Gharagozloo Marjan Phd 2019.Pdf

Université de Sherbrooke

The NOD-Like Receptors as Endogenous Inhibitors of Multiple Sclerosis

Par Marjan Gharagozloo Programme d’Immunologie

Thèse présentée à la Faculté de médecine et des sciences de la santé en vue de l’obtention du grade de philosophiae doctor (Ph.D.) en Immunologie

Sherbrooke, Québec, Canada January, 2019

Membres du jury d’évaluation Dr. Denis Gris, programme d’Immunologie Dr. Abdelaziz Amrani, programme d’Immunologie Dr. Xavier Roucou, external evaluator program Dr. Samuel David, external evaluator at the University

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This thesis is dedicated to

My dear husband, Behrooz, who inspired me for MS research. His endless sacrifice and love were a valuable source of strength in my life,

My lovely twin sons, who gave me joy and encouraged me to overcome all the challenges,

And my kind parents, who were always by my side to give me love, support, and confidence

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RÉSUMÉ Les récepteurs de type NOD en tant qu'inhibiteurs endogènes de la sclérose en plaques

Par Marjan Gharagozloo Programmes d’Immunologie

Thèse présentée à la Faculté de médecine et des sciences de la santé en vue de l’obtention du grade de philosophiae doctor (Ph.D.) en Immunologie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4

La sclérose en plaques (SEP) est une maladie chronique du système nerveux central (SNC) qui se manifeste par des rechutes dans la phase précoce de la maladie suivies d'une invalidité progressive dans la phase ultérieure. Les réponses immunitaires innées et adaptatives jouent un rôle crucial dans l'apparition et la progression de la maladie. Cependant, les traitements disponibles actuellement pour la SEP ciblent principalement la réponse immunitaire adaptative avec un effet limité sur la réponse immunitaire innée et l'inflammation du SNC. Par conséquent, les molécules anti-inflammatoires endogènes sont des cibles thérapeutiques potentielles pour prévenir la pathologie de la SEP. Les récepteurs de type NOD (NLR) sont une famille de récepteurs immunitaires innés qui sont des régulateurs de l'inflammation dans les cellules. Certains membres de cette famille, tels que NLRP1 et NLRP3, induisent des réponses inflammatoires, tandis que d'autres membres, comme NLRP12 et NLRX1, inhibent les voies inflammatoires. L'objectif de cette étude est d’investiguer si les NLRs anti- inflammatoires préviennent l'apparition et la progression de la SEP. Pour atteindre notre objectif, nous avons examiné les rôles anti-inflammatoires de NLRP12 et NLRX1 dans la pathogenèse de l’encéphalomyélite auto-immune expérimentale (EAE), le modèle murin de la SEP. NLRP12 est un inhibiteur de la voie NF-κB et est exprimé dans les cellules immunitaires, y compris les cellules myéloïdes et les cellules T. À l'aide de souris Nlrp12-/- , cette étude démontre la double fonction immunorégulatrice de NLRP12 dans la pathogenèse de l'EAE. NLRP12 inhibe la progression de l'EAE induite par l'immunisation avec un adjuvant à la myéline, mais n'empêche pas l'apparition de l’EAE spontanée (spEAE) chez les souris transgéniques du récepteur de cellules T spécifique à la myéline (TCR) (2D2). Des expériences in vitro démontrent la fonction inhibitrice de NLRP12 dans l'activation des microglies et des cellules T. NLRP12 inhibe les événements précoces en aval de la voie de signalisation du TCR, indiquant son rôle immunorégulateur de manière intrinsèque aux cellules T. Contrairement à NLRP12, NLRX1 est un NLR situé au niveau des mitochondries qui est exprimé de manière omniprésente. NLRX1 est un inhibiteur de la voie NF-κB et peut empêcher l’apparition de la spEAE chez les souris 2D2. Considérant que les cellules immunitaires innées sont les principaux activateurs de la réponse des cellules T, cette partie de l'étude se concentre sur l'effet anti-inflammatoire de NLRX1 dans les cellules immunitaires innées. Des expériences de transfert adoptif utilisant des souris Nlrx1-/- Rag-/- déficientes en lymphocytes démontrent que NLRX1 inhibe l'inflammation dans les compartiments immunitaires innés. Plus spécifiquement, au tout début de l'inflammation dans le SNC, NLRX1 inhibe l'activation des microglies et l'induction d'astrocytes v neurotoxiques qui provoquent la mort des oligodendrocytes et des neurones. Les résultats de cette étude démontrent que les processus neurodégénératifs au sein du SNC sont des déclencheurs possibles de la réponse des cellules T autoréactives. De plus, plusieurs polymorphismes de NLRX1 ont été découverts dans des familles affectées par la SEP, suggérant que NLRX1 serait un facteur de risque potentiel pour la SEP. Pris dans leur ensemble, ces résultats démontrent que les NLR sont des inhibiteurs endogènes de l'inflammation et peuvent être considérés comme des cibles thérapeutiques potentielles pour la prévention de l'inflammation du SNC dans la SEP.

