The Role of Type I Alpha/Beta Receptor Signaling and Sex in Responses to Viral Infection

by Maedeh Darzianiazizi (AKA Mahi Azizi)

A Thesis Presented to The University of Guelph

In Partial Fulfilment of Requirements for the degree of Doctor of Philosophy In Pathobiology

Guelph, Ontario, Canada ©Maedeh Darzianiazizi (AKA Mahi Azizi), January, 2020 ABSTRACT

THE ROLE OF TYPE I IFN ALPHA/BETA RECEPTOR SIGNALING AND SEX IN CYTOKINE RESPONSES TO VIRAL INFECTION

Maedeh Darzianiazizi (AKA Mahi Azizi) Advisors: The University of Guelph, 2020 Dr. Byram W. Bridle Dr. Khalil Karimi

Viruses are one of the main causes of morbidities and mortalities in humans. However, the outcome of a viral infection can be different between individuals and the cellular and molecular mechanisms underlying the discrepancy in the outcome of a viral infection are not well- understood. Host innate antiviral responses are largely controlled by type I (IFNs) signaling through their specific receptor, type I IFN α/β Receptor (IFNAR). Virus-induced aberrant

IFNAR signaling has been shown to be associated with excessive inflammatory responses.

Moreover, sex, as an intrinsic factor, has been implicated in differential host predisposition to severe viral infection. In this study, IFNAR-mediated antiviral cytokine responses of male andfemale mice were characterized during viremia of recombinant strain of vesicular stomatitis virus (rVSVΔm51). Our findings highlighted a crucial role for IFNAR signaling in the negative regulation of plasma and hepatic antiviral cytokine responses that could be influenced by sex while

IFNAR signaling was disrupted or absent. Further, hepatic IFNAR-mediated antiviral cytokine responses were shown to be influenced by sex which contributed to differential liver damage strongly biased against females. Our in-vitro studies on mouse bone marrow-derived mast cells

(BMMCs) infected with rVSVΔm51-infected, implicated mast cells as potential source of exaggerated cytokine responses to rVSVΔm51 when IFNAR-mediated signaling is impaired or absent. We showed that mice BMMCs are infected by VSV and produce pro-inflammatory in response to rVSVΔM51 infection and that IFNAR signaling is required to shut these responses down and protect the cells from virus-induced death. These findings highlight: 1) a crucial role for IFNAR signaling in negative regulation of systemic and local anti-viral cytokine responses which can be influenced by sex during viremia associated with the lack of IFNAR signaling. 2) A sexually dimorphic landscape of IFNAR-mediated antiviral responses which can make the host either susceptible or resistant to immune-mediated pathogenesis during viremia associated with the lack of IFNAR signaling. 3) Further understanding of sex-mediated antiviral pathways can lead to development of novel, sex-tailored anti-inflammatory therapeutics. 4) Mast cells might represent a therapeutic target to limit excessive anti-viral inflammatory responses resulted from disrupted IFNAR signaling, particularly in females.

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ACKNOWLEDGEMENTS

I would like to wholeheartedly express my gratitude to my advisors Dr. Byram Bridle and Dr.

Khalil Karimi for giving me the opportunity to think about the subject of my thesis and touch my capabilities and most importantly, for their genuine support and supervision throughout this programme, particularly during running of in-vivo experiments and thesis preparation. I also thank my advisory committee, Dr. Shayan Sharif and Dr. Ravi Kulkarni for their constructive comments and guidance throughout this programme. I would also like to thank Dr. Bridle Lab members for their assistance and support. My special thanks, however, go to our former technician, the legendarily kind and devoted Mary Ellen Clark, whose presence and support was heart warming for all of us. I would also like to express my gratitude to supportive personnel of central animal facility and isolation unit, in particular, Janet Cugan, Linda Groocock, and Abiran Sritharan for mice monitoring as well as mice sacrifice. I also want to express my thankfulness to my advisors,

Dr. Byram Bridle and Dr. Khalil Karimi for their time and effort for mice injections and bleedings.

My additional thanks go to my advisor, Dr. Khalil Karimi for monitoring mice inventory as well as mice sacrifice. My deepest deepest gratitude, however, goes to my beloved family whose genuine love and support from overseas made this step of my life easier. Baba, since your diagnosis with Parkinson’s disease, it has been a shame for me, as a researcher, that I could not be involved in research making your healing possible. But I bet I will be the Coco Chanel of new era! My gorgeous Maman, I have always been so grateful for the genes for honesty, ethics, discipline, integrity, diligence, intellect, and immense desire to learn new things passed down from you and

Baba that I believe helped me accomplish this step of my life, of which mainly occurred internally, within my precious self. And last but not least, I would like to loudly thank my darling, my beloved v husband, my genuine friend since childhood, for his love and whole-hearted support and preciously, for his attention to all those possibilities that I had imagined for my PhD project. I look forward to giving him my warmest, biggest hug and of course, the reddest kiss ever on his forthcoming success scene.

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TABLE OF CONTENTS

ABSTRACT ...... ii ACKNOWLEDGEMENTS ...... iv TABLE OF CONTENTS ...... vi LIST OF FIGURES ...... ix LIST OF TABLES ...... x LIST OF ABBREVIATIONS ...... xi Chapter 1: Review of the Literature ...... 1 1. Introduction ...... 2 2. Innate Immune Response to Viruses ...... 3 2.1. Recognition of Viral PAMPs by Innate Sensors ...... 3 2.1.1 Cytoplasmic Sensors ...... 3 2.1.2 Membrane-Localized Sensors ...... 4 2.2. Host Inflammatory Responses to Viral Infection ...... 5 2.3. Interferons (IFNs) ...... 6 2.3.1 Type I IFNs ...... 7 2.3.1.1. The Type I IFN Response to a Viral Infection ...... 8 2.3.1.2. The Early Phase of the Type I IFN Response: Production of IFN-β ...... 8 2.3.1.3. The Intermediate Phase of the Type I IFN Response: IFN-β Signaling ...... 8 2.3.1.4. The Late Phase of the Type I IFN Response: Amplification of IFN-α/-β Genes ...... 8 2.3.1.5. Regulation of Type I IFN Signaling ...... 9 2.3.1.6. Pathogenic Type I IFN Response: The Other Side of the Coin ...... 11 2.3.1.7. Host Mechanisms to Avoid Pathogenic Type I IFN Response ...... 12 2.4. Innate Cellular Response to Viral Infection ...... 15 2.5. Factors imlicated in development of severe Viral Infection ...... 16 Excessive Infiltrating Inflammatory Cells and Development of a Cytokine Storm ...... 16 Sex Disparity in Host Immune Responses to Viral Infections ...... 18 Impaired Type I IFN Response During a Viral Infection ...... 21 Rationale ...... 22 Overall Hypotheses and Research Objectives ...... 23 References ...... 27 Chapter 2: Virus-Induced Type I IFN Response: Disrupted IFNAR Signaling and Sexually Dimorphic Cytokine Response ...... 58 Highlights ...... 59 Graphical Abstract ...... 60 vii

Abstract ...... 61 Introduction ...... 62 Materials and Methods ...... 63 Results ...... 66 Discussion ...... 69 Conclusions ...... 73 References ...... 82 Chapter 3: Impaired Type I Interferon Receptor Signaling Drives Sexually Dimorphic Virus-Induced Liver Damage ...... 91 Highlights: ...... 92 Graphical Abstract ...... 93 Abstract ...... 94 Introduction ...... 95 Methods and Materials ...... 96 Results ...... 99 Discussion ...... 103 Conclusions ...... 106 References ...... 113 Chapter 4: Type I Interferon α/β Receptor-Mediated Signaling Negatively Regulated Antiviral Cytokine Responses in Mouse Bone Marrow-Derived Mast Cells ...... 120 Highlights: ...... 121 Graphical Abstract ...... 122 Abstract ...... 123 Introduction ...... 125 Materials and methods ...... 127 Results ...... 131 Discussion ...... 135 Conclusions ...... 137 References: ...... 158 Chapter 5: Summary of Research ...... 169 Virus-induced type I interferon response: disrupted type I interferon receptor-mediated signaling and dimorphic cytokine responses ...... 170 viii

Disrupted IFNAR signaling drove sexually dimorphic virus-induced liver damage ...... 172 Virus-induced differential liver damage during impaired IFNAR signaling was associated with dimorphic liver-infiltrating neutrophils and ...... 175 IFNAR signaling negatively regulated antiviral cytokine response in mast cells and protected the cells from virus-induced death ...... 176 Conclusions ...... 178 References: ...... 179

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LIST OF FIGURES

Figures of Chapter 1: ...... 25 Figure 1. Virus-induced inflammatory responses...... 25 Figure 2. The Triphasic Model of the Type I IFN genes Induction...... 26 Figures of Chapter 2: ...... 75 Figure 1. Mice develop a systemic inflammatory response to recombinant vesicular stomatitis virus (rVSV∆m51) that was characterized by an increase in the frequency of granulocytes in blood, which was potentiated by type I interferon receptor (IFNAR)-blockade...... 75 Figure 2. Female mice with impaired type I interferon receptor (IFNAR) signaling show acute reductions of body temperatures after viral infection...... 76 Figure 3. Virus-induced responses were exaggerated in females with disrupted type I interferon receptor-mediated signaling...... 77 Figure 4. Female mice had an impaired ability to negatively regulate cytokine responses to recombinant vesicular stomatitis virus (rVSV∆m51) during type I interferon receptor (IFNAR)-blockade...... 78 Figure 5. Type I interferon receptor (IFNAR) signaling is required for viral clearance...... 79 Figures of Chapter 3: ...... 107 Figure 1. Mice treated with a recombinant vesicular stomatitis virus (rVSV∆m51) have elevated concentrations of plasma cytokines compared to naïve mice, with evidence of sexual dimorphism...... 107 Figure 2. Intravenous administration of recombinant vesicular stomatitis virus (rVSV∆m51) result in infection of livers, along with local inflammatory cytokine responses but with no evidence of sexual dimorphism...... 108 Figure 3. A lack of type I interferon receptor (IFNAR) signaling causes dysregulation of cytokine responses to recombinant vesicular stomatitis virus (rVSV∆m51), which is influenced by sex...... 109 Figure 4. Male and female mice with impaired type I interferon receptor (IFNAR) signaling exhibit differential liver injuries following administration of recombinant vesicular stomatitis virus (rVSV∆m51)...... 111 Figures of Chapter 4: ...... 139 Figure 1. Mouse bone marrow-derived cells cultured in the presence of pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) contained granules and exhibited features of mast cells...... 139 Figure 2. Mouse bone marrow-derived mast Cells (BMMCs) produced (IL)-6 and tumour factor-α (TNF-α) in response to phorbol myristate acetate (PMA)/ Ionomycin...... 141 Figure 3. Mouse bone marrow-derived mast Cells (BMMCs) showe susceptibility to infection with vesicular stomatitis virus (VSV)...... 142 Figure 4. Mouse bone marrow-derived mast Cells (BMMCs) produce interleukin (IL)-6 and tumour necrosis factor-α (TNF-α) in response to recombinant strain of vesicular stomatitis virus (rVSVΔm51)...... 144 Figure 5. kinetics of cytokine production by bone marrow-derived mast Cells (BMMCs) after infection with a recombinant strain of vesicular stomatitis virus (rVSVΔm51)...... 145 Figure 6. Cytokine responses of bone marrow-derived mast Cells (BMMCs) to recombinant strain of vesicular stomatitis virus (rVSVΔm51) was dose-dependent...... 148 Figure 7. Infection of bone marrow-derived mast Cells (BMMCs) with recombinant strain of vesicular stomatitis virus (rVSVΔm51) resulted in cell death...... 150 Figure 8. Recombinant strain of vesicular stomatitis virus (rVSVΔm51)-induced cytokine production by bone marrow-derived mast Cells (BMMCs) was regulated by Type I IFN α/β Receptor (IFNAR) signaling...... 151 Figure 9. Type I IFN α/β Receptor (IFNAR) protected mice bone marrow-derived mast Cells (BMMCs) from Recombinant strain of vesicular stomatitis virus (rVSVΔm51)-induced cell death...... 156

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LIST OF TABLES

Table 1. Key Resource Table ...... 80 Table 2. Mice infected with a recombinant vesicular stomatitis virus (rVSV∆m51) with concomitant blockade of their type I interferon receptors showed signs of sickness...... 81

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LIST OF ABBREVIATIONS

AIM2 Absent in 2-like receptors ASC Apoptosis-associated speck-like AdV Adenovirus AEC Airway epithelial cells AFT2 Activating transcription factor 2 AREG Amphiregulin ANOVA Analysis of variance ALT Alanine aminotransferase BM Bone marrow BMMCs Bone marrow-derived mast cells CXCL CXC ligand cGAS Cyclic GMP-AMP (cGAMP) synthase DAI DNA-dependent activator of IFN-regulatory factor ds Double-stranded EBV Epstein-Barr Virus FBS Fetal bovine serum GPs Glycoproteins HIV-1 Human immunodeficiency virus-1 hpi Hours post-infection HBSS Hanks' balanced salt solution HSV-1 Herpes simplex virus-1 IFI16 IFN-inducible protein 16 IRF IFN regulatory factor IL Interleukin IFN Interferon IFNAR-/- IFNAR-knock out IFN-β Interferon-β IFNAR IFN-α/β receptor ISGs IFN-stimulated genes ISGF3 IFN-stimulated gene factor 3 ISREs IFN-stimulated response elements IFNGR IFN-γ receptor IAV Influenza A virus JAK/STAT /signal transducer and activator of transcription KC Keratinocyte chemoattractant LOD Limit of detection MΦs Macrophages MCMV Murine cytomegalovirus MHV68 Murine herpesvirus 68 MAVS Mitochondrial antiviral signaling MyD88 Myeloid differentiation primary response protein 88 MCP-1 Monocyte chemoattractant protein-1 xii

MIP-2 inflammatory protein 2 MMPs Matrix metalloproteases MT1 Membrane type I MCs Mast cells MDA5 Melanoma differentiation-associated protein 5 MOI Multiplicity of infection MCT Mast cell tryptase that produce tryptase MCTC Mast cell tryptase and chymase that produce tryptase and chymase. NAs Nucleic acids NLRs Nucleotide-binding oligomerization domain (NOD)-like receptors NLRP3 Nod-like receptor family pyrin domain containing 3 NF-κB Nuclear factor kappa--chain-enhancer of activated B cells NDV Newcastle disease virus PAMPs Pathogen-associated molecular patterns PRRs Pattern recognition receptors Pol Polymerase 5′-PPP 5′-triphosphate pDCs Plasmacytoid dendritic cells PIAS I Protein inhibitors of activated STAT PWM-SCM Pokeweed mitogen-stimulated spleen cell-conditioned medium PMA Phorbol myristate acetate PBS Phosphate-buffered saline PFU Plaque-forming units RIG-I Retinoic acid-inducible gene I RSV Respiratory syncytial virus rVSVΔm51 Recombinant vesicular stomatitis virus delta m51 STING Stimulator of interferon gene ss Single-stranded SOCS1 Suppressors of cytokine signaling SEOV Seoul hantavirus TLR Toll-like receptor TRIF TIR-domain-containing adapter-inducing interferon-β TRAM TRIF-related adaptor molecule TGF-β Transforming -β TNF-α -α TCID50 50% tissue culture-infected dose USP18 Ubiquitin carboxy-terminal hydrolase 18 VACV Vaccinia virus VSV Vesicular stomatitis virus VZV varicella-zoster virus WNV West Nile virus WT Wild-type

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Chapter 1: Review of the Literature

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1. Introduction

Hosts encounter a broad range of pathogenic and non-pathogenic microorganisms. While the harmless ones (e.g. commensal bacteria) are often attenuated, the immune system should mount an immediate response to pathogenic microorganisms. All aspects of such first and rapid lines of defense are referred to as innate immunity. The invading pathogen might be controlled by the innate immune response. Otherwise, another arm of the immune system, known as the adaptive immune system, consisting of highly specialized leukocytes is induced to eliminate the invading pathogen. The innate immunity is initiated with recognition of pattern-associated molecular patterns (PAMPs) by specific pattern recognition receptors (PRRs), or sensors, localized in different parts of cells of the immune system, including macrophages (MΦs) and neutrophils. The

PAMPs include conserved molecular structures of pathogenic microorganisms, such as nucleic acid (NA) motifs, glycoproteins (GPs), and cell wall components of bacteria such as lipopolysaccharides and proteoglycans, which are essential for their survival and/or infectivity. Several endogenous molecules including adenosine triphosphate [1], high mobility group box 1 [2], endogenous DNA [3], and mitochondrial components [4] can also be sensed by

PRRs. These molecules are known as danger-associated molecular patterns (DAMPs) providing alarm signals to the immune system in the event of sensing cell/tissue damage. Recognition of danger-associated molecular patterns, in the absence of pathogenic threats, can also trigger inflammatory pathways [5]. Many viruses replicate in the cytoplasm of cells following entry by direct fusion with the plasma membrane or with the membrane of endosomal compartments after endocytosis. The immune system can recognize viruses via detection of viral nucleic acids (NAs)

(DNA and RNA) or their replicative intermediates by innate sensors localized in different parts of a cell [6]. 3

2. Innate Immune Response to Viruses

2.1. Recognition of Viral PAMPs by Innate Sensors

2.1.1 Cytoplasmic Sensors

Within the cytoplasm of a cell, viral DNA can be recognized by a variety of sensors including nucleotide transferase cyclic GMP-AMP (cGAMP) synthase (cGAS) [7], DNA-dependent activator of IFN-regulatory factor (DAI) [8], absent in melanoma 2-like receptors (AIM2) [9], and

IFN-inducible protein 16 (IFI16) [10]. Cytoplasmic DNA is the main ligand of cGAS. cGAS is involved in defense against many DNA viruses, including herpes simplex virus-1 (HSV-1),

(VACV), MHV68, and adenovirus (AdV) [11-13]. Emerging evidence, however, also indicates the importance of cGAS and its stimulator of interferon gene (STING) adaptor protein in immunity against RNA viruses such as West Nile virus (WNV) and vesicular stomatitis virus (VSV) [11].

AIM2 appears to exclusively sense cytosolic dsDNA viruses, including murine cytomegalovirus

(MCMV) and VACV [14], whereas IFI16 has been reported to be capable of detecting viral dsDNA both in the nucleus and cytoplasm. IFI16 has also been found to cooperate with cGAS to detect atypical negative-sense ssDNA (-) carrying long stem-loop structures produced during reverse transcription of human immunodeficiency virus-1 within the cytoplasm [15, 16]. RNA:

DNA hybrids, which can be products of retroviral reverse transcription, are also thought to be potential ligands for cGAS [17]. Cytoplasmic viral RNAs can be sensed by nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) [18], as well as a well-studied family of receptors including retinoic acid-inducible gene I (RIG-I) and melanoma differentiation- associated protein 5 (MDA5) that distinguish between different classes of viral RNAs in the cytosol [19-21]. 4

RIG-I is activated following detection of short viral dsRNA molecules as well as (-) ssRNA viruses such as VSV, influenza virus, and viruses from the paramyxovirus family. RIG-I appears to distinguish non-self RNA from endogenous RNA by sensing the uncapped 5′-triphosphate (5′-

PPP) motif present in viral-derived RNA [22, 23]. RIG-I has also been shown to be capable of sensing DNA viruses such as Epstein-Barr virus (EBV) [24], HSV-1 [25] and AdV [26]. Cellular

DNA-dependent RNA polymerase (Pol) III, localized in the cytoplasm and nucleus, has also been found to mediate the induction of immune responses against EBV, HSV-1, and AdV by converting classical B-form DNA to a RIG-I-inducing dsRNA with uncapped 5′-PPP motif [27].

MDA5 is preferentially activated by long (>2kb) dsRNA, specifically sensing the masked 5′- ppp motif that is generated during replication of positive (+) ssRNA viruses such as encephalomyocarditis virus from the family of picornaviridae [20, 23]. HSV-1 DNA virus has also been shown to be recognized by MDA5 in human primary MΦs, independently of RNA Pol

III [28] (Figure 1).

2.1.2 Membrane-Localized Sensors

Within the endosomal compartment, viral ssRNA can be sensed by endosomal Toll-like receptor (TLR)7 and TLR 8 [29], whereas viral dsRNA can be recognized by TLR3 [30]. TLR9, on the other hand, senses (viral or bacterial) DNA bearing unmethylated CpG motifs [31, 32]. Other than NAs, viral proteins can also be sensed by PRRs. For instance, plasma membrane-localized sensors including TLR4 and TLR2 that are well-established in anti-bacterial responses, have also been found capable of sensing viral GPs during viral entry into cells. TLR2, in complex with TLR1 or TLR6, has been shown to sense GPs of HSV-1, EBV, VSV, and VACV. Despite an earlier paradigm of TLR2 being incapable of inducing antiviral responses, TLR2 has been shown to differentially respond to viral ligands as compared with bacterial PAMPs. respiratory syncytial 5 virus (RSV) fusion protein and VSV glycoprotein G can also be sensed by TLR4 on plasma membrane of a cell [33-38] (Figure 1).

