | Virology

This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2123 INVESTIGATING THE ROLE OF INNATE INFLAMMATORY PATHWAYS DURING INFECTION WITH MURINE CORONAVIRUS

Zachary B. Zalinger

A DISSERTATION

Cell and Molecular Biology

Presented to the Faculties of the University of Pennsylvania

Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

2015

Supervisor of Dissertation

______

Susan R. Weiss

Professor of Microbiology

Graduate Group Chairperson

______

Daniel S. Kessler, Associate Professor of Cell and Developmental Biology

Dissertation Committee:

Igor E. Brodsky, Assistant Professor of Pathobiology Christopher A. Hunter, Professor of Pathobiology Dennis L. Kolson, Professor of Neurology Glenn F. Rall, Professor, Fox Chase Cancer Center Luis J. Sigal, Professor, Jefferson University

INVESTIGATING THE ROLE OF INNATE INFLAMMATORY PATHWAYS DURING INFECTION WITH MURINE CORONAVIRUS

2015

Zachary B. Zalinger

This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License

To view a copy of this license, visit http://creativecommons.org/licenses/by-ny-sa/2.0 ACKNOWLEDGMENTS

I would like to thank my mentor, Dr. Susan Weiss, for her support and guidance, and the members of her laboratory for their patience.

I would also like to thank my friends and classmates in Philadelphia, who have made these long years tolerable, and the completion of my time here bittersweet. I will miss them all.

I am especially thankful for Dr. Cierra Casson, whose brilliance and love has made all the difference. Without her I would not be standing here today.

And finally I would like to thank my family for all their support and acceptance over the years. My parents, Wendy Bestor and Lee Zalinger, who taught me that knowledge has its own value. And my grandfathers, Dr. Alvin Zalinger, who I never knew but who I hope would have been proud of me, and Dr. Charles Bestor, who has always been a shining example of the value of the intellectual life.

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ABSTRACT

INVESTIGATING THE ROLE OF INNATE INFLAMMATORY PATHWAYS DURING

INFECTION WITH MURINE CORONAVIRUS

Zachary B. Zalinger

Susan R. Weiss

Multicellular organisms are constantly exposed to microorganisms, such as viruses and bacteria, many of which are infectious pathogens. The immune system evolved to provide protection against these organisms, and includes numerous components to defend against a diverse, rapidly evolving pool of pathogens. The immune system is classically divided into two arms, the innate and adaptive systems. The time and metabolic cost of activating the T and B cell responses are considerable, and can also have deleterious side effects if the response is not properly controlled. Therefore, the adaptive immune system is carefully regulated so as to be activated only when necessary. The innate immune system serves to control most pathogens in a more rapid, less energetically expensive manner than the adaptive immune system, and to help regulate subsequent immune responses, guiding and controlling them in order to most efficiently clear pathogens with a minimum of pathological side effects. Among the first elements of the innate system that pathogens encounter are pattern recognition receptors, invariant receptors that detect conserved pathogen associated molecular patterns. Upon recognizing its binding ligand, a pattern recognition receptor will activate a signaling cascade that often triggers production and/or release of one or more pro- inflammatory cytokines. These in turn modulate additional elements of both the innate and adaptive immune responses, and are often critical lynchpins of host defense. Due to this central role in the immune system we investigated the roles of two distinct innate inflammatory pathways, the inflammasome and the MDA5-dependent portion of the type 1 interferon response, during infection with the murine coronavirus mouse hepatitis virus (MHV). Utilizing transgenic mice deficient in Caspase-1 and -11, which catalyze all inflammasome processing, the IL-1 receptor, the IL-18 receptor, or MDA5, we characterize the disease course and subsequent immune response following infection with MHV, a model murine coronavirus. We find that inflammasome signaling is protective during infection, and that much or all of this protection is mediated by IL-18 signaling driving production of interferon gamma by T cells. We also demonstrate that MDA5 signaling controls viral tropism and replication, and that in its absence the host immune response becomes over active, likely leading to lethal pathology. Overall, our studies help define how innate inflammatory pathways can have diverse influences on pathogenesis and the immune response.

TABLE OF CONTENTS

Content Page

ACKNOWLEDGMENT…………………..…………………………………………..………….iii ABSTRACT……..…………………………………………………………………………..……iv TABLE OF CONTENTS…………………………………………………………...…………….v LIST OF ABBREVIATIONS…………………………………………………….……………...vii LIST OF ILLUSTRATIONS…..………………………………………………………………..viii

CHAPTER 1: INTRODUCTION…….………………………………………..…………………1

A. Structure and function of the inflammasome……………………………….………..3 B. Ligands of inflammasome sensors………………………………………….………...4 C. The non-canonical inflammasome……………………………………….……………6 D. Inflammasome-mediated cell death………………………………….………………..7 E. IL-1 signaling…………………………………………………………………………….8 F. IL-18 signaling…………………………………………………………………………...9 G. Role of the inflammasome in demyelination……………………….……………….10 H. Structure and function of MDA5…………………………………….………………..12 I. MDA5’s binding substrate(s) …………………………………….…………………..14 J. The type 1 interferon signaling pathway…………………………………………….14 K. The role of type 1 interferon during acute infection………………………………..15 L. The interferon stimulated gene response………………………………...…………17 M. The role of type 1 interferon in the memory T cell response……………...………18 N. Deleterious effects of MDA5 and type 1 interferon……………………...…………19 O. Taxonomy and natural history of mouse hepatitis virus…………………...………20 P. Course of mouse hepatitis virus infection……………………………………...……21 Q. Immunology of mouse hepatitis virus-induced demyelination………………..…..23 R. The blood brain barrier………………………………………………………..………25 S. Immunology of acute mouse hepatitis virus infection………………………..…….25 T. Dissertation aims…………………………………………………………………..…..27

CHAPTER 2: ROLE OF THE INFLAMMASOME-RELATED CYTOKINES IL-1 AND IL-18 DURING INFECTION WITH MURINE CORONAVIRUS

A. Abstract…………………………………………………………………………………31 B. Introduction……………………………………………………………………………..32 C. Results…………………………………………………………………...……………..35 D. Discussion………………………………………………………………..…………….43 E. Acknowledgments………………………………………………………..……………48 F. Materials and methods……………………………………………………..…………49

CHAPTER 3: ROLE OF MDA5 SIGNALING DURING INFECTION WITH MURINE CORONAVIRUS

A. Abstract………………………………………………………………...……………….62 B. Introduction………………………………………………..……………………………63

C Results……………………………………………………………………...…………..66 D. Discussion…………………………………………………………………..………….74 E. Acknowledgments………………………………………………………….………….80 F. Materials and methods…………………………………………………….………….81

CHAPTER 4: DISCUSSION

A. The inflammasome-related cytokines IL-1 and IL-18 have differential roles during MHV infection…………………………………………………………………….…….95 B. The infection specific-specific role of IL-1 signaling………………………….…….96 C. A model for explaining the discrepancy between the survival of Casp-1/11-/- and IL-18R-/- mice……………………………………………………………………….…..97 D. The protective role of IL-18 signaling during MHV infection is mediated by production of interferon gamma……………………………………………………...98 E. The role of the inflammasome in MHV-induced demyelination…………………100 F. MDA5 plays a protective role during MHV infection through negative regulation of the immune response…………………………………………………..……………102 G. The relative contributions of MDA5 and TLR7 to induction of the type 1 interferon response………………………………………………………………………………103 H. MDA5 signaling in maintenance of tropism barriers……………………………...104 I. PD-1 as a marker of defective T cells…………………………………...…………107 J. Immunopathology in the absence of MDA5 signaling……………………………108 K. MDA5 and type 1 interferon as negative regulators of the immune response...110 L. Concluding remarks………………………………………………………………….111

BIBLIOGRAPHY…..…………………………………………………………………………..113

LIST OF ABBREVIATIONS

ASC: Apoptosis-associated speck containing a CARD Casp-1/11-/- Caspase-1 and caspase-11 double knockout CMV: Cytomegalovirus CNS: Central nervous system BBB: Blood brain barrier DC: Dendritic cell EAE: Experimental autoimmune encephalitis IAV: Influenza A virus i.c.: Intracranial IFNα: Interferon subtype alpha IFNβ: Interferon subtype beta IFNAR: Type 1 interferon receptor IL-1R: IL-1 receptor IL-18R: IL-18 receptor i.p.: Intraperitoneal ISG: Interferon stimulated gene LCMV: Lymphocytic choriomeningitis virus MAVS: Mitochondrial antiviral signaling protein MDA5: Melanoma-differentiation associated protein 5 MHV: Mouse hepatitis virus MS: Multiple sclerosis NK: Natural killer NKT: Natural killer T PAMP: Pathogen associated molecular pattern PFU: Plaque forming unit PRR: Pattern recognition receptor TLR: Toll-like receptor TNFα: Tumor necrosis factor alpha WNV: West Nile virus WT: C57Bl/6 wild type

vii

LIST OF ILLUSTRATIONS

Figure Page

FIGURE 1-1. Caspase-1/11-/- mice are more susceptible to MHV infection…….……….52

FIGURE 1-2. IL-18R-/- mice are more susceptible to MHV infection while IL-1R-/- mice have increased survival but elevated viral loads……………………………………………53

FIGURE 1-3. IL-18 concentration is lower in the serum of Casp-1/11-/- mice compared to WT mice…..…………………………………………………………………………………..…54

FIGURE 1-4. Demyelination is unchanged in Casp-1/11-/- and IL-1R-/- mice compared to WTs but elevated in IL-18R-/- mice…..……………………………………………………55

FIGURE 1-5. Interferon gamma in serum…..…………………………………………….…56

FIGURE 1-6. Interferon gamma and perforin production in the spleen is IL-18 independent at the peak of infection…..…………………………………………………..…57

FIGURE 1-7. Interferon gamma and perforin production in the liver is IL-18 independent at the peak of infection….…………………………………………………………………..…58

FIGURE 1-8. T cells from WT and IL-18R-/- spleens have similar activation profiles at the peak of the response. …………………………………………………………………..…59

FIGURE 1-9. T cells from the spleens of IL-18R-/- mice at the peak of the T cell response exhibit reduced interferon gamma production compared to those from WT mice. ……………………………………………………………………………………….……60

FIGURE 2-1. MDA5-/- mice have heightened susceptibility to MHV infection……..……86

Figure 2-2. MDA5-/- mice have increased pathology in the liver………………………….87

FIGURE 2-3. Interferon, but not interferon stimulated gene, expression is reduced in MDA5-/- mice……………………………………………………………………………………88

FIGURE 2-4. Innate inflammatory cell recruitment is largely intact in MDA5-/- mice, but activation of dendritic cells is elevated……………………………………………………….89

FIGURE 2-5. Cytokine production by natural killer and natural killer T cells is similar or elevated in MDA5-/- mice compared to WT mice…………………………………………...90

FIGURE 2-6. T cell activation is independent of MDA5 signaling. ……………………….91

FIGURE 2-7. Interferon gamma production, but not perforin production, is elevated in T cells from MDA5-/- mice. ……………………………………………………………………...92

viii

FIGURE 2-8. Elevated cytokines, but not T cells, may explain decreased survival of infected MDA5-/- mice…………………………………………………………………………93

CHAPTER 1

INTRODUCTION

The innate immune system includes numerous different hematopoietic cell types, dozens of sensors expressed by both hematopoietic and stromal cells, and both pro- and anti-inflammatory cytokines. Among the first elements of the system that pathogens encounter, however, are pattern recognition receptors (PRR), invariant receptors that detect conserved pathogen associated molecular patterns (PAMPS). Upon recognizing its ligand, a PRR will activate a signaling cascade that often triggers production and/or release of one or more pro-inflammatory cytokines. These in turn modulate additional elements of both the innate and adaptive immune responses, and are often critical

lynchpins of host defense. Due to the central role of innate immunity in host defense we investigated the roles of two distinct innate inflammatory pathways, the inflammasome and the melanoma differentiation-associated gene 5 (MDA5)-dependent portion of the type 1 interferon response, during infection with the murine coronavirus MHV.

A. Structure and function of the inflammasome

The inflammasome is a multimeric protein complex that forms in the cytosol following detection of a diverse range of ligands, and which is responsible for activation of caspase-1 (Martinon, Burns, & Tschopp, 2002). Caspase-1 in turn processes members of the IL-1 family of cytokines into their active forms (Ghayur et al., 1997; Gu et al., 1997;

Howard et al., 1991; Thornberry et al., 1992). Unlike other innate signaling pathways, in which a single stimulus is necessary and sufficient for transcriptional upregulation and secretion, inflammasome signaling is more tightly controlled, and requires two signals.

Many, although not all, of the IL-1 family cytokines are not constitutively expressed, and must instead be upregulated transcriptionally by a first signal, such as detection of lipopolysaccharide by toll-like receptors (TLRs) (Chin & Kostura, 1993; Newton, 1986).

Cleavage by caspase-1 provides the second signal needed for the release of the cytokines, which are not secreted prior to processing (Keller, Rüegg, Werner, & Beer,

2008).

Numerous receptors for danger and PAMPs trigger activation of the inflammasome.

These receptors and their ligands will be discussed below. Following detection of a ligand most receptors recruit the apoptosis-associated speck-like protein containing a carboxy-terminal caspase recruitment domain (ASC). ASC acts as a scaffold, mediating

interaction between receptors and caspase-1. This structure allows for auto-processing of caspase-1, leading to its activation (Mariathasan et al., 2004; Srinivasula et al., 2002).

Caspase-1 is a cysteine protease with an active form that is produced by auto-lysis of its p45 subunit into the p10 and p20 subunits (Cerretti et al., 1992; Thornberry et al., 1992;

N. P. Walker et al., 1994; K. P. Wilson et al., 1994). Once in its active form, caspase-1 processes members of the IL-1 family of cytokines, including IL-1α, IL-1β, IL-18, IL-33, and IL-37 (Garlanda, Dinarello, & Mantovani, 2013).

The best studied of the IL-1 family cytokines are IL-1α, IL-1β, and IL-18. IL-1β and IL-18 are both translated as immature, pro-forms, and must be processed by caspase-1 to be active (Ghayur et al., 1997; Gu et al., 1997). IL-1α is active in its unprocessed form

(Mosley et al., 1987), and does not require processing from caspase-1. However, secretion of IL-1α is enhanced by caspase-1 (Howard et al., 1991) (Keller et al., 2008).

IL-1 family cytokines do not require the canonical ER/golgi trafficking utilized for secretion of other cytokines, such as tumor necrosis factor α (TNFα) (Monteleone, Stow,

& Schroder, 2015), and in fact lack the signal sequences required for this method of trafficking (Auron et al., 1984). The alternative method of secretion used by IL-1 family cytokines remains poorly understood.

B. Ligands of inflammasome sensors

The inflammasome can be activated by a wide range of stimuli, including both pathogen associated molecular patterns and danger associated molecular patterns. Multiple receptors recruit ASC following binding to their ligand or an intermediate receptor,

triggering inflammasome formation. This broad range of receptors allows the inflammasome to respond to a diverse set of insults.

Absent in melanoma 2 (AIM2) is a receptor capable of activating the caspase-1 dependent inflammasome (Hornung et al., 2009; T. L. Roberts et al., 2009). A cytosolic protein, it is capable of direct interaction with dsDNA (T. Jin et al., 2012a), and has been shown to be important for host defense against vaccinia virus, cytomegalovirus virus

(CMV), and Francisella tularensis (Fernandes-Alnemri et al., 2010; Rathinam et al.,

2010). Like several other inflammasome components, AIM2 is an interferon stimulated gene (ISG) (DeYoung et al., 1997).

The NOD-like receptor (NLR) family is conserved across both vertebrate and invertebrate species, suggesting an ancient lineage (Rast, Smith, Loza-Coll, Hibino, &

Litman, 2006). Many of the NLRs, including NLRC4 and NLRP 1, 2, 3, 6, and 12 can lead to caspase-1 activation (Agostini et al., 2004; Grenier et al., 2002; Martinon et al.,

2002; Poyet et al., 2001; L. Wang et al., 2002). NLRs do not always directly bind their ligands, but either directly or through adaptors are capable of detecting diverse structures, including bacterial RNA (Kanneganti et al., 2006) and uric acid crystals

(Martinon, Pétrilli, Mayor, Tardivel, & Tschopp, 2006).

Alum activates caspase-1 in an NLRP3-dependent manner (Eisenbarth, Colegio,

O'Connor, Sutterwala, & Flavell, 2008), and may contribute to vaccine responses. The

NLRP3 inflammasome also mediates the response to many fungal, bacterial and viral pathogens, including Saccharomyces cerevisiae (Lamkanfi, Malireddi, & Kanneganti,

2009), Candida albicans (Gross et al., 2009; Hise et al., 2009), Staphylococcus aureus

(Craven et al., 2009), Aeromonas hydrophila (Gurcel, Abrami, Girardin, Tschopp, & van der Goot, 2006), influenza A virus (IAV) (Allen et al., 2009; P. G. Thomas et al., 2009), adenovirus, herpes virus, and vaccinia virus (Delaloye et al., 2009; Muruve et al., 2008).

NLRP3 detects cytosolic dsRNA through the sensor helicase DHX33 in some cases

(Mitoma et al., 2013). NLRC4 and NLRP3 are predominately expressed in hematopoietic cell types, while NLRP1 is more widely expressed (Kummer et al., 2007). Bacillus anthracis (Boyden & Dietrich, 2006), and Toxoplasma gondii infection (Cavailles et al.,

2014; Cirelli et al., 2014; Ewald, Chavarria-Smith, & Boothroyd, 2014; Gorfu et al., 2014) activate the inflammasome in an NLRP1-dependent fashion.

C. The non-canonical inflammasome

The original caspase-1-/- mouse line had an unidentified mutation in the gene for caspase-11, which can also lead to inflammasome related cytokine release. This led to several roles being attributed to caspase-1, such as involvement in the septic shock response (S. Wang et al., 1996; 1998) that in fact belong to caspase-11. The second mutation was discovered in 2011, and the mouse line in question is now referred to as the Caspase-1/11-/- mouse (Kayagaki et al., 2011). Since then a caspase-11 dependent inflammasome, called the non-canonical inflammasome, has been characterized. Like the canonical inflammasome, it can mediate secretion of IL-1α, IL-1β, and IL-18, and trigger cell death (Aachoui et al., 2013; Broz et al., 2012; Gurung et al., 2012; Kayagaki et al., 2011; Rathinam et al., 2012).

D. Inflammasome-mediated cell death

In addition to processing pro- and anti-inflammatory cytokines, inflammasome activation also leads to a form of pro-inflammatory cell death called pyroptosis (Cookson &

Brennan, 2001; Kuida et al., 1995; Li et al., 1995). Unlike apoptosis, which is a form of programmed cell death that generally does not induce inflammation, and necrosis, an inflammatory form of non-programmed cell death, pyroptosis is carefully regulated, and produces danger associated molecular patterns. Interestingly, NMR imaging has demonstrated that the structure of the inflammasome is similar to that of the apoptosome, a multimeric protein structure assembled during programmed cell death

(Faustin et al., 2007). Caspase-11 mediated pyroptosis requires cleavage of Gasdermin

D (Kayagaki et al., 2015; J. Shi et al., 2015), but otherwise the pathway linking cell death and the inflammasome has not been well characterized.

Pyroptosis has several potential anti-pathogen functions. First, obligate intracellular pathogens such as all viruses and some bacteria and eukaryotic parasites rely on host cells for replication. Infections that trigger inflammasome activation lead to cell death, removing this replicative niche. Second, unlike apoptosis, which is immunologically

“quiet”, pyroptotic cell death provides danger signals that warn nearby cells of the infection, promoting an inflammatory response

(Bergsbaken, Fink, & Cookson, 2009; Kono & Rock, 2008). Caspase-1 mediated pyroptosis has been demonstrated to improve bacterial clearance (Miao et al., 2010).

E. IL-1 signaling

Early studies with IL-1α and IL-1β determined that they were capable of activating T cells, and they were designated lymphocyte-activating factors (Gery & Waksman, 1972;

Gery, Gershon, & Waksman, 1972). IL-1α and IL-1β signal through the same receptor, the IL-1 receptor (Dower et al., 1986), and were not differentiated as separate factors until 1985 (March et al., 1985).

Once activated and processed following inflammasome activation, IL-1 mediates a variety of immunoregulatory effects. IL-1 signaling upregulates expression of the chemokine KC, which recruits neutrophils to the site of infection (Y.-S. Lee et al., 2015).

IL-1 signaling can also polarize T cells to adopt a Th17 phenotype (Sato, Martinez,

Omura, & Tsunoda, 2011), and blockade of IL-1 has been found to reduce IL-17 production in vitro and during the development of autoimmune diseases in mouse models (Coccia et al., 2012; Joosten, 2010; Sutton et al., 2009; Sutton, Brereton, Keogh,

Mills, & Lavelle, 2006).

IL-1 signaling is also critical for host defense during IAV infection (Ichinohe, Lee, Ogura,

Flavell, & Iwasaki, 2009), with IL-1 receptor knockout (IL-1R-/-) mice exhibiting a defective T cell response. Similarly, IL-1R-/- mice infected with West Nile virus (WNV) exhibited a poor CD8 T cell response (Ramos et al., 2012). These findings were initially surprising, as IL-1 signaling was thought to polarize T cells to a Th17 phenotype, while the T cell response to IAV and WNV has a Th1 phenotype. Subsequent studies demonstrated that during infection with either virus IL-1 signaling was critical for promoting dendritic cell (DC) activation (Durrant, Robinette, & Klein, 2013; Pang,

Ichinohe, & Iwasaki, 2013), and that mice lacking the IL-1 receptor had poor T cell priming as a result of defective DC populations, demonstrating a role for IL-1 signaling in

DC function independent of the subsequent T helper phenotype.

F. IL-18 signaling

Like IL-1β, IL-18 becomes active after proteolytic cleavage by caspase-1. IL-18 binds to the IL-18 alpha chain (IL-18Rα). Binding of IL-18 and IL-18Rα is too low affinity to trigger downstream effects, and requires the co-receptor IL-18 chain beta (IL-18Rβ), which helps form a high affinity complex that is capable of signaling (Weber, Wasiliew, &

Kracht, 2010). IL-18Rα is widely expressed, but expression of IL-18Rβ is more restricted, and is primarily found on T cells and DCs (Kim, Han, Azam, Yoon, &

Dinarello, 2005). This limits the number of cell types that can be affected by IL-18.

Binding of IL-18 and its receptor triggers a signaling cascade that, like IL-1R signaling, requires the adaptor protein MyD88 (Adachi et al., 1998).