2.2. Host Inflammatory Responses to Viral Infection

Recognition of viral PAMPs, including viral NAs and GPs, by PRRs expressed by hematopoietic and non-hematopoietic cells of the immune system results in the activation of intracellular signaling pathways mediated by a number of interconnected adaptor proteins.

cGAS, the well-established cytoplasmic DNA sensor, signals through cGAMP-mediated interaction with STING adaptor protein located on endoplasmic reticulum. Cytoplasmic RNA sensors, RIG-I and MDA5, induce immune response through interaction with mitochondrial antiviral signaling (MAVS) adaptor protein. TLRs, on the other hand, signal through different adaptor proteins. While signals from activated TLR7 and 8 are transmitted via myeloid differentiation primary response protein 88 (MyD88)-mediated cascade [39], TLR3 and TLR4 signals are transmitted through TIR-domain-containing adapter-inducing interferon-β (TRIF) adaptor protein [40, 41]. TLR4, however, can signal through three other adaptor proteins, including

TRIF-related adaptor molecule (TRAM), MyD88, and Toll interleukin-1 receptor-associated protein (TIRAP) [42-44]. Following recognition of viral GPs, on plasma membrane, TLR2 is translocated into endosomal compartment wherein by interacting with MyD88 adaptor protein it activates TRAF3-mediated signaling cascade [45]. The virus-induced intracellular signaling pathways eventually lead to IFN regulatory factor (IRF)-mediated upregulation of IFNs and ISGs.

In addition, a group of other cytokines, belonging to a large family of pro-inflammatory cytokines including interleukin (IL) IL-6, tumer necrosis factor- α (TNF-α), IL-12, IL-1, IL-8, and the CC, CXC, CX3C, and the C family of , including monocyte chemoattractant protein-1 and macrophage inflammatory protein 2 are also induced through tumor necrosis factor 6 receptor-associated factor 6 (TRAF6)-mediated activation of nuclear factor kappa-light-chain- enhancer of activated B cells (NF-κB) pathway that play an important role in recruitment of the innate immune cells to infected tissues [6, 46-58]. In addition to induction of inflammatory signaling pathways, viral PAMPs have been shown to induce -mediated activation and release of pro-inflammatory cytokines and chemokines including IL-1β and IL-18 [59-61].

Inflammasomes are another component of the innate immune system involved in antiviral immune responses and consist of a complex of proteins, including a sensor (a PRR, for instance), apoptosis- associated speck-like (ASC) adaptor protein, and enzymatic element (procaspase-1) [62]. Upon sensing of viral stimuli, activated PRRs, including Nod-like receptor family pyrin domain containing 3 (NLRP3), RIG-I, AIM2, and IFI16, are capable of forming inflammasome complexes which ultimately result in the induction of inflammatory IL-1β and IL-18 cytokines, as well as inflammatory-mediated cell death known as pyroptosis [63-70] (Figure 1).

2.3. Interferons (IFNs)

IFNs are a large family of pleiotropic cytokines playing a key role in host antiviral defense.

IFNs have been classified into three distinct groups, type I, type II, and type III IFNs, based on sequence identity, cognate receptors, biological functions, and sources of origin [71-74].

In humans, type I IFN family consists of 13 IFN-α genes and a number of single gene products, including IFN-β, IFN-κ, IFN-ε, and IFN- ω [75]. In mice, there are at least 14 IFN-

α subtypes, a single IFN-β isoform, and multiple other subtypes [76]. Almost all nucleated cells are capable of producing type I IFNs, however, the natural IFN-producing cells, also known as plasmacytoid dendritic cells, produce more type I IFNs on a per-cell basis than any other cells in the body [77]. Type I IFNs are produced upon viral infection and exert 7 their antiviral effects through interaction with their cognate receptor, IFN-α/β receptor (IFNAR) expressed on almost all cells, including leukocytes [78-80].

Type II IFN family has a single member, IFN-γ, that interacts with the IFNGR that is expressed on a broad range of cell types. However, in contrast to type I IFNs, IFN-γ is mainly produced by cells of the immune system [71]. Type III IFNs, on the other hand, include four subtypes: IFN-λ1 (IL29), IFN-λ2 (IL28A), IFN-λ3 (IL28B) and IFN-λ4. Like type I IFNs, this class of cytokines is induced by viruses, however, compared to the broad impact of type I

IFNs, the antiviral activity type III IFNs appears to be restricted to epithelial cells. These cytokines have been shown to be important primarily in protection of the respiratory tract and the gut epithelium against viral infections [81-86]. The tissue-specific effects of these cytokines can be explained by the distribution of their receptor, IL28RA, which is mainly expressed on epithelial cell surfaces116. In spite of signaling through different unconnected receptors, type I and III IFNs trigger similar downstream signaling cascades and therefore represent similar biological functions

[87, 88].

2.3.1 Type I IFNs

Understanding the biology of type I IFNs has been a growing interest since the first description of IFNs mediating cellular resistance to viral infection by Issacs and Lindenmann in 1957 [89,

90]. Numerous functions have since been ascribed to these versatile cytokines, including the induction and modulation of immune responses against invading pathogens. These cytokines also play a critical role in maintaining cell homeostasis by contributing to regulation of cell proliferation, development, and differentiation [91, 92]. However, their critical role in host antiviral defense has long been established [93]. 8

2.3.1.1. The Type I IFN Response to a Viral Infection

A triphasic (early, intermediate, and late) model of type I IFN response has been proposed from extensive mice studies [94-96] which is depicted in Figure 2.

2.3.1.2. The Early Phase of the Type I IFN Response: Production of IFN-β

As mentioned, viral recognition by innate cells of the immune system results in the induction of intracellular signaling pathways culminating in upregulation of IFN-β gene and ISGs [55-58]

(Figure 2A).

2.3.1.3. The Intermediate Phase of the Type I IFN Response: IFN-β Signaling

Autocrine feedback of IFN-β with IFNAR on virus-infected cells mediates the intermediate phase of the response beginning with the activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway, leading to formation of IFN-stimulated gene factor 3 (ISGF3) transcription factor complex, which consists of phosphorylated

STAT1/STAT2 and IRF9. Binding of ISGF3 to ISREs in the promoter region of ISGs upregulates numerous IFN target genes, including IRF7. Like IRF3, activated cytoplasmic IRF7 translocates to the nucleus to induce the induction of IFN-α genes [78, 97-99] (Figure 2B).

2.3.1.4. The Late Phase of the Type I IFN Response: Amplification of IFN- α/-β Genes

The late phase of IFN response is mediated by a positive feedback loop, through which IRF7 and IRF3 cooperate to induce robust induction of IFN-α and -β genes, respectively. This, in turn, leads to the induction of numerous ISGs mediating type I IFN-driven antiviral responses [55-58,

97, 100-102] (Figure 2C). Amplification of IFN α/β genes in the late phase of the type I IFN response is associated with boosted pro-inflammatory responses, resulting in increased infiltration 9 of type I IFN-producing-effector cells and thereby further amplification of in inflammatory responses. Ultimately, type I IFNs not only promote apoptosis of virus-infected cells but also prevent virus spread into uninfected neighboring cells via paracrine interaction with IFNAR on the surface of those cells [103, 104] (Figure 2D). While IRF3 is essential for both early and late phases of IFN response, IRF7 is a critical component of the positive feedback loop during the late stage of IFN response. Unlike IRF3, which is constitutively expressed in uninfected cells, IRF7 is a transient, short-lived protein being produced and phosphorylated in response to IFN-α/-β signaling within the virus-infected cells [102].

In a steady state and in the absence of a viral invasion, intrinsically expressed transcription factors, IFN regulatory factor IRF1, NF-κB, Activating transcription factor 2 (AFT2)/c-Jun, and

IRF3, induces a basal expression of IFN-β and a subset of their target genes known as ISGs [55-

58]. Indeed, IRF3 was shown to be constitutively expressed in uninfected mouse embryonic. At this stage, IRF7 has been found to be expressed at a very low level as a result of constitutive, basal signaling of IRF3-induced IFN-β [94-96]. Given that IRF7 induces IFN-α gene expression, it is speculated that a basal expression of IFN-α and -β provides an uninvaded cell with some level of intrinsic protection against an invading virus (Figure 2E). However, a constitutive and IRF-3/IRF-

7-independent expression of IFN-α and -β genes in uninvaded cells has been reported to determine the efficacy of positive feedback mechanism upon viral invasion [102].

2.3.1.5. Regulation of Type I IFN Signaling

Type I IFN response has been proposed to develop gradually over time from early, limited to a full-blown response [96] (Figure 2). Such gradual development of the IFN response has also been observed in lungs of mice during infection with the WSN strain of IAV. While early stages of viral infection could be controlled by limited IFN responses, later stages of infection required a 10 robust response for ultimate control of viral infection, and this was associated with severe pathology driven by overly robust inflammatory responses [105]. The early phase of the type I

IFN response has been shown to be driven by IRF3 inducing low levels of type I IFNs, which is amplified over time through an autocrine feedback loop mediated by both IRF3 and IRF7 leading to robust induction of type I IFNs [96, 106].

Development of a robust type I IFN response can also be achieved by recruiting cells that secrete high concentrations of type I IFNs. For example, pDCs with constitutive expression of

IRF7, have been shown to rapidly produce massive amounts type I IFNs (IFN-α, β, ω) in response to viral challenges in vivo [77, 107, 108]. Moreover, immediately after production of such enormous amounts of IFNs, pDCs were shown to be differentiated (through autocrine type I IFNs and TNF-α feedback loop) into mature DCs capable of expanding the antiviral response by activation of other innate effector cells, including natural killer cells, immature myeloid DCs, as well as B and T from the adaptive arm of immune system [77]. Antiviral responses of pDCs have been shown to be downregulated by type I IFNs [109]. A negative feedback loop independent of type I IFNs for downregulation of antiviral responses of pDCs during non-viral inflammation in mice has also been described [110]. These findings suggest that potent inflammatory cells are tightly controlled in the context of acute inflammation.

Ly6C+ monocytes and myeloid DCs have also been reported to be involved in the production of type I IFNs during viral infection [33, 111].

To maintain homeostasis and limit the immune-mediated tissue damage, cellular response to type I IFNs is dynamically controlled by a complementary, interdependent network of negative regulators [112]. Among the inhibitory mechanisms downregulating type I IFN signaling are internalization and subsequent degradation of IFNAR to reduce cellular response to type I IFNs. 11

The magnitude and duration of type I IFN responses, however, are downregulated by the induction of negative feedback loop involving ubiquitin carboxy-terminal hydrolase 18 (USP18), suppressors of cytokine signaling 1 (SOCS1), and protein inhibitors of activated STAT (PIAS I) proteins. While SOCS interfere with JAK-mediated phosphorylation and activation of STATS,

USP18 dissociate JAK1 from cytoplasmic tail of the IFNAR. By occupying IFNAR binding sites,

SOCS proteins also prevent IFNAR-mediated phosphorylation of STAT proteins [113, 114]. PIAS

I, on the other hand, suppresses type I IFN signaling by targeting DNA binding site of STAT1 in the nucleus [115]. Differential expression of downstream stimulatory or inhibitory genes can also be induced by preferential activation of STAT proteins. Various homo- and heterodimer forms of

STAT proteins are formed in response to type I IFNs [116]. For instance, through the formation of STAT1 homodimers, IFNAR gives rise to transcriptional activation of numerous ISGs and therefore establishment of cellular antiviral state [117]. By activation of STAT3 homodimers, however, type I IFNs exert their immunomodulatory effects through inhibition of STAT1-mediated gene activation [106]. Post-translational modifications, including phosphorylation or de-phosphorylation of type I IFN signaling components are among other mechanisms to restrain type I IFN responses [118, 119].

2.3.1.6. Pathogenic Type I IFN Response: The Other Side of the Coin

In spite of their critical role in host defense against viral infections, type I IFNs have been implicated in the promotion of multiple viral- [120, 121] and non-viral infection-associated pathologies [122, 123], as well as immune-mediated inflammatory diseases [124]. The beneficial or detrimental outcome of type I IFN response during viral infections has been found to be context dependent . The duration and magnitude of the response appear to be critical for efficient viral clearance without destructive consequences of sustained activation of immune responses [120, 12

125-129]. In other words, a protective IFN response requires an intricate balance between stimulatory and modulatory responses. While it promotes effective clearance of infection, it also facilitates a return to homeostasis.

2.3.1.7. Host Mechanisms to Avoid Pathogenic Type I IFN Response

Given the ubiquitous expression of IFNAR and the prevalence of potent IFN-producing cells in the immune system, the body has a number of strategies allowing early, local antiviral responses to be triggered before transitioning to a systemic, robust, and potentially pathogenic type I IFN response. Intrinsic low-level expression of ISGs (independent of virus-induced IFNs) during homeostatic conditions, has been shown to protect hosts from viral infections [130]. Such constitutive expression of ISGs appears to determine susceptibility of a host to infection with viruses [131] such as IAV [132] and reovirus [133]. Moreover, as mentioned, type III IFNs play a major role in restraining viral infection of epithelial cells. This can result in diminishing the accumulation of type I IFN response-inducing viral PAMPs, thereby reducing the risk of developing systemic inflammatory responses and associated immunopathology. This phenomenon was demonstrated in an in-vivo study evaluating the treatment efficacy of IFN-α and IFN-λ in the setting of infection with IAV [134]. Therapeutic administration of IFN-α and IFN-λ differentially affected the outcome of infection with IAV. Treatment with exogenous IFN-α exacerbated infection-associated pathology. Specifically, viral replication was limited but concentrations of inflammatory cytokines in bronchoalveolar lavage fluids were augmented and associated with increased infiltration of inflammatory cells (including pDCs and inflammatory monocytes) into lungs, and increased apoptosis of airway epithelial cells (AEC). Administration of exogenous IFN-

λ, however, was shown to reduce viral spread without inflammatory side effects. This protective effect was attributed to restriction of both IAV replication and IFN-λ-induced responses of 13 pulmonary epithelial cells and an inability of IFN-λ, unlike IFN-α, to directly stimulate cells of the immune system. Moreover, IFN-α but not IFN-λ, was found to have a pro-inflammatory effect on bone marrow-derived MΦs, conventional DCs, and pDCs, in-vitro. Moreover, in-vitro cultures of

AEC were shown to induce antiviral genes but not inflammatory cytokines in response to both

IFNα and IFN λ, suggesting the importance of local and restricted antiviral responses in reducing side-effects of systemic inflammatory responses [134].

Another example of the restriction of IFN responses at mucosal sites would be physiological production of IFN-ε from the type I IFN family [135]. Despite sharing little homology with

IFN-α and –β, IFN-ε has been shown to use IFNAR for signaling [136, 137]. However, its expression in several mucosal tissues including the gut, lungs, vagina and cervix of Indian rhesus macaques has been found to be independent of viral invasion [138].

Moderate type I IFN responses with limited inflammatory responses, have also been described in the setting of viral evasion. Fusion of virus-like particles with cell membranes has been shown to activate a non-canonical IFN induction pathway driven by STING-IRF3. However, in contrast with what was expected, the STING-mediated IFN response was shown to be induced by virus- cell membrane fusion interrupting the uniformity of the cell membrane and independently of

PRR-mediated detection of viral NAs and capsid proteins [139]. The disturbance of membrane integrity had already been demonstrated to be sufficient for induction of innate antiviral responses in the presence or absence of IFN production [139-145]. Other instances of antiviral states have also been reported to be established in the absence of type I IFNs (IFNα/β) [140, 146, 147].

Recently, a STING- mediated antiviral pathway in epithelial cells against HSV-2 was reported to operate before the induction of type I IFN (IFN-α and-β) responses. The IFN-independent pathway was shown to be triggered by a mechanism independent of PRR-mediated sensing of HSV-2 but 14 relied on viral O-linked glycans. Even the disturbance of mucosal integrity was shown to be capable of triggering the pathway. The pathway was also found to be highly dependent on neutrophils recruited by CXCR3 chemokines, including CXCL9 and CXCL10. Moreover, mice that were unable to activate the neutrophil-dependent antiviral pathway were found to suffer from elevated inflammatory responses mediated by IFN-inducing TLR signaling pathways [148].

Exaggerated immune responses arising from excessive production of type I IFNs have also been observed in other settings where antiviral mechanisms functioning independently of IFNα/β, were abrogated [137, 149].

Regarding host IFN-dependent and -independent antiviral responses, peroxisomal and mitochondrial MAVS have been shown to function in a consecutive manner towards establishment of type I IFN-dependent and-independent antiviral state, respectively. In the event of viral invasion, peroxisomal MAVS launches an immediate antiviral cascade from peroxisome membrane leading to induction of early, interferon-independent genes restraining viral replication until a robust and sustained antiviral response is initiated via mitochondrial MAVS with delayed kinetics of IFN-α/-β gene expression. In addition to this, the IFN-independent peroxisomal MAVS pathway is thought to be important in restraining viruses that interfere with cellular type I IFN responses such as VSV. In line with this notion, cells expressing mitochondria-only MAVS showed the same susceptibility to VSV infection as MAVS-deficient cells [150, 151].

A consecutive order of local and systemic antiviral responses has also been reported in the case of IAV that preferentially invades and start replicating in the epithelial cells of mammalian upper respiratory tract. IFN-λ has been shown to establish a local, non-inflammatory defense against IAV within the epithelial cells, which could be followed by a systemic, inflammatory, and potentially pathogenic type I IFN responses [152]. The IFN-mediated inflammatory responses 15 were then proposed to occur predominantly in compartments beyond the epithelial cells. Consisten with this proposition, IFN-λ has recently been found to be more important than IFN-α/β in preventing influenza virus spread from upper respiratory tracts into the lungs of infected mice

[134].

In addition, ISGs have been reported to not only exert their antiviral effects via both

IFN-mediated and IFN-independent pathways [100], but also independently of these cytokines

[130, 153-158]. These findings together suggest how early, local antiviral mechanisms controlling early stages of a viral infection can prevent pathological outcomes of systemic type I IFN responses against viruses.

2.4. Innate Cellular Response to Viral Infection

Early recruitment of innate inflammatory cells into virus-infected sites is not only crucial for promoting inflammatory responses, but also for tissue regeneartion and establishment of a homeostatic state after ultimate control of a viral infection. Neutrophils are the first subset of immune cells mobilized to sites of virus infection [159]. In IAV-infected mice, lung neutrophilia was present one day post-infection and persisted for seven days [160]. Neutrophils trafficking to inflamed tissues are followed by infiltration of other cells of the immune system, including Mфs, dendritic cells, natural killer cells, and B and T lymphocytes [160-162]. Neutrophils and MΦs are major effector cells involved in promotion of inflammatory responses against a viral infection while also being involved in immune modulation and establishment of a homeostatic state following successful clearance of viruses [163-168]. In addition to infiltrating cells, residential cells such as lung- and liver-resident myeloid cells, particularly, alveolar MΦs and Kupffer cells play a major role in promotion of antiviral responses and restoration of lung homeostasis following 16 clearance of a viral infection by restraining lung-infiltrating inflammatory cells and subsequent resolution of inflammation [169-173].

2.5. Factors imlicated in development of severe Viral Infection

Viruses are one of the main causes of morbidities and mortalities in humans but the immune system can efficiently combat most viral infections. However, dysregulated immune responses predispose the host to development of immune-mediated pathologies in a variety of contexts, including viral infections. The outcome of a viral infection can be different between individuals, ranging from moderate to severe infections associated with moderate to severe immune-mediated tissue/organ damage, respectively. While intrinsic factors have been implicated in host predisposition to severe viral infections, the cellular and molecular mechanisms underlying the discrepancy in the outcome of a viral infection are not well-understood. Further understanding of host cellular and molecular antiviral responses could provide novel explanations for differential host predisposition to severe viral infections and development of immunopathology. This could facilitate the development of novel antiviral therapeutics. Of the factors that can be involved in development of severe viral infection, three factors are considered in the following.

Excessive Infiltrating Inflammatory Cells and Development of a Cytokine Storm

Despite their critical role in promotion of host antiviral responses, excessive infiltration of inflammatory cells into virus-infected and/or inflamed sites and persistent production of inflammatory cytokines create an extreme inflammatory environment leading to a severe condition where the exaggerated host immune response rather than viral cytopathic effect causes fatal tissue/organ damage [174-179]. Accordingly, comparison of HIN1 and H3N2 infection versus highly pathogenic pandemic infection of IAV strains, including H5N1, showed that early excessive inflammatory responses and massive lung- infiltrating inflammatory cells were found to 17 determine the lethal outcome of infection with H5N1 strain, compared with non-lethal H5N1 and seasonal IAV strains [180-182]. Moreover, a massive increase in plasma levels of inflammatory cytokines, particularly IL-6, and chemokines attracting neutrophil (CXC chemokine ligand

(CXCL) 8) and monocyte (CXCL10 and MCP-1) was found to underlie the fatal outcome of severe viral infections [180, 182-184].

The immune system has been programmed to maintain an optimum balance between inflammatory and anti-inflammatory responses to achieve homeostasis following a disease or trauma. While excessive inflammatory responses would secure viral clearance, it drives tissue injury. Predominate anti-inflammatory responses, on the other hand, would lead to viral persistence.

As mentioned, myeloid cells including neutrophils and macrophages are among the first leukocytes that are recruited to infection sites. They are largely involved in host inflammatory responses that have been demonstrated in substantial tissue/organ damages after severe viral infections [126, 160, 178, 185, 186].

A range of inflammatory mediators, including cytotoxic cytokines, reactive oxygen species, lipid mediators, and cationic proteins, released by neutrophils and MΦs have been reported to contribute to tissue damage during viral infections [187]. Recent data show the of matrix metalloproteases (MMPs) in irreparable destruction of lung tissues during IAV infection [188,

189]. MMPs are proteolytic enzymes, involved in remodeling of the extracellular matrix during physiological and pathological events. Under inflammatory conditions, however, substantial release of MMPs by infiltrating leukocytes have been shown to particularly contribute to pathogenesis, including in the pulmonary system [190, 191]. Accordingly, substantial release of

MMP9 and membrane type I (MT1)-MMP/MMP-14 enzymes by neutrophils and myeloid cells, 18 respectively, have been reported to significantly contribute to IAV-induced pathology and mortality [186, 188]. The importance of neutrophil-derived MMP9 had already been addressed in lung pathogenesis secondary to induced pancreatitis in rats [189]. Autopsies from victim of pandemic influenza infection revealed substantial destruction of lung tissues [192, 193]. Fatal consequences of infection with IAV have been shown to be independent of viral or bacterial burden but arise from host failure to tolerate or repair the massively damaged lung tissue. Accordingly, promoting tissue maintenance by selective depletion of aforementioned MΦ-derived MT1-MMP or upregulation of epithelial growth factor family member, AREG, amphiregulin (AREG), rescued the mice with no influence on viral burden [194, 195].

Sex Disparity in Host Immune Responses to Viral Infections

Sexual dimorphism in immune responses to non-self and self-antigens is well-documented among various species, including mammals; and the immune response in all of the studied species have been found to be generally of higher magnitude in females than in males. Accordingly, the expression of pro-inflammatory genes involved in innate immunity and development of adaptive immune responses, have consistently been found to be higher in females than males of both adult humans and rats following vaccination and virus challenge, respectively. Moreover, the frequency, severity, and the outcome of infectious diseases, including the ones mediated by viruses vary between the sexes of mammalian species [184, 196].

In the case of IAV, for instance, in spite of a higher frequency of infection in males, females were predisposed to experience greater morbidity from IAV infections and higher IAV-associated mortality [197]. Females tend to mount robust antiviral responses. While higher magnitude immune responses may allow them to accelerate clearance of an invading pathogen, it can also make them more susceptible to the development of severe pathologies driven by excessive 19 inflammatory responses. Males and females have also been shown to differentially respond to IAV vaccines. Human adult females vaccinated with a half-dose of an inactivated IAV vaccine were found to generate titers of antibodies that were equivalent to their male counterparts who received the full dose of the vaccine [198].

Differential regulation of immune responses appears to determine differential outcomes of viral infections in males and females. In the setting of imbalanced or dysregulated immune responses during a pathogenic viral infection, Seoul hantavirus (SEOV)-infected female rats showed a tendency towards development of excessive inflammatory responses, resulting in immune-mediated tissue injuries. By contrast, dysregulated responses in male rats were found to be biased towards upregulation of anti-inflammatory and regulatory responses and, therefore, viral persistence [199]. Whether the same trend would be observed with other viral infections remains to be elucidated. However, upregulated inflammatory responses and regulatory T cells in female and male rodents during SEOV infection, respectively, would support the differential regulation of immune responses in males and females [184, 199, 200].

Interactions between endocrine and immune system have been shown to play a role in differential immune responses to viral infections [201-203]. Immune response to viral infections can be influenced by variations in concentrations of sex hormones. Downregulation of sex hormones in IAV-infected female mice has been shown to be associated with higher levels of inflammatory responses, which made them more susceptible to development of pathogenic IAV infection than their male counterparts [184, 197, 204].

Generally, androgens have been found to have a suppressive effect on the activation of leukocytes and, accordingly, serum levels of testosterone have been found to be negatively correlated with antibody responses of human adult males to vaccination against IAV [205]. 20

Estradiol, on the other hand, has been shown to differentially impact immune responses, with low concentrations promoting proinflammatory and Th1 responses and high concentrations upregulating Th2-mediated antibody responses [206] while attenuating the influx of inflammatory leukocytes to infected tissues, including lungs [207]. The attenuating effect of estradiol is partially caused by a reduction in NF-ҡB-mediated inflammatory responses [208]. Accordingly, reduced levels of circulating sex steroid hormones, including testosterone and estradiol (E2), were detected in HIN1-infected male and female mice, respectively. While the protective effect of the circulating androgens remains to be clarified, the pathogenic and protective role of low and high concentrations of circulating estradiol was documented in H1N1-infected female mice [184].

Consistent with the protective effect of high concentrations of female sex hormones (E2) treatment of adult female mice with exogenous progesterone was found to promote lung homeostasis following severe IAV infection by changing the immune setting of the lungs from inflammatory to a regulatory milieu characterized by increased concentrations of IL-22, transforming growth factor (TGF)-β, IL-6, the number of regulatory CD39+ Th17 cells, and upregulated pulmonary epithelial cell-derived AREG, which promoted repair of pulmonary tissues, without affecting viral burden [209]. Treatment of female mice and humans with either exogenous E2 or P4 reduced inflammatory responses and restored lung homeostasis, was found to be protective against influenza virus pathogenesis and auto-immune diseases [197, 209-211].

In addition, sex hormones have been shown to influence host immune responses at the gene expression level. The promoters of several genes encoding innate components of the immune system have been reported to contain androgen and estrogen response elements [212]. Moreover, the host gut microbiome has also been proposed to be influenced by sex hormones and the composition of gut microbiome, in turn, regulates the levels of sex hormones, thereby influencing 21 the expression of immune-related regulatory genes in a sex-specific manner [213]. The aforementioned factors can experience unwelcome changes throughout life [196], which can also underlie age-biased immunity to viral infections.

Impaired Type I IFN Response During a Viral Infection

As mentioned, host innate antiviral response is largely controlled by type I IFNs which exert their antiviral and immunomodulatory effects by interaction with IFNAR [214-217]. As intracellular obligatory parasites, viruses have evolved strategies to compromise host type I

IFN-mediated antiviral responses. Despite their well-established protective roles against invading pathogens [218, 219], virus-induced aberrant type I IFN responses have been associated with toxic inflammatory responses and development of immunopathology [120, 125, 152, 220-227]. IAV- induced type I IFN responses have been shown to be directly associated with high levels of inflammatory cytokines/chemokines, massive infiltration of inflammatory cells, including excessive IFN- producing pDCs, and potent expression of TNF-related apoptosis-inducing ligand and on inflammatory monocytes and epithelial cells, respectively, which all together were shown to fuel substantial lung tissue damage [120]. While the pathogenic role of type I IFNs has been demonstrated in the setting of IAV infection [120, 134, 152], protective virus- induced type I IFN response has also been reported in the context of infection with IAV strain

A/Puerto Rico/8/34 (PR8/H1N1). PR8/H1N1-infected IFNAR KO mice experienced severe lung inflammation and pathology characterized by massive lung infiltrating neutrophils mediated by

Keratinocyte chemoattractant (KC)-producing Ly6Chi monocytes [126]. This could be explained by differences in routes of virus administration, the dosage of virus, and different model of IFNAR deficient mice [120], it can also be attributed to the strain and/or type of a virus. Viruses have evolved a myriad of strategies to sabotage host antiviral defences. In turn, hosts mount antiviral 22 responses through a wide variety of parallel pathways. The strategy used by an infecting virus to interfere with host immunity seems to influence the antiviral pathway or pathways utilized by a host [100, 228-230].

Rationale

Cytokine production following recognition of an invading pathogen is part of the host immune response for ultimate clearance of the pathogen. However, excessive induction of pro- inflammatory cytokines may result in the development of acute immunopathology and potential death of the host. Indeed, many deaths associated with severe infectious diseases including viral infections [120, 125, 221] are not directly caused by the virus itself but by a dramatic increase in inflammatory responses and subsequent immunopathology. Despite strong evidence for deaths secondary to toxic cytokine responses in a wide variety of severe acute (infectious or non- infectious) diseases, the molecular and cellular mechanisms by which toxic cytokine responses contribute to pathogenesis of severe acute viral infections are not well-understood.

Host innate antiviral responses are largely controlled by type I IFNs signaling through their specific receptor, IFNAR; and viruses, as intracellular obligatory parasites, have a myriad of strategies to compromise host type I IFN-mediated antiviral responses [231].

Virus-induced aberrant IFNAR signaling has been associated with excessive and/or anomalous infiltration of effector cells into tissues, thereby promoting toxic inflammatory responses and development of immune-mediated pathologies. Moreover, sex is an important biological factor in determining the nature of antiviral responses, with females tending to mount higher magnitude pro-inflammatory cytokine responses against viruses. However, to the best of our knowledge, evidence of a direct association between sex and IFNAR-mediated antiviral cytokine responses in host predisposition to virus-induced immune-mediated pathologies is not 23 available. This study aimed to investigate the roles of IFNAR signaling and sex on cytokine responses to viral infection.

Overall Hypotheses and Research Objectives

Hypothesis 1. Compromised IFAR signaling would be associated with dysregulated proinflammatory cytokine responses.

Objective 1. Characterize the impact of antibody-mediated IFNAR-blockade or the IFNAR- knockout phenotype on the cytokine response to an attenuated, safe virus administered intravenously to mice to model viremia.

Hypothesis 2. Female mice mount more robust inflammatory responses to viruses, and these would become exaggerated in the absence of IFNAR signaling, compared to males.

Objective 2. Investigate sex-biased differences in virus-induced cytokine responses when

IFNAR signaling is disrupted during viremia.

Hypothesis 3. Dysregulated cytokine responses during impaired IFNAR signaling is associated with immunopathology.

Objective 3. Investigate the potential for dysregulated cytokine responses in the development of immunopathology by impairing IFNAR signaling during viremia.

Hypothesis 4. The innate immune cells that respond to viremia contribute to the severity of inflammation and cytokine responses while IFNAR signaling is impaired. 24

Objective 4. Identify the immunological cell subset(s) and their role(s) in dysregulated cytokine responses to viremia with concomitant IFNAR blockade.

25

Figures of Chapter 1:

Figure 1. Virus-induced inflammatory responses.

Recognition of viral pathogen associated molecular patterns (PAMPs) by innate cells of the immune system results in

Inflammatory Responses. Activation of innate cells of the immune system via pattern recognition receptors (PRRs) that recognize viral PAMPs in different parts of an innate cell of the immune system, gives rise to a number of intracellular signaling cascades mediated by various interconnected adaptor proteins which eventually leads to IFN regulatory factor (IRF)-mediated upregulation of IFNs and IFN-stimulated genes (ISGs), as well as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor-mediated induction of inflammatory cytokines and chemokines. In addition to the induction of inflammatory signaling pathways, upon sensing of viral

PAMPs-activated sensors, including nod-like receptor family pyrin domain containing 3 (NLRP3), retinoic acid- inducible gene I (RIG-I), absent in melanoma 2-like receptors (AIM2), and IFN-inducible protein 16 (IFI16), are capable of forming inflammasome complexes which ultimately result in the induction of inflammatory IL-1β and IL-

18.

26

Figure 2. The Triphasic Model of the Type I IFN genes Induction.

The expression of IFN-β gene is immediately upregulated following recognition of viral PMAPs (A). If the virus is successfully eliminated, no additional IFNs is induced. Otherwise further production of IRF3-induced IFN-β results in a continuous signaling of IFN-α/β receptor (IFNAR) signaling and thereby IRF-7-mediated upregulation of IFN-α gene. (B). Then, in late phase of viral infection, the newly produced IRF7 in cooperation with IRF3 activates a positive feedback loop to amplify the induction of IFN-α/β genes to efficiently eliminate the invading virus (C). Via paracrine interaction with IFNAR, type I IFNs create an antiviral state in bystander cells and therefore, prevent virus spread to neighboring cells (D). In a steady state and in the absence of a viral invasion, basal expression and signaling of IFN-

β through IFNAR is speculated to intrinsically protect healthy cells from invading viruses (E).

27

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58

Chapter 2: Virus-Induced Type I IFN Response: Disrupted IFNAR Signaling

and Sexually Dimorphic Cytokine Response

Maedeh Darzianiazizi‡, Katrina E. Allison‡, Ravi Kulkarni, Shayan Sharif, Khalil Karimi†,* and

Byram W. Bridle†,*

Affiliation: Department of Pathobiology, University of Guelph, 50 Stone Rd. E., Guelph,

Ontario, Canada, N1G 2W1.

‡contributed equally

†contributed equally

*Corresponding Author: Byram W. Bridle, [email protected]

Keywords: type I interferon, type I interferon receptor, interferon signaling, cytokine response, vesicular stomatitis virus, sex

This manuscript was drafted by M. Darzianiazizi and subsequently edited by B. Bridle and

K. Karimi in preparation for submission to the journal Cytokine.

59

Highlights

• Mice infected with a recombinant vesicular stomatitis virus (rVSV∆m51) developed

signs of sickness if type I interferon receptors (IFNAR) were systemically blocked with

an antibody.

• Morbidity was greater in females than in males.

• Inflammatory cytokine responses induced by rVSV∆m51 were exaggerated in females

with disrupted IFNAR-mediated signaling compared to males.

• Type I interferon signaling was required by females to down-regulate cytokine

responses to rVSV∆m51 and avoid immune-mediated morbidity.

This study describes a mechanism that could account for dysregulated cytokine responses to viruses that potently interfere with type I IFN responses.

60

Graphical Abstract

61

Abstract

Type I interferons (IFNs) play a crucial role in the establishment of an antiviral state via signaling through their cognate type I IFN receptor (IFNAR). In this study, a replication-competent but highly attenuated strain of VSV (rVSVΔm51) carrying a deletion at position 51 of the matrix protein to remove suppression of anti-viral type I IFN responses was used to explore the effect of disrupted IFNAR signaling on inflammatory cytokine responses in mice. The kinetic responses of interleukin-6, tumor necrosis factor-α and interleukin-12 were evaluated in virus-infected male and female mice with or without concomitant antibody-mediated IFNAR-blockade. Unlike controls, both male and female IFNAR-blocked mice showed signs of sickness by 24-hours post-infection. Female IFNAR-blocked mice experienced greater morbidity as demonstrated by a significant decrease in body temperature. This was not the case for males. In addition, females with IFNAR blockade mounted prolonged and exaggerated systemic inflammatory cytokine responses to rVSVΔm51. This was in stark contrast to controls with intact IFNAR signaling and males with IFNAR-blockade; they were able to down-regulate virus-induced inflammatory cytokine responses by 24-hours post-infection. Exaggerated cytokine responses in females with impaired IFNAR signaling was associated with more effective control of viremia than their male counterparts. However, the trade-off was greater immune-mediated morbidity. The results of this study demonstrated a role for IFNAR signaling in the down-regulation of antiviral cytokine responses, which was strongly influenced by sex. Our findings suggest that the potential to mount exessive cytokine responses to a virus with concomitant disruption of IFNAR signaling is heavily biased towards females.

62

Introduction

Interferons (IFNs) are a large family of pleiotropic cytokines that can be subdivided into three distinct groups and are produced by a variety of cells after viral infections. They play a dominant role in initiating host canonical antiviral defenses. Type I IFNs, including IFN-α and β, play a crucial role in the early establishment of an antiviral state by interacting with their cognate heterodimeric receptor, IFNAR, which is expressed on nucleated cells [1-9]. Cellular responses to type I IFNs are initiated by IFNAR-mediated signaling and include boosting of the late phase of antiviral immunity via a positive feedback loop [10]. Type I IFN-mediated antiviral responses are also characterized by robust local induction of pro-inflammatory cytokines and chemokines that attract inflammatory cells of the innate immune system. The ability of type I IFNs to overcome viral infections relies on a finely tuned interplay of inflammatory and anti-inflammatory responses of both the innate and adaptive immune systems [11-17]. Severe viral infections are sometimes associated with a phenomenon known as a “cytokine storm” or “hypercytokinemia”, which is caused by an overly robust inflammatory response that can become toxic [18-36]. Coincidentally, these viruses that induce excessive, toxic cytokine responses are associated with mechanisms that facilitate potent suppression of type I IFN responses. Antiviral immunity can be influenced by intrinsic host factors such as sex [37, 38] and genetic background [3-5], the type of virus, the strategies used by viruses to evade immune responses, and the interplay between viruses and the host’s immune system [3, 8, 33, 39-47]. There is a sex-related disparity in antiviral responses, with a bias towards females experiencing a higher proportion of virus-associated morbidities and mortalities than males [37, 38, 48]. We sought to test the hypothesis that females would have more exaggerated cytokine responses to viral infection during IFNAR blockade than males. To accomplish this, a replication-competent but highly attenuated recombinant strain of vesicular 63 stomatitis virus (rVSVΔm51) was used [49, 50]. This virus had the methionine at position 51 of the matrix protein deleted to remove suppression of anti-viral type I IFN responses. This safe virus was developed to be administered intravenously (i.v.) at high doses to treat patients with cancers.

Intravenous delivery of rVSVΔm51 into male and female mice was used in conjunction with i.v. administration of an IFNAR-blocking antibody to simulate potent inhibition of type I IFN responses during viremia. The results of this study indicated a role for IFNAR in the negative regulation of antiviral cytokine responses. Notably, this was influenced by sex, with a bias towards females.

Materials and Methods

A summary of materials can be found in Key Resources Table (table 1).

Mice. Age-matched six-to-eight-week-old male and female Balb/c mice (Charles River

Laboratories, USA; strain code #028) were used in all experiments. Mice were housed in an isolated pathogen-free environmentally-controlled facility at the University of Guelph. Mice were accommodated to the facility for one week prior to the initiation of experiments and were monitored regularly and provided with food and water ad libitum. Mouse studies adhered to the guidelines provided by the Canadian Council on Animal Care and were approved by the University of Guelph’s Animal Care Committee (animal utilization protocol #3807).

Virus. The highly attenuated replication-competent rVSVΔm51 that was used in this study was kindly provided by Dr. Brian Lichty (McMaster University, Hamilton, Ontario, Canada) and has been previously described [49]. The virus was propagated in Vero cells (American Type

Culture Collection; #CCL-81), concentrated by ultracentrifugation and purified by centrifugation on a sucrose density gradient followed by dialysis. Determination of viral titers was done using a standard plaque assay. Doses of 1x109 pfu of rVSV∆m51 in 200 µL of phosphate-buffered saline 64

(PBS) were administered to mice via tail veins. The use of this virus was approved by the

University of Guelph’s Biosafety Committee (biohazard permit #A-367-04-19-05).

IFNAR blockade. To block type I IFN receptors, mice received 1 mg of a mouse anti-mouse

IFNAR-1 (clone MAR1-5A3, Leinco Technologies; cat. #0516L270) via tail veins two-hours before viral infection. Mice in control groups received intravenous injections of 1 mg of an IgG isotype control immunoglobulin (Leinco Technologies, USA; cat. #I-536).

Quantification of Ly6G+ granulocytes. Blood samples were drawn from the retro-orbital sinus of mice using heparinized capillary tubes into 1.5 mL microtubes containing 5 µL of heparin at a concentration of 3 µg/mL. Erythrocytes were removed from blood samples using a red blood cell lysing buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA). Leukocytes had their Fc gamma receptors blocked by treatment with anti-CD16/CD32 (clone 93; BioLegend, USA; cat.

#101320) in flow cytometry staining buffer (PBS + 0.1% bovine serum albumin) for 15 minutes at 4°C. Cells were then stained with anti-Ly6G-PE (clone 1A8; BioLegend; cat. #127608) in flow cytometry staining buffer for 20 minutes in the dark at 4°C. After washing, the cells were stained with a fixable viability dye (Zombie Aqua; BioLegend; cat. #423102) as per the manufacturer’s directions to facilitate exclusion of dead cells. Cells were washed, re-suspended in flow cytometry staining buffer and analyzed with a FACSCanto II flow cytometer (Becton Dickinson, Canada) using FACS Diva version 8 software for data acquisition. The resultant data were then analyzed using Flowjo version 10 software (FlowJo LLC, Ashland, Oregon, USA). Doublets and dead cells were excluded from the analyses.