Like IL-1, IL-18 has a variety of immunomodulatory effects. The cytokine was originally characterized as a co-stimulatory factor that enhanced production of interferon gamma by T cells, and was named interferon gamma inducing factor (IGIF) (Kohno et al., 1997;

Okamura et al., 1995). IL-12 has a similar function, and the combination of IL-18 and IL-

12 leads to the development of interferon gamma producing Th1 T cells (D. Robinson et al., 1997). Each cytokine upregulates the receptor for the other, leading to a feed forward loop. However, in some infectious contexts IL-12 or IL-18 alone is sufficient to maintain the Th1 response so the requirement for IL-18 in production of interferon gamma is context specific. Additional studies have shown that IL-18 signaling can also

act on natural killer (NK) and natural killer T (NKT) cells to promote interferon gamma production (Hunter et al., 1997; Nagarajan & Kronenberg, 2007; W. Walker, Aste-

Amezaga, Kastelein, Trinchieri, & Hunter, 1999).

In addition to promoting interferon gamma production, IL-18 signaling can also stimulate the cytotoxic functions of T cells (Dao, Ohashi, Kayano, Kurimoto, & Okamura, 1996) and NK cells (Okamura et al., 1995; Tsutsui et al., 1996). This can be mediated through promotion of perforin production (Dao, Mehal, & Crispe, 1998) as well as induction of

Fas ligand (Tsutsui, Matsui, Okamura, & Nakanishi, 2000).

G. Role of the inflammasome in demyelination

Multiple sclerosis (MS) is a complex autoimmune disease in which the myelin that sheaths neuronal axons is lost, leading to loss of neuromuscular control and coordination. The initial events that lead to development of MS are completely unknown, and the inflammatory factors that drive pathogenesis have not been fully characterized.

However, studies on patients with MS have suggested that the inflammasome may be involved in pathogenesis. IL-18 and its ability to drive interferon gamma may underlie the disease state (Karni, Koldzic, Bharanidharan, Khoury, & Weiner, 2002; Losy &

Niezgoda, 2001). IL-1β is observed in the cerebrospinal fluid of patients with MS

(Cannella & Raine, 1995), and polymorphisms in the IL-1 gene can alter the severity of

MS (Mann et al., 2002).

Interestingly, the two front line therapies for treatment of MS, glatiramer acetate and interferon beta, both negatively regulate inflammasome signaling. Glatiramer acetate, a

synthetic subunit of myelin basic protein, leads to elevated serum levels of the secreted form of the IL-1 receptor antagonist, potentially reducing IL-1 signaling (Burger et al.,

2009). Interferon beta therapy may act by dampening the NLRP3 and NLRP1 pathways and inhibiting IL-1β signaling (Guarda et al., 2011). Patient response to interferon beta therapy is inconsistent, and those who respond well to the therapy generally have elevated levels of NLRP3 and IL-1β prior to treatment, while those who do not respond have normal levels (Malhotra et al., 2015). This finding may suggest that the inflammasome is a pathogenic driver in only a subset of MS cases, which is unsurprising given the heterogeneity of the disease (Denic et al., 2011).

Animal models have been key in understanding the pathogenesis of MS. Work with the experimental autoimmune encephalitis (EAE) model, which is induced by vaccination with whole or subunits of myelin basic protein, and the cuprizone induced model of demyelination suggests that the inflammasome may be important for pathogenesis. Mice lacking IL-18 signaling were found to experience less severe EAE symptoms, and to have fewer interferon gamma and IL-17 producing cells (F. D. Shi, Takeda, Akira,

Sarvetnick, & Ljunggren, 2000). Similarly, IL-1 signaling can also promote EAE severity, possibly through polarization of αβ T cells (Chung et al., 2009) or γδ T cells (N. J. Wilson et al., 2007) toward a Th17 phenotype. Both IL-1β and IL-18 alone are sufficient to promote DCs to polarize T cells toward a Th17 phenotype (Lalor et al., 2011), suggesting a possible role in regulation of T cell priming during inflammatory diseases.

The cuprizone model of demyelination, which is believed to be a Th1 driven disease, is partially dependent on IL-18 signaling, with mice lacking this pathway demonstrating delayed disease onset (Jha et al., 2010).

Demyelination can also be induced following infection with either MHV or Theiler’s encephalomyelitis virus. The role for the inflammasome in virus-induced models of demyelination has not yet been studied. Due to the heterogeneity of MS (Denic et al.,

2011) we cannot assume that results from one model will be recapitulated in other models or in the actual disease, and it would be beneficial to study pathogenic drivers in as diverse a range of models as possible.

H. Structure and function of MDA5

MDA5 was originally characterized as a melanoma-differentiation associated gene

(Kang et al., 2004; 2002), and was shown to play a role in nuclear remodeling during apoptosis (Kovacsovics et al., 2002). Its role in promoting type 1 interferon production during infection was observed later.

MDA5 is a cytosolic RIG-I-like helicase that detects viral double stranded RNA (dsRNA).

Like RIG-I and LGP2, the other RIG-I-like helicases, MDA5 contains a DECH-box helicase domain, through which it binds dsRNA and catalyzes hydrolysis of ATP. The catalytic core of the DECH-box domain is composed of two ReA-like domains, Hel1 and

Hel2 (Cordin, Banroques, Tanner, & Linder, 2006). MDA5 has a low affinity for its RNA substrate, and may be assisted by LGP2, a RIG-I-like helicase that lacks the CARD domain necessary for interaction with the adaptor protein mitochondrial antiviral signaling protein (MAVS) (Rodriguez, Bruns, & Horvath, 2014). Despite their names, there is little evidence suggesting that MDA5 or the other RIG-I-like helicases are actually capable of unwinding nucleic acid duplexes. The ATP hydrolysis catalyzed by

the DECH-box domain is essential for MDA5 signaling, however (Bamming & Horvath,

2009).

Once MDA5 binds to dsRNA multiple MDA5 molecules will assemble into a filamentous structure around the RNA. This aggregation allows for oligomerization of MDA5’s N- terminal CARD domain, which in turn allows it to interact with its adaptor protein MAVS

(Hou et al., 2011; H. Xu et al., 2014b). A minimum of 11 MDA5 molecules appears to be necessary for this structure to form (Peisley, Wu, Yao, Walz, & Hur, 2013).

MAVS, which is also called IFNβ promoter stimulator-1 (IPS-1), virus-induced signaling adaptor (VISA), and CARD adaptor inducing IFNβ (Cardif), is the adaptor protein linking both MDA5 and RIG-I to downstream signaling molecules (Jiang et al., 2011; Kowalinski et al., 2011; Luo et al., 2011; Zeng et al., 2010). MAVS is a membrane bound protein found on both peroxisomes and mitochondria (Dixit et al., 2010). Much like how MDA5 forms aggregates upon binding of RNA, interaction with MDA5 leads MAVS to transition from a soluble form to a helical fiber that is similar to amyloid fibers and prions in that once it begins to aggregate it can propagate itself further without needing additional stimuli from upstream in the pathway (Cai et al., 2014).

Once activated, MAVS in turn propagates the signal to the cytosolic protein kinase IKK and to the transcription factor NF-κB. IKK associates with TBK1 to phosphorylate and activate the transcription factor IRF3 (Yoneyama et al., 2004). These transcription factors promote transcription of various type 1 interferon subtypes as well as a large number of ISGs. The type 1 interferon pathway is discussed below.

I. MDA5’s binding substrate(s)

MDA5 is not capable of detecting all dsRNA, but the exact nature of its binding specificity remains unclear. Studies have shown that MDA5 mediates the response to

Poly(I:C), encephalomyocarditis virus, murine norovirus, picornaviruses, polioviruses, and coronaviruses (Gitlin et al., 2006; Kato et al., 2006; McCartney et al., 2008; Roth-

Cross, Bender, & Weiss, 2008). Given the complicated nature of RNA from actual viruses, as well as the variable nature of Poly(I:C), more recent studies have focused on manipulated RNA. When RNA is isolated from digested populations or from virus- infected cells and separated by weight, only the high molecular weight fractions, in general those above 2 kbp, induce MDA5 signaling (Kato et al., 2008; Pichlmair et al.,

2009). Viruses lacking the ability to cap the 2’-O position of their genome induce higher interferon during infection in an MDA5-dependent manner, so it has been hypothesized that MDA5 discriminates between host RNA, which has a 2’ cap, and viral RNA in this manner (Züst et al., 2011). Ultimately the exact structure of RNA that MDA5 recognizes has not been fully elucidated.

J. The type 1 interferon signaling pathway

The downstream effect of MDA5 signaling is the induction of the type 1 interferon response, and the antiviral and immunomodulatory roles of this pathway must be addressed in order to fully understand the relevance of MDA5. Type 1 interferons, which include the IFNβ and multiple IFNα subtypes, signals through the type 1 interferon receptor (IFNAR), which is composed of two components, IFNAR1 and IFNAR2. Binding of IFNAR leads to activation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, which phosphorylates STAT1 and STAT2 (Dhib-Jalbut,

2002; Kasper & Shoemaker, 2010). These in turn dimerize and associate with interferon regulatory factor 3 (IRF3), and the complex binds to interferon stimulated response elements (ISREs) in the nucleus (Bekisz et al., 2013). The end result of this pathway is the transcriptional upregulation of type 1 interferon subtypes as well as hundreds of

ISGs. IFNβ and IFNα4 are produced first, in an initial, IRF3-dependent wave of transcription. This upregulates IRF7, which facilitates a feedback loop, leading to a second wave of transcription, which includes additional IFNα subtypes as well as ISGs

(Honda, Takaoka, & Taniguchi, 2006; Tamura, Yanai, Savitsky, & Taniguchi, 2008).

Interferon produced by infected cells is secreted, and signals through IFNAR in both an autocrine and paracrine manner. This signaling induces an antiviral state in uninfected bystander cells, and helps control viral replication in cells that are already infected (Yan

& Chen, 2012). The importance of this pathway in host defense is evident in the number of viruses that encode antagonists against one or more elements of the pathway

(Versteeg & García-Sastre, 2010). It is further supported by data showing that IFNAR-/- mice are susceptible to numerous viruses, including MHV, Vesicular stomatitis virus,

Semliki forest virus, vaccinia virus, and lymphocytic choriomeningitis virus (LCMV)

(Durbin et al., 2000; Garcia-Sastre et al., 1998; Koerner, Kochs, Kalinke, Weiss, &

Staeheli, 2007; Price, Gaszewska-Mastarlarz, & Moskophidis, 2000).

K. Role of type 1 interferon during acute infection

In addition to stimulating production of ISGs, type 1 interferon can have additional direct effects, and is capable of modulating the behavior of many arms of the immune response. It is arguably a central regulator of the host immune response. While originally

considered a pro-inflammatory cytokine, it has since become evident that type 1 interferon can also have anti-inflammatory properties, adding additional complexity to its role.

Type 1 interferon can promote the function of NK cells, although this has proven to be dependent on the infectious model. During IAV and vaccinia virus infection interferon enhances the cytotoxicity and interferon gamma production of NK cells (Hwang et al.,

2012; J. Martinez, Huang, & Yang, 2008), while during murine CMV infection interferon signaling is necessary for the recruitment and cytolytic activity of NK cells, but not for interferon gamma production (Nguyen et al., 2002).

Type 1 interferon also signals directly to DCs, causing upregulation of MHC, CD80, and

CD86 (Hahm, Trifilo, Zuniga, & Oldstone, 2005; Ito et al., 2001; Montoya et al., 2002) and leading to an improved capacity to prime T cells. It can also enhance cross presentation during viral infection (Le Bon, Durand, et al., 2006a; Le Bon et al., 2003;

Spadaro et al., 2012), and enhance migration of DCs to lymph nodes by upregulating chemokine receptors (Parlato et al., 2001; Rouzaut et al., 2010). A synergy between

PRR stimulation and interferon signaling leads DCs to produce IL-12. However, high levels of type 1 interferon can instead inhibit IL-12 production in some contexts

(Cousens, Orange, Su, & Biron, 1997; Dalod et al., 2002), suggesting that the pro- and anti-inflammatory roles of interferon are highly context specific.

Evidence for this bi-modal role of interferon signaling is evident in studies of T cells.

Type 1 interferon signaling promotes survival and clonal expansion of CD4 (Havenar-

Daughton, Kolumam, & Murali-Krishna, 2006) and CD8 (Aichele et al., 2006; Curtsinger,

Valenzuela, Agarwal, Lins, & Mescher, 2005; Kolumam, Thomas, Thompson, Sprent, &

Murali-Krishna, 2005; Marrack, Kappler, & Mitchell, 1999) T cells in some contexts, but can have an anti-proliferative effect on CD8 T cells in others (Bromberg, Horvath, Wen,

Schreiber, & Darnell, 1996; Kaser, Nagata, & Tilg, 1999; C. K. Lee, Smith, Gimeno,

Gertner, & Levy, 2000; Marshall, Urban, & Welsh, 2011; Petricoin et al., 1997; Tanabe et al., 2005). CD4 T cells exposed to type 1 interferon are better able to help B cells (Le

Bon, Thompson, et al., 2006b), while during LCMV infection interferon signaling can block lymphocyte egress from lymphoid organs (Shiow et al., 2006). Concentration of interferon, as well as the kinetics of signaling, seems to dictate the ultimate effect on

CD8 T cells (Thompson, Kolumam, Thomas, & Murali-Krishna, 2006), but in other contexts the factors involved in the opposing effects of type 1 interferon remain unclear, and whether the cytokine is pro- or anti-inflammatory during any given infection, and indeed during any particular phase of an infection, must be determined empirically.

L. The interferon stimulated gene response

In addition to the effects of direct type 1 interferon signaling, the pathway also leads to transcriptional upregulation of hundreds of ISGs. It should be noted that signaling by

MAVS located on peroxisomes is capable of upregulating ISGs in an interferon- independent manner (Dixit et al., 2010), so the ISG response is downstream of both

MDA5/MAVS acting through interferon and MDA5/MAVS signaling acting independently.

Many ISGs have direct antiviral effects, including APOBEC3, TRIM5α, tetherin, and viperin. APOBEC3 edits viral genomes, potentially leading to deleterious effects on viral fitness (Sheehy, Gaddis, Choi, & Malim, 2002; Vartanian, Meyerhans, Asjö, & Wain-

Hobson, 1991), while TRIM5α blocks replication of retroviruses by binding to the viral nucleocapsid to prevent uncoating (D. Wolf & Goff, 2008). Tetherin anchors virions to the host cell membrane, preventing their release (77 from yun12r). Viperin also inhibit viral release (Nasr et al., 2012; Tan et al., 2012; X. Wang, Hinson, & Cresswell, 2007), although it appears to act by altering plasma membrane fluidity and disrupting lipid rafts

(Tan et al., 2012; X. Wang et al., 2007). ISG15 prevents virion release through interference with the ubiquitination pathway (Malakhova & Zhang, 2008; Okumura, Lu,

Pitha-Rowe, & Pitha, 2006; Okumura, Pitha, & Harty, 2008). STAT1, STAT2, MDA5 and

RIG-I are themselves ISGs, creating a positive feedback loop that enhances the interferon response. The combined effects of these hundreds of genes provide antiviral activity against a wide range of viruses, making the interferon response a broad spectrum defense mechanism.

M. The role of type 1 interferon in the memory T cell response

Type 1 interferon signaling is capable of affecting the development of the memory T cell pool, as well as the function of these cells. Research to date suggests that, unlike during primary infections, the effects of type 1 interferon signaling are primarily pro- inflammatory in the context of memory cells. Interferon seems to promote a more robust memory response during infection with vesicular stomatitis virus and LCMV (Kolumam et al., 2005; Ramos et al., 2009; Thompson et al., 2006), although the mechanism for this effect is expansion of the initial T cell pool during acute infection, so the effect is arguably on effector cells, not on memory cells.

Interferon signaling can also promote the function of T cells during reinfection, including by improving the cytotoxicity of cells during Sendai virus infection (Kohlmeier,

Cookenham, Roberts, Miller, & Woodland, 2010). Exposure to interferon also causes T cells to upregulate inhibitory NK cell receptor ligands, reducing NK cell-mediated attrition

(Crouse et al., 2014; H. C. Xu et al., 2014a). Many of interferon’s effects are indirect, acting on non-T cells in ways that improve the T cell response. These effects include promoting production of chemokines to aid in proper trafficking of central memory lymphocytes (Sung et al., 2012), and driving production of IL-15 and IL-18 from inflammatory monocytes, which aid in CD8 T cell survival and function (Soudja, Ruiz,

Marie, & Lauvau, 2012).

N. Deleterious effects of MDA5 and type 1 interferon

As with many pro-inflammatory pathways, MDA5 and type 1 interferon signaling can be harmful when improperly regulated. Polymorphisms in MDA5 in humans have been linked to type 1 diabetes (Smyth et al., 2006) and lupus (T. Robinson et al., 2011). Gain of function mutations in humans are thought to underlie some cases of Aicardi-Goutieres syndrome (Kovacsovics et al., 2002), an autoimmune condition characterized by constitute expression of ISGs (Miner & Diamond, 2014).

Type 1 interferon signaling can also be pathological, and numerous inflammatory diseases in humans have now been classified as “type 1 interferonopathies”. These diseases are characterized by inappropriate activation of type 1 interferon signaling, and can have a wide variety of clinical symptoms (Lee-Kirsch, Wolf, Kretschmer, & Roers,

2015). Work in murine disease models suggests some possible mechanisms for the

pathological action of this pathway. Type 1 interferon induces production in TNF-related apoptosis inducing ligand (TRAIL) in inflammatory monocytes (Högner et al., 2013).

TRAIL has been shown to induce damage in epithelial cells during influenza infection

(Herold et al., 2008), and blocking interferon signaling can reduce inflammation and lung pathology (Davidson, Crotta, McCabe, & Wack, 2014).

O. Taxonomy and natural history of mouse hepatitis virus

Coronaviruses are a family of single strand, positive sense RNA viruses (Lai &

Stohlman, 1978). With a 31 kilobase genome they are among the largest of the RNA viruses (H. J. Lee et al., 1991). They were first recognized as a distinct family within the

Nidoviridae order in 1967 (Almeida & Tyrrell, 1967), and their name is derived from their unusual, crown-like appearance under microscopy, which is caused by their surface proteins (Almeida & Tyrrell, 1967).

The Coronavirus family is composed of numerous individual strains, with diverse organ tropism. Various strains can infect humans, mice, livestock and poultry, and other domestic animals. This broad host tropism has become a potential public health concern in recent years. Coronaviruses capable of infecting humans, including the HCoV-229E

(Hamre & Procknow, 1966) and HCoV-OC43 (McIntosh, Dees, Becker, Kapikian, &

Chanock, 1967) strains were identified early on, but the virus family was not considered a serious threat until the emergence of severe acute respiratory syndrome (SARS)-CoV in 2003, which caused a world wide epidemic (Drosten et al., 2003; Ksiazek et al., 2003).

Since then several new human respiratory coronaviruses have been discovered; HCoV-

NL63 (van der Hoek et al., 2004) and HCoV- HKU1 (Woo et al., 2005). Most recently the

Middle East respiratory syndrome (MERS)-CoV, which causes severe life threatening respiratory disease, has emerged in the Middle East (Bermingham et al., 2012), providing another reminder of the importance of studying this viral family.

The research described herein focuses on the study of MHV, a murine coronavirus that has proven to be a tractable model for studying general coronavirus biology. MHV is a naturally occurring coronavirus that can be found among wild rodent populations as well as research vivariums (Perlman, 1998). Although originally spread by a fecal-oral route, lab strains of MHV have evolved to lose their ability to spread naturally, and have acquired new tropisms. The strain of MHV used herein, A59 (referred to here simply as

MHV), was originally cultured from a mouse with leukemia in 1961 (MANAKER, PICZAK,

MILLER, & STANTON, 1961). A59 is capable of infecting a wide range of cells in the brain, spinal cord, liver, and lungs (Weiss & Leibowitz, 2011), and is therefore a useful model for studying pathogenesis in a variety of organs as well as mechanisms of tropism barriers. There are several different MHV strains used by the field and much of the literature, including much of the work cited here, is for strains other than A59.

Characteristics of strains can differ from each other, and results from one strain do not necessarily apply to others.

P. Course of mouse hepatitis virus infection

MHV strain A59 exhibits an inoculation site-dependent organ tropism. When the virus is inoculated intracranially (i.c.) it replicates in the brain and spinal cord, and also reaches the periphery by an unknown mechanism, where it infects the liver, spleen, and lungs

(Weiss & Leibowitz, 2011). In contrast, when MHV is inoculated intraperitoneally (i.p.) it

replicates in the liver, spleen, and lungs, but only rarely is able to cross the blood brain barrier (BBB) and infect the central nervous system (CNS). Kinetics of infection, including both replication and lethality, are unchanged by site of inoculation.

MHV has a broad cellular tropism. In the brain it infects oligodendrocytes, astrocytes

(Lavi et al., 1987), neurons (Parra, Hinton, Lin, Cua, & Stohlman, 1997), microglia and infiltrating macrophages. In the liver the virus infects hepatocytes (Navas et al., 2001; L.

P. Weiner, 1973), endothelial cells and Kuppfer cells (Even, Rowland, & Plagemann,

1995) (unpublished data from Weiss lab). T cells have not been observed to be infected, although B cells can be (Coutelier et al., 1994).

In the brain and liver viral load, as determined by plaque assay, peaks at five days post inoculation (Navas et al., 2001), and is cleared entirely by fourteen days post inoculation

(Matthews et al., 2001). During acute infection MHV can cause encephalitis and meningitis in the CNS and hepatitis in the liver (Perlman, 1998), which can be lethal depending on the dose of virus, strain, and immune status of the mouse.

An unusual aspect of MHV infection is its ability to persist in the CNS. This ability is shared by a small number of other viruses, including HIV, measles, and John

Cunningham virus (Elsner & Dörries, 1992; Haase et al., 1981; Kleinschmidt, Neumann,

Möller, Erfle, & Brack-Werner, 1994; Tominaga, Yogo, Kitamura, & Aso, 1992).

Infectious virions cannot be isolated from the CNS by fourteen days after infection, but a low level of viral antigen is detectable up to thirty days after infection. Viral RNA is detectable ten to twelve months after infection (Lavi, Gilden, Wroblewska, Rorke, &

Weiss, 1984), and is persistent in the form of a swarm of quasispecies (Adami et al.,

1995).