Assessment of body temperatures. A certified digital thermometer with a rectal probe

(Microtherma 2 Type "T" Thermometer; Braintree Scientific, USA; cat. #TW2- 107) was used to measure the body temperature of mice. Proper calibration of the thermometer was confirmed by 65 cross-referencing the room temperature with a second certified digital thermometer (Traceable

Excursion-Trac Datalogging Thermometer, ITM Instruments, Canada).

Quantification of plasma-derived cytokines. Blood samples were acquired from the retro- orbital sinus of mice using heparinized capillary tubes. Blood was collected in 1.5 mL microtubes containing 5 µL of heparin at a concentration of 3 µg/mL. Blood was centrifuged at 4oC for 10 minutes at 2,000 x g and the plasma was immediately collected, aliquoted into polypropylene tubes and archived at -80oC until further analysis. The inflammatory cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-12(p40) were quantified in plasma using ProcartaPlex Mouse

Simplex kits (Invitrogen eBioscience, USA; cat. #s EPX01A-20603-901, EPX01A-20607-901 and

EPX01A-26033-901) according to the manufacturer’s instructions and a Bio-Plex 200 system

(Bio-Rad, Canada).

Determination of virus titers. Plasma samples were serially diluted in complete Dulbecco's

Modified Eagle Medium (Hyclone) supplemented with 10% bovine calf serum (Hyclone, USA) and used for determination of 50% tissue culture-infected dose (TCID50) and converting the values to plaque-forming units (pfu) by using the conversion factor 0.69, as previously described [51].

Statistics. GraphPad Prism version 8 was used for all graphing and statistical analyses. Graphs show means and standard errors. If required, data were normalized by log transformation. Data were analyzed using one- or two-way analysis of variance with Sidak’s multiple comparisons test when assessing one or two variables, respectively. Statistical significance was defined as p≤0.05.

Statement of work performed:

KK, BWB, RK and SS conceived and designed the study; KA, KK, and BWB conducted experimental work; BWB analyzed data and performed the statistical analyses; MD wrote the first 66 draft of the manuscript; KK and BWB wrote sections of the manuscript and edited the final version.

All authors contributed to reading and revising the manuscript and approving the submitted version.

Results

Mice develop a systemic inflammatory response to recombinant vesicular stomatitis virus

(rVSV∆m51) that is characterized by an increase in the frequency of granulocytes in blood, which was potentiated by type I interferon receptor (IFNAR)-blockade.

Neutrophils are the most prevalent granulocytes and are usually the first cells to traffic to sites of infection. As such, they can be used as a surrogate marker of acute inflammation. Male and

9 female mice were infected intravenously with 1x10 pfu rVSVΔm51 to simulate viremia. This was done with or without concomitant IFNAR-blockade. Blood Ly6G+ granulocytes were then quantified by flow cytometry ten hours post-infection (Figure 1). Regardless of sex, the frequency of circulating granulocytes in mice with intact IFNAR signaling was increased by approximately two-fold relative to uninfected controls. Interestingly, this frequency was significantly increased in both males and females by antibody-mediated blockade of IFNARs. This suggested that VSV- induced inflammatory responses were being modulated by type I interferon signaling.

Mice infected with rVSV∆m51 with concomitant IFNAR-blockade show signs of sickness, with females having acute decreases in body temperature.

Due to its clinical development as an oncolytic virus, rVSV∆m51 has been shown to be safe when administered to mice [52]. However, since mice treated with an IFNAR blocking antibody had evidence of an exaggerated acute inflammatory response to this virus, they were monitored beyond ten hours to determine if any adverse events developed. Although all mice survived 67 infection with rVSV∆m51, both male and female IFNAR-blocked mice showed multiple signs of morbidity at 24-hours post-infection (Table 2). This was in stark contrast to control mice with intact IFNAR signaling, which did not show any signs of sickness. In addition, female mice that had been infected during IFNAR-blockade had significant reductions in body temperature (from

37.10°C pre-infection to 32.05°C and 32.90°C at 10.5- and 24-hours post-infection, respectively) compared to males and IFNAR-intact females (Figure 2). This suggested that female mice with compromised IFNAR signaling might be mounting more severe inflammatory responses than their male counterparts that were not reflected in circulating neutrophils. This prompted an investigation of acute pro-inflammatory cytokine responses.

Inflammatory cytokine responses induced by rVSV∆m51 are exaggerated in females with disrupted IFNAR-mediated signaling.

The cytokines IL-6, TNF-α and IL-12(p40) were selected as representative soluble markers of inflammation. At 10-hours post-infection with rVSV∆m51, female mice with blocked IFNARs showed an exaggerated production of all three of these cytokines in the blood compared to female controls and male IFNAR-blocked mice (Figure 3). Specifically, females with IFNAR-blockade, females with intact IFNAR signaling and males with IFNAR blockade, respectively, had 6,337 versus 3,766 versus 2,674 pg/mL of IL-6, 1,784 versus 366 versus 442 pg/mL of TNF-α, and 880 versus 159 versus 201 pg/mL of IL-12(p40). This demonstrated a strong female bias towards more inflammatory cytokine responses against rVSV∆m51 if IFNAR signaling was compromised.

68

Female mice have an impaired ability to negatively regulate cytokine responses to recombinant vesicular stomatitis virus (rVSV∆m51) during type I interferon receptor

(IFNAR)-blockade.

Finding evidence of dysregulated cytokine responses 10-hours after infection with a virus in female mice that had concomitant IFNAR-blockade prompted a more detailed kinetic study over a period of 24-hours. Plasma samples were collected just prior to intravenous administration of rVSV∆m51 and at 2.5, 5, 10, and 24 hours post-infection. Groups included male and female mice with or without pre-treatment with an intravenously administered IFNAR-blocking antibody.

Concentrations of IL-6, TNF-α and IL-12(p40) were determined (Figure 4A). In all mice with intact IFNAR signaling, IL-6 and TNF-α responses peaked at 2.5-hours post-infection and had returned to baseline by 24 hours after infection. For IL-12(p40), the response peaked 5-hours after infection. Interestingly, the IL-6 response was blunted at 2.5-hours post-infection in males with

IFNAR blockade, but this represented the only significant effect of interfering with IFNAR signaling in male mice. In stark contrast, females with IFNAR blockade experienced massively dysregulated cytokine responses that not only failed to return to baseline by 24 hours post- infection, they were still increasing in concentration at this time point for IL-6 and IL12(p40).

Converting these kinetic cytokine response data to areas under the curves confirmed that females with IFNAR-blockade mounted exaggerated responses for all three cytokines compared to both groups of males and female controls with intact IFNAR signaling (Figure 4B). These results provided evidence that females with disrupted IFNAR signaling were able to mount normal acute inflammatory cytokine responses to a virus but were subsequently unable to properly down-regulate these responses, especially for IL-6 and IL-12(p40). This suggested a critical role 69 for IFNAR signaling in the modulation of cytokine responses and unveiled a scenario in which females experienced a higher degree of inflammation.

IFNAR signaling was required for viral clearance.

Type I interferon responses have been reported to be critical for clearance of viruses. To confirm this, rVSV∆m51 was quantified in plasma 24 hours after infecting male and female

IFNAR-intact versus IFNAR-blocked mice (Figure 5). Mice with intact IFNAR signaling did not have virus titers above the limit of detection of the TCID50 assay. As expected, mice that had been infected with concomitant IFNAR-blockade had difficulty controlling acute viremia. Notably, however, females with IFNAR-blockade had significantly lower viral titers than their male counterparts, which correlated with their more robust cytokine responses (Figure 4). These findings confirmed the importance of type I interferon responses in the acute control of viral infections and suggested that exaggerated production of other virus-induced cytokines in females

(Figre 4) could partially compensate for this, but at the expense of greater morbidity (Figure 2).

Discussion

Sex disparity in type I IFN production by cells of the immune system [37, 53-59] in the context of viral infection [38], cancer [60], and immunological disorders [61-63] has been reported.

Furthermore, sex disparity in other cytokine responses is a well-established phenomenon in a variety of contexts [64-67], including viral infections [38, 48, 68, 69]. However, to the best of our knowledge, this study provides the first evidence of a direct, sex-biased link between IFNAR signaling and the regulation of an array of pro-inflammatory cytokine responses to a viral infection.

Indeed, in this study, female mice had exaggerated concentrations of the inflammatory cytokines

IL-6, TNF-α, and IL-12(p40) in plasma after infection with rVSVΔm51 when there was 70 concomitant disruption of IFNAR signaling (Figures 3 and 4). IL-6 and IL-12(p40) were the most dysregulated of the three cytokines in females, implying a particularly strong association with the sex of the host. Consistent with our findings, a significant association has previously been reported between serum concentrations of IL-6, female sex, and autoimmune toxicity of anti-cytotoxic T- -associated protein-4-mediated checkpoint blockade therapy in patients with cancers

[64]. While the present study provides additional evidence of sex-biased inflammatory responses, more importantly, it also shows a role for IFNAR signaling in the dimorphic regulation of antiviral cytokine responses, which might also be applicable to other contexts, including autoimmune diseases that feature differential IFN responses in females [61, 62, 70, 71]. A synergistic interplay between estrogen and IL-6 has also been reported to be associated with the severity of experimental, trifluoroacetyl chloride-haptenated liver proteins-induced liver injury in female

Balb/c mice. Specifically, estrogen-induced IL-6 was shown to promote sex-disparity in severity of drug-induced liver injury by reducing the expansion of splenic regulatory T cells in females

[72].

While mechanisms underlying differential immune response between males and females are complex, the synergistic functions of the endocrine and immune systems, together with dimorphic gene expression and viral factors have been implicated in differential outcomes of viral infections between sexes [37, 47, 48, 73]. Many hematopoietic and non-hematopoietic cells express receptors for sex steroid hormones [74-76] and immunity to viral infections can be impacted by variations in the levels of sex hormones. For example, female mice infected with influenza A virus mounted more robust inflammatory responses than their male counterparts, which made them more susceptible to infection-associated morbidities [36, 38, 77] . Furthermore, the presence of androgen or estrogen response elements within the promoters of several genes implicated in innate immunity 71 represent one of the potential mechanisms whereby hormones can introduce sex-related dimorphism in immune responses [78].

Another potential mechanism to introduce sex-related biases into immune responses is the differential encoding of immunoregulatory genes in sex chromosomes [37, 61]. In contrast to a limited number of immunoregulatory genes encoded within the Y chromosome, the X chromosome encodes more than 1,100 genes, constituting 5% of the human genome, including some crucial regulatory genes that can introduce a sex bias in immune responses at both transcriptional and translational levels [37]. For example, Toll-like receptor (TLR) 7, is encoded on chromosome X and failure of second-X inactivation results in higher levels of expression of

TLR7 in cells. This is associated with higher concentrations of cytokines and chemokines in females, than in males, in response to TLR7-mediated signaling [79]. TLR7-mediated type I IFN responses of plasmacytoid DCs of healthy females have also been shown to be influenced by the number of X chromosomes, as well as serum concentrations of sex hormones [53].

Physiological levels of male and female sex hormones have been shown to differentially affect inflammatory responses during viral infections. A hypothetical mechanism by which sex hormones potentially affect the outcomes of infections with influenza A virus and responses to influenza virus vaccines in both humans and mice was proposed by Klein et al. [38]. Specifically, infections in females, which cause a decrease in the levels of estradiol, are associated with upregulation of inflammatory cytokines and chemokines and excessive infiltration of leukocytes into inflamed sites, which could have severe pathological consequences in the lungs, and even potentiate death.

Virally-suppressed testosterone in males, on the other hand, was not associated with potentiation of inflammatory responses and inflammation-associated morbidities. Similar associations have been made for responses to influenza virus vaccines, with females experiencing more adverse 72 events. Although the present study did not assess concentrations of sex hormones in rVSVΔm51- infected mice, it unveiled a novel association between sex and the severity of a viral infection. In the event of disrupted IFNAR signaling, male mice were as capable as control mice in down- regulating inflammatory cytokine responses at 24-hours post-infection, whereas females with impaired IFNAR signaling failed to control the cytokine responses (Figures 3 and 4), which was associated with an acute decrease in body temperature as an adverse event (Figure 2). Similar to hormone levels, this implicates a reduction in type I interferon signaling with a female bias towards increased inflammation and adverse events during a viral infection.

Both male and female mice failed to clear rVSVΔm51 by 24-hours post-infection when there was a concomitant blockade of IFNAR signaling (Figure 5). This agrees with other studies that have shown the indispensable role of IFNAR signaling in viral clearance [4, 17, 80]. Type I IFN responses have been reported to be particularly important in host protection against infections with

VSV, as compared with infections with vaccinia virus or lymphocytic choriomeningitis virus-

Armstrong strain that can be cleared by both type I and type II IFN responses [3, 42, 80]. Other viruses, like NDV, are also able to activate IFN-independent antiviral pathways in addition to IFN- mediated responses [41]. With that said, mice with IFNAR-blockade were able to eventually clear the virus, as evidenced by their long-term survival. The evidence at 24-hours post-infection showed that males with IFNAR blockade had greater viremia than females with IFNAR blockade

(Figure 5). This inversely corelated with cytokine responses (Figures 3 and 4) the number of adverse events (Table 1 and Figure 2). These results suggest that VSV-mediated viremia can be controlled by cytokine responses other than type I interferons, albeit less efficiently. Indeed, host antiviral defenses against VSV appear to be influenced by the type of cells that are infected. Studies 73 of mouse embryonic fibroblasts and a human respiratory epithelial cell line indicated a role of type

I IFN-independent mechanisms in providing protection against infection with VSV [41, 43].

Conclusions

This study modeled viremia using an attenuated virus to determine how blockade of type I

IFN signaling impacted the immune response. This was based on the observation that pathogenic viruses that are associated with induction of pathogenic inflammatory responses often express proteins that can potently interfere with type I IFN responses. The results presented here highlight a crucial role for IFNAR signaling in the negative regulation of antiviral cytokine responses.

Indeed, blocking IFN responses during infection with rVSVΔm51 resulted in a sexually dimorphic dysregulated cytokine response that delayed viral clearance. Importantly, viral clearance was less delayed in females with compromised IFN signaling, but this was at a cost of exaggerated pro- inflammatory cytokine responses that were associated with greater morbidity than their male counterparts. In contrast, male mice were able to down-modulate inflammatory responses to VSV during impaired IFNAR signaling. These findings have implications for infections with viruses that have mechanisms to potently interfere with type I IFN signaling. Many viruses need to evade innate immune responses to survive in hosts, with type I IFN responses playing a dominant role in controlling their replication. The evidence presented here supports a direct link between IFNAR blockade and dysregulated cytokine responses. Although speculative, this could potentially be a mechanism of action contributing to cytokine storms that are induced by some pathogenic viruses.

Of greater novelty, this study documented a scenario in which immune responses mounted by females caused greater morbidity, despite more efficiently clearing the virus that initiated the responses in the first place. Specifically, infection with a virus with concomitant blockade of type

I IFN signaling could drive a female bias towards dysregulated cytokine responses. This concept 74 warrants further investigation in the context of both infectious and non-infectious inflammation- mediated diseases that have a higher incidence in females.

75

Figures of Chapter 2:

Figure 1. Mice develop a systemic inflammatory response to recombinant vesicular stomatitis virus

(rVSV∆m51) that was characterized by an increase in the frequency of granulocytes in blood, which was potentiated by type I interferon receptor (IFNAR)-blockade.

Male and female Balb/c mice received intravenous injections of 1 mg of an isotype control immunoglobulin or a type

9 I interferon receptor (IFNAR)-blocking antibody two hours before intravenous administration of 1x10 pfu of

+ rVSV∆m51. Ten hours post-infection blood-derived Ly6G granulocytes were quantified by flow cytometry. Bars

+ represent the mean fold-increase in the frequency of Ly6G cells relative to uninfected control mice. Standard errors are shown; n=4/group. Data are representative of three experimental replicates and were assessed using a one-way analysis of variance with Sidak’s multiple comparisons test.

s l

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Figure 2. Female mice with impaired type I interferon receptor (IFNAR) signaling show acute reductions of body temperatures after viral infection.

Male and female Balb/c mice received intravenous injections of 1 mg of an isotype control immunoglobulin or a type

9 I interferon receptor (IFNAR)-blocking antibody two hours before intravenous administration of 1x10 pfu of a recombinant vesicular stomatitis virus (rVSV∆m51). Rectal temperatures were measured at multiple time points post- infection. Data are shown as means with standard errors; n=4/group. Data are representative of three experimental replicates and were assessed using a two-way analysis of variance with Sidak’s multiple comparisons test. NS=not significant, *p=0.0372, **p=0.0152.

38

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31 0 3 5 10.5 24

Hours Post-Infection

77

Figure 3. Virus-induced inflammatory cytokine responses were exaggerated in females with disrupted type I interferon receptor-mediated signaling.

Male and female Balb/c mice received intravenous injections of 1 mg of an isotype control immunoglobulin or a type

9 I interferon receptor (IFNAR)-blocking antibody two hours before intravenous administration of 1x10 pfu of a recombinant vesicular stomatitis virus (rVSV∆m51). Plasma samples were collected ten hours post-infection to quantify the cytokines interleukin (IL)-6 (left panel), tumor necrosis factor (TNF)-α (middle panel) and IL-12 (p40)

(right panel) using a multiplex assay. Data are shown as means with standard errors; n=4/group. Data are representative of three experimental replicates and were assessed using a one-way analysis of variance with Sidak’s multiple comparisons test.

78

Figure 4. Female mice had an impaired ability to negatively regulate cytokine responses to recombinant vesicular stomatitis virus (rVSV∆m51) during type I interferon receptor (IFNAR)-blockade.

Male and female Balb/c mice received intravenous injections of 1 mg of an isotype control immunoglobulin or an

9 IFNAR-blocking antibody two hours before intravenous administration of 1x10 pfu of rVSV∆m51. Plasma samples were collected 0, 2.5, 5, 10, and 24 hours post-infection to quantify the cytokines interleukin (IL)-6 (left panels), tumor necrosis factor (TNF)-α (middle panels) and IL-12(p40) (right panels) using a multiplex assay. Data were graphed as

(A) curves and (B) areas under the curves and shown as means with standard errors; data were pooled from two experiments; n=8/group. Data were assessed using (A) two-way analysis of variance with Tukey’s multiple comparisons test, or (B) one-way analysis of variance with Sidak’s multiple comparisons test.

24 24 24

79

Figure 5. Type I interferon receptor (IFNAR) signaling is required for viral clearance.

Male and female Balb/c mice received intravenous injections of 1 mg of an isotype control immunoglobulin or an

9 IFNAR-blocking antibody two hours before intravenous administration of 1x10 pfu of a recombinant vesicular stomatitis virus (rVSV∆m51). Plasma samples were collected 24-hours post-infection to quantify titers of circulating viruses using a standard assay to determine plaque-forming units (pfu). Data are shown as means with standard errors; n=4/group. Data are representative of three experimental replicates and were assessed using a one-way analysis of variance with Sidak’s multiple comparisons test. Mice that had been infected without IFNAR-blockade had titers below the limit of detection (LOD) of the assay (i.e. approximately 10 pfu/mL)

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80

Tables:

Table 1. Key Resource Table

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies Mouse IgG1 Isotype Control (clone HKSP84) Leinco Technologies Catalogue #I-117 Anti-Mouse Interferon Alpha/Beta Receptor-1 (clone Leinco Technologies Catalogue #I-401 MAR1-5A3) PE Rat Anti-Mouse Ly6G (clone 1A8) BD Biosciences Catalogue #551461 Anti-Mouse CD16/CD32 (clone 93) BioLegend Catalogue #103020 Virus Strain Recombinant Indiana Strain of Vesicular Stomatitis Dr. Brian Lichty, McMaster University, Cancer Cell . 2003 Oct;4(4):263-75. Virus (rVSVΔm51) Hamilton, Ontario, Canada PMID: 14585354 Biological Samples Bovine Calf Serum VWR Catalogue #2100-500

Chemicals Heparin Sodium Salt from Porcine Intestinal Mucosa Sigma-Aldrich Catalogue #H3149 Ammonium Chloride (NH4CL) Fisher Scientific Cat#A649500 Potassium Bicarbonate (KHCO3) Fisher Scientific Catalogue #P184-500 EDTA Disodium Salt (Na2EDTA) Fisher Scientific Catalogue #BP120500 Trypan Blue Stain 0.4% Gibco Catalogue #15250 Phosphate Buffer Saline Hyclone Catalogue #SH0325601 Bovine Serum Albumin Fisher Scientific Catalogue #BP1600100 Zombie Aqua Fxable Viability Kit BioLegend Catalogue #423101/423102 Dulbecco's Modified Eagle Medium Hyclone Catalogue #SH3002201

Experimental Models: Organisms/Strains

Female and Male Balb/c mice Charles River Laboratories strain code 028

Experimental Models: Cell Line Vero cells America Type Culture Collection #CCL-81 Critical Commercial Assays ProcartaPlex Mouse IL-12/IL-23p40 Simplex ThermoFisher Catalogue #EPX01A-26033-901 ProcartaPlex Mouse TNF alpha Simplex ThermoFisher Catalogue #EPX01A-20607-901 ProcartaPlex Mouse IL-6 Simplex ThermoFisher Catalogue #EPX01A-20603-901 Software and Algorithms

Flowjo Software version 10 Becton Dickinson https://www.flowjo.com/solutions/flowjo https://www.graphpad.com/scientific- GraphPad Prism version 8 TreeStar software/prism/

Other

FACS Canto II Flow Cytometer Becton Dickinson Heparinized Micro-Hematocrit Capillary Tubes Fisher Scientific Catalogue #22362566 Microtherma 2 Type "T" Thermometer Braintree Scientific Catalogue #TW2- 107 Traceable Excursion-Trac Datalogging Thermometer ITM Instruments 81

Table 2. Mice infected with a recombinant vesicular stomatitis virus (rVSV∆m51) with concomitant blockade of their type I interferon receptors showed signs of sickness.

Male and female Balb/c mice received intravenous injections of 1 mg of an isotype control immunoglobulin or a type 9 I interferon receptor (IFNAR)-blocking antibody two hours before intravenous administration of 1x10 pfu of rVSV∆m51. The mice were then monitored at 12- and 24-hours post-infection (hpi) to determine if clinical signs were present (+) or absent (-).

Clinical Signs

Ruffled Laboured Lethargic Isolated Hunched Fur Breathing

Isotype - - - - - control 12 hpi Anti------IFNAR1 Male Isotype - - - - - control 24 hpi Anti- + + + + + IFNAR1

Isotype - - - - - control 12 hpi Anti------IFNAR1 Female Isotype - - - - - control 24 hpi Anti- + + + + + IFNAR1 82

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91

Chapter 3: Impaired Type I Interferon Receptor Signaling Drives Sexually

Dimorphic Virus-Induced Liver Damage

Maedeh Darzianiazizi, Robert C. Mould, Jacob P. van Vloten, Ashley A. Stegelmeier, Geoffrey

A. Wood, Shayan Sharif, Ravi Kulkarni, Byram W. Bridle†,*and Khalil Karimi†,*

Department of Pathobiology, University of Guelph, Guelph, Ontario, Canada

†contributed equally

*Correspondence: Khalil Karimi [email protected]

Byram W. Bridle [email protected]

Key words:

Sex disparity, liver damage, cytokine storm, tumor necrosis factor-α, interleukin-6.

This manuscript was drafted by M. Darzianiazizi and edited by B. W. Bridle and K.

Karimi in preparation for submission to Frontiers in Immunology – Inflammation.

92

Highlights:

• A sexually dimorphic role for IFNAR signaling in downregulation of both systemic

and local antiviral inflammatory responses to recombinant vesicular stomatitis virus

(rVSV∆m51).

• Both male and female IFNAR-/- mice relied on presence of IFNAR signaling for

negative regulation of both systemic and local antiviral inflammatory responses to

rVSV∆m51. However, they showed a differential reliance on IFNAR signaling for

coping with virus-induced liver damage and therefore, showing a sexually dimorphic

predisposition to liver damage during viremia concomitant with the absence of

IFNAR signaling.

• These findings together imply a sexually dimorphic landscape of IFNAR-mediated

antiviral cytokine responses in mice.

93

Graphical Abstract

94

Abstract

Viral infections can cause acute liver failure due to excessive anti-viral responses. Innate antiviral responses are largely controlled by type I interferon (IFN)-α/β receptor (IFNAR)- mediated signaling. Further, a role for virus-induced aberrant type I IFN responses in the development of pathogenic cytokine responses that can cause organ failure are now well- described. The liver is a sex-dimorphic organ implicated in host anti-viral immune responses; and sex-biased immunological responses underlie differential predisposition of sexes to pathogenesis of diseases, including viral infections. Understanding sex-dependent factors underlying virus- induced liver injuries opens new avenues towards development of therapeutics tailored to sexes.

We investigated sex disparity in IFNAR-mediated cytokine responses and its implication in liver pathogenesis in the context of a non-hepatotropic virus infection. Recombinant vesicular stomatitis virus (rVSV∆m51) administered to wild-type (WT) mice resulted in the presence of viruses in the liver, which was associated with local and systemic inflammatory cytokine responses. Notably,

IFNAR-knockout (IFNAR-/-) mice mounted higher magnitude hepatic and systemic pro- inflammatory cytokine responses, which were exaggerated in females. Moreover, female

IFNAR-/- mice developed severe liver pathogenesis, unlike their male counterparts and WT controls. Consistent with a critical role for early cellular responses to IFNAR signaling in the control of viral infections, IFNAR-/- mice, especially males, showed higher titers of the virus in livers than WT mice. Sex disparity, however, was not observed in liver viral titers of WT mice.

Conclusion: Our findings indicate that IFNAR-mediated antiviral cytokine responses are controlled by sex-discriminating factors and this can cause disparate sex-dependent liver pathogenesis. 95

Introduction

The liver is implicated in host immune responses to viral infections, which can sometimes result in acute liver failure due to excessive anti-viral responses to either hepatotropic viruses (e.g. hepatitis B virus or hepatitis C virus) [1] or non-hepatotropic viruses (e.g. human cytomegalovirus, vaccinia virus, Epstein-Barr virus, herpes simple virus, influenza A virus, severe acute respiratory syndrome-coronavirus, etc.) [2-9]. Innate antiviral responses are largely controlled by type I interferons (IFNs) [10, 11], which exert their antiviral and immunomodulatory effects through engaging their cognate heterodimeric receptor, IFNAR, expressed at the surface of all nucleated cells [12-14]. As intracellular obligatory parasites, many viruses have efficient strategies to compromise host type I IFN-mediated antiviral responses. Despite their well-established protective roles [15, 16], virus-induced type I IFN responses have also been implicated in some scenarios in the development of toxic cytokine responses leading to immunopathogenesis and organ failure, including the liver [17-22]. The liver is a sex-dimorphic organ and a growing body of evidence emphasizes a noticeable sex bias in liver metabolism, gene expression, and host predisposition to liver morbidities and mortalities [23-34]. Moreover, it is well-documented that immune responses to viral infections are influenced by host intrinsic factors, including sex. Sex- biased immunological responses and sexual dimorphism in predisposition to development of virus- induced immune-mediated pathogenesis clearly emphasizes sex as a biological variable driving differential outcomes of infections [35-37]. In spite of a higher incidence of viral infections in males, females are more liable to develop immune-mediated diseases following viral infections

[38]. Moreover, females have also been reported to be highly susceptible to chemical- and drug- induced liver pathogenesis [27, 39]. Type I IFNs play an integral role in liver homeostasis and hepatic antiviral immunity [2, 17, 40, 41]. However, little is known about sex-driven IFNAR 96 signaling and its potential role in liver pathogenesis has not been explored. The study presented here used a mice model of disrupted IFNAR signaling to explore sex disparity in IFNAR-mediated antiviral cytokine responses and its implication in promoting liver pathogenesis in the context of infection with a highly attenuated recombinant vesicular stomatitis virus (VSV∆m51). This virus was designed to elicit robust type I IFN responses by virtue of a deletion of the methionine at position 51 of the matrix protein, thereby abrogating the ability to block protein transport [42]. Our observations indicated a sexually dimorphic regulatory role for IFNAR signaling in both hepatic and systemic cytokine responses to this non-hepatotropic virus infection. Furthermore, our findings aligned with recent reports on the requirement of IFNAR signaling for protection against liver immunopathology during non-hepatotropic viral infections [2, 6, 43]. This study demonstrated that virus-induced liver injuries during impaired IFNAR signaling can be a sexually dimorphic event that is heavily biased towards females.

Methods and Materials

Mice. In order to extend our work to second strain of mice, age-matched six-to-eight-week- old male and female wild-type (WT) C57BL/6 mice (Charles River Laboratories, USA) were used as controls in experiments. IFNAR-knockout (IFNAR-/-) mice on the C57BL/6 background were kindly provided by Dr. Yonghong Wan, McMaster University, Hamilton, Ontario, Canada (with permission kindly granted by Dr. Laurel L. Lenz, University of Colorado School of Medicine,

Aurora, Colorado, USA) to facilitate the establishment of a local breeding colony. All mice were kept in an isolated pathogen-free environment at the University of Guelph (Guelph, Ontario,

Canada). WT mice were adapted to the facility one week before initiating experiments and were monitored regularly and provided with food and water ad libitum. All mouse-based studies met 97 the Canadian Council on Animal Care guidelines and were approved by the University of Guelph’s

Animal Care Committee (animal utilization protocol #3807).

Virus. A replication-competent but highly attenuated recombinant strain of vesicular stomatitis virus carrying a deletion at position 51 of the matrix protein to remove suppression of anti-viral type I IFN responses (rVSVΔm51) has been described previously[44]. The virus was propagated in Vero cells (American Type Culture Collection; #CCL•81), concentrated by ultracentrifugation and purified by centrifugation on a sucrose gradient followed by dialysis. rVSV∆m51 was administered to mice via tail veins at a dose of 1x109 pfu in 200 µL of phosphate- buffered saline (PBS). The use of this virus was approved by the University of Guelph’s Biosafety

Committee (biohazard permit # A-367-04-19-05).

Quantification of cytokines and alanine aminotransferase (ALT). Blood samples were acquired from mice via the retro-orbital sinus using heparinized capillary tubes and collected in microtubes containing 5 μL of heparin at 3 μg/mL. Samples were taken at five- and 18-hours post- infection. Blood was centrifuged at 4oC for 10 minutes at 2,000 x g and the supernatant was immediately collected, aliquoted and archived at -80°C until further analysis for quantifying plasma-derived inflammatory cytokines and ALT using a Bio-Plex 200 system (Bio-Rad) and a

Cobas 6000 c501 biochemistry analyzer (Roche), respectively.

Liver cytokine measurement, virus titration, and histology. Mice were euthanized 18 hours after infection with rVSVΔm51. Livers were extracted and cut into slices to be used in three assays. For detection of inflammatory cytokines, liver slices were placed in 1.5 mL microtubes containing no buffer and snap-frozen in liquid nitrogen followed by immediate archival at −80°C until further analysis for cytokines. Liver slices were gradually thawed on ice and after determining their weights, they were placed in 2 mL Lysing Matrix tubes containing one ¼-inch ceramic bead 98

(MP biomedicals) and 750 μL of radioimmunoprecipitation assay lysis buffer (150 mM NaCl, 5 mM ethylenediaminetetraacetic acid [pH 8.0], 50 mM Tris [pH 8.0], 1.0% NP-40 [IGEPAL CA-

630], 0.5% sodium deoxycholate, 0.1% SDS and milli-Q H2O) containing 1% Protease and

Phosphatase Inhibitor Single-Use Cocktail (Thermo Scientific) and homogenized using a Precellys

24 tissue homogenizer (Bertin Instruments) for 60 seconds at 6,000 rpm. The tubes were then centrifuged at 18,659 x g for 15 min at 4°C. The resultant supernatants were transferred into clean

1.5 mL microtubes and used for quantifying inflammatory cytokines using a LEGENDplexTM

Mouse Inflammation panel (BioLegend) according to the manufacturer’s instructions. For a second application, a slice of each liver was placed in a PBS-containing 1.5 mL microtube on ice and immediately archived at −80°C until being analyzed for infectious titers of rVSV∆m51. Liver slices were gradually thawed on ice and after determining their weights, they were placed in 2 mL-

Lysing Matrix tubes containing one ¼ inch ceramic bead (MP biomedicals) and 750 μL of

Dulbecco's modified Eagle’s medium (DMEM; Gibco) supplemented with 1% penicillin/streptomycin (Hyclone) + 1% antibiotic-antimycotic (Gibco) and homogenized using a

Precellys 24 tissue homogenizer for 10 seconds at 5,000 rpm. The tubes were then incubated on ice for three minutes and were subject to a second round of homogenization for 15 seconds at

5,000 rpm, followed by centrifugation for five minutes at 9,520 x g at 4°C. The resultant supernatants were carefully removed and transferred into sterile 1.5 mL microtubes to be serially diluted in complete DMEM (supplemented with 10% bovine calf serum [BCS] and 1% penicillin/streptomycin) and used for determination of 50% tissue culture infected dose (TCID50) and converting the values to plaque-forming units (pfu) using a conversion factor of 0.69, as described [45]. A third piece of each liver was placed into a histology cassette and submerged in

10% formalin solution to be fixed and stained with hematoxylin and eosin using standard 99 procedures, followed by blinded interpretation by a board-certified veterinary pathologist (G. A.

Wood).

Statistics. GraphPad Prism version 8 was used for all graphing and statistical analyses. Graphs show means and standard errors. Student's t-tests were used for comparing two independent groups. One- and two-way analysis of variance (ANOVA) with Sidak's multiple comparisons test was used when means of more than two independent groups were subject to comparison across one or more time points, respectively. Statistical significance was defined as p≤0.05.

Statement of work performed:

KK, BWB, RK and SS conceived and designed the study; MD, RCM, JPvV, AAS, KK, and

BWB conducted experimental work; GAW performed blinded analysis of tissue sections; MD,

KK and BWB analyzed data and performed the statistical analyses; MD wrote the first draft of the manuscript; KK and BWB wrote sections of the manuscript and edited the final version. All authors contributed to reading and revising the manuscript and approving the submitted version.

Results

Mice treated with a recombinant vesicular stomatitis virus (rVSV∆m51) had elevated concentrations of plasma cytokines compared to naïve mice, with evidence of sexual dimorphism.

To assess the impact of type I IFN signaling on cytokine responses to viremia, a model was adopted in which C57BL/6 mice received intravenous injections of 1x109 pfu of the highly attenuated, non-pathogenic rVSV∆m51. Both males and females were treated to determine if there were any sex-related effects. Cytokines were quantified in the plasma of infected mice using a multiplex assay (Figure 1). Within five hours of infection there were dramatic elevations of key pro-inflammatory cytokines relative to baseline. These cytokines included IL-6, TNF-α and IL-12. 100

Interestingly, sex had a significant impact on the magnitude of the cytokine responses. At five hours post-infection, females had 1.7- and 2.3-fold higher concentrations of circulating IL-6 and

TNF-α, respectively, compared to males. In contrast, males had 1.5-fold more IL-12. The differences between sexes in the concentrations of IL-6 and TNF-α was no longer apparent at 18- hours post-infection. However, the concentration of IL-12 decreased in males between five and

18-hours post-infection, but not in females. This resulted in sustained IL-12 in females than males at this timepoint, which was the opposite of what was observed five hours after infection. With the exception of IL-12 at five hours post-infection, these results suggested that females mounted higher magnitude pro-inflammatory cytokine responses than their male counterparts, which was consistenwith the well-established concept of sexual dimorphism in anti-viral immunity [35].

Intravenous administration of recombinant vesicular stomatitis virus (rVSV∆m51) results in infection of livers, along with local inflammatory cytokine responses but with no evidence of sexual dimorphism.

Having characterized a cytokine profile in the blood of rVSV∆m51-infected mice and determined that it was impacted by sex, the next goal was to ascertain whether these observations extended to the tissue of primary interest, which was the liver. Viral plaque assays confirmed the presence of infectious viruses in the livers of mice 18 hours after infection with rVSV∆m51 (Figure

2A). There were no differences in viral titers between males and females. Virus accumulation in the liver was associated with hepatic pro-inflammatory cytokine responses (Fig. 2B). However, in contrast to the cytokine profile in plasma, which differed between males and females, there was no evidence of sex-associated differences between hepatic cytokine profiles 18 hours after infection.

101

A lack of type I interferon receptor (IFNAR) signaling causes dysregulation of cytokine responses to recombinant vesicular stomatitis virus (rVSV∆m51), which is influenced by sex.

With plasma and hepatic cytokine profiles and baseline hepatic titers of rVSV∆m51 having been defined, the next step was to determine if any of these parameters would be modulated if type

I IFN signaling was disrupted. Many pathogenic viruses have efficient mechanisms to disrupt type

I IFN signaling. To simulate this with rVSV∆m51, which promotes robust type I IFN responses, the virus was administered to IFNAR-/- mice. Within only five hours after infection, the virus- induced cytokine responses in the blood of IFNAR-/- mice were massively increased relative to

WT controls and this dysregulation became even more exaggerated by 18 hours post-infection

(Figure 3A). Of note, the dysregulation was consistently more severe in female IFNAR-/- mice.

Disconcertingly, at 18-hours post-infection IL-6 was upregulated 1,171- and 2,632-fold in the blood of male and female IFNAR-/- mice, respectively, compared to their WT counterparts.

Similarly, TNF-α was 220- and 296-fold higher and IL-12 was 29- and 36-fold higher. These dramatic differences in the cytokine profiles extended to the liver where excessive responses were common to the IFNAR-/- mice, with hepatic concentrations of IL-6 and TNF-α being higher in females (Figure 3B). Specifically, 18-hours after infection with rVSV∆m51, the hepatic concentration of IL-6 was 14- and 17-fold higher in male and female IFNAR-/- mice, respectively, compared to WT controls. The TNF-α was 38- and 57-fold higher for male and female IFNAR-/- mice, respectively, and IL-12 was four-fold higher for both sexes. Of concern, IFNAR-/- mice with grossly exaggerated cytokine concentrations could not survive infection with highly attenuated rVSV∆m51, which was safe in WT mice. Indeed, IFNAR-/- mice succumbed to the infection within only eighteen hours (Figure 3C).

102

Male and female mice with impaired type I interferon receptor (IFNAR) signaling exhibit differential liver injuries following administration of recombinant vesicular stomatitis virus (rVSV∆m51).

Infection with rVSV∆m51 was acutely lethal in all IFNAR-/- mice regardless of sex, presumably due to an overwhelming systemic cytokine storm. However, when these mice reached endpoint 18 hours post-infection, interesting differences were noted between males and females with respect to liver damage. Anomalies in concentrations of liver enzymes are often used as surrogate markers of liver damage. This includes the elevation of ALT in plasma or serum, which is commonly reported in extrahepatic viral infections [3]. Concentrations of ALT in plasma were quantified in male and female WT and IFNAR-/- mice with or without intravenous infection with rVSV∆m51 (Figure 4A). Infected WT mice had no elevation of ALT in their plasma compared to uninfected mice and this was irrespective of sex. Nor did infected male IFNAR-/- mice have an increase in plasma ALT concentrations. In stark contrast, female IFNAR-/- mice had an almost two-fold greater concentration of in ALT in plasma post-infection compared to all other groups

(p<0.0001). Importantly, this increase in ALT was associated with severe liver damage (Figure

4B). While male IFNAR-/- mice had some evidence of mild coagulation, neutrophil margination, and mild liver necrosis, liver damage was far more severe in female IFNAR-/- mice and was characterized by thrombosis, massive necrotic lesions and prominent apoptotic cells. To determine whether uncontrolled replication of the virus might be directly responsible for the severe liver damage in female IFNAR-/- mice, rVSVΔm51 titers were quantified in liver homogenates from mice in all groups (Figure 4C). All IFNAR-/- mice struggled to eliminate rVSVΔm51 from their livers, with titers being approximately four-logs higher than those observed in WT mice.

Interestingly, viral titers in the livers of female IFNAR-/- mice were 44% lower than those in 103

IFNAR-/- males. This meant the differential liver damage between male and female IFNAR-/- mice correlated with cytokine responses, not viral titers. These findings support the concept that a more potent immune response may facilitate accelerated clearance of an invading pathogen in females but it can also predispose them to development of severe pathogenesis driven by excessive inflammation [35].

Discussion

This study demonstrated that the modulatory effect of type I IFN signaling on anti-viral cytokine responses is regulated or controlled by sex. Further, virus infections with concomitant disruption of IFNAR signaling can lead to a sex-biased toxic anti-viral cytokine response contributing to differential liver pathogenesis between the sexes. Experimental administration of rVSV∆m51 to WT mice resulted in the presence of the virus in the liver, which was accompanied by increased local and systemic inflammatory cytokine responses. Consistent with well- established sexual dimorphic anti-viral responses [35], WT female mice tended to mount more potent systemic inflammatory cytokine responses than their male counterparts at both five- and

18-hours post-infection. The only exception was that males had more IL-12 in circulation five hours after infection. However, these cytokine responses were dwarfed by those mounted by

IFNAR-/- mice. Indeed, the magnitude of the response was so exaggerated in IFNAR-/- mice that it was likely a bona fide cytokine storm. Notably, the excessive production of pro-inflammatory cytokines in IFNAR-/- mice was strongly biased towards females. This reduced ability of female mice to negatively regulate anti-viral cytokines during disrupted IFNAR signaling implies a relationship between the virus and host sex determinants that can influence the outcome of infections. Understanding the mechanisms by which IFNAR-mediated anti-viral cytokine responses are controlled or regulated by sex-associated factors and identification of potential viral 104 strategies to compromise sex-specific factors could provide strategies to develop novel therapeutics tailored to the sexes.