Mice that survive MHV infection develop a demyelinating disease following clearance of the virus. The link between viral persistence and demyelination remains unclear. This demyelination, which is specific to the CNS (Lampert, Sims, & Kniazeff, 1973), involves loss of the myelin sheaths from neuronal axons (Lavi, Gilden, Highkin, & Weiss, 1986) as well as axonal degeneration (Marro, Blanc, Loring, Cahalan, & Lane, 2014). Mice experience ataxia and hind limb paralysis (Lavi et al., 1984), after which they generally recover. The disease state also includes cycles of remyelination, in which myelin loss is reversed (Kristensson & Norrby, 1986; Takahashi, Goto, Matsubara, & Fujiwara, 1987).

Lesions, regions in which myelin has been lost, remain visible up to ninety days after infection (Stohlman & Weiner, 1981). Demyelinating human diseases, including MS, remain poorly understood, and viral-induced models of demyelination in mice can provide insights into disease development and pathogenesis.

Q. Immunology of mouse hepatitis virus-induced demyelination

The pathogenic mechanisms underlying MHV-induced demyelination are not entirely understood. Numerous immunological cell subsets and effectors have been shown to be superfluous for demyelination. Mice lacking B cells still undergo demyelination

(Matthews, Lavi, Weiss, & Paterson, 2002), as do mice lacking perforin and the Fas-

FasL pathway, both mechanisms for cytolytic activity (Lin, Stohlman, & Hinton, 1997;

Parra et al., 2000). TNFα, interferon gamma, and IL-10 are also unnecessary (Lin,

Hinton, Parra, Stohlman, & van der Veen, 1998; Parra et al., 1999; Stohlman et al.,

1995b). Although demyelination is an immune-mediated process, the severity of demyelination correlates with the intensity of the acute viral infection, with the extent of viral spread during acute phase being an accurate predictor of development of demyelination (MacNamara, Chua, Nelson, Shen, & Weiss, 2005; Marten et al., 2000;

Mitoma et al., 2013).

Early research found that RAG1-/- mice do not demyelinate, but that transferring whole splenocytes from immunocompetent mice would restore disease (Kang et al., 2002; F. I.

Wang, Stohlman, & Fleming, 1990; G. F. Wu & Perlman, 1999). More recent studies have shown that purified CD4 or CD8 T cells are sufficient to restore demyelination

(Lane et al., 1998; Roth-Cross et al., 2008; G. F. Wu, Dandekar, Pewe, & Perlman,

2000), demonstrating a role for T lymphocytes in the disease state. The precise role of T cells in this disease remains unknown.

In EAE, an induced murine model of demyelination, infiltrating macrophages appear to be drivers of pathology (Kato et al., 2006; Ransohoff, 2012). Recent work suggests that infiltrating macrophages initiate demyelination while microglia clear debris (Sirén et al.,

2006; Yamasaki et al., 2014). Macrophages also play a role in MHV-induced demyelination (Lavi et al., 1986; McCartney et al., 2008; Stohlman & Weiner, 1981; G. F.

Wu & Perlman, 1999), although the differential roles of infiltrating macrophages and microglia has not been studied in this context.

R. The Blood brain barrier

The CNS is separated from circulating blood by the BBB, a selectively permeable barrier formed by brain endothelial cells. The BBB prevents entry of infectious pathogens as well as serum antibodies in the absence of inflammation (Cserr, DePasquale, Harling-

Berg, Park, & Knopf, 1992; Lazear et al., 2013), while allowing entry of activated T cells

(Brabb et al., 2000; Y. H. Jin et al., 2012b). Infection with MHV and other neurotropic viruses causes a loss of integrity of the BBB, allowing access to serum antibodies and other elements of the immune system.

S. Immunology of acute mouse hepatitis virus infection

Mice lacking humoral immunity clear virus with similar kinetics to wild type (WT) mice, suggesting that B cells and antibody do not play a role in host defense during acute infection. However, virus does reemerge from the CNS shortly before clearance, indicating that humoral immunity is critical for complete clearance (Lavi et al., 1984; Lin,

Hinton, Marten, Bergmann, & Stohlman, 1999; Matthews et al., 2001; Ramakrishna,

Stohlman, Atkinson, Shlomchik, & Bergmann, 2002). Interestingly, virus does not reemerge from the liver (Matthews et al., 2001), suggesting there may be organ-specific control mechanisms.

Shortly after MHV infection of the CNS, memory T cells specific to irrelevant antigens are recruited into the brain. Their role is largely unclear, and their numbers are quickly overwhelmed by MHV specific T cells (80). These virus-specific T cells are critical to control of the virus. Depletion of either CD4 or CD8 T cells compromises control of viral replication (Williamson & Stohlman, 1990). Adoptive transfer of CD8 T cells into SCID

mice suppressed viral replication in the CNS (Bergmann et al., 2004), and CD4 T cells have been shown to be critically important for CD8 T cell function (Phares et al., 2012).

Following infection of the CNS, an initial expansion of T cells is observed in the cervical lymph nodes, the draining lymph nodes of the CNS, followed by a secondary expansion in the spleen. T cells then appear in the CNS, peaking seven days after infection

(Marten, Stohlman, Zhou, & Bergmann, 2003). The kinetics and location of T cell expansion following infection of the liver has not been examined. Much of the work on the T cell response to MHV was performed with a strain of MHV, called JHM, that infects only neurons, and it is unknown if its more restricted cellular tropism alters the immune response, compared to that induced by A59.

Interferon gamma appears to be the most important element of the adaptive immune response during MHV infection. Mice lacking this cytokine are more susceptible to infection, with increased mortality following infection in the periphery (Kyuwa et al., 1998;

Schijns, Wierda, van Hoeij, & Horzinek, 1996). Depletion of CD8 T cells from interferon gamma deficient mice further increases this mortality, suggesting interferon gamm- independent mechanisms also contribute to protection (Kyuwa et al., 1998; Roth-Cross et al., 2008). CD8s isolated from infected brains produce granzyme B in addition to interferon gamma, and have cytolytic killing function (Bergmann, Altman, Hinton, &

Stohlman, 1999; Ramakrishna, Stohlman, Atkinson, Hinton, & Bergmann, 2004; Roth-

Cross et al., 2008). Fas/FasL pathway has no effect on pathogenesis, clearance or pathology (Cervantes-Barragan et al., 2007; Parra et al., 2000). Interestingly, different immunological mechanisms appear to underlie viral clearance from different cell types in the CNS. Mice lacking interferon gamma have increased viral replication in

oligodendrocytes (Bergmann et al., 1999; 2003; Parra et al., 1999; Roth-Cross et al.,

2008). Mice with decreased signaling through the interferon gamma receptor on oligodendrocytes have defective clearance, indicating that interferon gamma signaling in infected oligodendrocytes is involved in viral control (Cervantes-Barragan et al., 2007;

González et al., 2005). Mice lacking perforin have uncontrolled replication in macrophages, microglia, and astrocytes, but normal replication in oligodendrocytes

(Stohlman, Bergmann, van der Veen, & Hinton, 1995a). Perforin, along with granzyme

B, mediates cytolytic activity of CD8 T cells and NK cells, and it is reasonable to hypothesize that cytolytic killing of infected oligodendrocytes or neurons, both of which are difficult to replace, would be suppressed. Macrophages, microglia and astrocytes are, by comparison, easily replaceable, and cytolytic elimination of infected cells would be advantageous. These data suggest that the CNS may have elaborate regulatory mechanisms in place to control viral infections while mitigating tissue damage.

T. Dissertation aims

MHV has a wide organ tropism that is partially dependent on host immune mechanisms, and its clearance is reliant on the type 1 interferon response, as well as both interferon gamma production and cytotoxicity by the cellular immune response. It is therefore a tractable model for studying both determinants of organ tropism and the role of innate inflammatory pathways on multiple elements of the innate and adaptive immune responses. Furthermore, acute MHV infection induces hepatitis in the liver and encephalitis in the CNS, while mice that have cleared MHV undergo demyelination. The infection can therefore be used to model the pathogenesis of different diseases, and insights into the relationship between disease course and inflammatory pathways could

aid in development of new therapies. Although extensive investigations of the adaptive immune response to murine coronaviruses have been conducted, there have been fewer studies done on the role of innate inflammatory pathways in both the pathogenesis of infection and subsequent immune responses. Therefore, the aims of the work presented in this dissertation are as follows:

1) To study the role of the inflammasome and two inflammasome substrate cytokines,

IL-1 and IL-18, during infection with MHV.

Previous studies have shown that the NLRP3 inflammasome is activated during infection with human coronaviruses, but there has not been any follow up of published work using this system, and no studies have examined the role of the inflammasome during murine coronavirus infection. Other studies have found that the inflammatory cytokines IL-1 and

IL-18 can have a significant impact on host defense during infection with other murine viruses, but their role during coronavirus infection is largely unclear. As such, we have examined the pathogenesis of and immune response against MHV infection in mice lacking components of the inflammasome or the receptors for IL-1 or IL-18. In contrast to previous work showing a protective role for the cytokine during viral infections, we find that IL-1 signaling appears to be largely dispensable, and possibly pathogenic, during

MHV infection. The inflammasome as a whole and IL-18 signaling specifically are found to be important for host defense, and the specific role of IL-18 is suggested to be in promoting production of the pro-inflammatory cytokine interferon gamma.

2) To investigate the role of the cytosolic RNA sensor MDA5 during infection with MHV.

Previous work has demonstrated that the type 1 interferon response is critically important for host defense during MHV infection, and that this response is initiated by the RNA sensors MDA5 and TLR7 (Züst et al., 2011). TLR7-/- mice have previously been characterized, and were found to be more susceptible to MHV infection than immunocompetent mice, but less so than mice lacking all type 1 interferon signaling, suggesting a protective role for MDA5-dependent interferon. We characterized the disease course and immune response during MHV infection of MDA5-/- mice and found that the mice were significantly more susceptible to infection than WT mice. This susceptibility seems to stem from excessive inflammation that occurs in the absence of

MDA5, suggesting a regulatory role for the sensor or its downstream products.

CHAPTER 2

ROLE OF THE INFLAMMASOME-RELATED CYTOKINES IL-1 AND IL-18 DURING INFECTION WITH MURINE CORONAVIRUS

This chapter contains unpublished data generated by Zachary B. Zalinger, Ruth Elliott, and Susan R. Weiss. It was submitted to the journal mSphere in November 2015.

A. Abstract

The inflammasome, a cytosolic protein complex that mediates the processing and secretion of pro-inflammatory cytokines, is one of the first responders during viral infection. The cytokines secreted following inflammasome activation, which include IL-1 and IL-18, regulate cells of both the innate and adaptive immune system, guiding the subsequent immune responses. In this study we use MHV infection of the CNS and liver to assess of the role of the inflammasome and its related cytokines on pathogenesis and host defense during a viral infection. Mice lacking all inflammasome signaling due to the absence of caspase-1 and -11 were more vulnerable to infection, with poor survival and elevated viral replication. Mice lacking IL-1 signaling experienced elevated viral replication but improved survival, indicating a pathological role for IL-1 signaling. In the absence of IL-18 mice had elevated viral replication and poor survival, and this protective effect of IL-18 was found to be due to promotion of interferon gamma production in αβ T cells. These data suggest that inflammasome signaling is largely protective during murine coronavirus infection, in large part due to the pro-inflammatory effects of IL-18.

B. Introduction

During infections, pathogens first encounter the innate immune system, which includes germline encoded PRRs that detect conserved PAMPs and induce production of pro- inflammatory cytokines and chemokines. These messengers, which include type 1 interferon, IL-6, TNFα and others can have direct anti-pathogen effects and help induce and guide the subsequent components of the innate immune response as well as the adaptive immune response. Innate inflammatory pathways are therefore key regulators of much of the host response to pathogens. Many pathways can also drive damaging pathological effects when improperly regulated, so studying these pathways can aid in both treatment of inflammatory diseases and improvement of host responses to pathogens.

One innate inflammatory pathway is the inflammasome. The inflammasome is a multimeric protein complex that forms in the cytosol following detection of a diverse range of ligands, and which is responsible for activation of caspase-1 (Martinon et al.,

2002). Caspase-1 in turn processes members of the IL-1 family of cytokines into their active forms (Ghayur et al., 1997; Gu et al., 1997; Howard et al., 1991; Thornberry et al.,

1992), leading to their secretion. These cytokines, including IL-1α, IL-1β, and IL-18, are pro-inflammatory and induce a variety of immunomodulatory effects, capable of leading to both host protection and damaging pathological response. IL-1α and IL-1β induce recruitment of neutrophils (Y.-S. Lee et al., 2015), polarize T cells to adopt a Th17 phenotype (Chung et al., 2009), and promote DC activation for priming (Ichinohe et al.,

2009; Ramos et al., 2012). IL-18 signaling promotes production of interferon gamma

(Bellora et al., 2012; Kohno et al., 1997; Okamura et al., 1995; Serti et al., 2014) and

cytotoxicity (Dao et al., 1996; Okamura et al., 1995; Tsutsui et al., 1996) in a variety of cell types. Characterization of the role of the inflammasome and its related cytokines during viral infection is still ongoing, and little work has been done in the context of coronavirus infection.

MHV strain A59 is positive strand RNA virus of the coronavirus family, and is a tractable model for studying pathogenesis of and host responses to coronaviruses. It has a wide organ tropism, and is capable of infecting the brain, spinal cord, liver, spleen, lungs and other organs. Infection can cause hepatitis in the liver, and encephalitis and meningitis in the CNS. Following viral clearance mice develop a demyelinating disease with phenotypic similarities to MS. MHV can therefore be used to model a variety of pathologies.

MHV infection induces a strong innate inflammatory response, which includes induction of type 1 interferon (Roth-Cross et al., 2008), TNFα, IL-6 and IL-12 (Parra et al., 1997;

Pearce, Hobbs, McGraw, & Buchmeier, 1994; Rempel, Murray, Meisner, & Buchmeier,

2004; Rempel, Quina, Blakely-Gonzales, Buchmeier, & Gruol, 2005), as well as neutrophil, macrophage and NK infiltration of infected sites (Bergmann et al., 1999;

Zhou, Stohlman, Hinton, & Marten, 2003). Clearance is reliant on the interferon response (Zalinger, Elliott, Rose, & Weiss, 2015) as well as interferon gamma production by CD4 and CD8 T cells, and perforin-mediated cytotoxicity by CD8 T cells

(Bergmann et al., 1999; 2003; Lin et al., 1997; Ramakrishna et al., 2004). Therefore,

MHV is a tractable model for studying the role of inflammatory pathways on the regulation of many aspects of both the innate and adaptive immune response.

In this study we systematically examined the role of the inflammasome as a whole as well as the specific contributions of the inflammasome-related cytokines IL-1 and IL-18 to disease course and immune response during MHV infection. We demonstrate a protective role for the inflammasome as a whole, and a hybrid protective/pathogenic role for IL-1 signaling. Mice lacking IL-18 signaling were found to have poor survival and elevated viral replication compared to wild type mice. IL-18 signaling was demonstrated to promote interferon gamma production in activated T cell populations, and to be crucial for global interferon gamma responses, and we therefore conclude that the inflammasome-related protein IL-18 improves host defense through the promotion of interferon gamma production in T cells.

C. Results

Caspase-1/11-/- mice have heightened susceptibility to MHV infection

To determine the role of inflammasome signaling during murine coronavirus infection we used a well characterized model of i.c. inoculated MHV. Wild type C57Bl/6 or C57Bl/6 mice lacking caspase-1 and -11 (Casp-1/11-/-) were challenged with 5,000 plaque forming units (PFU) of MHV, inoculated i.c., and survival was monitored. Casp-1/11-/- mice were found to be acutely vulnerable to this infection, with roughly 40% survival compared to 90% survival among WT mice (Figure 1-1A). These data demonstrate that inflammasome signaling is critical to host defense during MHV infection. To better understand what role the inflammasome plays in host defense the magnitude of viral replication in infected organs was assessed. Mice were inoculated i.c. with 500 PFU of

MHV. Three, five, and seven days after infection mice were euthanized and the magnitude of viral replication in the brain, spinal cord, liver and spleen was determined by plaque assay (Figure 1-1B). Replication in the brain and spinal cord were unchanged, suggesting that overall inflammasome signaling is not relevant for control of infection within the CNS. Viral replication was elevated in the liver five days after infection, and in the spleen three and five days after infection, suggesting that infection of peripheral organs may be partially controlled by inflammasome signaling.

IL-18 signaling controls viral replication and improves survival

Caspase-1 and -11 cleave a variety of substrates, the best characterized of which are IL-

1α, IL-1β, and IL-18. IL-18 has been shown to promote production of interferon gamma and perforin in a variety of cells types (Bellora et al., 2012; Dao et al., 1996; Kohno et al.,

1997; Okamura et al., 1995; Serti et al., 2014; Tsutsui et al., 1996), both of which are

important for host defense against MHV infection (Lin et al., 1997; Parra et al., 1999). IL-

18 signaling is partially redundant with IL-12 signaling, and the ability of each cytokine to compensate for the loss of the other varies between infection models. Therefore, while it is known that the downstream effects of IL-18 signaling, production of interferon gamma and perforin, contribute to host defense during MHV infection, it is unknown if IL-18 is required.

While the poor survival of Casp-1/11-/- mice could be due to a protective role of IL-18 signaling, the inflammasome also processes other cytokines. To determine the role of IL-

18 specifically we infected IL-18 receptor knockout (IL-18R-/-) mice with 500 PFU of

MHV, a dose that is usually sublethal, and monitored their survival (Figure 1-2A). While roughly 90% of WT mice survived the infection, only 10% of IL-18R-/- survived, demonstrating a critical role for IL-18 signaling in host defense.

Impact of IL-18 signaling on replication of the virus was also assessed. WT and IL-18R-/- mice were infected with 500 PFU of MHV. A randomly determined portion of the mice were euthanized 3, 5, or 7 days after infection and the viral load in their livers, spleens, brains, and spinal cords was determined by plaque assay (Figure 1-2B). Viral load was elevated in IL-18R-/- mice compared to WT mice at early time points in the liver and spleen, and there was a slight trend toward poor clearance at later time points in these organs. Replication in the spinal cord was unchanged until seven days after infection, which may also be indicative of a clearance defect. Replication was also elevated in the brain at all tested time points, with the difference between IL-18R-/- and WT mice growing larger at seven days after infection, when WT mice began clearing virus. These

data suggest that the lack of IL-18 signaling produces some immunodeficiency that limits the ability to clear the virus.

IL-1 signaling promotes control of viral infection but is detrimental to survival

IL-1α and IL-1β are also caspase-1 substrates, and the susceptibility of Casp-1/11-/- mice could be due in part to the loss of IL-1 signaling as well. Although IL-1α and IL-1β can have different effects, both signal through the IL-1 receptor. We employed IL-1R-/- mice to assess the role of IL-1 signaling during MHV infection. These mice lack both IL-

1α and IL-1β signaling. Initial research showed that IL-1 promoted Th17 T cell response

(Chung et al., 2009), while subsequent research has shown that it can also promote activation of DC during viral infection, even when the subsequent T cell response adopts a Th1 phenotype (Ichinohe et al., 2009; Ramos et al., 2012).

To assess the role of IL-1 signaling we infected IL-1R-/- mice with 5,000 PFU of MHV and observed survival. Roughly 25% of WT mice survived the infection, while 50% of IL-1R-/- did (Figure 1-2C). This suggests that unlike IL-18, IL-1 does not play a protective role during MHV infection. It may also suggest that IL-1 is pathogenic during infection, as mice lacking IL-1 signaling had slightly improved survival over those with the pathway intact. To determine if this change in susceptibility was related to changes in viral replication we tested viral load in the liver, spleen, brain and spinal cord by plaque assay

(Figure 1-2D). IL-1R-/- mice had similar viral loads in the liver compared to WT mice, but had a slight clearance defect in the spleen, and slightly elevated loads in the brain and spinal cord. These data indicate that IL-1 signaling provides some control of viral

replication, but also has detrimental effects, as demonstrated by the improved survival in mice lacking this pathway.

The majority of IL-18 secretion is Caspase-1/11 dependent

Lethality was higher among IL-18R-/- mice than Casp-1/11-/- mice (Figures 1-1A and 1-

2B). If Caspase-1 is required for activation of IL-18, we would expect that Casp-1/11-/- and IL-18R-/- mice would phenocopy each other. However, our findings show that mice lacking IL-18 signaling are more susceptible to infection than those lacking caspase-1.

We propose two hypotheses to explain this discrepancy. First, caspase-1 processes numerous substrates (Garlanda et al., 2013), and it is possible that one of these other substrates has a deleterious effect on animals during MHV infection. Therefore, its loss in the Casp-1/11-/- mice would improve survival somewhat, partially countering the loss of IL-18 signaling. Our data suggests that IL-1 may be a candidate for this hypothesis.

Alternatively, inflammasome independent sources of IL-18 (Omoto et al., 2006; 2010;

Sugawara et al., 2001) may provide partial protection to the Casp-1/11-/- mice. IL-18R-/- mice lack all signaling regardless of the source of IL-18, depriving them of this partial protection. To address this hypothesis we isolated serum from infected WT and Casp-

1/11-/- mice and determined the concentration of IL-18. While Casp-1/11-/- mice had significantly lower levels of IL-18, a limited amount was observed five days after infection

(Figure 1-3). This low level of the cytokine may provide limited protection.

IL-18 signaling may influence MHV-induced demyelination, but overall inflammasome activation and IL-1 signaling do not

MHV infection of the CNS induces a chronic demyelinating disease following viral clearance. This disease is defined by loss of myelin from neuron axons in the spinal cord, which is phenotypically similar to MS. Several reports demonstrate that the inflammasome plays a role in demyelination in MS (Karni et al., 2002; Losy & Niezgoda,

2001) and in murine models of the disease (Inoue & Shinohara, 2013; Jha et al., 2010;

F. D. Shi et al., 2000). To determine if this is also the case for MHV induced demyelination, we infected WT, Casp-1/11-/-, IL-1R-/-, and IL-18R-/- mice with 500 PFU of

MHV inoculated i.c., then removed their spinal cords thirty days after infection, which is believed to be the peak of demyelination in this model. Cords were sectioned and stained with luxol blue, which binds to myelin. Sections were scored for percent quadrants exhibiting demyelination (Figure 1-4). Casp-1/11-/- and IL-1R-/- had similar levels of demyelination compared to WT mice, demonstrating that in the MHV-induced demyelination model overall inflammasome activation, and IL-1 signaling, do not play a protective or pathogenic role. IL-18R-/- suffered higher degrees of demyelination compared to WT mice, which may suggest a protective role for IL-18 signaling during

MHV-induced demyelination. However, previous studies have shown that more severe acute MHV infection of the CNS lead to more severe demyelination, and IL-18R-/- mice have higher viral loads in the brain and spinal cord compared to WT mice (Figure 1-2B), which could account for the increased demyelination. Therefore the role of IL-18 in MHV- induced demyelination is difficult to assess.

IL-18 signaling promotes interferon gamma production at the peak of infection but is dispensable at later time points

IL-18R-/- mice are highly susceptible to MHV infection, with poor survival (Figure 1-2A) and poor control of viral replication (Figure 1-2C). We next sought to determine why the loss of IL-18 signaling leads to this phenotype. IL-18 acts on T cells, NK cells, and NKT cells to promote interferon gamma production (Bellora et al., 2012; Nagarajan &

Kronenberg, 2007; Okamura et al., 1995; Serti et al., 2014) as well as cytotoxicity, often through promoting perforin production (Dao et al., 1996; Okamura et al., 1995; Tsutsui et al., 1996). Both interferon gamma and perforin-mediated cell killing are important for host defense during MHV infection (Bergmann et al., 1999; 2003; Lin et al., 1997;

Ramakrishna et al., 2004), so we hypothesize that in the absence of IL-18 signaling mice develop an immunodeficiency that makes them vulnerable to MHV infection. We began by determining the concentration of interferon gamma in the serum of infected WT and

IL-18R-/- mice, hypothesizing that if one or more cell types produce interferon gamma in an IL-18 dependent manner during MHV infection, we might be able to observe a global decrease in interferon gamma production. Mice were infected with 500 PFU of MHV, and three, five, and seven days after infection serum was isolated. Seven days after infection the concentration of interferon gamma was higher in IL-18R-/- mice (Figure 1-5). We believe that this phenotype is due to the elevated viral loads in IL-18R-/- mice at this time point, which likely induces high levels of interferon gamma, overwhelming any defect caused by the loss of IL-18 signaling. However, five days after infection, when the differences in viral load between WT and IL-18R-/- mice are evident but not as large as by seven days after infection, we see a significantly lower concentration of interferon gamma in the IL-18R-/- mice compared to WTs.

IL-18 has no detectable effect on cytokine production by T cells, natural killer cells, or natural killer T cells at the peak of infection

While interferon gamma levels in the serum are low in IL-18R-/- mice, we do not know what cell types contribute to this poor response. To address this we assessed production of interferon gamma by multiple cell types five days after infection. This time point was chosen because it is the peak of the interferon gamma response, interferon gamma levels are lower in IL-18R-/- mice compared to WT at this time, and because it is the peak of viral replication in WT mice (Figure 1-2B). Production of perforin was also assessed, as its production can also be IL-18 dependent, and a poor perforin response could also make mice more susceptible to infection. Mice were infected with 500 PFU of MHV, and five days after infection cells were isolated from the spleen (Figure 1-6) or the liver

(Figure 1-7), incubated with MHV peptides and brefeldin A, then identified by flow cytometry. Production of interferon gamma (Figures 1-6A and 1-7A) and perforin

(Figures 1-5B and 1-6B) in αβ T cells (CD3+NK1.1-β TCR+), γδ T cells (CD3+NK1.1- γδ

TCR+), NK cells (CD3-NK1.1+), and NKT cells (CD3+NK1.1+) was determined by intracellular staining. No differences were found in the percent of any given cell type producing either interferon gamma or perforin. This suggests that these processes are independent of IL-18 signaling, and defects in this aspect of the immune response are unlikely to underlie the poor survival of IL-18R-/- mice.

Interferon gamma production by αβ T cells is partially dependent on IL-18 signaling by seven days after infection

The αβ T cell response is not expected to be fully developed by five days after MHV infection, so we also phenotyped T cells isolated seven days after infection, to determine

if these T cells exhibited IL-18 dependent deficiencies in interferon gamma or perforin production. WT and IL-18R-/- mice were infected with 500 PFU MHV, and cells were isolated from the spleen seven days later. Splenocytes were incubated with MHV peptides and brefeldin A, then phenotyped by flow cytometry. To assess the activation status of the T cell response, the percent (Figure 1-8A) and total number (Figure 1-8B) of CD4 and CD8 T cells expressing elevated levels of the activation markers CD44 and

CD11a were quantified. All subtypes showed similar activation status between cells from

WT and IL-18R-/- mice.

The interferon gamma and perforin production of T cell populations was also assessed.

CD4 and CD8 T cells with elevated surface expression of CD44+ or CD11a+ were gated on, and the percent (Figure 1-9A) and total number (Figure 1-9B) of these cells with elevated levels of interferon gamma or perforin were quantified. Production of interferon gamma and perforin were similar between cells from WT and IL-18R-/- mice with the exception of two subsets. The percent of CD44high CD4 T cells with elevated interferon gamma levels was higher in cells from WT mice compared to those from IL-18R-/- mice, and the number of CD11ahigh CD8 T cells with elevated interferon gamma levels was higher in WT than IL-18R-/- mice. Although serum concentrations of interferon gamma are higher in IL-18R-/- mice at this time point, it is possible that production of this cytokine by other cell types in response to the elevated viral loads is masking defective production by T cells, and that the reduction of interferon gamma production by T cells in

IL-18R-/- mice could lead to the poor survival and elevated viral replication observed in these mice.

D. Discussion

We have demonstrated a complex role for the inflammasome in host defense during murine coronavirus infection. Although mice lacking caspase-1 and caspase-11, and therefore both the canonical and non-canonical inflammasomes, are more vulnerable to

MHV infection (Figure 1-1), examination of the individual roles of two inflammasome – related cytokines, IL-1 and IL-18, demonstrated a greater complexity. IL-1 signaling was found to control viral replication to some degree (Figure 1-2D), but appeared to exert a pathological effect, with mice lacking IL-1 signaling more likely to survive infection than those with an intact pathway (Figure 1-2C). We have shown that IL-18 is protective, improving both survival and control of viral replication (Figures 1-2A and 1-2B). IL-18 signaling increased concentrations of interferon gamma in the serum (Figure 1-5), although by seven days after infection, when viral titers are significantly higher in IL-18R-

/- mice, IL-18 signaling is no longer necessary, and elevated levels of the cytokine are observed, suggesting that IL-18-independent sources of interferon gamma respond to out of control viral replication. We have shown that certain T cell subsets have decreased production of interferon gamma in IL-18R-/- mice compared to WT mice, and

IL-18-dependent interferon gamma production from T cells may account for the reduced cytokine levels in the serum. This interferon gamma defect likely accounts for the poor survival of both Casp-1/11-/- and IL-18R-/- animals.

While Casp-1/11-/- mice have reduced survival compared to WT mice (Figure 1-1A), they have better survival compared to IL-18R-/- mice. We believe that this is due to one of two factors; inflammasome independent processing of IL-18, likely by calpains and other proteases on the surface of other cells that process immature IL-18 secreted by dying

cells (Omoto et al., 2006; 2010; Sugawara et al., 2001) may be protective, or the loss of pathogenic inflammasome substrates in Casp-1/11-/- mice. The former hypothesis is supported by the presence of IL-18 in the serum of Casp-1/11-/- mice (Figure 1-3).

However, the levels of inflammasome-independent IL-18 observed are quite low, and may not be sufficient to produce any protective effects. The latter hypothesis is supported by the observed pathogenic effect of IL-1 signaling (Figure 1-2C). It is possible that IL-1 and IL-18, despite being coordinately regulated, have opposing effects during MHV infection.

Our finding that IL-1 exerts a pathogenic effect (Figure 1-2C) was surprising, as recent work with IAV (Ichinohe et al., 2009) and WNV (Ramos et al., 2012) has found that IL-1 signaling is critical to induction of a protective T cell response during infection with these viruses. IL-1 mediates this effect through promotion of DC activation, and mice lacking

IL-1 signaling possessed defective DCs and a poorly primed T cell response (Durrant et al., 2013; Pang et al., 2013). As IL-1R-/- actually have improved survival during high dose

MHV infection (Figure 1-2C), we do not believe that IL-1 signaling is critical for T cell priming in our model, although the slight elevation in viral replication (Figure 1-2D) could be due to a minor T cell defect. The mechanistic explanation for why IL-1 signaling is critical for DC function during some infections but not others is unknown. During IAV infection IL-1 signaling appears to replace the need for PRR signaling on DCs (Pang et al., 2013). It is possible that during MHV infection PRR signaling is sufficient to induce activation, removing the need for IL-1 signaling.

The mechanism by which IL-1 signaling is pathogenic during high dose MHV infection has not been elucidated. Prior to the discovery that IL-1 could improve priming of Th1 responses (Ichinohe et al., 2009) it was largely considered to be a TH17 inducing cytokine (Chung et al., 2009). It is possible that while the T cell response induced by

MHV infection is predominately Th1 polarized that IL-1 signaling induces some level of

IL-17 production, which could have a deleterious effect on survival. IL-1 is also capable of inducing recruitment of neutrophils to the site of infection through upregulation of the chemokine KC (Y.-S. Lee et al., 2015). While often protective, neutrophils are also capable of mediating damaging immunopathologies. Mice lacking IL-1 signaling may experience reduced neutrophil recruitment and therefore less immunopathology, improving their survival. Unfortunately, the pathogenic role of IL-1 is beyond the scope of this paper.

Due to the difficulties inherent in research on human patients, inquiries into the pathogenic factors involved in demyelinating diseases such as MS have been mostly conducted in murine disease models. Work with both humans

(Karni et al., 2002; Losy & Niezgoda, 2001) and mice (Chung et al., 2009; Jha et al.,

2010; F. D. Shi et al., 2000) has suggested a role for the inflammasome and its related cytokines in the pathogenesis of demyelination. We assessed the severity of MHV- induced demyelination in mice lacking Caspase-1 and -11, IL-1 signaling, or IL-18 signaling, in order to determine the role of the inflammasome in this demyelination model

(Figure 1-4). An important element of the MHV-induced demyelination model is that the more severe the acute infection is in the CNS, the more severe we expect subsequent demyelination to be (Marten et al., 2000). We found that Casp-1/11-/- animals had

similarly severe demyelination as compared to WT mice, as well as similar viral loads in the brain and spinal cord (Figure 1-1B). This suggests that the inflammasome as a whole does not effect MHV-induced demyelination. This does not rule out possible roles for individual inflammasome-associated cytokines, however. IL-1R-/- mice had similar demyelination severity compared to WT mice, but transiently elevated viral titers in the brain and spinal cord. It is possible that this increase in viral load should lead to more severe demyelination, and that the similar severity in fact indicates a reduced demyelination. If this is the case then IL-1 signaling is a pathogenic factor during both

MHV-induced demyelination and during acute disease. However, without further study this remains purely speculative. IL-18R-/- mice experienced more severe demyelination compared to WT mice. However, they also had significantly higher viral loads in the brain and spinal cord, so elevated demyelination is expected. Further study will be necessary to determine if IL-18 plays a pathogenic role in MHV-induced demyelination, as it does in other murine models (Jha et al., 2010).

IL-18 has been shown in previous studies to promote cytotoxicity and interferon gamma production in numerous cell types, including T cells and NK cells (Bellora et al., 2012;

Dao et al., 1996; Kohno et al., 1997; Okamura et al., 1995; Serti et al., 2014; Tsutsui et al., 1996). We observed no differences in perforin production in any assayed cell type

(Figure 1-6B and 1-7B), suggesting cytotoxicity is IL-18-independent during MHV infection, but did observe a significant decrease in the concentration of interferon gamma in the serum of IL-18R-/- mice five days after infection. By seven days after infection interferon gamma levels were higher in IL-18R-/- mice compared to WT mice, which we attribute to IL-18-independent source of the cytokine responding to the

increased viral replication. However, the decreased interferon gamma levels five days after infection, at the pea of interferon gamma production, suggests that interferon gamma production is partially dependent on IL-18 signaling during MHV infection. This may account for the poor survival and control of viral replication of IL-18R-/- mice

(Figures 1-2A and 1-2B). In order to determine which cell types specifically require IL-18 signaling for full interferon gamma production we isolated αβ T cells, γδ T cells, NK cells, and NKT cells from the spleen and liver five days after infection, and αβ T cells from the spleen seven days after infection, and assessed their production of interferon gamma by intracellular staining and flow cytometry. We found reduced cytokine production in

CD44+ CD4 T cells and CD11a+ CD8 T cells from the spleen seven days after infection.

Although these defects were detectable only seven days after infection, when interferon gamma levels were higher in IL-18R-/- mice than WT mice, we speculate that they are indicative of a general defect in the T cell response that leads to the susceptibility of IL-

18R-/- mice to MHV infection. The elevated interferon gamma levels in the serum may come too late, or from the wrong cell types in the wrong organs, to compensate for the reduced interferon gamma production by activated T cells, suggesting that IL-18 signaling may play a cell type, organ, and/or temporally specific, but nonetheless critical, role during MHV infection.

E. Acknowledgments

This work was supported NIH grant R01-AI-060021 to S.R.W.

Z.B.Z. was supported in part by training grant T32-NS007180.

Thanks to Drs. Cierra N. Casson and Sunny Shin, University of Pennsylvania,

Department of Microbiology, Philadelphia, for technical support and transgenic mouse lines, and to Dr. Igor Brodsky, University of Pennsylvania, Department of Pathobiology,

Philadelphia, for transgenic mouse lines.

F. Materials and methods

Virus and mice

Recombinant MHV strain A59 (referred to herein as MHV) has been described previously (MacNamara et al., 2005; J. J. Phillips, Chua, Lavi, & Weiss, 1999). Titer was determined by plaque assay on murine L2 cell monolayers, as described previously

(Hingley, Gombold, Lavi, & Weiss, 1994). Wild type C57BL/6 (WT) mice and IL-1R-/- mice were purchased from Jackson Laboratories (Bar Harbor, ME). IL-18R-/- mice were a generous gift from Dr. Sunny Shin (University of Pennsylvania, department of

Microbiology). Mice deficient in caspase-1 and caspase-11 (Casp-1/11-/-) were a generous gift from Dr. Igor Brodsky (University of Pennsylvania, department of

Pathobiology).

All mice were genotyped and bred in the animal facilities of the University of

Pennsylvania. Four-to-six week old mice were used for all experiments. For infections, virus was diluted in phosphate-buffered saline (PBS) supplemented with 0.75% bovine serum albumin (BSA) and inoculated i.c. All experiments were approved by the

University of Pennsylvania Institutional Animal Care and Use Committee.

Viral replication burden

To quantify viral replication, mice were inoculated i.c. with 500 PFU of MHV and sacrificed 3, 5, and 7 days after infection. Following cardiac perfusion with phosphate buffered saline, organs were harvested and placed in gel saline (an isotonic saline solution containing 0.167% gelatin), weighed, and frozen at -80°C. Organs were later

homogenized, and plaque assays were performed on L2 fibroblast monolayers as previously described (Hingley et al., 1994).

Serum collection

To isolate serum for ELISAs, blood was incubated for one hour at 37oC, then spun at maximum speed in a tabletop centrifuge for ten minutes to pellet red blood cells. Serum was removed from the upper phase, and frozen at -20oC until needed.

IL-18 and Interferon gamma ELISA

Levels of IL-18 or interferon gamma in serum isolated from infected mice was assayed using capture and detection antibodies specific for IL-18 (MBL) or interferon gamma

(BD).

Quantification of demyelination

In order to quantify severity of demyelination, spinal cords were isolated from mice that were euthanized 30 days after infection with MHV. Cords were divided into 4-6 segments and embedded in paraffin and sectioned. Slides were produced, each of which contained a slice of each segment taken from a single spinal cord, and stained with luxol blue. Each section was divided into quadrants, and each quadrant was scored plus or minus for myelin loss.

Cell isolation from the spleen and liver

WT and IL-18 deficient (IL-18R-/-) mice were infected i.c. with 500 PFU of MHV, and organs were subsequently harvested after cardiac perfusion with PBS. Splenocytes

were rendered into single-cell suspensions through a 70-micron filter, after which red blood cells were selectively lysed by incubating for 2 minutes in 0.206% tris HCl, 0.744%

NH4Cl solution. Livers were homogenized mechanically, then lysates were centrifuged through a Percoll® gradient to obtain a single-cell suspension, followed by RBC lysis as above.

Surface marker and intracellular cytokine staining

Intracellular cytokine staining was performed on single cell suspensions of splenocytes or hepatocytes following a four-hour incubation with brefeldin A (20 µg/ml, Sigma) and the MHV peptides M133 (MHC class II; 4 µg/ml, Biosynthesis) and S598 (MHC class I;

9.3 µg/ml, Biosynthesis). Cells isolated five days after infection were stained with the following antibodies: CD3 (eBioscience, clone 17A2, NK1.1 (eBioscience, clone PK136),

βTCR (eBioscience, clone H57-597), and γδTCR (Biolegend, clone GL3). Cells isolated seven days after infection were stained with the following antibodies: CD3 (eBioscience, clone 17A2), CD4 (eBioscience, clone GK1.5), CD8 (eBioscience, clone 53-6.7), CD44

(eBioscience, clonse IM7), and CD11a (Biolegend, clone M17/4). Staining for interferon gamma and perforin was performed after permeabilization with Cytofix/cytoperm Plus

Fixation/Permeabilization kit (BD). Following staining, cells were analyzed by flow cytometry on an LSR II (Becton Dickinson), and the resulting data were analyzed using

FlowJo software (Treestar).

A 100 WT 50 Casp-1/11-/-

0 Percent survival Percent 0 5 10 15 20 25

Days post infection

B Brain Spinal cord

10 8 ) ) 8 10 10 6 6 4 4 PFU (log PFU PFU (log PFU 2 2 0 0 3 5 7 3 5 7 Days post infection Days post infection

Liver Spleen

8 * 6 **** *** ) ) 10 10 6 4 4 PFU (log PFU PFU (log PFU 2 2 0 0 3 5 7 3 5 7 Days post infection Days post infection

WT Casp-1/11-/-

Figure 1-1. Caspase-1/11-/- mice are more susceptible to MHV infection. WT and Casp-1/11-/- Mice were inoculated i.c. with 5,000 PFU of MHV, and survival was monitored (A). Data is pooled from two independent experiments, and is significant by the Mantel-Cox and the Gehan-Breslow-Wilcoxon tests. To determine differences in viral replication WT and Casp-1/11-/- mice were inoculated i.c. with 500 PFU of MHV. Three, five, and seven days post infection mice were euthanized and viral loads in their brains, spinal cords, spleens and livers were assessed by plaque assay (B). Data is pooled from two independent experiments. Statistical significance was determined by a two-part test using a conditional t test and proportion test.

Spinal cord A IL-18R-/- survival B Brain

100 10 * ** **** 10 *** 8 8 ) ) 10 6 10 6 50 4 4 2 2 Percent survival Percent PFU (log PFU PFU (log PFU 0 0 0 0 5 10 15 20 25 3 5 7 3 5 7 Days post infection Days post infection Days post infection Liver Spleen WT IL-18R-/ 8 ** 6 *** ) 6 ) 4 10 10 4 WT 2 2 IL18R-/- PFU (log PFU PFU (log PFU 0 0 3 5 7 3 5 7

Days post infection Days post infection

C IL-1R-/- survival D Brain Spinal cord 100 8 **** * 7 *** ) 6 ) 6 10 10 50 4 5 2 4 PFU (log PFU PFU (log PFU

Percent survival Percent 0 0 3 0 5 10 15 20 25 3 5 7 3 5 7 Days post infection Days post infection Days post infection Liver Spleen WT IL-1R-/ 6 6 ** ) ) 10 4 10 4

WT 2 2 PFU (log PFU IL1R-/- (log PFU 0 0 3 5 7 3 5 7 Days post infection Days post infection

Figure 1-2. IL-18R-/- mice are more susceptible to MHV infection while IL-1R-/- mice have increased survival but elevated viral loads. WT and IL-18R-/- mice were inoculated i.c. with 500 PFU of MHV (A), or WT and IL-1R-/- were inoculated i.c. with 5,000 PFU of MHV (C), and survival was monitored. To determine differences in viral replication WT and IL-18R-/- mice (B) or WT and IL-1R-/- mice (D) were inoculated i.c. with 500 PFU of MHV. Three, five, and seven days post infection mice were euthanized and viral loads in their brains, spinal cords, spleens and livers were assessed by plaque assay. Data is pooled from two independent experiments for each genotype, with the exception of date on IL-1R-/- spleens, which was performed once. Statistical significance was determined by a two-part test using a conditional t test and proportion test.

1500 **** **** ****

1000

IL-18 (pg/ml) 500

0 3 5 7 Days post infection

WT Casp-1/11-/-

Figure 1-3. IL-18 concentration is lower in the serum of Casp-1/11-/- mice compared to WT mice. To determine if Casp-1/11-/- mice produced caspase-independent IL-18, WT and Casp- 1/11-/- mice were infected with 500 PFU of MHV and serum was collected three, five, and seven days post infection. The concentration of IL-18 in the serum was determined by ELISA. Data is pooled from two independent experiments. Statistical significance was determined by a two-part test using a conditional t test and proportion test.

Casp-1/11-/- IL-1R-/- IL-18R-/- 150 100 50 * WT Knockout 80 40 100 60 30 50 40 20 % Demyelination % Demyelination 20 % Demyelination 10 0 0 0

Figure 1-4. Demyelination is unchanged in Casp-1/11-/- and IL-1R-/- mice compared to WTs but elevated in IL-18R-/- mice. To determine the role of the inflammasome and its related cytokines during MHV- induced demyelination WT mice were inoculated i.c. with MHV along with either Casp- 1/11-/-, IL-1R-/-, or IL-18R-/- mice. Casp-1/11-/- and controls WT mice received 500 PFU, IL-1R-/- and controls received 10,000 PFU, and IL-18R-/- and controls received 25 PFU. Thirty days post infection mice were euthanized and their spinal cords fixed, sectioned, and stained with luxol blue. Spinal cords were divided into quadrants, and each quadrant was scored as either healthy or undergoing myelin loss. Each data point represents a single mouse. Casp-1/11-/- data is pooled from three independent experiments, IL-1R-/- is pooled from two independent experiments, and IL-18R-/- data is pooled from two independent experiments. Statistical significance of was determined by an unpaired, two-tailed t test (for Casp-1/11-/- and IL-1R-/- experiments) or by a two-part test using a conditional t test and proportion test (for IL-18R-/- experiments).