Consistent with a well-defined critical role for early type I IFN responses in controlling virus replication [46-49], the livers of IFNAR-/- mice had higher titers of the infectious virus following intravenous administration of rVSV∆m51 than that of IFNAR-intact mice. Increased hepatic viral titers in IFNAR-/- mice was accompanied by higher levels of local inflammatory cytokines, implying that cellular sensing of type I IFNs and subsequent response of the given cells to IFNAR signaling was required to counteract virus replication and down-modulate inflammatory responses.

Accordingly, signaling through the type I IFN receptor has been shown to negatively regulate production of inflammatory cytokines by various cells of the immune system [15, 50-52]. IFN responsiveness of hematopoietic and non-hematopoietic cells of the immune system has also been shown to be critical in protection against virus-induced acute liver failure [2, 6]. Comparisons of hepatic viral titers in IFNAR-/- mice revealed a bias towards males. Male IFNAR-/- mice with lower systemic cytokine responses experienced higher viral loads in their livers but developed less severe pathogenesis in their livers than their female counterparts. In contrast, female IFNAR-/- mice with exaggerated systemic cytokine responses showed lower liver viral loads but developed massive liver pathology. This finding highlights excessive inflammatory responses, rather than direct viral cytopathic effects, as a possible underlying mechanism for virus-induced organ failure, suggesting that down-modulation of acute inflammation is crucial for restoring organ and tissue homeostasis, thereby protecting the host from pathogenic immune responses [53-55]. Following administration of rVSV∆m51 into mice, an absence of IFN signaling was associated with gross systemic and hepatic inflammatory responses and development of a severe acute hepatitis characterized by sinusoidal thrombosis and hepatic necrosis reminiscent of virus-induced fulminant hepatitis in 105 humans [56]. However, higher infectious titers of rVSV∆m51 in IFNAR-/- mice was not correlated with severity of liver injuries. Our results are in line with previous studies showing that excessive production of inflammatory cytokines, notably TNF-ɑ, but not necessarily viral burden, fuel the severity of liver pathogenesis in humans [57, 58] and mice [59-61]. Nonetheless, liver pathogenesis may not be exclusively ascribed to host toxic cytokine responses and virus replication likely played an essential part in triggering development of the acute cytokine storm, culminating in severe liver damage in IFNAR-/- mice. Consistent with this concept, a certain level of viremia was shown to be required for development of a full-blown hyperinflammatory syndrome, hemophagocytic lymphohistiocytosis; and non-replicative virus was shown to fail to culminate in immunopathogenesis, suggestive of a synergistic effect of viruses and host immune responses in promotion of a pathogenic event [62]. Further studies are required to investigate the contribution of virus replication versus induced inflammatory cytokines to liver pathogenesis in IFNAR-/- mice.

Identification and therapeutic targeting of cellular sources of IFNAR-mediated dysregulated cytokine responses during viral infection would provide a sensible approach to lessen the intensity of or even control immune-mediated pathogenesis of viral infections. To mount a balanced yet efficacious antiviral responses, type I IFNs exert tuned inflammatory and immunomodulatory effects. During acute virus-induced inflammation, type I IFNs have been shown to mediate temporary replacement of liver-resident macrophages, known as Kupffer cells that attenuate hepatic inflammation, with inflammatory monocyte-derived macrophages until virus clearance has occurred. Then the immunomodulatory effect of type I IFNs becomes such that it helps to restore liver homeostasis. Temporary loss of Kupffer cells during acute inflammation has been found to correlate with positive outcomes of acute hepatitis [2, 7, 63]. Such a finely tuned regulation of immune responses, however, can be disrupted during viral infections and result in acute liver 106 failure. Further studies are required to dissect the cellular sources of dysregulated cytokine responses implicated in rVSVΔm51-induced liver pathogenesis.

Using a mouse model of infection with influenza type A viruses, Polakos, et al. presented an example of collateral liver damage where in the absence of viral antigens, virus-specific CD8+ T cell responses were shown to result in damage of hepatocytes [7]. This observation, however, does not apply to our experimental model where IFNAR-/- mice mounted exaggerated cytokine responses to rVSV∆m51 and reached endpoint in only eighteen hours post-infection, when adaptive immune responses would not have developed yet.

Conclusions

To summarize, our findings demonstrated a) A critical role for IFNAR signaling in down- modulation of anti-viral cytokine responses, which could otherwise result in immunopathogenesis of viral infections. b) Sex-associated factors drove IFNAR-mediated anti-viral cytokine responses. c) Sex-biased IFNAR-mediated anti-viral cytokine responses may underlie sexually dimorphic predisposition of hosts to liver pathogenesis over the course of a non-hepatotropic viral infection.

Overall, these findings may have clinical relevance in the context of sex-biased IFNAR-mediated immunopathogenesis in livers, since several common viral infections that are associated with interference of IFNAR signaling are also often accompanied by hepatitis that can potentially result in sexually dimorphic liver failures [4, 6, 57]. Future studies to elucidate the sex-associated factors that influence IFNAR-mediated anti-viral cytokine responses could provide novel explanations for sex-biased predispositions to virus-induced immunopathologies and facilitate development of sex- tailored prevention or treatment strategies.

107

Figures of Chapter 3:

Figure 1. Mice treated with a recombinant vesicular stomatitis virus (rVSV∆m51) have elevated concentrations of plasma cytokines compared to naïve mice, with evidence of sexual dimorphism.

Six-to-eight-week-old male and female C57BL/6 mice were treated with 1x109 plaque-forming units (pfu) of rVSV∆m51 via tail vein injections. Blood was collected from the retro-orbital sinus at 5- and 18-hours after administration of the virus. Plasma-dervied cytokines were quantified using multiplex assays. Graphs show mean concentrations of the pro-inflammatory cytokines interleukin (IL)-6 (left panel), tumor necrosis factor (TNF)-α

(middle panel) and IL-12 (p40) (right panel). Standard errors are shown. A total of four mice per group were used for each independent serial sampling experiment. Four independent experiments were conducted. Representative results from one of the expeirments are shown. Statistical significance was assessed by two-way analysis of variance with

Sidak's multiple comparisons test.

108

Figure 2. Intravenous administration of recombinant vesicular stomatitis virus (rVSV∆m51) result in infection of livers, along with local inflammatory cytokine responses but with no evidence of sexual dimorphism.

Six-to-eight-week-old male and female C57BL/6 mice were infected with 1x109 plaque-forming units (pfu) of rVSV∆m51 via tail veins. The mice were monitored until endpoint signs were observed, which was at 18-hours post- infection. Livers were then harvested and assayed for titers of rVSV∆m51and cytokines. Graphs represent mean (A) virus titers and (B) concentrations of the pro-inflammatory cytokines interleukin (IL)-6 (left panel), tumor necrosis factor (TNF)-α (middle panel) and IL-12 (right panel) in liver homogenates. Data were pooled from three independent experiments (n=13/group). Standard errors are shown. Statistical significance was assessed via unpaired two-tailed t- tests.

109

Figure 3. A lack of type I interferon receptor (IFNAR) signaling causes dysregulation of cytokine responses to recombinant vesicular stomatitis virus (rVSV∆m51), which is influenced by sex.

Six-to-eight-week-old male and female C57BL/6 wild-type and IFNAR-knockout (IFNAR-/-) mice received 1x109 plaque-forming units (pfu) of rVSV∆m51 via tail veins and were monitored until endpoint signs were observed 18- hours later. Blood samples were drawn from the retro-orbital sinus to quantify concentrations of inflammatory cytokines in plasma. The livers were harvested and assayed for cytokine concentrations in homogenates. Graphs represent mean concentrations of (A) systemic (i.e. plasma-derived) and (B) hepatic pro-inflammatory cytokines interleukin (IL)-6 (left panels), tumor necrosis factor (TNF)-α (middle panels) and IL-12 (right panels) in wild-type

(WT) and IFNAR-/- male and female mice that had been infected with rVSV∆m51. (C) Overall survival of WT and

IFNAR-/- mice was also assessed after administration of the virus. Data were pooled from three independent experiments (n=13 for wild-type male and female groups; n=19 for IFNAR-/- males and n=21 for IFNAR-/- females).

Standard errors are shown. Statistical significance was assessed via two-way analysis of variance with Sidak's multiple comparisons test.

110

A

3 7×10 p<0.0001 6 70000 Male 4×10 p<0.0001 Male Male p<0.0001 5×103 Female 3×106 Female Female 50000 2×106 3×103 p=0.0035

6 L

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111

Figure 4. Male and female mice with impaired type I interferon receptor (IFNAR) signaling exhibit differential liver injuries following administration of recombinant vesicular stomatitis virus (rVSV∆m51).

Six-to-eight-week-old male and female C57BL/6 wild-type and IFNAR-knockout (IFNAR-/-) mice were treated with

1x109 plaque-forming units (pfu) of rVSV∆m51 via tail veins. Retro-orbital blood and livers were harvested from all mice when the IFNAR-knockouts reached endpoint at 18-hours post-infection. A) Alanine aminotransferase (ALT) concentrations were quantified in plasma. (B) Pieces of livers were fixed in formalin, embedded in paraffin, sectioned

(4 μm thickness) and stained with hematoxylin and eosin. Representative images are shown. Livers from females had signs of thrombosis (e.g. blue oval), apoptotic cells (e.g. orange arrows), inflammatory cell infiltrates (e.g. green arrow), and necrotic areas (e.g. brown circle); an example of healthy liver tissue is shown by the white circle. (C)

Liver slices were snap-frozen in liquid nitrogen and analyzed for cytokine concentrations. (D) Plaque assays were employed to titer rVSV∆m51 in liver homogenates. Graphs show means and standard errors. Experiments were replicated three times. Results from one representative experiment are shown (n=4/group). Statistical significance was assessed via two-way analysis of variance with Sidak's multiple comparisons test.

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113

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120

Chapter 4: Type I Interferon α/β Receptor-Mediated

Signaling Negatively Regulated Antiviral Cytokine Responses in Mouse Bone

Marrow-Derived Mast Cells

121

Highlights:

• IFNAR signaling negatively regulates BMMCs cytokine responses to rVSVΔm51.

• The lack of IFNAR signaling during viremia can lead to excessive production of

antiviral cytokines that can be associated with increased chance of virus-induced cell

death in mast cells.

122

Graphical Abstract

123

Abstract

Mast cells (MCs) are among the first sentinel cells that respond to invading pathogens and their potent inflammatory responses have been demonstrated to play a key role in the development of pathogenic viral infections. Type I interferons (IFNs) play a central role in host cellular responses to viruses and have been reported to potently induce MC-mediated inflammatory responses to a number of viruses, in-vitro. Type I IFNs exert their antiviral functions by interacting with the type I IFN receptor (IFNAR). Given that virus-induced excessive toxic inflammatory responses are associated with aberrant IFNAR signaling and considering MCs as an early source of inflammatory cytokines during viral infection, this study sought to determine whether IFNAR signaling plays a role in antiviral cytokine responses of MCs. Therefore, IFNAR-intact, IFNAR- blocked, and IFNAR-knock out (IFNAR-/-) bone marrow-derived MCs (BMMCs) were treated in- vitro with a recombinant vesicular stomatitis virus (rVSVΔm51) to assess cytokine production by these cells.

All groups of cells showed a trend for increased production of cytokines until 16 hours post- infection with rVSVΔm51. However, there was a significant difference in the amounts of cytokines produced by cells at later time points. Between 16- and 20-hours post-infection, production of IL-6 and TNF-α was notably decreased in IFNAR-intact cells as compared with

IFNAR-/- cells with a steady increase in production of both cytokines. Although the production of

IL-6 and TNF-α in IFNAR-blocked cells was less than that of IFNAR-/- cells, it was still significantly higher than IFNAR-intact cells at 20 hours post-stimulation. Furthermore, rVSVΔM51 infection was shown to induce death of BMMCs and functional IFNAR signaling was important in protecting the cells from this virus-induced cell death. Conclusions: the findings of this study show that mouse BMMCs produce pro-inflammatory cytokines in response to 124 rVSVΔM51 and that IFNAR signaling is required to shut these responses down and protect the cells from dying.

125

Introduction

The biological role of mast cells (MCs) was initially attributed to allergic inflammation [1, 2].

However, as time passed by, research findings highlighted MCs as important sentinel cells in tissues for priming and coordinating host defenses to various pathogens, including viruses [3-17].

Immature MC precursors originate from the common myeloid lineage in the BM and migrate to peripheral tissues where they mature [18, 19]. Mouse MCs are classified as connective-tissue type

MCs or mucosal-type MCs based on their tissue localization and granule content. Human MCs, however, have been classified based on protease contents of their granules: mast cell tryptase

(MCT) that produce tryptase and mast cell tryptase and chymase (MCTC) that produce tryptase and chymase. MCs are highly plastic cells whose phenotype and function are profoundly influenced by their surrounding milieu [20, 21]. Widely spread throughout the body, MCs mainly reside in tissues interfacing with the external environment, like skin, intestines, airways, and the genitourinary tract [4, 22-24]. Such strategic locations enable MCs to promptly respond to pathogens, allergens or other intrusions. MCs have developed a wide range of mechanisms to deal with the event of intrusions. These cells can be activated via complement receptors [15, 25], crosslinking of IgE bound to high affinity FCεRI receptors [26, 27], and recognition of pathogen- associated molecular patterns via pattern recognition receptors [28-30]. Activation of MCs leads to degranulation and release of granular components, including a variety of pre-formed inflammatory mediators including histamine, heparin, proteases, chymase, antimicrobial peptides, and TNF-α. Shortly after degranulation, de novo synthesis of other mediators begins, including leukotriene C4 (LTC4) and prostaglandin D2 [31, 32]. Finally, over the course of hours, a wide range of inflammatory cytokines and chemokines are produced. MC inflammatory mediators enhance mobilization of other effector leukocytes to the original site of the insult [3, 4, 29, 33-44]. 126

The nature of the stimulus has been reported to influence the nature of the MC response. For example, recognition of Escherichia coli adhesion fimbriae FimH by CD48 leads to the release of

TNF-α by mouse BMMCs [45], whereas sensing fungal β-glucan by Dectin-1 releases LTC4 by human MCs [46]. Furthermore, various TLR ligands induce inflammatory cytokines and chemokines in BMMCs [47-51], of which TLR2- and TLR4-mediated inflammatory responses have been reported to occur independently of degranulation [18, 49, 51, 52]. Induction of antiviral cytokines and chemokines by MCs appears to occur via RIG-I/MAVS/Mda5-mediated recognition of viral products leading to the induction of IFN-α and β, and chemokines [3, 9, 53, 54], independently of classical MC degranulation [9, 14, 55-58]. Human MCs have also been shown to selectively induce certain types of inflammatory cytokines and chemokines in response to treatment with IFN-α2 and IFN-γ independently of degranulation [59, 60]. An antiviral response by MCs can also occur indirectly through detection of IL-33 produced by infected epithelial cells, leading to induction of TNF-α and IL-6 without degranulation [8]. Human MCs induce type I IFNs in response to several viruses, including herpes simplex virus [8], respiratory syncytial virus [61,

62], reovirus [44, 61, 63], influenza virus [61] dengue virus [3, 53, 64], Newcastle disease virus

[50], Hantavirus [65] and Sendai virus [66]. It has also been shown that IFNs have a profound impact on cytokine and chemokine production by human MCs treated with reovirus and respiratory syncytial virus in-vitro [59]. Type I IFNs, which signal through IFNARs, are crucial for host defense against viruses and virus-induced impaired IFNAR signaling is correlated with immune- mediated pathogenesis of a variety of viral infections [67-76]. While MCs can be protective in some viral infections [8, 15], they can also be detrimental due to excessive production of inflammatory mediators that promote immunopathogenesis of viral infections [9, 12, 14, 77-88]. 127

Given the limited information on the role of type I IFN-mediated signaling on the modulation of antiviral responses of MCs, this was explored in this in-vitro study using mouse BMMCs.

Materials and methods

Mice. Six- to 8-week-old female C57BL/6 mice were purchased from Charles River

Laboratories, USA. IFNAR-/- mice on the C57BL/6 background were kindly provided by Dr.

Yonghong Wan (McMaster University, Hamilton, Ontario Canada) with the permission of

Dr. Laurel Lenz (University of Colorado, Colorado, United States) and were used as bone marrow donors for in- vitro culture and development of BMMCs. All mice were kept in an isolated specific pathogen-free facility at the University of Guelph (Guelph, Ontario, Canada).

Viruses. A replication-competent WT recombinant Indiana strain of vesicular stomatitis virus

(VSV) carrying a transgene encoding GFP and a highly attenuated version with a deletion at position 51 of the matrix protein to remove suppression of anti-viral type I IFN responses

(rVSVΔm51) [89], were kindly provided by Dr. Brian Lichty, McMaster University. These were used to study antiviral cytokine responses of BMMCs.

In-vitro differentiation of bone marrow-derived cells into MCs. BMMCs were generated according to a previously described method [90]. Femurs and tibias were harvested from mice and the marrow was flushed aseptically with HBSS (Hyclone; Cat# SH3058802) using a syringe needle

23G (BD Syringe; cat. #14-826-6B) and 18G (BD Syringe; cat. #148265D) for flushing and mixing, respectively followed by passing the homogenized single cells through a 70 μm cell strainer (Fisher Scientific; cat. #22363548) and centrifugation at 500 × g for 5 minutes. The resultant cell pellet was resuspended in a complete BMMC medium (RPMI-1640 (Hyclone; cat.

#SH3002701) medium supplemented with, 0.1 mmol/L MEM nonessential amino acid solution

(Corning, cat. #MT25025CI), 0.1 mmol/L sodium pyruvate (Gibco cat. #11360-070), 2- 128 mercaptoethanol (1:1000) (Life Technologies; cat. #21-985-023), (100U/ml) penicillin/ (100U/ml) streptomycin (Hyclone; cat. #SV30010) and 10% (V/V) fetal bovine serum (VWR Life Science

Seradigm; cat. #97068-085) supplemented with 20% (V/V) pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) to have 1×105 cells/mL in tissue-culture flasks incubated at 37°C in a 5% CO2 humidified atmosphere. Cell culture media was refreshed once a week by centrifugation and resuspension of resulting cell pellets in fresh complete BMMC medium.

Preparation of PWM-SCM. PWM-SCM was prepared as described [91] with minor modifications. Specifically, 2106 splenocytes per mL of BMMC media were cultured from

C57BL/6 mice and stimulated with 8 μg/mL lectin (Sigma; cat. # L8777) for five days at 37°C in a 5% CO2 humidified incubator. Afterwards, the medium was collected, centrifuged for 15 min at

3005 × g and filtered through a 0.2 micron-filter and archived in small aliquots in a -20°C freezer until being used for the development of mature BMMCs.

Characterization and treatment of BMMCs. In-vitro-cultured BMMCs (1× 105 cells) were cytospun and stained with Wright-Giemsa for assessment of granularity via light microscopy.

Purity of BMMCs was confirmed by flow cytometric analysis of the surface markers using FITC anti-mouse CD117 (c-Kit) (clone 2B8, eBioscience; cat. #105806), APC/Cy7 anti-mouse FCεRIα

(clone MAR-1, BioLegend; cat. #134326), and PerCP/Cy5.5 anti-mouse IL-33Rα (IL1RL1, ST2)

(clone DIH9, BioLegend; cat. #145312). Five- to 10-week-old BMMCs that were more than 95% double-positive for c- and FcεRI-α were used for experimental purposes as follows:

Viable cells were counted using 0.4% trypan blue dye (Gibco; cat. #15250) exclusion and

9×105 cells were plated in fresh complete BMMC media containing 20% PWM-SCM in a flat- bottom 96-well-plate and stimulated with a range of doses of rVSVΔm51. To detect intracellular

IL-6 and TNF-α, the cells were incubated with brefeldin A (1:1000) (BD Biosciences; cat. 129

#420601) 10 hours before the end of the experiment to retain cytokines within the cells.