2500 * *** WT 2000 IL18R-/- 1500 1000 500

Interferon gamma (pg/ml) Interferon gamma 0 Mock 3 5 7 Days post infection

Figure 1-5. Interferon gamma in serum To quantify production of interferon gamma WT and IL-18R-/- mice were inoculated i.c. with 500 PFU of MHV. Three, five, and seven days after infection serum was isolated and concentration of interferon gamma was assessed by ELISA. Data is pooled from two (for days 3 and 7) or four (for day 5) independent experiments. Statistical significance was determined by a two-part test using a conditional t test and proportion test.

A αβ T cell Natural killer cell B αβ T cell Natural killer cell 10 8 3 5 + 8 6 + 4 2 + + 6 3 γ γ 4 4 1 2 2 % Perforin % Perforin % IFN % IFN 2 1 0 0 0 0

γδ T cell Natural killer T cell γδ T cell Natural killer T cell 4 6 4 10 3 3 8 + 4 + + + 6 γ γ 2 2 2 4 1 1 % IFN % IFN 2 % Perforin 0 0 0 % Perforin 0

WT WT IL18R-/- IL18R-/-

Figure 1-6. Interferon gamma and perforin production in the spleen is IL-18 independent at the peak of infection. To test whether IL-18R-/- mice have a defect in interferon gamma or perforin production WT and IL-18R-/- mice were inoculated i.c. with 500 PFU of MHV. Five days after infection, the peak of MHV replication, cells were isolated from the spleen and imunophenotyped by flow cytometry. Interferon gamma (A) and perforin (B) production was determined by intracellular staining of αβ T cells (CD3+NK1.1-β TCR+), γδ T cells (CD3+NK1.1- γδ TCR+), NK cells (CD3-NK1.1+), and NKT cells (CD3+NK1.1+). Data is expressed as percent of total cells with elevated expression of interferon gamma or perforin. Data is pooled from two independent experiments. Statistical significance was determined by an unpaired, two-tailed t test.

αβ T cell Natural killer cell B αβ T cell Natural killer cell A 15 25

+ 20 10 + 20 8 + + 15 γ 10 γ 15 6 10 10 % Perforin 4 % Perforin % IFN 5 % IFN 2 5 5 0 0 0 0

γδ T cell Natural killer T cell γδ T cell Natural killer T cell 4 15 25 15 + + 3 20 + + 10 10 γ γ 2 15 5 10 % Perforin % Perforin 5 % IFN % IFN 1 5 0 0 0 0

WT WT IL18R-/- IL18R-/-

Figure 1-7. Interferon gamma and perforin production in the liver is IL-18 independent at the peak of infection. To test whether IL-18R-/- mice have a defect in interferon gamma or perforin production WT and IL-18R-/- mice were inoculated i.c. with 500 PFU of MHV. Five days after infection, the peak of MHV replication, cells were isolated from the liver and immunophenotyped by flow cytometry. Interferon gamma (A) and perforin (B) production was determined by intracellular staining of αβ T cells (CD3+NK1.1-β TCR+), γδ T cells (CD3+NK1.1- γδ TCR+), NK cells (CD3-NK1.1+), and NKT cells (CD3+NK1.1+). Data is expressed as percent of total cells with elevated expression of interferon gamma or perforin. Data is pooled from two independent experiments. Statistical significance was determined by an unpaired, two-tailed t test.

A CD4+ CD8+ B CD4+ CD8+ 25 30 3×106 6×105 20 + + 6 5 + 2×10 + 4×10 15 20 10 10 1×106 2×105 % CD44 % CD44 # CD44 # 5 CD44 # 0 0 0 0

CD4+ CD8+ CD4+ CD8+ 30 40 4×106 5×106 6 4×106 + + + 30 3×10 + 20 3×106 20 2×106 2×106 10 6 # CD11a # # CD11a # 6 % CD11a % CD11a 10 1×10 1×10 0 0 0 0

20141209 zz IL18 stim 2 spln.jo Layout: for thesis WT WT Sample % Specimen_001_KO infected A_015.fcs 33.9 IL18R-/-Specimen_001_WT infected A_010.fcs 32.8 IL18R-/- Specimen_001_WT mock A_001.fcs 25.9 C 100 WT, uninfected 80 WT, infected IL-18R-/-, infected 60 % of max % of Max 40

0 0 102 103 104 105 <610/20 Green-A> CD8+ Interferon gamma

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Figure 1-8. T cells from WT and IL-18R-/- spleens have similar activation profiles at the peak of the response. To assess if activation of T cells is IL-18 dependent seven days after infection, the peak of the αβ T cell response, mice were inoculated i.c. with 500 PFU of MHV. Seven days after infection cells were isolated from the spleen and imunophenotyped by flow cytometry. The percent (A) and number (B) of CD4 and CD8 T cells with elevated expression of the activation markers CD44 or CD11a was determined. Representative interferon gamma staining in CD8 T cells is shown in (C). Data is pooled from three independent experiments. Statistical significance was determined by an unpaired, two- tailed t test.

+ + CD11a+ CD4+ CD44+ CD4+ A CD11a+ CD4+ CD44 CD4 B 20141209 zz20141209 IL18 stim zz2 spln.joIL18 stim 2 spln.jo Layout: for thesisLayout: 2 for thesis 2 20 8 ** 5×105 1.5×105 4×105 + + + 15 + 6 5 γ γ γ γ 3×105 1.0×10 10 4 5 # IFN # # IFN # 2×10 4 % IFN % IFN 5.0×10 5 2 1×105 0 0 0 0.0

+ + + + CD11a CD8 CD44 CD8 CD11a+ CD8+ CD44+ CD8+ 105 105 1.08 1.08 25 2.0×105 * 5×104 20 104 104 4 + + + 20 + 5 4×10 γ γ γ γ 15 1.5×10 Red-A> 4 15 Red-A> 3×10 103 5 103 1.0×10 # IFN # 10 IFN # 4 % IFN 10 % IFN 2×10 <670/30 20141209 zz IL18 stim 2 spln.jo <670/30 5 5.0×104 Layout: for thesis4 2 5 2 1×10 2 10 0 10 0 0.0 0 0 0 WT WT 2 3 4 5 2 3 0 104 105 10 10 IL18R-/- 0 10 10 10 <450/5010 Violet-A> IL18R-/- CD11a+<450/50 (CD8, Violet-A> by eye) CD11a+ (CD8, by eye)Specimen_001_WT mock B_002.fcs Specimen_001_WT mockEvent B_002.fcs Count: 646 Event Count: 646 C Mock WT infected IL-18R-/- infected

5 5 5 5 10 11 10 5 10 10 11 1.08 10 16.4 16.4

4 4 4 10 10 104 10 104 Red-A> Red-A> Red-A> Red-A>

CD8 3 CD8 CD8 3 Red-A> 3 10 10 103 10 103 <670/30 <670/30 <670/30 <670/30 <670/30 2 2 2 10 10 102 10 102 0 0 0 0 0

2 3 4 5 2 3 4 5 2 3 0 104 105 10 10 0 10 10 10 10 0 102 103 104 105 0 10 10 10 10 0 102 103 104 105 <450/50 Violet-A> <450/50 Violet-A> <450/50 Violet-A> CD11a+<450/50 (CD8, Violet-A> by eye) CD11a+ (CD8, by eye) + + CD11a+<450/50 (CD8, Violet-A> by eye) + + CD11a+ (CD8, by eye) + + CD11a CD8 CD11a+ (CD8, by eye) CD11a CD8 CD11aSpecimen_001_KO CD8 infected A_015.fcs Specimen_001_WT mock B_002.fcs Specimen_001_WT infected A_010.fcs Specimen_001_KO infectedEvent A_015.fcsCount: 500 Event Count: 646 Specimen_001_WT infectedEvent Count:A_010.fcs 1848 Event Count: 500 Event Count: 1848

-/- 5 Figure5 1-9. T cells from the spleens of IL10 -18R mice11 at the peak of the T cell 10 16.4 response exhibit reduced interferon gamma production compared to those from 104 WT mice.104 Red-A> Red-A> To assess if interferon gamma production 10by3 αβ T cells is IL-18 dependent seven days 103

after infection, the peak of the αβ T cell response,<670/30 mice were inoculated i.c. with 500 <670/30

PFU of MHV. Seven days after infection cells102 were isolated from the spleen and 102 0 immunophenotyped0 by flow cytometry. Production of interferon gamma was determined 11/6/15 3:31 PM Page 1 of 1 high high (FlowJo v9.6.4) 11/6/15 3:31 PM Page2 1 3of 1 4 5 (FlowJo v9.6.4) by intracellular2 staining.3 4 The5 percent (A) and number0 10 10 (B)10 of CD11a10 or CD44 CD4 0 10 10 10 10 <450/50 Violet-A> <450/50 Violet-A> CD11a+ (CD8, by eye) and CD11a+CD8 (CD8, T by eye)cells with elevated interferon Specimen_001_KOgamma infectedproduction A_015.fcs was determined. Specimen_001_WT infected A_010.fcs Event Count: 500 high + RepresentativeEvent Count: 1848 staining for interferon gamma in CD11a CD8 populations is shown in (C). Data is pooled from three independent experiments. Statistical significance was determined by an unpaired, two-tailed t test.

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CHAPTER 3

ROLE OF MDA5 SIGNALING DURING INFECTION WITH MURINE CORONAVIRUS

This chapter appeared as a published peer-reviewed article titled “MDA5 is critical to host defense during infection with murine coronavirus” by Zachary B. Zalinger, Ruth Elliott, Kristine M. Rose, and Susan R. Weiss. Journal of Virology 2015.

A. Abstract

Infection with the murine coronavirus MHV activates the PRRs MDA5 and TLR7 to induce transcription of type 1 interferon. Type 1 interferon is crucial for control of viral replication and spread in the natural host, but the specific contributions of MDA5 signaling to this pathway, as well as to pathogenesis and subsequent immune responses, are largely unknown. In this study, we use MHV infection of the liver as a model to demonstrate that MDA5 signaling is critically important for controlling MHV- induced pathology and regulation of the immune response. Mice deficient in MDA5 expression (MDA5–/– mice) experienced more severe disease following MHV infection, with reduced survival, increased spread of virus to additional sites of infection, and more extensive liver damage. Although type 1 interferon transcription decreased in MDA5–/– mice, the ISG response remained intact. Cytokine production by innate and adaptive immune cells was largely intact in MDA5-/- mice, but perforin induction by NK cells, and serum levels of interferon gamma, IL-6, and TNFα were elevated. These data suggest that MDA5 signaling reduces the severity of MHV-induced disease, at least in part by reducing the intensity of the pro-inflammatory cytokine response.

B. Introduction

Eukaryotic cells use a variety of molecular sensors to detect pathogens, allowing them to rapidly respond to infections. These sensors are called pattern recognition receptors

(PRRs), while the structures they detect are called PAMPs. The known critical PRRs for

RNA viruses are the RIG-I-like receptors (RLR), RIG-I and MDA5; non-RLR helicases such as DHX33 (Mitoma et al., 2013); and toll-like receptors (TLRs, in particular TLR3,

TLR7, and TLR8). Since these pathways are among the earliest host responses triggered by infection, studying them is critically important for understanding tropism, virulence, and regulation of host defense during viral infections.

RLRs are expressed in many cell types throughout the body and are therefore the first sensors likely to detect many viral infections, regardless of route of entry or cellular tropism. RIG-I and MDA5 detect different conformations of RNA, and not all RNA viruses are detectable by both. Although first identified in the context of cancer (Kang et al.,

2002), MDA5 has since been shown to have roles in host defense against a wide variety of viruses. MDA5 is critical for type-1 interferon induction following coronavirus (Roth-

Cross et al., 2008), picornavirus (Kato et al., 2006), and IAV (Sirén et al., 2006) infection as well as for cytokine production in DCs during norovirus infection (McCartney et al.,

2008). Type 1 interferon constitutes an important component of the early innate response by inducing a large number of ISGs encoding antiviral effectors. Type 1 interferon also plays a role in regulating the adaptive immune response in that animals lacking MDA5 signaling (MDA5-/-) demonstrate a variety of immunological defects, including dysregulation of the adaptive immune response during WNV (Lazear et al.,

2013) and Theiler’s encephalomyelitis virus infection (Y. H. Jin et al., 2012b).

The murine coronavirus MHV is a positive sense RNA virus of Betacoronavirus lineage

2a. Laboratory strains of MHV have a diverse range of cellular and organ tropisms, making them useful model organisms for studying host pathways involved in tropism barriers and virulence (Weiss & Leibowitz, 2011). MHV strain A59 (MHV) is dual tropic, infecting primarily the liver and the CNS, causing moderate hepatitis and mild encephalitis followed by chronic demyelinating disease (Lavi et al., 1984). Intraperitoneal inoculation of MHV leads to infection of the liver, spleen, and lungs in immunocompetent mice. MHV can also replicate in the CNS when inoculated i.c.; however, it cannot spread more than minimally from the periphery to the CNS in immunocompetent mice. MHV causes hepatitis when it infects the liver and acute encephalitis and chronic demyelination when it infects the CNS. Other MHV strains infect the lung and gastrointestinal tracts, making MHV infection a model for multiple human diseases

(Bender, Phillips, Scott, & Weiss, 2010; Weiss & Leibowitz, 2011). The tropism and virulence of MHV infection are partially determined by immunological factors, as infection of mice lacking type 1 interferon signaling (IFNAR1-/-) results in expanded organ tropism and greatly reduced survival (Cervantes-Barragan et al., 2007; Roth-Cross et al., 2008).

Although MDA5, but not RIG-I, is known to be necessary for induction of type I interferon in cultured bone marrow derived macrophages (Roth-Cross et al., 2008) and microglia

(data not shown), the importance of MDA5 in induction of interferon during MHV infection has not been well characterized.

Although type 1 interferon signaling is crucial for host defense against MHV and MDA5 contributes to production of type 1 interferon in cell culture, it is unclear to what extent

MDA5 contributes to host defense during in vivo infections. In this study we found that

mice lacking MDA5 had similar levels of viral replication in the liver and spleen as wild type C57BL/6 (WT) mice but were nevertheless more susceptible to infection, experiencing decreased survival and increased hepatitis. Furthermore, the ISG response was intact, suggesting a more complicated role for MDA5-induced interferon. Production of pro-inflammatory cytokines was elevated in MDA5-/- mice which, taken together with increased hepatitis, suggests that MDA5 signaling limits the damage from pathological pro-inflammatory immune responses.

C. Results

MDA5-/- mice are more susceptible to MHV infection.

MHV strain A59 is a dual tropic strain infecting primarily the liver and the CNS of WT

C57BL/6 mice. We (Roth-Cross et al., 2008) and others (Cervantes-Barragan et al.,

2007) have shown previously that mice lacking the type 1 interferon receptor are acutely susceptible to MHV infection. Infection of Ifnar1-/- mice results in spread to multiple organs not usually infected in immunocompetent mice and 100% lethality by day two post-infection. MHV has been shown to induce type 1 interferon via two pathways,

MDA5 (Roth-Cross et al., 2008) and TLR7 (Cervantes-Barragan et al., 2007) signaling.

To determine whether mice lacking MDA5 signaling were more or less vulnerable to infection with MHV, we infected MDA5-/- mice i.p. with 500 PFU of dual tropic MHV, approximately one tenth of a 50% lethal dose of this virus in wild type C57BL/6 mice. As expected, this dose was nonlethal in all but a small minority of WT mice but lethal in roughly 75% of the MDA5-/- animals by 7 days post infection (Figure 2-1A).

Following i.p. inoculation, MHV replicates in the liver and several other organs of

C57BL/6 (WT) mice, with replication peaking at day 5 post-infection. Although capable of replicating in the CNS, MHV does not readily cross the BBB of most animals following i.p. inoculation of WT mice (Lavi et al., 1986; Mitoma et al., 2013). Inflammation and necrosis peak in the liver at day 7 post infection (Kang et al., 2004; Navas et al., 2001;

Navas & Weiss, 2003). To determine if the absence of MDA5 expression results in increased viral replication relative to WT mice, we infected WT and MDA5-/- animals with

500 PFU of MHV. On days 3, 5, and 7 post infection animals were sacrificed and their livers, spleens, brains, spinal cords, hearts, kidneys, and lungs were assessed for viral

replication by plaque assay. Mean viral titers in the liver and spleen were similar between WT and MDA5-/- mice (Figure 2-1B), suggesting that MDA5 is not required to control the infection in those organs. However, in the absence of MDA5, increased viral titers were observed in other organs. Replication was increased in the lungs, kidney, and heart (Figure 2-1C), with a larger number of MDA5-/- mice than WT mice having infection at those sites (Figure 2-1C). Similarly, spread from the periphery to the CNS was increased in MDA5-/- mice (Figure 2-1B), suggesting that MDA5 signaling contributes to the BBB and other tropism barriers.

The severity of liver damage was compared between WT and MDA5-/- mice by hematoxylin and eosin staining of liver sections from mice sacrificed on days 3, 5, and 7 post-infection. The number of inflammatory foci in four to five non-overlapping fields of view, each 9x106 microns2, was counted and the mean number was determined. In some samples the foci had combined to form a continuous confluence covering the entirety of the section. These samples were scored as too numerous to count (TNTC).

Despite the similarity in viral replication in the liver between MDA5-/- and WT mice, the prevalence of inflammatory foci was higher in MDA5-/- mice (Figure 2-2A). The increased severity of liver damage despite unchanged viral replication in MDA5-/- mice compared to

WT mice suggests that an immunopathological mechanism may underlie the greater susceptibility of MDA5-/- mice to MHV infection.

The mRNA expression of type 1 interferon, but not interferon-stimulated genes, is reduced in MDA5-/- mice.

The primary role of MDA5 is to upregulate type 1 interferon expression in response to viral dsRNA. We hypothesized that mice lacking MDA5 would express less interferon, which should in turn limit the activation of transcription of interferon-stimulated genes

(ISGs). To test this hypothesis we isolated RNA from the livers of infected WT and

MDA5-/- mice collected 3, 5 and 7 days post infection and performed quantitative reverse transcription PCR (qRT-PCR). Induction of IFNβ and –α4 mRNA expression in the livers of infected mice relative to mock infected mice was lower for MDA5-/- mice than for WT mice on days 3 and 5 after infection, although this difference was transient, and was no longer evident by day 7 post-infection (Figure 2-3A). We hypothesized that a biologically relevant decrease in interferon expression would have downstream effects, most notably on induction of anti-viral ISG expression, which could lead to the observed disease phenotype, and therefore performed qRT-PCR to measure the expression of a representative panel of ISGs in the liver. Surprisingly, ISG expression in MDA5-/- mice was intact for almost all genes at almost all time points despite the reduced induction of type I interferon mRNA (Figure 2-3B). These results suggest that if the increased susceptibility of MDA5-/- mice to MHV infection is due to decreased interferon expression, it is not a result of reduced ISG expression.

Innate inflammatory cell recruitment is largely intact in MDA5-/- mice, but activation of dendritic cells is elevated.

Shortly after infection, a diverse set of innate inflammatory cells infiltrate the sites of viral replication, where they are often instrumental in restricting infection, regulating the

adaptive immune response, and causing immunopathology. We hypothesized that the loss of MDA5 signaling might cause dysregulation of the inflammatory infiltrate. A delay or reduction in magnitude of the recruitment could allow the virus to replicate to higher levels or in different organs or cell types, while over-exuberant recruitment could lead to tissue damage, as observed in the livers of MDA5-/- mice (Figure 2-2).

To determine if there were differences in the kinetics or magnitude of recruitment we isolated and immunophenotyped cells from the livers of infected mice shortly after i.p. inoculation with 500 pfu of MHV (Figure 2-4A). We observed no differences in the kinetics or magnitude of recruitment of macrophages (CD3-CD19-NK1.1-CD45+Ly6-C-

Ly6-G-CD11c-CD11b+F4/80+), DCs (CD3-CD19-NK1.1-CD45+Ly6-C-Ly6-G-CD11c+), plasmacytoid DCs (CD3-CD19-NK1.1-CD45+Ly6-C-Ly6-G-CD11c+B220+PDCA-1+), neutrophils (CD3-CD19-NK1.1-CD45+Ly6-C-Ly6-G+), NK cells (CD3-CD19-

NK1.1+CD45+Ly6-C-Ly6-G-), or inflammatory monocytes (CD3-CD19-NK1.1-

CD45+CD11b+Ly6-C+Ly6-G-), indicating that early infiltration is largely independent of

MDA5 signaling.

We also assessed the activation phenotype of plasmacytoid, lymphoid, and myeloid

DCs. CD80 and CD86, co-activation markers utilized by DCs during T cell priming

(Rudd, Taylor, & Schneider, 2009; Ueda et al., 2003), are used as a marker of activation.

Plasmacytoid DCs (pDCs) are a major producer of type 1 interferon during MHV infection that rely on TLR7 rather than MDA5 to detect MHV (Cervantes-Barragan et al.,

2007; Kang et al., 2004). pDC-dependent type 1 interferon production should be intact in

MDA5-/- mice, which may account for the intact ISG response. The co-expression of

CD80 and CD86 on pDCs was compared between MDA5-/- and WT mice. The number of

CD80+CD86+ pDCs was lower on day 1, equal on day 2, and higher on day 3 post infection in the MDA5-/- mice (Figure 2-4B). We speculate that the larger number of activated plasmacytoid DCs at later time points may lead to higher production of interferon driving expression of ISGs even in the absence of MDA5-dependent interferon. However, TLR7-dependent interferon may also account for the interferon present in MDA5-/- mice, independent of pDC activation. Lymphoid and myeloid DCs are involved in T cell priming. The co-expression of CD80 and CD86 was higher on these populations in MDA5-/- mice compared to WT mice (Figure 2-4B). This may suggest that

T cell priming is more robust in MDA5-/- animals.

Although the magnitude and kinetics of NK cell infiltration into the liver was independent of MDA5 signaling (Figure 2-4A), the function of these cells, as well as that of the related

NKT cells, is dependent on their production of interferon gamma and perforin, and an

MDA5-dependent defect in cytokine production could alter their efficacy without affecting the size of the populations. To test whether cytokine production in either cell population was dependent on MDA5 signaling we isolated cells from the spleen and liver five days after infection and assessed their cytokine production by intracellular staining and flow cytometry (Figure 2-5). The percent of each population producing interferon gamma or perforin was similar between genotypes, with the exception of NK cells isolated from the spleen, which exhibited elevated perforin production in mice lacking MDA5 signaling.

Perforin is a driver of harmful pathology in many contexts (Amrani et al., 1999; Gebhard et al., 1998; Kägi et al., 1997), and may be in the context of MHV infection as well.

Elevated perforin production could lead to either the increased liver damage, decreased survival, or both.

Effect of MDA5 on the T cell response.

A functional T cell response is essential for clearance of MHV infection. Both CD4 and

CD8 T cells, as well as perforin and granzyme and the pro-inflammatory cytokine interferon gamma, have been established to be necessary (Bergmann, Lane, &

Stohlman, 2006; Roth-Cross et al., 2008). B cells appear to be relevant only during a later stage of acute infection, at a time point after we see death among MDA5-/- mice (Lin et al., 1999; Ramakrishna et al., 2002). Given that MDA5 can in some contexts be important for regulating the T cell response (Y. H. Jin et al., 2012b; Kato et al., 2005;

Lazear et al., 2013) and that DCs express higher levels of the co-activating receptors

CD80 and CD86 in MDA5-/- mice, we hypothesized that CD4 and/or CD8 T cells are dysregulated in MDA5-/- animals. This could result in either a less effective T cell response that is unable to control the virus or an over-exuberant T cell response that produces pathology. To determine whether the T cell response was intact we compared the activation states of CD4 and CD8 T cell populations isolated from the spleens of WT and MDA5-/- mice 7 days post infection. Isolated cells were stimulated with the M133

(class II) and S598 (class I) MHV encoded peptides, which are the immunodominant class I and II peptides during MHV infection (Castro & Perlman, 1995; Xue, Jaszewski, &

Perlman, 1995). Following stimulation the presence of the surface activation markers

CD44 and CD11a were analyzed by flow cytometry. The CD4 and CD8 T cell populations from the spleens of MDA5-/- animals were similar to those from WT mice in terms of both the percentage of cells positive for each activation marker (Figure 2-6A) as well as the total number of cells positive for each (Figure 2-6B). Statistically significant differences between the genotypes are present in mock samples. However, these are minimal and we believe them to have no biological significance.

We also found that CD11a+ CD4, CD44+ CD4, and CD11a+ CD8 T cells from MDA5-/- mice displayed a higher mean fluorescence intensity (MFI) of PD-1 staining (Figure 2-6C and 6D). While PD-1 is a marker of functionally defective, exhausted T cells during chronic infection (Barber et al., 2005; Y. H. Jin et al., 2012b), its role in acute infections remains unclear, with it marking both more and less functional T cells, depending on the infectious context (Brown, Freeman, Wherry, & Sharpe, 2010). The observation of more severe liver pathology despite comparable viral replication in MDA5-/- mice suggests that

PD-1 could be a marker of pathologically overactive T cells during MHV infection.

However, a less functional T cell response that is incapable of controlling viral replication could also explain the increased viral spread and decreased survival, so PD-1 may instead be a marker of less activated T cells in this context.

Cytokine production is a major effector function of both CD4 and CD8 T cells. Reduced cytokine production in T cells from MDA5-/- mice could suggest that the adaptive immune response is too weak to control virus, while elevated cytokine levels could suggest a damaging, pathological T cell response. Production of both interferon gamma and perforin was assessed. Perforin production from CD8 T cells in both the spleen and liver was similar in WT and MDA5-/-, indicating that perforin production by T cells is independent of MDA5 signaling (Figure 2-7B). Production of interferon gamma was also similar between the genotypes in both CD4 and CD8 T cells in the spleen (Figure 2-7C).

However, a larger proportion of CD4 and CD8 T cells from the livers of MDA5-/- animals expressed interferon gamma (Figure 2-7D). Since viral replication is similar in the liver between genotypes, this may suggest that the adaptive immune response is over exuberant and may be pathological.

Mice depleted of T cells are more susceptible to MHV infection.

To test the hypothesis that T cells are pathological in the absence of MDA5 signaling, we depleted infected WT and MDA5-/- mice of CD4 and CD8 T cells by administering a cocktail of two depleting antibodies 2 days prior to and 4 and 6 days after infection.

While we expected to observe improved survival in mice without T cells, mice depleted of both CD4 and CD8 T cells had reduced survival compared to those mice given a control antibody (Figure 2-8A). This suggests that either T cells are not pathological, or their pathological role is outweighed by their antiviral role. Regardless, the poor survival of mice lacking MDA5 signaling is likely caused by a factor independent of the T cell response.

Pro-inflammatory cytokines are elevated in the serum of MDA5-/- mice.

As T cells do not appear to be the cause of the low survival of MDA5-/- mice, we considered other drivers of inflammation. Pro-inflammatory cytokines, such as IL-6 and

TNFα, are produced early during infection by a variety of cell types. These cytokines can contribute to control of pathogens, both before and after development of the adaptive immune response, as well as drive damaging pathological responses. Inflammatory cytokines in the serum of WT and MDA5-/- mice were assayed to determine if levels were altered in the absence of MDA5 signaling. Levels of interferon gamma, IL-6, and TNFα were all elevated in MDA5-/- mice as early as five days after infection (Figure 2-8B). This potent response may be the causative agent of both increased pathology and decreased survival in MDA5-/- mice, suggesting that MDA5 has a regulatory role, inhibiting excessively strong pro-inflammatory cytokine (Figure 2-8B) and perforin-mediated cellular (Figure 2-5) responses in order to protect the host.

D. Discussion

Induction of type 1 interferon expression is an early host response to viral invasion. In the case of some viral models, including MHV infection of its natural host, type I interferon signaling is vital to host survival (Cervantes-Barragan et al., 2007; Ireland,

Stohlman, Hinton, Atkinson, & Bergmann, 2007; Lavi et al., 1984; Roth-Cross et al.,

2008; Weiss & Leibowitz, 2011). Alpha and beta interferon activate the type 1 interferon receptor in an autocrine and paracrine manner to induce the expression of ISGs. This set of genes includes additional type 1 interferon subtypes, various pro-inflammatory cytokines and chemokines, MDA5 itself, and many other effectors (Cervantes-Barragan et al., 2007; Loo et al., 2008). Through these ISGs, type 1 interferon constitutes a key component of the innate immune response. In addition, interferon is often involved in regulating adaptive immune responses: it can facilitate antigen presentation (Stark, Kerr,

Williams, Silverman, & Schreiber, 1998), promote clonal expansion among activated T cells (Curtsinger et al., 2005; Kolumam et al., 2005), and enhance humoral responses

(Hingley et al., 1994; Le Bon et al., 2001; Mitoma et al., 2013; Roth-Cross et al., 2008).

Type 1 interferon expression can be induced through numerous signaling pathways, including RLR and TLR signaling. The interplay of these different pathways and the extent to which they are redundant vary by context. We aimed to study the role of MDA5 signaling specifically during MHV infection. Both MDA5 and TLR7 detect MHV infection

(Züst et al., 2011), and mice lacking TLR7 but not MDA5 are more susceptible to infection than WT mice but more resistant than IFNAR-/- mice (Cervantes-Barragan et al., 2007; Kang et al., 2002; 2004), consistent with our findings here that interferon induced by MDA5 signaling contributes to host protection.

We show here that MDA5-/- mice are highly susceptible to MHV infection, with the majority of MDA5-/- animals dying by 7–8 days post infection while roughly 90% of WT animals survived. Following i.p. inoculation viral replication levels in the liver and spleen were similar between the genotypes, but virus more readily infected additional sites, including the CNS, in MDA5-/- mice, demonstrating a role of MDA5 signaling in maintaining organ tropism barriers. Despite similar levels of replication, inflammation and pathology in the liver were more severe in the absence of MDA5 signaling.

We compared the host immune response to MHV between WT and MDA5-/- mice and found that many facets of the immune response were largely unchanged in the absence of MDA5 expression. While induction of type 1 interferon expression is reduced in the livers of MDA5-/- mice, the ISG response was largely intact. The magnitude and kinetics of the innate cellular infiltration of the liver were similar, as was the activation and cytokine production of the T cell response in the spleen. This suggests that, unsurprisingly, many aspects of the immune response are independent of type 1 interferon signaling.

Despite decreased levels of interferon β (IFNβ) and interferon α4 (IFNα4) in MDA5-/- mice relative to WT (Figure 2-3A), transcriptional induction of almost all tested ISGs was similar between the genotypes. This demonstrates that interferon induced by TLR7 is sufficient to maintain ISG induction even in the absence of MDA5 signaling, highlighting the redundancy within the interferon response. However, despite this redundancy in ISG induction, MDA5-/- mice still succumbed rapidly to infection, suggesting that other downstream effects of MDA5 signaling are necessary for full protection of the host. In

addition, these observations indicate that some downstream effects of type I interferon signaling were more susceptible to the loss of MDA5-dependent interferon than others.

The dependence of organ tropism on route of inoculation makes MHV infection a useful model for studying tropism barriers during viral infection. There is extensive literature demonstrating that type 1 interferons play an important role in viral tropism (McFadden,

Mohamed, Rahman, & Bartee, 2009; Roth-Cross et al., 2008). However, infection studies in mice genetically deficient in interferon signaling components are often confounded by the difficulty of distinguishing between tropism changes caused by disruption of barriers and tropism changes caused by increased virus replication at primary sites of infection leading to spillover into other organs. MDA5-/- mice infected with MHV lack that confounding factor because viral replication in the liver and spleen is unchanged. MHV inoculated i.c. replicates in the CNS as well as in the liver, spleen and lungs. However, when inoculated i.p. it does not readily cross the BBB and remains restricted to the liver, spleen and lungs in most infected mice. We have shown that in

MDA5-/- mice, MHV was able to expand its tropism despite similar titers in the primary sites of replication, frequently crossing the BBB to infect the CNS after i.p. inoculation and also spreading into and replicating in the heart and kidneys. This demonstrates that the BBB and other tropism barriers depend in part on MDA5 signaling. The effect of increased tropism during infection of MDA5-/- mice remains to be elucidated, but we hypothesize that it may lead to increased pathogenesis, and may account for the increased virulence observed in mice lacking MDA5. Pathology in organs that are infected at higher rates in MDA5-/- mice than in WT mice may lead to death.

Lethality and liver damage were substantially higher in MDA5-/- than WT mice, but, surprisingly, there was not a corresponding difference in viral replication in the liver. This is notably different from previous studies from our lab in which we compared hepatitis induced by several strains of MHV and found an association between pathology and viral replication in the liver in WT mice (Cervantes-Barragan et al., 2007; Kato et al.,

2006; Navas et al., 2001; Navas & Weiss, 2003). Since the increased pathology in the livers of MDA5-/- mice is not associated with a higher viral load in comparison with WT mice, we hypothesize that the liver damage, and possibly the lethality, observed in

MDA5-/- mice is immunopathological in nature. A similar phenomenon, in which effector functions normally used to clear pathogens also damage host sites, leading to often severe pathology, has been reported during infection with many other pathogens, including hepatitis B and C viruses (Lavi et al., 1986; Mitoma et al., 2013; Sirén et al.,

2006; Zignego, Piluso, & Giannini, 2008), Dengue virus (Kang et al., 2004; Kurane &

Ennis, 1992; McCartney et al., 2008; Navas et al., 2001; Navas & Weiss, 2003), and

Toxoplasma gondii (Israelski et al., 1989; Lazear et al., 2013; Rudd et al., 2009; Ueda et al., 2003). MDA5-/- mice infected with Theiler’s encephalomyelitis virus generated higher levels of IL-17 and interferon gamma, both of which can contribute to pathology

(Cervantes-Barragan et al., 2007; Y. H. Jin et al., 2012b; Kang et al., 2002). Taken in this context, our data suggest MDA5 may be important for negative regulation of immune responses.

We had expected to observe a difference in the T cell response between MDA5-/- and

WT mice due to the elevated expression of activation markers on DCs. However, by many metrics, including expression of activation markers, perforin expression, and

interferon gamma expression from cells in the spleen, the T cell response was similar between genotypes. A similar observation was made during WNV infection, in which the

T cell response was also seemingly independent of MDA5 signaling (Amrani et al., 1999;

Gebhard et al., 1998; Kägi et al., 1997; Lazear et al., 2013; Novais et al., 2013; Weiss &

Leibowitz, 2011). This suggests that despite elevated expression of co-activation markers by DCs priming was largely unchanged, although elevated production of interferon gamma by T cells in the liver was observed, and may be a direct result of improved priming.

We found that T cells from MDA5-/- mice had elevated expression of PD-1. PD-1/PD-L1 signaling is a driver of T cell exhaustion during chronic infections (Barber et al., 2005;

Bergmann et al., 2006; Lavi et al., 1984; Roth-Cross et al., 2008), but its role during acute infections is less clear. PD-1 expression correlated with a defective T cell response during WNV infection (Bender et al., 2010; Lazear et al., 2013; Lin et al., 1999;

Ramakrishna et al., 2002; Weiss & Leibowitz, 2011), and PD-1 signaling was found to weaken T cell responses during acute infection with Histoplasma capsulatum

(Cervantes-Barragan et al., 2007; Y. H. Jin et al., 2012b; Kato et al., 2006; Lazear et al.,

2013; Lázár-Molnár et al., 2008; Roth-Cross et al., 2008) and rabies virus (Castro &

Perlman, 1995; Lafon et al., 2008; Roth-Cross et al., 2008; Xue et al., 1995), suggesting that PD-1 has similar roles in acute and chronic infections. However, PD-1/PD-L1 signaling was shown to improve T cell responses during acute infection with Listeria monocytogenes (Barber et al., 2005; Y. H. Jin et al., 2012b; MacNamara et al., 2005; J.

J. Phillips et al., 1999; Rowe, Johanns, Ertelt, & Way, 2008) and influenza (Brown et al.,

2010; Hingley et al., 1994; Talay, Shen, Chen, & Chen, 2009), indicating that it can

positively regulate T cells in the context of some infections. We hypothesize that during

MHV infection PD-1 expression indicates strong activation of T cells and that these T cells induce pathology in the liver, possibly accounting for the increased pathology. Jin et al. similarly found higher PD-1 ligand, PD-L1, expression on DC during Theiler’s encephalomyelitis virus infection of MDA5-/- mice, as well as increased pathology and elevated levels of interferon gamma and IL-17 (Cervantes-Barragan et al., 2007; Hingley et al., 1994; Ireland et al., 2007; Y. H. Jin et al., 2012b; Lavi et al., 1984; Roth-Cross et al., 2008; Weiss & Leibowitz, 2011), both of which can be drivers of pathology. We see no IL-17 production by T cells (data not shown), and minimal changes in production of interferon gamma, so we speculate that while the absence of MDA5 leads to immunopathology during both MHV and Theiler’s encephalomyelitis virus infection, the mechanism of pathology is different for each virus.

Survival of MDA5-/- mice depleted of T cells was lower than those with T cells (Figure 2-

8A), indicating that if the susceptibility of these mice to MHV infection is due to a lethal immunopathological response, it is not driven by T cells. We do however observe elevated expression of perforin by NK cells (Figure 2-5), and increased levels of the pro- inflammatory cytokines interferon gamma, IL-6, and TNFα in the serum (Figure 2-8B) of

MDA5-/- mice. We speculate that the low survival of MDA5-/- mice is due to an immunopathological response driven by one or more of these factors. These findings suggest a context-dependent role for MDA5 as a negative regulator of the immune response that limits immunopathology.

E. ACKNOWLEDGMENTS. This work was supported NIH grant R01-AI-060021 and

NS-054695 to S.R.W. Z.B.Z. was supported in part by training grant T32-NS007180.

K.M.R. was supported by T32-AI007324 and a diversity supplement to grant NS-054695.

Thanks to Drs. Helen Lazear and Michael S. Diamond, Washington University, St Louis, for MDA5-/- mice and help at early stages of the project, and to Drs. Cierra Casson and

Sunny Shin, University of Pennsylvania, for technical assistance.

F. Materials and methods

Virus and mice

Recombinant MHV strain A59 (referred to herein as MHV) has been described previously (MacNamara et al., 2005; J. J. Phillips et al., 1999). The titer was determined by plaque assay on murine L2 cell monolayers, as described previously (Hingley et al.,

1994). Wild type C57BL/6 (WT) mice were purchased from Jackson Laboratories (Bar

Harbor, ME). MDA5 deficient (MDA5-/-) mice were a generous gift from Dr. Michael S.

Diamond (Washington University, St. Louis). Mice were genotyped and bred in the animal facilities of the University of Pennsylvania. Four-to-six week old mice were used for all experiments. For infections, virus was diluted in phosphate-buffered saline (PBS) supplemented with 0.75% bovine serum albumin (BSA) and inoculated i.p. All experiments were approved by the University of Pennsylvania Institutional Animal Care and Use Committee.

Viral replication burden

To monitor viral replication, mice were inoculated i.p. with 500 PFU of MHV and sacrificed 3, 5, and 7 days after infection. Following cardiac perfusion with phosphate buffered saline, organs were harvested and placed in gel saline (an isotonic saline solution containing 0.167% gelatin), weighed, and frozen at -80°C. Organs were later homogenized, and plaque assays were performed on L2 fibroblast monolayers as previously described (Hingley et al., 1994).

Quantitative real time RT-PCR

Total RNA was purified from the lysates of homogenized livers using Trizol Reagent

(Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The resulting

RNA was treated with TURBO DNase (Ambion),. A custom Taqman Low Density Array

(LDA) was designed to include ISG probes (IRF-7, IRF-9, STAT1, STAT2, ISG15,

ISG56, IFNb, IFNa4, RIG-I) and 2 control probes (18S and RPL13a). RNA (1 µg) was reverse-transcribed to cDNA using the High Capacity RNA-to-cDNA kit (Applied

Biosystems). The 384-well custom LDA plates were loaded with 50 µL of cDNA and 50

µL of 2X Taqman Universal PCR Master Mix and run on a 7900 HT Blue instrument

(Applied Biosystems). The data were analyzed using SDS 2.3 software. The same genes were also assayed by qRT-PCR as follows. RNA was reverse-transcribed using

Superscript III reverse transcriptase (Invitrogen) to produce cDNA, then amplified using primers obtained from Integrated DNA Technologies (Coralville, IA). The sense and antisense primer sequences are available upon request. Real time PCR was performed using iQ SYBR Green Supermix (Biorad) on an iQ5 Multicolor Real-Time PCR detection system (Biorad). mRNA was quantified as ΔCT values relative to beta actin or 18S rRNA mRNA levels. ΔCT values of infected samples were expressed as fold changes over ΔCT values of mock samples (log10).

Cell isolation from the spleen and liver

WT and MDA5 deficient (MDA5-/-) mice were infected i.p. with 500 PFU of MHV, and organs were subsequently harvested after cardiac perfusion with PBS. Splenocytes were rendered into single-cell suspensions through a 70-micron filter, after which red blood cells were selectively lysed by incubating for 2 minutes in 0.206% tris HCl, 0.744%

NH4Cl solution. Livers were homogenized mechanically, then lysates were centrifuged through a Percoll® gradient to obtain a single-cell suspension.

Surface marker and intracellular cytokine staining

Intracellular cytokine staining was performed on single cell suspensions of splenocytes following a four-hour incubation with brefeldin A (20 µg/ml, Sigma) and the MHV peptides M133 (MHC class II; 4 µg/ml, Biosynthesis) and S598 (MHC class I; 9.3 µg/ml,

Biosynthesis). T cells, NK and NKT cells were stained with the following antibodies: CD3

(eBioscience, clone 17A2), CD4 (eBioscience, clone GK1.5), CD8 (eBioscience, clone

53-6.7), CD44 (eBioscience, clonse IM7), CD11a (Biolegend, clone M17/4), PD-1

(eBioscience, clone RMP1-30), βTCR (eBioscience, clone H57-597), γδTCR

(Biolegend, clone GL3), perforin (eBioscience, clone MAK-D), and interferon gamma

(eBioscience, clone XMG1.2). For innate cell immunophenotyping cells were stained with the following antibodies: CD45 (Biolegend, clone 30-F11), Ly6G (Biolegend, clone

1A8), Ly6C (Biolegend, clone HK1.4), CD11b (eBioscience, clone M1/70), CD11c

(Biolegend, clone N418), CD3 (Biolegend, clone 145-2C11), CD19 (BD, clone 1D3), and

NK1.1 (eBioscience, clone PK136). For DC phenotyping experiments cells were stained with the following antibodies: CD11b (eBioscience, clone M1/70), CD11c (Biolegend, clone N418), CD3 (eBioscience, clone 17A2), CD19 (eBioscience, clone 1D3), NK1.1

(eBioscience, clone PK136), CD45 (eBioscience, 30-F11), CD86 (Biolegend, clone GL-

1), CD80 (Molecular Probes, clone 16-10A1), MHC class II (eBioscience, clone

M5/114.15.2), B220 (Life Technologies, clone RA3-6B2), and PDCA-1/CD317

(eBioscience, 129c). Staining for interferon gamma was performed after permeabilization with Cytofix/cytoperm Plus Fixation/Permeabilization kit (BD). Following staining, cells

were analyzed by flow cytometry on an LSR II (Becton Dickinson), and the resulting data were analyzed using FlowJo software (Treestar).

Liver histology

Livers were harvested from sacrificed mice and fixed for 24 hours in 4% phosphate buffered formalin, embedded in paraffin, and sectioned. Sections were stained with hematoxylin and eosin. Four to five non-overlapping fields each 9x106 microns2 in area were selected, the number of inflammatory foci on each field was counted, and the mean was determined. Samples in which foci had coalesced into a continuous confluence were scored as too numerous to count (TNTC).

Serum collection

IL-18 and Interferon gamma ELISA

Levels of IL-6, interferon gamma, or TNFα in serum isolated from infected mice was assayed using capture and detection antibodies specific for IL-6 (Biolegend), interferon gamma (BD), or TNFα (Biolegend).

T cell depletions

Mice were inoculated with antibody i.p. two days prior to, and four and six days after, infection with MHV. Mice received either a negative control antibody (LTF-2, Bio X Cell),

or a cocktail of a CD4 depleting antibody (GK1.5, Bio X Cell) and a CD8 depleting antibody (2.43, Bio X Cell). All antibodies were at 250µg/ml concentration.