Stimulation of cells with 20 ng/mL Phorbol myristate acetate (PMA) (Sigma; cat. #P1585) and

500 ng/mL ionomycin (Sigma; cat. # I9657) was used as a positive control for detection of intracellular IL-6 and TNF-α in BMMCs. To study the role of IFNAR signaling in cytokine responses of BMMCs to rVSVΔm51, WT BMMCs were treated with 10 μg/mL of anti-mouse

IFNAR-1 (clone MAR1-5A3, Leinco Technologies; cat. #0516L270) two hours before stimulation with rVSVΔm51 or PMA and ionomycin. Three groups of cells were used in these experiments:

WT BMMCs with intact IFNAR signaling, WT BMMCs treated with anti-IFNAR1 to block

IFNAR signaling, and IFNAR-/- BMMCs without IFNAR gene expression.

Flow cytometric detection of intracellular cytokines in BMMCs. Stimulated BMMCs were transferred into a U-bottom 96-well-plate to be stained for surface markers and intracellular cytokines for further analysis by flow cytometry. To that end, the cells were spun down at 500 ×g at 4°C for five minutes, the supernatant was removed and cells were re-suspended in flow cytometry staining buffer containing phosphate buffer saline (PBS) (Hyclone; cat. #SH0325601) and 0.1% bovine serum albumin (BD Biosciences; cat. #BP1600100) containing anti-mouse

CD16/32 (FC-blocking Ab) (clone 93, BD Biosciences; cat. #101321) for 15 minutes at 4°C to block FC receptors. The cells were then stained in flow cytometry staining buffer containing FITC anti-mouse c-Kit, APC/Cy7 anti-mouse FCεRIα, and PerCP/Cy5.5 anti-mouse ST2. After a 20- minute incubation at 4°C in the dark, the cells were washed with flow cytometry staining buffer, stained with a fixable viability dye (Zombie Aqua; BioLegend, cat. #423101/423102), and incubated at 4°C in the dark for an additional 20 minutes. BMMCs were washed twice with PBS and re-suspended in intracellular fixation buffer (BioLegend, cat. # 420801) for 30 minutes at room temperature in the dark. Afterwards, cells were washed once with intracellular staining 130 permeabilization buffer (1X) (BioLegend; cat. #421002) and re-suspended in permeabilization buffer containing PE anti-mouse TNF-α- (clone MP6-XT22, BioLegend; cat. #506306); APC anti- mouse IL-6 (clone MP5-20F3, BioLegend; cat. #504508). After 50 minutes incubation at room temperature in the dark, the cells were washed with permeabilization buffer and re-suspended again in permeabilization buffer and incubated at 4°C in the dark for an extra 15 minutes. The cells were then spun down and re-suspended in flow cytometry staining buffer and run on a FACS Canto

II flow cytometer (Becton Dickinson) using FACS Diva Software Version 8 for data acquisition.

The resultant data were then analyzed using Flowjo Software version 10 (FlowJo LLC, Ashland,

Oregon, USA). An example of gating strategies for flow cytometry analyses of BMMCs is shown in figure 1A.

Time-lapse images of VSV-infected BMMCs. BMMCs were infected with WT VSV expressing a GFP transgene at a multiplicity of infection (MOI) of 10 in a 25-mm cell culture petri dish and real-time images were recorded over the course of 24 hours using time-lapse-microscopy

(LumaScope, Bioimager).

Statistics. GraphPad Prism version 8 was used for all graphing and statistical analyses. Graphs show means and standard errors. Two-way analysis of variance (ANOVA) was used when means of more than two independent groups were subject to comparison across one or more time points, respectively. Statistical significance was defined as *P < 0.05; **P < 0.001; ****P < 0.0001).

Statement of work performed:

Khalil Karimi (KK) and Byram W Bridle (BWB) designed the study; Maedeh Darzianiazizi

(MD) conducted experimental work; BWB and MD analyzed data and performed the statistical analyses; MD wrote the first draft of the manuscript; KK and BWB edited the final version.

131

Results

Bone marrow cells cultured in the presence of PWM-SCM contained granules and exhibited features of MCs

In order to conduct In-vitro studies on MCs to explore their antiviral cytokine responses to rVSVΔm51 and potential influence of IFNAR signaling on antiviral cytokine responses of these cells, MCs were differentiated, In-vitro, from mouse bone marrow in the presence of pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM). After 5 weeks, the cells showed the features of mature MCs. Flow cytometric analysis of the cultured cells revealed that more than 95% of the cells expressed IL-33 receptor ST2, c-kit, and FcεRIα, all phenotypical markers of mast cells (Fig.1A). The granularity of bone marrow-derived cells cultured in PWM-

SCM-containing medium was assessed using the Wright-Giemsa staining method. As expected, five-week-old BMMCs featured large cytoplasmic secretory granules when assessed on Wright

Giemsa stained cytospun slides (Figure 1B).

BMMCs produced IL-6 and TNF-α in response to stimulation with phorbol myristate acetate (PMA) and ionomycin

In order to study cytokine responses of MCs to rVSVΔm51, PMA and ionomycin were used as stimuli for maximal activation and induction of cytokine response in BMMCs via protein kinase

C activation and increased intracellular Ca++ , respectively [92]. 16 hours post-stimulation, flow cytometry analysis showed that c-kit+/FcεRIα+ BMMCs were positive for the inflammatory cytokines IL-6 (92.85±1.88% of total cells) and TNF-α (94.92±1.46%) (Figure 2).

BMMCs could be infected by VSV

To explore the potential for MCs infection with rVSVΔm51, BMMCs were either treated with medium or WT VSV-GFP at a multiplicity of infection (MOI) of 10. Flow cytometry data showed that 14.37±1.02% of c-kit+/FcεRIα+ BMMCs were positive for GFP expression at 10 hours after 132 exposure to the virus (Figure 3A). This suggested that at least some population of BMMCs could be infected with VSV and support VSV-mediated GFP transgene expression. In addition, time- lapse video microscopy of BMMCs confirmed that these cells could be infected by VSV as demonstrated by the fluorescing green by 10 hours post-treatment. BMMCs were positive for GFP expression at 6 hours after adding VSV-GFP to the cells. GFP expression peaked at 10 hours and started to diminish at 12 hours. No more GFP-positive cells were visible 24 hours after being exposed to the virus (Figure 3B-F). The gradual disappearance of GFP-positive cells implied the possibility of virus-induced cell death.

BMMCs produced IL-6 and TNF-α in response to rVSVΔm51

BMMCs infected with rVSVΔm51 were assessed for the production of IL-6 and TNF-α using flow cytometry after intracellular staining. At 16 hours post-infection, 16.05 (mean)±2.51%

(standard deviation) and 15±1.21% of BMMCs were positive for IL-6 and TNF-α, respectively

(Figure 4). To study the kinetics of virus-induced cytokine production in BMMCs, the cells was infected with rVSVΔm51 at a MOI of 10. PMA and ionomycin-mediated stimulation were used as a control for detecting intracellular IL-6 and TNF-α. At 12 hours post-infection with the virus,

IL-6 and TNF-α were detectable in BMMCs (4.90±0.67% and 10.4±0.66%, respectively). The production of IL-6 and TNF-α increased at 16 hours (16.05±2.51% and 15.17±1.21%, respectively), but declined at 20 hours post-infection (5.87±0.64% and 7.92±0.88%, respectively)

(Figure 5). This provided a baseline cytokine profile for rVSVΔm51-treated BMMCs.

Virus-infected BMMCs produced cytokines in a virus dose-dependent manner

In order to investigate whether rVSVΔm51-induced cytokine response in MCs can be influenced by the dose of virus, BMMCs were infected with rVSVΔm51 at MOIs of 0.01, 0.1, 1, 133

10, 1 or 100. Sixteen hours post-infection, the frequencies of IL-6- and TNF-α-positive cells correlated with the dose of the virus (Figure 6). The lowest and highest frequency of IL-6

(7.16±0.62% vs. 66.95±1.20%) and TNF-α (13.8±0.78% vs. and 77.77±1.94%) were detected in cells treated with the lowest (0.01) and highest (100) MOI of virus, respectively. The highest frequency of IL-6- and TNF-α-positive cells was comparable to cells treated with PMA and ionomycin, with 92.85±1.88% and 94.92±1.46% IL-6- and TNF-α-positive cells, respectively.

rVSVΔM51 infection of BMMCs resulted in cell death in a time- and virus dose-dependent manner

In order to study the potential effect of virus-induced cytokine responses on viability of cells,

BMMCs were treated with medium and MOI 10 of rVSVΔm51. After 12, 16, and 20 hours, the cells were stained with a fixable viability dye to look at the number of viable cells using flow cytometry. In contrast to medium-treated cells, cell viability in virus-treated cells showed a decreasing trend over the course of rVSVΔm51 infection. There was a gradual but significant reduction in the number of viable cells between 12- and 20-hours post-infection as compared with controls (6,572±220.52 vs. 9,834.5±47.9 at 12 hours; 4,741.75±193.91 vs. 9,408.75±318.48 at 16 hours; and 3,316.25±645.55 vs. 9,781.5±1023.995 at 20 hours, respectively) (Figure7A).

Furthermore, cell viability had an inverse relationship with the dose of the virus. BMMCs infected with a MOI of 100 of rVSVΔm51 had the lowest number of viable cells (1,361.75±577.54) compared with cells infected with a MOI of 10 (3,316.25±645.55) at 20 hours post-infection

(Figure 7B).

IFNAR signalling facilitated down-regulation of cytokine production by BMMCs

In order to explore the potential effect of IFNAR signaling on MCs’ antiviral cytokine responses, IFNAR-intact, IFNAR-blocked, and IFNAR-/- BMMCs were treated with rVSVΔm51 134 at a MOI of 10 and assessed for cytokine production. PMA and ionomycin were used as control stimuli for detecting cytokines in BMMCs. All groups of cells showed an increase in production of cytokines between 12- and 16-hours post-infection. However, there was a significant difference between BMMCs regarding IL-6 and TNF-a production at later time points. Production of IL-6 and TNF-a decreased between 16- and 20-hours post-infection in IFNAR-intact cells from

16.05±2.51% and 15±1.21% to 5.87±0.64% and 7.92±0.88%, respectively. In contrast, IFNAR-/-

BMMCs showed a steady increase in production of both cytokines between 16- and 20-hours post- infection (17.27±1.61% and 25.90±1.79%, respectively). Although the production of both cytokines in IFNAR-blocked BMMCs was lower than that of IFNAR-/- cells, it was still significantly higher than IFNAR-intact cells, at 20 hours post-infection (9.30±0.79% IL-6 and

14.75±1.20% TNF-α, respectively) (Figure 8). Together, these findings highlight the importance of IFNAR signaling in the ability of BMMCs to down-regulate antiviral cytokine responses after an infection. Specifically, disruption of type I IFN signaling during a viral infection can result in over-production of cytokines by MCs.

IFNAR signaling protected BMMCs from rVSVΔM51-induced cell death

In order to explore the potential effect of IFNAR signaling on virus-induced cell death,

IFNAR-intact, IFNAR-blocked, and IFNAR-/- BMMCs were treated with medium and rVSVΔm51 at a MOI of 10. After 12, 16, and 20 hours, the cells were stained with a fixable viability dye to look at the number of viable cells using flow cytometry. In contrast to medium-treated cells, all groups of virus-treated cells showed a reduction in cell viability after infection with rVSVΔm51

(Figure 9A). At 20 hours post-infection, IFNAR-/- BMMCs, which over-expressed cytokines

(Figure 8), showed lower number of viable cells than IFNAR-blocked and -intact BMMCs (Figure

9A) that had relatively lower-magnitude cytokine responses (Figure 8) (651.75±364.06 vs. 135

2,545.75±520.85 vs. 3,316.25±645.55, respectively). There was not a significant difference in viability of cells between IFNAR-intact and-blocked BMMCs at 20 hours post-infection.

IFNAR- /- cells did not show a significant difference in the reduction of viable cells after 20 hours post-infection with rVSVΔM51 at MOIs of 10 versus 100 (651.75±364.06 vs. 151±45.32, respectively) (Figure 9B). This suggested that virus-induced cytotoxicity was not dose-dependent in these cells. In contrast, the reduction in viability of IFNAR-blocked cells did correlate with the dose of the virus (2,545.75±520.85 vs. 1,035±304.64 after treatment with rVSVΔM51 at MOIs of

10 and 100, respectively).

Discussion

Pro-inflammatory mediators produced by MCs against invading pathogens can profoundly shape the nature of host immune responses. In some cases potent MC-mediated inflammatory responses have been demonstrated to play a key role in the development of virus-associated pathologies [93]. Given that virus-induced toxic inflammatory responses are associated with aberrant IFNAR signaling and considering MCs as an early source of inflammatory cytokines during viral infection [54, 94], this study sought to determine whether IFNAR signaling plays a role in antiviral cytokine response of MCs.

Their prevalence at common points of entry for pathogens, expression of a broad array of potential entry receptors, and a wide range of inflammatory mediators have made MCs ideal targets for a number of viruses. The susceptibility of MCs to viral infection varies between viruses [3, 9,

12, 14, 15, 53, 95]. However, viral infection of MCs may not necessarily lead to the release of infectious virions [9, 62, 95]. While this study shows that BMMCs are infected with VSV, whether the infection was productive is a question that future studies may address. 136

In-vitro, MCs have been reported to produce inflammatory cytokines and chemokines in response to different stimuli, including lipopolysaccharide [96, 97], synthetic viral double- stranded RNA as well as infection of viruses [12, 50] including VSV [30, 54]. In this study, in- vitro-cultured BMMCs were shown to produce pro-inflammatory IL-6 and TNF-α cytokines in response to rVSVΔM51. Further, IFNAR signaling was required to control this cytokine production and protecting the cells from death. Relatively lower production of cytokines in

IFNAR-blocked BMMCs than that of IFNAR-/- cells may infer that antibody-mediated blockade of IFNARs might have been incomplete and that some level of IFNAR signaling might have been maintained during infection with rVSVΔm51 infection. If true, this would support the importance of functional IFNAR signaling in regulation of antiviral cytokine responses.

rVSVΔM51-induced cell death in BMMCs observed in this study aligns with previous reports on attenuated strains of VSV that were found to be highly lytic in IFN-non-responsive on a number of NCI-60 human tumor cell lines (, colon carcinoma, non-small-cell lung carcinoma, melanoma, ovarian carcinoma, renal carcinoma, prostate, and breast cancer), as compared with

IFN-responsive cells [98, 99]. Importantly, our findings highlight rVSVΔM51-induced cell death in primary cultures of MCs. This may be particularly relevant to oncolytic virotherapies that rely on rhabdoviruses, especially since these are versatile innate sentinel cells that can profoundly shape host immune responses to a viral infection. Infection and killing of MCs during rhabdovirus- mediated oncolytic virotherapies might be an unappreciated off-target effect. Further, mast cells might contribute to adverse events that are associated with exaggerated cytokine responses in some patients treated with oncolytic viruses. These concerns deserve thorough investigation in the future. 137

Moreover, influenza A virus-induced apoptosis in the P815 mouse mastocytoma cell line was associated with virus replication and production of pro-inflammatory cytokines and chemokines, including IL-6 and TNF-α. This virus-induced apoptosis was implicated in the pathogenesis of influenza A virus infections [12]. MC apoptosis has also been reported to be induced by other viruses, including dengue virus [100] and rhinovirus [101]. Dengue virus-induced apoptosis in human MCs and MC lines was also associated with production of pro-inflammatory cytokines and chemokines [102]. This current study, however, did not evaluate the type of cell death programme that in-vitro-cultured BMMCs underwent during rVSVΔM51 infection.

Activation of MCs has been associated with cell degranulation [94]. Whether rVSVΔM51- induced cytokine responses in BMMCs were accompanied by degranulation remains to be determined. However, VSV infection and cytokine production of BMMCs have been reported to be associated with cell degranulation. Given MC degranulation was detected at six hours after infection with VSV, the authors proposed that MC infection by the virus was probably required for cell degranulation [54]. However, in another study, infection with dengue virus was not required to trigger human, monkey, and rodent MC degranulation [3].

Conclusions

Taken together, the results of this study emphasize the role of IFNAR signaling in controlling the magnitude of antiviral cytokine responses by MCs. Given that MCs are among the early cytokine-producing cells during viral infections [54] and given their well-established contributions to toxic cytokine responses during severe pathogenic viral infections [103], it is important to define the early virus-induced events that lead to unbridled inflammatory responses by these versatile cells. According to the findings of this study, it is tempting to speculate that virus-induced type I

IFN responses in epithelial and mucosal cells modulate tissue-resident MCs. In the event of viral 138 invasion associated with impaired IFNAR signaling, unrestrained inflammatory responses of MCs could lead to excessive infiltration of inflammatory cells and subsequent generation of a cytokine storm, thereby causing the development of immune-mediated pathogenesis. Further understanding of IFNAR-mediated antiviral cytokine responses of MCs might facilitate the rational development of novel strategies to prevent or treat pathogenesis associated with excessive inflammatory responses by MCs during viral infections.

139

Figures of Chapter 4:

Figure 1. Mouse bone marrow-derived cells cultured in the presence of pokeweed mitogen-stimulated spleen

cell-conditioned medium (PWM-SCM) contained granules and exhibited features of mast cells.

Bone marrow-derived cells were obtained from five-week-old C57BL/6 mice and cultured in 20% (V/V) PWM-

SCM-containing media. After five weeks, the purity of BMMCs was assessed using flow cytometry by staining them

with surface-staining antibodies against FcεRIα, CD117 (c‐Kit), and IL-33 receptor ST2. (A) A side Scatter (SSC)/

forward Scatter (FSC) gate was initially applied to exclude any electronic noise, followed by a singlet gate to exclude

any doublets, then a viability gate to exclude dead cells. Cells that were double-positive for FcεRIα and c-kit were

defined as mast cells and were also shown to be ST2-positive. Cell granularity was assessed using (B) Wright-Giemsa

staining of a cytospin prepared from 1x105 cells.

A

it

k

- c

FcεRIα

FSC(width)

Side Scatter SideScatter (SSC) Fixable Viability Viability Fixable Dye

Forward Scatter (FSC) FS FS C C ST2

FSC

140

B

141

Figure 2. Mouse bone marrow-derived mast Cells (BMMCs) produced interleukin (IL)-6 and tumour necrosis factor(TNF)-α in response to phorbol myristate acetate (PMA)/ ionomycin.

BMMCs were treated with PMA (20 ng/mL) and ionomycin (500 ng/mL). Sixteen hours later, the cells were stained for surface markers FcεRIα and c-kit and intracellular cytokines, IL-6 and TNF-α. The cells were analyzed by flow cytometry. Fluorescence minus one controls were used to set gates to identify IL-6+ or TNF-α+ cells. Shown are dot plots that are representative of >10 similar experiments, showing means and standard errors of IL-6+ or TNF-α+ cells among FcεRIα+c-Kit+ mast cells

A

6 -

IL 92.85±1.88%

FcεRIα

B

α

-

TNF 94.92±1.46%

FcεRIα 142

Figure 3. Mouse bone marrow-derived mast Cells (BMMCs) showed susceptibility to infection with vesicular stomatitis virus (VSV).

BMMCs were left untreated in the cell culture medium or treated with a multiplicity of infection of 10 of WT VSV that carried a transgene encoding GFP. (A) Ten hours later, BMMCs were harvested and stained for FCεRIα and c-

Kit. GFP + cells, representative of VSV-infected BMMCs were detected using flow cytometry. Dot plots are representative of four similar experiments showing means and standard errors of GFP + FCεRIα+c-Kit+ mast cells. (B-

F) Fluorescent microscopy of VSV-GFP-treated BMMCs (from tree biological replicates) at (B) 5 minutes, (C) 6 hours, (D) 10 hours, (E) 12 hours, and (F) 24 hours.

143

144

Figure 4. Mouse bone marrow-derived mast Cells (BMMCs) produce interleukin (IL)-6 and tumour necrosis factor-α (TNF-α) in response to recombinant strain of vesicular stomatitis virus (rVSVΔm51).

BMMCs were infected with rVSVΔm51 at a multiplicity of infection of 10. Sixteen hours later, the cells were stained for surface markers FcεRIα and c-kit and intracellular cytokines, IL-6 and TNF-α. The cells were analyzed by flow cytometry. Fluorescence minus one controls were used to set gates for identifying IL-6+ or TNF-α+ cells. Dot plots that are representative of >10 similar experiments, showing means and standard errors of IL-6+ or TNF-α+ cells among

FcεRIα+c-Kit+ mast cells.

145

Figure 5. kinetics of cytokine production by bone marrow-derived mast Cells (BMMCs) after infection with a recombinant strain of vesicular stomatitis virus (rVSVΔm51).

BMMCs were infected with rVSVΔm51 at a multiplicity of infection of 10. Twelve, 16, and 20 hours later, the cells were stained for the surface markers FcεRIα and c-kit, as well as intracellular cytokines, interleukin (IL)-6 and tumour necrosis factor-α (TNF-α). The cells were then analyzed using flow cytometry. Fluorescence minus one (FMO) controls were used to set gates to identify IL-6 + and TNF-α + cells. PMA and ionomycin were used as control stimuli.