Liver Spleen A B Liver Spleen 10 10 * MDA5 KO ) 100 ) ) 8 ) 8 B6 WT 10 10 10 10 WT -/- 6 MDA56 50 4 4 PFU (log PFU PFU (log PFU pfu/g (log 2 pfu/g (log 2

Percent survival Percent 0 0 0 0 5 10 15 3 5 7 3 5 7 Daysday postpost infection Daysday postpost infection infection WT MDA5-/- Brain Spinal cord C Brain Spinal cord 10 * * * 10 **** 10 **** MDA5 KO MDA5 KO ) ) ) ) ) B6 WT) B6 WT 10 10 10 8 8 8 10 10 10 6 6 6 4 4 4 PFU (log PFU PFU (log PFU PFU (log PFU pfu/g (log pfu/g (log 2 2 pfu/g (log 2 0 0 0 Lung Heart Kidney 3 5 7 3 5 7 day post infection day post infection Days post infection Days post infection WT WT MDA5-/- MDA5-/-

Figure 2-1. MDA5-/- mice have heightened susceptibility to MHV infection. Survival was monitored after i.p. inoculation of 500 PFU MHV. Results were statistically significant by the Mantel-Cox test (A). Viral titers in the liver, spleen, brain and spinal cord were determined by plaque assay three, five and seven days after i.p. inoculation with 500 pfu MHV (B) and at five days post inoculation in the lungs, heart and kidney (C). For panels B and C, statistical significance was determined by a two-part test using a conditional t-test and proportion test. Bars represent the mean. Data for all panels were pooled from two independent experiments.

A B 5 dpi 7 dpi ** TNTC 20 WT 15 10 5 0 3 5 7 Inflammatory foci/field Inflammatory Days post infection

WT MDA5-/- MDA5-/-

Figure 2-2. MDA5-/- mice have increased pathology in the liver. Three, five and seven days post i.p. inoculation with 500 PFU MHV livers were removed, fixed, sectioned and stained with hematoxylin and eosin. Four to five non-overlapping fields of view, each 9x106 microns2, were chosen at random and the number of inflammatory foci was determined and the mean was calculated (A). Statistical significance was determined by a two-part test using a conditional t-test and proportion test. Samples in which foci had coalesced into a continuous confluence were scored as too numerous to count (TNTC). Representative fields are shown in B. Scale bar is 500 microns. Data are from one experiment.

) A ) 10 Interferon β 10 Interferon α4 IFNβ IFNα4 5 ** * 4 ** * WT 4 3 MDA5-/- 3 2 2 1 1 0 0 -1 -1 -2 3 5 7 3 5 7 Expression over mock (log mock over Expression Expression over mock (log mock over Expression Days post infection Days post infection ) ) ) ) 10

10 B 10 10 STAT1STAT1 STAT2STAT2 IRF7IRF7 IRF9IRF9 2 1 2 * 1

1 1 0 0 0 0

-1 -1 -1 -1 3 5 7 3 5 7 3 5 7 3 5 7 Expression over mock (log mock over Expression Expression over mock (log mock over Expression Expression over mock (log mock over Expression Days post infection Days post infection Days post infection (log mock over Expression Days post infection ) ) ) ) 10 10 10 10 ISG15ISG15 ISG56ISG56 RIG-IRIG-I Viperin 3 3 ** 1 2 2 2 1 1 1 0 0 0 0 -1 -1 -1 -1 3 5 7 3 5 7 3 5 7 3 5 7 Expressionover mock (log mock Expressionover Expression over mock (log mock over Expression Expression over mock (log mock over Expression Days post infection Days post infection Days post infection (log mock over Expression Days post infection

Figure 2-3. Interferon, but not interferon stimulated gene, expression is reduced in MDA5-/- mice. On days 3, 5, and 7 days post i.p. inoculation with 500 PFU MHV, RNA was isolated from the liver. Gene expression for type I interferons (A) and ISGs (B) was quantified by qRT-PCR and expressed as fold over expression from mock-infected mice. Statistical significance was determined by an unpaired, two-tailed t test. Outliers were eliminated by a ROUT test (Q=1%). Bars represent the mean. Data were pooled from two independent experiments, with the exception of viperin mRNA quantification, which was performed once.

A Macrophages Inflammatory monocytes Dendritic cells, total 1×106 108 ** WT 107 1×106 -/- 5 MDA5 1×10 106 105 1×104 1×104 104 3 2 1×103 10 1×10 102 #cells/gram tissue #cells/gram #cells/gram tissue #cells/gram 1×102 tissue #cells/gram 101 1×100 mock 1 2 3 mock 1 2 3 mock 1 2 3 Days post infection Days post infection Days post infection

Natural killer Neutrophils Dendritic cells, plasmacytoid 1×108 1×104 1×106 1×106 1×103 1×104 1×104 1×102

2 1×10 1×102 1×101 #cells/gram tissue #cells/gram #cells/gram tissue #cells/gram 1×100 tissue #cells/gram 1×100 1×100 mock 1 2 3 mock 1 2 3 mock 1 2 3 Days post infection Days post infection Days post infection

B Plasmacytoid DC Lymphoid DC Myeloid DC Plasmacytoid DC Lymphoid DC Myeloid DC 5 * ** * 4 * * *** WT 2.0×10 2.5MDA5× 10KO MDA5 KO4 + + + 8×10 MDA5 KO -/- WT 4 WT MDA5 1.5×105 2.0×10 4 WT 4 6×10 CD86 CD86 CD86 5 1.5×10 + + + 1.0×10 4 1.0×104 4×10 4 5.0×10 5.0×103 2×104 #CD80 #CD80 #CD80 0.0 0.0 0 mock 1 2 3 mock 1 2 3 mock 1 2 3 Days post infection Days post infection Days post infection

Figure 2-4. Innate inflammatory cell recruitment is largely intact in MDA5-/- mice, but activation of dendritic cells is elevated. On days 1, 2, and 3 post i.p. inoculation with 500 PFU MHV, mice were sacrificed, organs harvested and single cell suspensions derived from the liver (A) or spleen (B) and immunophenotyped by staining with cell type specific antibodies and analysis by flow cytometry. The total number of macrophages (CD3-CD19-NK1.1-CD45+Ly6-C-Ly6-G-CD11c-CD11b+F4/80+), neutrophils (CD3-CD19- NK1.1-CD45+Ly6-C-Ly6-G+), NK cells (CD3-CD19-NK1.1+CD45+Ly6-C-Ly6-G-), inflammatory monocytes (CD3-CD19-NK1.1-CD45+CD11b+Ly6-C+Ly6-G-), and total DCs (CD3-CD19-NK1.1-CD45+Ly6-C-Ly6-G-CD11c+) are shown, normalized by the mass of the tissue (A). Surface expression of the activation markers CD80 and CD86 was assessed on splenic plasmacytoid (CD3-CD19-NK1.1-CD45+CD11c+B220+PDCA-1+), lymphoid (CD3-CD19-NK1.1-CD45+CD11c+CD11b-), and myeloid (CD3-CD19-NK1.1- CD45+CD11c+CD11b-) DCs, and total number of CD80+CD86+ DCs of each subtype is shown (B). Statistical significance was determined by an unpaired, two-tailed t test. Bars represent the mean. Data were pooled from two independent experiments.

Spleen Liver Natural killer Natural killer 30 15 40 20 WT 30 15 20 10 MDA5-/- cells

+ 20 10 γ 10 5 10 5

% IFN % 0 0 0 0

40 * 8 50 20 40 cells 30 6 15 + 30 20 4 10 20 10 2 10 5 0 0 0 0 % Perforin%

Figure 2-5. Cytokine production by natural killer and natural killer T cells is similar or elevated in MDA5-/- mice compared to WT mice. WT and MDA5-/- mice were sacrificed five days after i.p. inoculation with 500 PFU MHV, spleens and livers were harvested and single cell suspensions derived from the organs were incubated with brefeldin A for three hours, and immunophenotyped by staining with cell type specific antibodies. Cells were permeabilized and stained with antibody specific to interferon gamma and perforin, then analyzed by flow cytometry. NK (CD3-NK1.1+) and NKT (CD3+NK1.1+) were assessed. Statistical significance was determined by an unpaired, two-tailed t test. Bars represent the mean. Data were pooled from two independent experiments.

A CD4+ T cell CD8+ T cell + + + + CD11aCD4 % 11a CD44CD4 % 44 CD11aCD8 % 11a CD44CD8 % 44 25 ** 40 40 80 **** 20 30 30 60 15 20 20 40 10 10 10 20 %CD44+ CD8 T cell %CD11a+ CD8 T cell

5 % CD44+ CD4 T cells % CD11a+ CD4 T cells % of cells of % % of cells of % % of cells of % 0 0 cells of % 0 0 Mock Infected Mock Infected Mock Infected Mock Infected B CD4 # 11a CD4 # 44 CD8 # 11a CD8 # 44 4×106 6×106 * 6×106 *** 1.5×107 3×106 4×106 4×106 1.0×107 2×106 2×106 2×106 5.0×106 1×106 # CD44+ CD4 T cell # CD44+ CD8 T cell #CD11a+ CD8 T cell # CD11a+ CD4 T cell # of cells # of cells # of cells # of cells 0 0 0 0.0 Mock Infected Mock Infected Mock Infected Mock Infected C D CD11a+ CD44+ 100 2000 ** 2000 *** 1500 1500 80 1000 1000 T cell

+ 500 500 60 Mean fluorescence intensity Mean fluorescence Mean fluorescence intensity Mean fluorescence 0 0 intensity Mock Infected intensity Mock Infected CD4 % of Max 40 PD-1Fluorescence PD-1Fluorescence PD-1Fluorescence PD-1Fluorescence

1500 2000 20 * 1500 1000 1000 0 T cell 500 0 102 103 104 105 + 500 <695/40 Blue-A>

PD-1 expression intensity Mean fluorescence 0 intensity Mean fluorescence 0 intensity Mock Infected intensity Mock infected CD8 WT infected PD-1Fluorescence PD-1Fluorescence MDA5-/- infected PD-1Fluorescence WT mock WT MDA5-/- mock MDA5-/-

Figure 2-6. T cell activation is independent of MDA5 signaling. Day 7 post i.p. inoculation with 500 PFU MHV, mice were sacrificed, spleens harvested and single cell suspensions were derived from the spleens of WT and MDA5-/- mice. Cells were immunophenotyped by staining with cell type specific antibodies and analysis by flow cytometry. The percent (A) and total number (B) of CD4 (CD3+CD4+) and CD8 (CD3+CD8+) T cells with elevated surface expression of CD44 and CD11a are shown. Expression of PD-1 (representative image, C) was assessed on CD44+ and CD11a+ CD4 and CD8 T cells (D). Statistical significance was determined by an unpaired, two- tailed t test. Bars represent the mean. Data are representative of two independent experiments.

20141203 zz MDA5 stim 2.jo LAYOUT-1

WT infected, 4 KO infected, 4

5 5 10 20141203 zz MDA5 stim 2.jo 10 LAYOUT-1

104 4 WT infected, 4 10 KO infected, 4 Green-A> 3

Green-A> 10 103 23.8 22.8 <780/60 <780/60 102 2 10 0

0 0 102 103 104 105 5 <670/30 Red-A> 5 10 10 0 102 103 104 105 CD44+ (CD4, by eye) <670/30 Red-A> Specimen_001_MDA5 KO infected C_018.fcs CD44+ (CD4, by eye) Event Count: 4934 Specimen_001_WT4 infected D_014.fcs Event 10Count: 6670 104 Green-A> 3

Green-A> 10 103 23.8 <780/60 22.8 20141203 zz MDA5 stim 2.jo LAYOUT-1 <780/60 102 102 0 WT infected, 4 KO infected, 4 0 WT mock, 4 0 102 103 104 105 KO mock, 4 <670/30 Red-A> 0 102 103 104 105 CD44+ (CD4, by eye) <670/30 Red-A> Specimen_001_MDA5 KO infected C_018.fcs CD44+ (CD4, by eye) Event Count: 4934 Mock Infected Specimen_001_WT infected D_014.fcs Event Count: 6670 A C Spleen

5 105 10 105 5 10 6 4 30 4 1.5×1010 10 104

20141203 zz MDA5 stim 2.jo LAYOUT-1104 + cells γ CD4 Green-A> WT 3 25 Green-A>

Green-A> 10

+ 6 3 3 1.0×10 23.8

10 10 γ Green-A> WT infected, 4 2.82 KO infected, 422.8 103 <780/60 <780/60 <780/60 3.03 20 WT mock, 4 KO mock, 4 102 102 102 <780/60 5 5.0×100 IFN % 102 15 0 0 # IFN 0 0 102 103 104 105 <670/30 Red-A> 2 3 4 5 0 102 103 104 105 0 10 10 10 10 CD44+0.0 (CD4, by eye) 10 Specimen_001_MDA5 KO infected C_018.fcs <670/30 Red-A> <670/30 Red-A> 2 3 4 5 CD44+ (CD4, by eye) CD44+ (CD4, by eye) 0 10 10 10 Event10 Count: 4934 Specimen_001_WT mock D_004.fcs Specimen_001_WT infected D_014.fcs <670/30 Red-A> CD44+ (CD4, by eye) 5 Event Count: 9834 Event Count: 6670 6 10 Specimen_001_MDA5 KO B_007.fcs 2.5×10 20 5 5 5 10 10 Event Count: 7884 10 6 +

CD8 γ cells 18 -/- 2.0×10 104 + 4 6 104 10 γ 1.5×10 16 104 6/4/15 11:34 AM Page 1 of 1 (FlowJo v9.6.4) Green-A> 6 103

Green-A> 14

Green-A> 1.0×10

3 3 IFN % 2.82 MDA5 Green-A> 10 10

<780/60 3 3.03 23.8 5 10 WT mock, 4 # IFN 5.0×10 KO mock, 4 12 <780/60 102 22.8<780/60 <780/60 2 102 0.0 10 0 10 2 10 0 0

0 102 103 104 105 0 <670/30 Red-A> 2 3 4 5 0 102 103 104 105 CD44+ (CD4, by eye) 0 10 10 10 10 5 <670/30 Red-A> 10 <670/30 Red-A> Specimen_001_WT mock D_004.fcs 2 3 4 5 5 CD4$ CD44+ (CD4, by eye) CD44+ (CD4, by eye) 10 Event Count: 9834 0 10 10 10 10 <670/30 Red-A> Specimen_001_MDA5 KO B_007.fcs Specimen_001_MDA5 KO infected C_018.fcs Event4 Count: 4934 CD44+ (CD4, by eye) Event Count: 7884 10 Specimen_001_WT infected D_014.fcs 104 Event Count: 6670 IFNγ$ Green-A> Perforin3 10

D Green-A> 6/4/15 11:34 AM Page 1 of 1 (FlowJo v9.6.4) 2.82 103 <780/60 3.03Liver

B Spleen Liver <780/60 102 2 0 10 *

+ 7 7 6 * 1.5×10 2.0×10 2.0×0 10 30 0 102 103 104 105 +

<670/30 Red-A> cells 2 3 4 5 CD44+ (CD4, by eye) 7 CD4 0610 10 10 10 γ Specimen_001_WT mock D_004.fcs + 1.5×10 <670/30 Red-A> 1.5×10 CD44+ (CD4, by eye) WT mock, 4 7 Event Count: 9834 γ 1.0×10 KO mock, 4 Specimen_001_MDA5 KO B_007.fcs 20 Event Count: 7884 cells 1.0×107 1.0×106 6 % IFN % 10

# Perforin 5.0×10 5 6/4/15 11:345.0 AM×106 Page 1 of 1 # IFN 5.0×10 (FlowJo v9.6.4) 0.0 0.0 0.0 0 105 105 + 4 * 10 4×106 15 104 60

20 + cells Green-A> CD8 6 γ 103 + 3×10 Green-A>

γ 10 2.82 15 103 40 cells <780/60 3.03 2×106 102 10 <780/60 2 % IFN % 5 0 Perforin% 10 20 6 # IFN 1×10 5 0

0 102 103 104 105 <670/30 Red-A> 2 3 4 5 0 0 CD44+ (CD4, by eye) 0 0 10 100 10 10 Specimen_001_WT mock D_004.fcs <670/30 Red-A> CD44+ (CD4, by eye) Event Count: 9834 Specimen_001_MDA5 KO B_007.fcs Event Count: 7884 WT MDA5-/- 6/4/15 11:34 AM Page 1 of 1 (FlowJo v9.6.4)

Figure 2-7. Interferon gamma production, but not perforin production, is elevated in T cells from MDA5-/- mice. WT and MDA5-/- mice were sacrificed seven days after i.p. inoculation with 500 PFU MHV, spleens and livers were harvested and single cell suspensions derived from the organs were incubated with immunodominant MHV peptides S598 (class I) and M133 (class II), and immunophenotyped by staining with cell type specific antibodies. Cells were permeabilized and stained with antibody specific to interferon gamma and perforin, then analyzed by flow cytometry (representative image, A). CD8 (CD3+CD8+) T cells, cells from the spleen and liver were assessed for expression of perforin (B). CD4 (CD3+CD4+) and CD8 (CD3+CD8+) T cells from the spleen (C) and liver (D) were also assessed for expression of interferon gamma. Statistical significance was determined by an unpaired, two-tailed t test. Bars represent the mean. Data are representative of two independent experiments.

A 100 WT neg. cntrl. WT 4&8 depleted 50 MDA5-/- neg. cntrl. MDA5-/- 4&8 depleted Percent survival Percent 0 0 5 10 15 Days post infection

B Interferon γ IL-6 TNFα 15000 * ** **** 10000 *** **** 400 ** ** 8000 300 10000 6000 4000 200 pg/ml pg/ml 5000 pg/ml 2000 100 0 0 0 mock 3 5 7 mock 3 5 7 mock 3 5 7

Days post infection Days post infection Days post infection WT -/- MDA5

Figure 2-8. Elevated cytokines, but not T cells, may explain decreased survival of infected MDA5-/- mice. MDA5-/- mice were treated with CD4 and CD8 depleting antibodies or a negative control -2, 4, and 6 days after infection with 500 PFU MHV inoculated i.p. Survival was monitored (A). Data were pooled from two independent experiments. Results are non-significant by the Mantel-Cox test. Serum was collected from WT and MDA5-/- mice with intact T cell compartments 3, 5, and 7 days after i.p. inoculation of 500 PFU MHV, and analyzed for levels of interferon gamma, IL-6, and TNFα by ELISA (B). Statistical significance was determined by a two-part test using a conditional t-test and proportion test. Data were pooled from four independent experiments, n of 3-6.

CHAPTER 4

DISCUSSION

In the findings presented in this dissertation we have defined roles for two innate inflammatory pathways, the inflammasome and the MDA5/type 1 interferon axis, during infection with MHV. We have shown that the inflammasome plays an overall protective role during infection, but that two of its substrate cytokines, IL-1 and IL-18, have differential roles. IL-1 restricts viral replication but diminishes survival of infected mice, while IL-18 plays a protective role by promoting production of interferon gamma.

Additionally, we have characterized a negative regulatory role for MDA5 signaling that prevents an overly strong, pathological immune response from developing.

A. The inflammasome-related cytokines IL-1 and IL-18 have differential roles during MHV infection

In this study we characterized the role of caspase-1 and -11, IL-1, and IL-18 in host defense during infection with MHV. We showed that Casp-1/11-/- mice were more susceptible to infection, with decreased survival and increased viral replication compared to WT mice (Figure 1-1). IL-1 signaling was found to decrease survival but improve control of replication, while IL-18 signaling was critically important for host defense, with IL-18R-/- mice losing control of viral replication and rapidly succumbing to infection (Figure 1-2). The role of IL-18 signaling was suggested to be promotion of protective levels of interferon gamma (Figures 1-5 and 1-9).

The absence of IL-1 and IL-18 signaling, in addition to Caspase-1/11, had different effects on viral replication in different organs and on survival. The cause of death during

MHV infection has never been definitively proven, but is often believed to be related to viral loads. These data suggest that the link between viral load and survival may be

more complex, as Casp-1/11-/- mice had WT levels of replication in the CNS but poor survival, and IL-1R-/- mice had higher viral load in the CNS but similar or improved survival. The cause of death remains unknown.

B. The infection-specific role of IL-1 signaling

It has recently been found that during infection with IAV (Ichinohe et al., 2009) or WNV

(Ramos et al., 2012), IL-1 signaling was required for full activation of DCs, and that without it, the T cell response, which has a Th1 polarization, was defective due to improper priming. Further studies found that during IAV infection, DCs that did not receive IL-1 signaling failed to migrate from the lungs to lymphoid organs (Pang et al.,

2013), and that during WNV infection DCs would not fully upregulate co-stimulatory markers without IL-1 signaling (Durrant et al., 2013). Normally DCs need to detect pathogens through PRR recognition in order to activate fully, but IL-1 signaling seems to replace this need (Pang et al., 2013), allowing uninfected, bystander DCs to be activated without needing to become infected.

It is unknown why IL-1 signaling is critically important during IAV and WNV infection but is less important during MHV infection. We hypothesize that IAV and WNV may inhibit the function of infected DCs more fully than MHV does, making bystander activation through the IL-1 pathway a necessity. To test this we propose to isolate DCs, infect them with each virus, and pulse them with a model antigen. They will then be co-cultured with

T cells, and the induced proliferation of the T cells, compared to that induced by uninfected DCs, will be used as a measure of priming capacity. If our hypothesis is correct, we expect DCs infected with IAV or WNV to induce less proliferation than

uninfected or MHV infected cells. Some lesser defect may be seen in MHV infected cells, which could account for the poor viral clearance in IL-1R-/- mice (Figure 2-1), but we hypothesize that MHV-infected DCs will have a higher capacity for priming than cells infected with IAV or WNV.

C. A model for explaining the discrepancy between the survival of Casp-1/11-/- and

IL-18R-/- mice

Caspase-1 and/or caspase-11 are responsible for the majority of IL-18 release during

MHV infection (Figure 1-5). Based on this data we would hypothesize that Casp-1/11-/- mice and IL-18R-/- mice should closely phenocopy each other, as neither genotype has

WT levels of IL-18 signaling. Instead, we observe a 60% fatality rate among Casp-1/11-/- mice when infected with 5,000 PFU of MHV (Figure 1-1), but a 100% fatality rate among

IL-18R-/- mice when infected with one tenth the dose (Figure 1-2). This suggests that

Casp-1/11-/- mice are less susceptible to MHV than IL-18R-/- mice, despite their low levels of IL-18.

We propose two possible explanations for this phenotype. First, the minimal levels of inflammasome independent IL-18 may be sufficient to partially protect the host. Pro-IL-

18 can be processed extracellularly by an inflammasome independent mechanism(Omoto et al., 2006; 2010; Sugawara et al., 2001), likely accounting for the remaining IL-18 in Casp-1/11-/- mice, and this concentration of cytokine may be sufficient for protection. To test this, we propose to cross Casp-1/11-/- mice with IL-18R-/- mice and observe the survival of the resulting progeny following MHV infection. We expect that if

the low level of inflammasome independent IL-18 is the only factor altering the survival of Casp-1/11-/- mice, the survival of the progeny should exactly phenocopy IL-18R-/- mice, as the remaining IL-18 will be unable to signal.