Graphs and representative dot plots show means and standard errors (n=4) of (A, B) IL-6+ and (C, D) TNF-α+ FcεRIα

+ c-Kit + mast cells at 12-, 16-, and 20-hours post-infection. Two-way analysis of variance (ANOVA) with Tukey’s multiple comparison test was used to define statistical significance as *P < 0.05; **P < 0.001; ****P < 0.0001.

146

Intact IFNAR

147

D

148

Figure 6. Cytokine responses of bone marrow-derived mast Cells (BMMCs) to recombinant strain of vesicular stomatitis virus (rVSVΔm51) was dose-dependent.

BMMCs were infected with rVSVΔm51 at a multiplicity of infection (MOI) of 0.01,0.1, 1, 10 or 100. After 16 hours, the cells were stained for the surface markers FcεRIα and c-kit as well as intracellular cytokines, interleukin (IL)-6 and tumour necrosis-α (TNF-α). The cells were then analyzed by flow cytometry and fluorescence minus one (FMO) controls were used to set gates to identify IL-6 + and TNF-α + cells. Graphs and representative dot plots show means and standard errors (n =4) of (A) IL-6 and (B) TNF-α expression in FcεRIα+c-Kit+ mast cells are shown after infection with rVSVΔM51 or treatment with PMA/ionomycin. Two-way analysis of variance (ANOVA) with Tukey’s multiple comparison test was used to define statistical significance as *P < 0.05; **P < 0.001; ****P < 0.0001.

149

rVSVΔM51 (MOI)

FMO PMA /Ionomycin 0.01 0.1 1 10 100

α - 94.92±1.46% 13.80±0.78% 16.10±1.68% 22.50±2.84% 39.65±2.34% 77.77±1.96% TNF FCεRIα

150

Figure 7. Infection of bone marrow-derived mast Cells (BMMCs) with recombinant strain of vesicular stomatitis virus (rVSVΔm51) resulted in cell death.

BMMCs were infected with rVSVΔm51 at a multiplicity of infection (MOI) of 10 and at various time points, the cells were stained for the surface markers FcεRIα and c-kit. A cell viability dye to discriminate live and dead BMMCs was applied to the cells before flow cytometry analysis. Graphs show means and standard errors (n=4) of the number of viable cells among total FcεRIα+c-Kit+ mast cells at (A) 20 hours post-infection with 10 or 100 MOI of the virus and

(B) at 12, 16, and 20 hours post-infection with 10 MOI of the virus. Two-way analysis of variance (ANOVA) with

Tukey’s multiple comparison test was used to define statistical significance as *P < 0.05; **P < 0.001; ****P <

0.0001.

151

Figure 8. Recombinant strain of vesicular stomatitis virus (rVSVΔm51)-induced cytokine production by bone marrow-derived mast Cells (BMMCs) was regulated by Type I IFN α/β Receptor (IFNAR) signaling.

BMMCs were left untreated (intact-BMMCs) or incubated with an antibody that blocked the type I IFN receptor

(IFNAR-blocked-BMMCs), or were derived from IFNAR-knock out (IFNAR-/-) mice (IFNAR-/- BMMCs) were infected with rVSVΔm51 at a multiplicity of infection of 10. After 12, 16, and 20 hours, the cells were stained for the surface markers FcεRIα and c-kit, as well as intracellular cytokines interleukin (IL)-6 and tumour necrosis factor-α

(TNF-α). The cells were then analyzed by flow cytometry and fluorescence minus one (FMO) controls were used to

+ + + + set gates to identify IL-6 and TNF-α cells. (A) IL-6 and (B) TNF-a expression in FcεRIα c-Kit mast cells are shown after infection with rVSVΔM51 or treatment with PMA/ionomycin. Line graphs (upper panel) and representative flow cytometry dot plots (lower panels) show means and standard errors (n=4 (intact- and blocked-

BMMCs) and n=6 (IFNAR-/- BMMCs)). Two-way analysis of variance (ANOVA) Tukey’s multiple comparison test was used to define statistical significance as *P < 0.05; **P < 0.001; ****P < 0.0001.

152

153

IFNAR-/-

154

155

IFNAR-/-

156

Figure 9. Type I IFN α/β Receptor (IFNAR) protected mice bone marrow-derived mast Cells (BMMCs) from

Recombinant strain of vesicular stomatitis virus (rVSVΔm51)-induced cell death.

-/- IFNAR-intact, IFNAR-blocked, and IFNAR-knock out (IFNAR ) BMMCs were infected with rVSVΔm51 at a multiplicity of infection (MOI) of 10 or 100. Twelve, 16, and 20 hours later, the cells were stained for the surface markers FcεRIα and c-kit. A cell viability dye was used to discriminate live and dead BMMCs by flow cytometry analysis. Graphs represent mean and standard errors (n=4 [IFNAR intact and IFNAR-blocked BMMCs] and n=6

-/- + + [IFNAR BMMCs]). of the number of viable cells among FcεRIα c-Kit mast cells at (A) 20 hours post-infection with 10 or 100 MOI of the virus and (B) at 12, 16, and 20 hours post-infection with 10 MOI of the virus. Two-way analysis of variance (ANOVA) with Tukey’s multiple comparison test was used to define statistical significance as

*P < 0.05; **P < 0.001; ****P < 0.0001.

157

158

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169

Chapter 5: Summary of Research

170

Virus-induced type I interferon response: disrupted type I interferon receptor-mediated signaling and dimorphic cytokine responses

The present study investigated the role of IFNAR and sex in cytokine responses to viral infection. Our findings indicate a crucial role for IFNAR signaling in viral clearance and establishment of an anti-viral state, as well as host survival from viremia caused by a recombinant strain of VSV. IFNAR signaling was also found to play a crucial role in the negative regulation of antiviral cytokine responses. Further, disrupted IFNAR signaling during a viral infection was associated with sexually dimorphic dysregulated cytokine responses. Based on the findings of this study, it is tempting to speculate that a strategy to protect rVSVΔm51-infected mice without risk of overly robust inflammation could be accomplished by diverting type I IFN-mediated antiviral responses to alternative antiviral pathway(s) that function independently of IFNAR signaling. This could involve pathways mediated by other types of IFNs or by inducing host intrinsic IFN- independent antiviral mechanisms. A number of anti-inflammatory strategies have recently been discussed for their potential in reducing virus-induced excessive inflammation [1-9]. However, exploring strategies to induce alternative host intrinsic antiviral mechanisms could be a more efficient approach to prevent excessive inflammatory responses associated with severe viral infections. In the model presented in this study, one could speculate that blocking intracellular signaling pathways leading to induction of IFN-β or infecting mice with a virus capable of blocking host induction of IFN-β might rescue the mice from excessive inflammation and death. In a setting where mice are incapable of producing IFN-β, an early type I IFN, cells might be forced to upregulate alternative type I IFN-independent antiviral pathways to overcome a viral infection

[10]. Importantly, these pathways might not develop excessive cytokine responses if IFNAR signaling is compromised. VSV is sensitive to host antiviral defenses. This is despite the viral M protein making the virus efficient in hindering host type I IFN responses, particularly the late phase 171 of the IFN response in which expression of IFN-α and IFN-β genes is amplified [10-17]. This implies that type I IFN-independent mechanisms can protect a host against infection with VSV.

Accordingly, in-vitro studies of mouse embryonic fibroblasts and a human respiratory epithelial cell line showed induction of potent antiviral genes that protected cells from VSV infection independently of type I IFN [10, 18]. Type I IFN-independent mechanisms that control infections with VSV have also been reported in-vitro [19-21]. WT VSV, which has an intact M protein was reported to be capable of blocking the host type I IFN system, causing virus-infected cells to use a bypass mechanism to survive viral infection independently of the IFN system [10]. However, in the current study, intact IFNAR signaling was found to be required for mice to properly control infections with both WT and Δm51-mutant strains of VSV with intact and mutant M proteins, respectively. In this study, IFNAR knock-out mice infected with rVSVΔm51, which lacks the ability to interfere with host type I IFNs due to a mutation in the matrix protein, succumbed to infection with this highly attenuated virus. While these findings together support an important role for type I IFNs in host antiviral defense against VSV, further studies are needed to elucidate whether protective antiviral defenses against VSV are restricted to the type I IFN system or whether sufficient upregulation of alternative pathway(s) could compensate for the absence of type

I IFN signaling.

Regarding the effect of sex on IFNAR signaling, our findings infer a potential for sex- dependent factors to enable the host to bypass excessive virus-induced inflammatory responses during impaired IFNAR signaling. The ability of male mice to downmodulate inflammatory responses (in the case of mice treated with anti-IFNAR1) or produce lower concentrations of inflammatory cytokines (in the case of IFNAR-KO mice) compared with their female counterparts, raises conjectures regarding factors unique to males that can compensate for disrupted IFNAR 172 signaling during a viral infection. Further, our findings also imply a strict versus flexible reliance of females and males, respectively, on intact IFNAR signaling for the negative regulation of antiviral cytokine response. The results of this study also raise questions about the roles of sex hormones in the regulatory arm of antiviral IFNAR signaling cascades and whether male hormones modulate IFNAR signaling in a way that enables them to efficiently regulate antiviral cytokine responses irrespective of the status of IFNAR signaling. Alternatively, female sex hormones might represent a downstream component of the IFNAR signaling cascade and, therefore, cannot be involved in immunomodulatory responses without having a license from an upstream functional

IFNAR. While further studies may be worthwhile to address these conjectures/queries, a better understanding of the interaction between sex hormones and IFNAR signaling pathways could provide new concepts to support the development of sex-tailored anti-inflammatory therapeutics.

Considering sex disparity in the development of pathogenic viral infections and the natural tendency of male species to mount relatively moderate anti-inflammatory responses during a viral infection, characterization of antiviral signaling pathways mediated by male sex hormones could potentially identify novel biological markers associated with prevention of virus-induced cytokine- mediated toxicities. Mimicking or targeting of those biological markers in females might confer them with the ability to moderate inflammatory responses, thereby preventing them from developing immunopathogenesis.

Disrupted IFNAR signaling drove sexually dimorphic virus-induced liver damage

The current study also demonstrated that IFNAR-mediated hepatic antiviral cytokine response is influenced by sex and that virus-induced inflammatory cytokine response in the absence of

IFNAR signaling can lead to a biased excessive cytokine response contributing to differential liver damage. Experimental administration of rVSV∆m51 virus to WT mice demonstrated presence of 173 the virus in the liver which was accompanied by increased local and systemic inflammatory cytokine responses. Consistent with well-established sexual dimorphic anti-viral responses [22], female mice mounted more plasma IL-6 and TNF-α than their male counterparts at 5 hours post- infection. IFNAR-/- mice, on the other hand, mounted exaggerated levels of both systemic and local pro-inflammatory cytokines at 18 hours post-infection which was strongly biased towards females.

Such a reduced ability of female mice to negatively regulate antiviral cytokine response during disrupted IFNAR signaling implies an association/correlation between virus and host sex- discriminating factors that determine the outcome of infection. Our findings are also aligned with previously reported sex-biased hepatic inflammatory gene expression [23] and a protective role of type I IFN(s) and IFNAR signaling against viral and/or non-viral pathogenesis of the liver [24-36].

However, to the best of our knowledge, we showed for the first time that impaired IFNAR signaling during a viral infection can result in a sex-biased deregulation of inflammatory responses which can lead to a sexually dimorphic development of liver damage, which was most severe in females. There was an inverse correlation between viral load and liver damage between sexes, suggesting that liver damage was likely not a direct effect of rVSV∆m51 in IFNAR-/- mice. It is well-documented that deregulated inflammatory responses contribute to liver pathology [29, 37].

IL-6 is a versatile cytokine involved in hepatic response to infections and/or systemic inflammation

[38]. Regarding interaction between sex hormones, inflammatory responses, and development of liver pathogenesis, similar to DILI (drug-induced liver injury) patients, female BALB/c mice with more inflammatory cytokines, in particular IL-6, have been reported to develop more severe experimental DILI than males. Elevated levels of estrogen and IL-6 were found to be associated with severity of DILI. Estrogen-induced IL-6 in female mice was concluded to promote sex- disparity in severity of DILI by reducing the expansion of splenic T regs in females [39]. 174

However, an inverse disparity in estrogen and IL-6 interaction was observed in a mouse model of hepatocellular carcinoma. Estrogen-mediated inhibition of IL-6 production by hepatic KCs was shown to inhibit chemically induced liver carcinogenesis in females. In response to a chemical carcinogen, serum IL-6 and liver IL-6 mRNA were higher in male mice than females. Estradiol

(E2) administration to male mice reduced liver IL-6 mRNA and liver injury while ovariectomy lead to increased accumulation of liver IL-6 mRNA and liver injury in females.

In spite of discrepancies in the literature regarding the correlation between estrogen and IL-6 production/function and their contribution to liver damage, these findings, at least, emphasize the concept that sex-biased inflammatory responses are implicated in dimorphic liver damage. The present study has not looked at the concentration of sex hormones in WT and IFNAR-/- mice during rVSVΔm51infection to conclude any correlation/interaction between sex hormones and hepatic inflammatory responses contributing to differential liver damage.

The findings of this study also highlight clinical importance of sex-biased IFNAR-mediated immunopathogenesis of the liver since several common viral infections often accompanied with hepatitis [25, 40, 41] which can potentially result in sex-biased liver failure. Understanding the mechanisms by which IFNAR-mediated antiviral cytokine response is influenced by sex and identification of potential viral strategies compromising the interplay between host sex and antiviral response not only provides novel explanations for sex-biased predisposition to virus- induced immunopathology but also opens new doors towards development of novel sex-tailored ant-inflammatory therapeutics.

Type I IFNs play a critical role in viral clearance and downmodulation of antiviral inflammatory responses, thereby protection against virus-induced acute liver failure [24, 25, 42-

47]. Accordingly, anti-inflammatory effect of IFNAR signaling and subsequent protection against 175 liver pathogenesis have also been reported [27, 29, 30, 33, 34, 48]. Consistent with these findings, at 18 hours post-infection, IFNAR-/- mice still showed far higher hepatic virus titers and higher levels of both systemic and local inflammatory cytokines than control mice. However, an inverse correlation was observed between plasma (data not shown) and liver virus titers and mounted inflammatory cytokines in IFNAR-/- mice. Male and female IFNAR-/- mice with lower and higher systemic and local cytokine responses, experienced higher and lower systemic and local viral load, respectively. This finding supports the well-established concept saying that excessive inflammatory response, rather than viral cytopathic effect, underlies virus-induced organ failure and that down-modulation of acute inflammation is crucial for restoring organ/ tissue homeostasis, thereby protecting the host from pathogenic immune responses [49-51].

Virus-induced differential liver damage during impaired IFNAR signaling was associated with dimorphic liver-infiltrating neutrophils and macrophages.

Identification and therapeutic targeting of cellular sources of cytokines during IFNAR- mediated cytokine responses to a viral infection could provide a sensible approach to lessen the intensity or even control immune-mediated pathogenesis of viral infections. The current study did not directly confirm a cellular source of deregulated inflammatory cytokines implicated in rVSVΔm51-induced liver pathogenesis in the absence of IFNAR signaling. However, we found that male IFNAR-/- mice with minor liver damage showed a higher frequency and number of hepatic neutrophils and MΦs as compared with females that had severe liver damage but lower frequencies and numbers of neutrophils and MΦs in the liver (data not shown). Given the inflammatory roles of neutrophils and MΦs and given their implication in virus-associated cytokine storms, it would be interesting to determine how hepatic neutrophils and MΦs of males and females differ during viral infections in the absence of IFNAR signaling. Whether quantity or 176 quality of either neutrophils or MΦs determined the sex-disparity of liver damage in IFNAR-/- mice remains to be explored. Given the importance of IFNAR signaling in cell proliferation, development, differentiation [52, 53], and trafficking into infected sites [54, 55], virus-induced impairment of IFNAR signaling might be implicated in the development of immature and highly active leukocytes infiltrating to infected sites, thereby promoting the development of immune- mediated pathogenesis. In malaria infections, for instance, type I IFN signaling has been shown to be implicated in liver damage by infiltration of highly inflammatory low density neutrophils [56] whose signature has been found in a variety of other pathological conditions [57-60]. Furthermore, considering that IFNAR signaling is influenced by sex, sex-disparity in virus-induced impaired

IFNAR-signaling could be the result of a sexually dimorphic leukocyte profile, including alterations in the frequency and/or type of neutrophils and MΦs. Future studies could characterize hepatic neutrophils and MΦs of male and female IFNAR-/- mice during infection with rVSV∆m51 to try to elucidate their relevance to the development of virus-induced differential liver damage in the absence of IFNAR signaling.

IFNAR signaling negatively regulated antiviral cytokine response in mast cells and protected the cells from virus-induced death

This study also discovered a role for IFNAR signaling in the negative regulation of antiviral cytokine responses in in-vitro-cultured BMMCs. MCs are among the first sentinel cells that respond to invading pathogens and their potent inflammatory responses have been demonstrated to play a key role in the development of pathogenic viral infections. Type I IFNs with a central role in host cellular responses to viral infections, have been reported to potently induce MC- mediated inflammatory responses to a number of viruses, in-vitro. To explore whether IFNAR signaling plays a role in antiviral cytokine responses of MCs, IFNAR-intact, IFNAR-blocked, and 177

IFNAR-/- BMMCs were treated in-vitro with rVSVΔm51 to assess cytokine production by these cells. Although all groups of cells showed increased production of IL-6 and TNF-α until 16 hours post-infection with rVSVΔm51, the cells showed a significant difference in the production of cytokines at later time points. While production of IL-6 and TNF-α was significantly decreased in

IFNAR-intact cells, IFNAR-/- cells showed a steady increase in production of both cytokines.

Although the production of IL-6 and TNF-a in IFNAR-blocked cells was less than that of IFNAR-

/- cells, it was still significantly higher than IFNAR-intact cells at 20 hours post-stimulation. Such a relatively lower production of cytokines in IFNAR-blocked BMMCs may reflect partial antibody-mediated blockade of IFNARs maintaining some level of IFNAR signaling during infection with rVSVΔm51 infection. If true, this would support the importance of functional

IFNAR signaling in regulation of antiviral cytokine responses. In addition, rVSVΔM51 infection was shown to induce the death of BMMCs and that functional IFNAR signaling was important in protecting the cells from this virus-induced cell death. At 20 hours post-infection, cell viability was significantly reduced in all groups of cells. IFNAR-/- BMMCs with higher production of cytokines, showed lower number of viable cells than IFNAR-blocked and -intact BMMCs with relatively lower-magnitude cytokine responses. These findings are aligned with previous reports on higher susceptibility of IFN-non-responsive human tumor cell lines to death induced by attenuated strains of VSV as compared with IFN-responsive cells [15, 61]. Our findings also, to some extent, can support recent reports on an association between virus-induced apoptosis in MCs and virus replication and production of pro-inflammatory cytokines and chemokines, including

IL-6 and TNF-α [62, 63]. Future studies may characterize the type of cell death programme that in-vitro-cultured BMMCs underwent during rVSVΔM51 infection concomitant with functional and disrupted IFNAR signaling. Our findings, however, highlight rVSVΔM51-induced cell death 178 in primary cultures of MCs which might be particularly relevant to oncolytic virotherapies that rely on rhabdoviruses. Considering MCs as versatile innate sentinel cells that can profoundly shape host immune responses to a viral infection, infection and killing of these cells during rhabdovirus- mediated oncolytic virotherapies might be an unappreciated off-target effect. Further, mast cells might contribute to adverse events that are associated with exaggerated cytokine responses in some patients treated with oncolytic viruses. These concerns deserve thorough investigation in the future. Given that MCs are among the early cytokine-producing cells during a viral infection [64] and given their well-established signature in excessive cytokine responses during some pathogenic viral infections [55], it would be worthwhile to investigate how they contribute to virus-induced cytokine storms. MCs might represent ideal therapeutic targets to limit excessive inflammatory responses that result from disrupted IFNAR signaling during viral infections, especially in females.

Conclusions

Taken together, the findings of this study indicate: 1) A crucial role for IFNAR signaling in negative regulation of systemic and local anti-viral cytokine responses which can be influenced by sex during viremia associated with the lack of IFNAR signaling. 2) A sexually dimorphic landscape of IFNAR-mediated antiviral responses which can make the host either susceptible or resistant to immune-mediated pathogenesis during viremia associated with the lack of IFNAR signaling. 3) Mast cells as potential therapeutic target to limit excessive anti-viral inflammatory responses resulted from disrupted IFNAR signaling, particularly in females. Moreover, rVSVΔm51-induced cell death in primary cultures of mast cells might particularly be relevant to oncolytic virotherapies relying on rhabdoviruses which deserve future investigations.

179

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