Our second possible explanation for the discrepancy between the survival of Casp-1/11-/- and IL-18R-/- mice is that some other inflammasome substrate is negatively affecting survival during MHV infection. The inflammasome processes numerous IL-1 family cytokines besides IL-1 and IL-18, including IL-33 and IL-37 (Garlanda et al., 2013). If any one of these cytokines, including IL-1, is more pathological than protective during MHV infection then its loss in Casp-1/11-/- mice would result in improved survival, which could partially offset the reduced survival caused by the loss of IL-18. We have demonstrated here that despite the role of IL-1 in controlling viral replication it appears to have a detrimental effect on survival, with IL-1R-/- mice surviving at higher rates than WT mice

(Figure 1-2). It is therefore possible that the result of losing both IL-1 and IL-18 signaling in Casp-1/11-/- mice results in an intermediate phenotype. To examine this hypothesis we intend to cross IL-1R-/- and IL-18R-/- mice and compare the survival of their progeny to Casp-1/11-/- mice. If the survival phenotypes were similar it would support our hypothesis, although it would not preclude the possibility that additional members of the

IL-1 family are also influencing survival.

D. The protective role of IL-18 signaling during MHV infection is mediated by production of interferon gamma

IL-18 signaling has two known roles; to promote cytotoxicity and to promote production of interferon gamma by a variety of cell types. We have found perforin production by αβ

T cells, γδ T cells, NK cells, and NKT cells to be independent of IL-18 signaling (Figures

1-6 and 1-7). This suggests that during MHV infection cytotoxicity is promoted or licensed by other mediators. However, while perforin levels are an important factor in cytotoxicity, they are not the only factor, and it is possible that cells from IL-18R-/- mice are defective in cytolytic killing even with WT levels of perforin. To address this, we propose to perform ex vivo cell killing assays in which cells are isolated from WT and IL-

18R-/- mice, sorted to separate different cell types, and then incubated with infected target cells to determine the relative cytolytic ability of each cell type from each genotype. This would provide a more direct measure of cytolytic ability and could expose currently undiscovered defects.

We did observe a significant change in the concentration of interferon gamma in the serum of IL-18R-/- mice compared to WT mice (Figure 1-5). Five days after infection, at the peak of interferon gamma production, IL-18R-/- mice had significantly lower cytokine concentrations, despite slightly higher viral loads in several organs. This suggests that

IL-18R-/- mice have a defect in interferon gamma production. Although this defect can be reversed at later time points, when the immune system has failed to begin clearing virus, it is likely that the transient defect at the peak of cytokine production is sufficient to make the mice highly susceptible to infection.

Additionally, our study suggests that αβ T cells rely on IL-18 signaling for full production of interferon gamma, while production by γδ T cells, NK cells, and NKT cells is independent of IL-18 (Figure 1-9). This defect likely underlies the decreased cytokine concentration in the serum, and in light of the importance of interferon gamma to host

defense against MHV (Kyuwa et al., 1998; Schijns et al., 1996), likely accounts for the poor survival and increased viral loads of IL-18R-/- mice.

In future studies we would like to determine if adding IL-18 responsive T cells to IL-18R-/- mice would improve their survival. IL-18R-/- mice lack the ability to respond to IL-18 signaling, but are expected to have normal levels of IL-18 itself. Therefore, WT T cells transferred into IL-18R-/- mice should be exposed to endogenous IL-18. If the survival of

IL-18R-/- mice improves when WT cells are transferred into them, this would demonstrate that IL-18 is capable of driving a protective role of T cells. As an additional experiment, T cells from interferon gamma-/- mice could be transferred into IL-18R-/- mice. If these mice show poor survival comparable to that of IL-18R-/- mice given IL-18R-/- T cells, we could conclude that promoting interferon gamma production is the protective role of IL-18. If the mice that received interferon gamma-/- T cells instead have improved survival compared to mice that received IL-18R-/- T cells, and survival comparative to those mice given WT T cells, we could conclude that IL-18 mediates its protection through other factors instead of, or in addition to, interferon gamma.

E. The role of the inflammasome in MHV-induced demyelination

Mice that survive infection with neurotropic MHV develop an immune mediated demyelination following viral clearance (Lampert et al., 1973). This disease is characterized by destruction of the myelin sheaths of neuronal axons and can lead to limb paralysis. It shares some pathological similarities to MS, a demyelinating disease in humans. MS is a heterogeneous disease, and its etiology and pathogenesis remain unclear, in part due to the limited means available to study diseases in humans. A

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number of murine models of demyelination have been developed in order to facilitate research into this disease state. Among them are EAE, which is induced by vaccination with whole or subunits of myelin basic protein, a cuprizone induced model, and the virus- induced models, which include neurotropic MHV and Theiler’s encephalomyelitis virus.

Given the heterogeneous nature of MS (Denic et al., 2011), we believe that parallel studies repeated across multiple models are most likely to produce clinically useful data.

No single model is likely to recapitulate MS pathogenesis, but it is possible that those pathogenic or protective factors found to be common to multiple murine models will represent elements universal to all or most forms of demyelination. As such, we have examined the role of the inflammasome during MHV-induced demyelination.

IL-1 has been shown to be a pathological driver in EAE (Chung et al., 2009; N. J. Wilson et al., 2007), and IL-18 appears to be pathological in both EAE and cuprizone induced models (Jha et al., 2010; F. D. Shi et al., 2000), suggesting a fundamental role for the cytokines in demyelination. We tested the role of both cytokines in MHV-induced demyelination by infecting receptor knockout mice and quantifying the severity of demyelination thirty days later, at the peak of the disease. Our data suggest that there is no role for IL-1 in MHV-induced demyelination, suggesting a possible difference between the pathogenesis of EAE and MHV-induced disease. Results from infection of IL-18R-/- mice were inconclusive (Figure 1-4), as severity of acute infection is believed to produce a more severe demyelination (MacNamara et al., 2005; Marten et al., 2000). IL-18R-/- mice have both higher viral loads in the CNS and more severe demyelination, as would be expected. It is possible that these mice have more or less severe demyelination compared to WT mice with a similarly higher viral burden, but further study is required. In

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order to test the effects of IL-18 signaling without altering initial infection, in future studies we propose to treat WT mice with an anti-IL-18 antibody (Plater-Zyberk et al.,

2001) after MHV has been fully cleared. This should eliminate IL-18 signaling during the bulk of the demyelinating phase, allowing us to compare the severity of demyelination to

WT mice treated with control antibody, without the confounding factor of altered viral loads. Given the central role of IL-18 in the EAE and cuprizone-induced models, we hypothesize that mice without IL-18 signaling should experience a less severe disease.

If this is correct, we will characterize the immune response and pathology in the CNS of mice with and without IL-18 signaling to begin determining its exact role in MHV-induced demyelination.

F. MDA5 plays a protective role during MHV infection through negative regulation of the immune response

We have demonstrated a critical role for MDA5 signaling during infection with MHV

(Figure 2-1). Previous findings showed that type 1 interferon signaling was essential for host defense (Cervantes-Barragan et al., 2007), and that both MDA5 and TLR7 contributed to induction of this pathway (Cervantes-Barragan et al., 2007; Roth-Cross et al., 2008; Züst et al., 2011). While the role of TLR7 had been examined, the role of

MDA5 during MHV infection had not been extensively studied prior to now. We have shown that mice lacking MDA5 signaling had decreased survival compared to WT mice, as well as elevated viral replication in several organs, spread of virus to additional organs (Figure 2-1), and elevated hepatitis (Figure 2-2). Interferon transcription was reduced but the ISG response was intact (Figure 2-3), and the T cell response appeared largely intact as well based on cell numbers, activation markers, and cytokine production

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(Figures 2-6 and 2-7). However, perforin production was elevated among NK cells

(Figure 2-5), and interferon gamma production was elevated among T cells in the liver

(Figure 2-7). Levels of IL-6, interferon gamma, and TNFα were drastically higher in serum from MDA5-/- mice compared to WT mice (Figure 2-8). We conclude from these data that in the absence of MDA5 signaling, mice develop an overly strong immune response that leads to lethal immunopathology.

G. The relative contributions of MDA5 and TLR7 to induction of the type 1 interferon response

In vivo studies with IFNAR-/- mice have demonstrated that these mice are highly susceptible to MHV infection, with 100% lethality within 48 hours of infection(Cervantes-

Barragan et al., 2007), and that mice lacking both MDA5 and TLR7 are similarly vulnerable (Züst et al., 2011). Our work with MDA5 (Zalinger et al., 2015) and other work with TLR7 (Cervantes-Barragan et al., 2007) have shown that while both sensors are important for host defense, the loss of either does not recapitulate the severe susceptibility phenotype of IFNAR-/- mice. This suggests that MDA5 and TLR7 can partially compensate for the loss of the other, but that the single knockout mice are less susceptible than mice entirely lacking interferon induction. This has been demonstrated in a previous study that found that viral replication in MDA5-/-TLR7-/- mice was similar to that of IFNAR-/- mice (Züst et al., 2011).

MDA5 has been shown to directly upregulate certain ISGs in a type 1 interferon- independent manner (Dixit et al., 2010). For this reason we cannot conclude that the vulnerability to infection seen in MDA5-/- mice is solely due to defects in the type 1

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interferon response. While comparing induction of ISGs in WT, MDA5-/-, and IFNAR-/- mice could indicate which ISGs are MDA5-dependent and IFNAR-independent during

MHV infection, the exceptionally poor survival of IFNAR-/- mice makes this experiment difficult to perform.

H. MDA5 signaling in maintenance of tropism barriers

MHV is a useful model for studying tropism barriers due to its broad, but often conditional, cellular and organ tropism patterns. MHV strain A59 is capable of infecting a wide range of organs, including the brain and spinal cord, which make up the CNS, and peripheral organs such as the liver, spleen, and lungs (Weiss & Leibowitz, 2011). Its ability to infect the CNS however is limited by its route of inoculation. Virus inoculated directly into the CNS, or intranasally, replicates in the CNS and spreads to peripheral organs. Virus inoculated in the periphery infects peripheral organs but crosses the BBB to enter the CNS only in a small minority of animals.

MHV is capable of infecting a wide variety of cell types, including hepatocytes (Navas et al., 2001; L. P. Weiner, 1973), liver sinusoidal endothelial cells (Pereira, Steffan, & Kirn,

1984), and Kuppfer cells in the liver (Even et al., 1995), neurons (Parra et al., 1997), astrocytes (Lavi et al., 1987), and all other major cell types of the CNS, and macrophages and DCs. Based on this wide cellular tropism we would expect that MHV is capable of infecting almost any, if not all, organs, barring effects of differential expression of the known MHV receptor CEACAM1 (Miura et al., 2008). However, our findings and the findings of others demonstrate that MHV’s tropism is not determined entirely by the availability of cell types that are susceptible to infection. Instead, several

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factors contribute to tropism barriers that prevent viral infection of certain organs. Our work suggests the influence of two different factors; MDA5-independent protection provided by the BBB and an MDA5-dependent host defense that protect the heart and kidney.

The BBB is composed of endothelial cells and astrocytes, and provides a selectively permeable barrier around the CNS. The ability of the BBB to prevent viral spread is partially regulated by interferon signaling, and in IFNAR-/- mice MHV is capable of trafficking from the periphery into the CNS within 48 hours (Cervantes-Barragan et al.,

2007). Additional studies with MHV (Bleau, Filliol, Samson, & Lamontagne, 2015) as well as WNV (Lazear et al., 2015) have confirmed that both type 1 and type 3 interferon signaling strengthens the BBB, reducing permeability and restricting viral spread into the

CNS. As such, we had originally hypothesized that MDA5-/- mice would also have increased viral replication in the CNS.

Seven days after infection we do see the expected phenotype, with no detectable virus in the brain or spinal cord of WT mice but detectable virus in the brain of 55% of MDA5-/- mice, and in the spinal cord in 44% of mice. However, five days after infection 25% of

WT and MDA5-/- mice have detectable viral loads in both the brain and spinal cord. This suggests that the role of MDA5 in infection of the CNS has two components. If the effect of MDA5 were purely to restrict viral spread to the CNS, we would expect higher rates of infection at both five and seven days after infection. Instead, we see similar rates early on, followed by increased rates among MDA5-/- mice and no infection among WTs

(Figure 2-1). This suggests that in the course of infection in WT mice a minority (25%) of

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mice experience CNS infection, but that by seven days post infection this virus has been cleared, leading to a 0% infection rate. Without MDA5 however, mice are unable to clear the infection between day five and seven. Although this suggests the role of MDA5 is purely in clearance, infection rates do increase between five and seven days post infection in knockout animals, suggesting that virus may also access the CNS more frequently at later time points in the absence of MDA5. We therefore speculate that

MDA5 signaling protects the CNS by two distinct mechanisms; maintaining the tropism barrier function of the BBB and by facilitating viral clearance from the brain and spinal cord.

The ability of MHV to infect the heart and kidney is strongly influenced by MDA5 signaling. Only 25% of mice with MDA5 signaling had detectable infection in the heart and kidney five days after infection, while 83% of mice lacking MDA5 signaling did

(Figure 2-1). Thus, MDA5 signaling appears to prevent infection of the heart and kidney in roughly 60% of animals. The mechanism by which MDA5 signaling prevents infection in this model remains unknown. We hypothesize that MDA5-dependent inflammatory pathways involved in tropism restriction are most important in either epithelial and endothelial cells or in macrophages. If MDA5 signaling in epithelial and endothelial cells blocks infection, it would suggest that in immunocompetent mice virus is unable to access certain organs, being unable to infect the cell types exposed to the circulatory system. If instead MDA5 signaling in macrophages blocks infection, it would suggest that virus is capable of accessing the heart and kidney, but that early inflammatory responses rapidly clear the infection and prevent further infection. These two hypotheses are not mutually exclusive. To address this, mice with MDA5 conditionally knocked out

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only in epithelial or endothelial cells or myeloid cells by the Cre/Flox system could be infected, and the subsequent patterns of infection assessed. Although this experiment is complicated by the lack of organ-specific drivers to regulate Cre expression, meaning that the mice will lack MDA5 signaling in a given cell type in multiple organs, it may be possible to draw conclusions from shifts in tropism.

I. PD-1 as a marker of defective T cells

Among the only differences we detected between T cells from WT and MDA5-/- mice was an elevation in PD-1 expression in T cells from MDA5-/- mice. PD-1/PD-L1 signaling is a driver of T cell exhaustion during both acute (Israelski et al., 1989; Lafon et al., 2008) and chronic infections (Barber et al., 2005). PD-1high exhausted T cells lose various effector functions, including interferon gamma production, over the course of infection, and are less capable of clearing pathogens. It is possible that during infection of MDA5-/- mice T cells become functionally defective, as would be expected of PD-1high cells, and that this could contribute to the poor survival of MDA5-/- mice. Depletion of CD4 and CD8

T cells decreased survival of MDA5-/- mice, suggesting that the T cells from MDA5-/- mice are at least somewhat protective, but it is still possible that these cells have a defect compared to T cells from WT mice, even if that defect is not so severe that the mice phenocopy T cell depleted mice. However, our analysis of the T cell response in MDA5-/- mice showed no signs of defect, with similar expression of activation markers and similar or elevated cytokine production compared to WT mice (Figures 2-6 and 2-7). While these data would seem to suggest that the T cell response is intact, and that the PD-1 expression is irrelevant, it should be noted that similar results have been obtained during

WNV infection of MDA5-/- mice. Lazear et al. (2013) found that MDA5-/- mice were more susceptible to WNV infection, but that T cells isolated from these mice showed no signs

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of functional defects based on activation marker expression and cytokine production.

CD8 T cells did possess higher levels of PD-1 on their surface, however. Lazear et al.

(2013) performed adoptive transfer experiments, in which infected CD3-/- mice were given T cells from infected WT or MDA5-/- mice, and found that T cells from WT mice would protect CD3-/- mice while those from MDA5-/- mice would not, suggesting the presence of an “invisible” T cell defect that was not evident during flow cytometric analysis of cells (Lazear et al., 2013). Repeating Lazear et al.’s adoptive transfer experiments with MHV could prove whether or not MHV infected MDA5-/- mice have a similar T cell defect that increases their susceptibility to infection.

J. Immunopathology in the absence of MDA5 signaling

We have shown that MDA5-/- mice have poor survival compared to WT mice when infected with MHV (Figure 2-1). While this demonstrates that MDA5 signaling is protective during MHV infection, it remains unclear what role MDA5 signaling actually plays in host defense. A closely related question is: “What is the cause of death in

MDA5-/- mice?” If the immune response in MDA5-/- mice is incapable of controlling viral replication, then pathology induced directly by the virus is likely to be the cause of death.

If MDA5 signaling is instead acting as a negative regulator of inflammation, then immunopathology is expected to be the cause of death. Unfortunately, it is difficult in this model to move beyond correlative relationships between damage to an organ and death, as the presence of pathology in an organ does not prove that said pathology is lethal.

MHV is a lytic virus, and it is suspected that virus-induced hepatitis or encephalitis is usually the cause of death, although this has not been proven. If MDA5-/- mice were

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unable to control viral replication, we could speculate that elevated viral loads would cause lethal pathology in one or more organs. There is no detectable change in the ISG response, and no detectable defect in T cells, but our survey of the immune response is by no means complete. It is also possible that an “invisible” T cell defect, like that seen during WNV infection, could lead to out of control viral replication. However, viral load in the liver is independent of MDA5 signaling, and load in the spleen is only transiently higher. It is possible that infection of the CNS leads to lethal encephalitis, and that the subset of mice in which virus penetrates the CNS could be the same subset that die.

However, virus was detectable in the brains of only 55% of MDA5-/- mice, and in the spinal cords of 44% of mice, while 74% of MDA5-/- died, suggesting this may not be the case. It is also possible that spread to additional organs such as the heart and kidney could lead to lethal pathology, and we cannot rule out the effects of elevated replication in the spleen or lungs. Histological analysis to detect excessive pathology in all infected organs, and in particular the organs infected mostly in MDA5-/- mice, such as the brain, heart, and kidneys, could provide insights into the cause of death, and therefore the ultimate role of MDA5 signaling.

Elevated pathology in the liver of MDA5-/- mice (Figure 2-2) despite the similar viral loads compared to WT mice is suggestive of an immunopathological response, in which the immune system itself causes tissue damage. If this hypothesis were correct, inflammatory mechanisms would be the cause of death, instead of direct viral damage. A similar paradigm is observed during infections with a diverse range of pathogens, including Dengue virus (Kurane & Ennis, 1992), IAV virus (Peiris, Hui, & Yen, 2010), and

Toxoplasma gondii (Israelski et al., 1989; Liesenfeld, Kosek, Remington, & Suzuki,

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1996). Elevated expression of the co-activation receptors CD80 and CD86 by lymphoid and myeloid DCs suggested that priming of T cells might be over-exuberant (Figure 2-

4B), however depletion of T cells reduced survival of MDA5-/- mice instead of improving it

(Figure 2-8A), which is the expected result if T cells were more protective than pathological. While T cells would seem to be an unlikely driver of pathology in this context, we also observed elevated production of perforin by NK cells (Figure 2-5) as well as substantially higher concentrations of IL-6, interferon gamma, and TNFα in the serum of MDA5-/- mice (Figure 2-8B). Both over-activated NK cells and pro-inflammatory cytokines could potentially lead to immunopathology that could account for the elevated liver pathology and the poor survival observed in MDA5-/- mice. A similar result was observed during infection of MDA5-/- mice by Theiler’s encephalomyelitis virus, in which more severe disease was attributed to elevated interferon gamma and IL-17 production by T cells (Y. H. Jin et al., 2012b). To determine which, if any, inflammatory factor is the lethal pathological driver during MHV infection of MDA5-/- mice, we could deplete NK cells or neutralize IL-6, interferon gamma, or TNFα and observe survival. If survival were to improve we would conclude that the neutralized cytokine or depleted cell type was responsible for death. Any improved survival would likely be transitory, as many of the proposed immunological factors are important for host defense, and in their absence control of viral replication could be lost, leading to virus-induced death.

K. MDA5 and type 1 interferon as negative regulators of the immune response

Elevated pro-inflammatory cytokines and NK cell activation in MDA5-/- mice suggest that the role of MDA5 during MHV infection is at least partially anti-inflammatory, as in the absence of MDA5 some elements of the inflammatory response are stronger. While

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MDA5 has type 1 interferon independent functions (Dixit et al., 2010), type 1 interferon has a well established role as an anti-inflammatory cytokine. While type 1 interferon was originally considered to be a purely pro-inflammatory mediator, a growing body of evidence has shown that it can also negatively regulate immune responses. Type 1 interferon inhibits the transcription of CXCL8 (Nozell, Laver, Patel, & Benveniste, 2006;

Oliveira, Sciavolino, Lee, & Vilcek, 1992), a chemokine that recruits neutrophils and leukocytes to sites of inflammation (P. Romagnani, Lasagni, Annunziato, Serio, &

Romagnani, 2004), as well as matrix metalloprotease 9 (Ma, Qin, & Benveniste, 2001;

Stuve, Chabot, Jung, Williams, & Yong, 1997), which facilitates migration of T cells into the CNS (Agrawal, Lau, & Yong, 2008). Type 1 interferon can also upregulate production of tristetraprolin (TTP), a negative regulator of TNFα transcription (Sauer et al., 2006), and IL-10 (Chang, Guo, Doyle, & Cheng, 2007). We speculate that the loss of MDA5- dependent interferon, while having no effect on ISG transcription, reduces interferon production to the point where it is unable to function as an anti-inflammatory modulator, at which point mice experience an out of control inflammatory response that is ultimately lethal.

L. Concluding remarks

The studies performed in this dissertation have described diverse roles for the inflammasome and the MDA5/type 1 interferon axis on both the pathogenesis of MHV infection and the regulation of the innate and adaptive immune system. Our work suggests that the role of the inflammasome related cytokine IL-1 is specific to the infectious model, and that IL-18 signaling drives protection through interferon gamma production. MDA5 signaling was shown to influence viral tropism and to provide an anti-

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inflammatory influence to control a pathological immune response. Together, these data describe a complex, fundamental role in immune modulation for the earliest responders to infection. Changes in even the first steps of an immune response can alter disease course, subsequent stages of the immune response, and the ultimate resolution of infection, reinforcing why study of these early inflammatory pathways is so essential.

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