SUMMARY The NOD-Like Receptors as Endogenous Inhibitors of Multiple Sclerosis

By Marjan Gharagozloo Immunology Program

Thesis presented at the Faculty of medicine and health sciences for the obtention of Doctor of Philosophy (Ph.D.) in Immunology, Faculty of medicine and health sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4

Multiple sclerosis (MS) is a chronic disease of the CNS, manifested by relapses in the early phase followed by progressive disability in the later phase. Innate and adaptive immune responses play a critical role in the onset and progression of the disease; however, the current MS treatments mainly target adaptive immune response with limited effect on innate immune response and inflammation within the CNS. Therefore, the endogenous anti-inflammatory molecules are potential therapeutic targets to prevent MS pathology. NOD-like receptors (NLRs) are a family of innate immune receptors that are smart regulators of inflammation within the cells. Some members of the NLR family induce inflammatory responses such as NLRP1 and NLRP3, while some other members inhibit inflammatory pathways such as NLRP12 and NLRX1. The focus of this study is to evaluate the hypothesis that anti- inflammatory NLRs prevent the onset and progression of MS. The hypothesis was addressed by investigating the anti-inflammatory roles of NLRP12 and NLRX1 in the pathogenesis of Experimental Autoimmune Encephalomyelitis (EAE), the mouse model of MS. NLRP12 is a negative regulator of NF-κB pathway and is expressed in immune cells including myeloid cells and T cells. Using Nlrp12-/- mice, this study demonstrates the dual immunoregulatory function of NLRP12 in the pathogenesis of EAE. NLRP12 inhibits the progression of EAE induced by myelin-adjuvant immunization but does not prevent the onset of spontaneous EAE (spEAE) in myelin-specific T cell receptor (TCR) transgenic mice (2D2). In vitro experiments demonstrate the negative regulatory function of NLRP12 in the activation of microglia and T cells. NLRP12 inhibits the very early events downstream of TCR signaling pathway, indicating its immunoregulatory role in a T cell-intrinsic manner. Unlike NLRP12, NLRX1 is a mitochondria-located NLR that ubiquitously expressed. NLRX1 is an inhibitor of NF-κB pathway and can prevent the onset of spEAE in 2D2 mice. Considering the innate immune cells as the crucial activator of T cell response, this part of the study focuses on the anti-inflammatory effect of NLRX1 in innate immune cells. Adoptive transfer experiments using lymphocyte-deficient Nlrx1-/-Rag-/- mice demonstrate that NLRX1 inhibits the inflammation in innate immune compartments. More specifically, in the very early stages of CNS inflammation, NLRX1 inhibits the activation of microglia and the induction of neurotoxic astrocytes that cause oligodendrocytes and neuronal death. The results of this study support the inside-out model of MS, in which neurodegenerative processes within the CNS are the possible triggers of autoreactive T cell response. Additionally, several polymorphisms of NLRX1 have been discovered in MS-affected families that suggest NLRX1 as a potential risk factor for MS. Taken together, these findings demonstrate that NLRs are endogenous inhibitors of inflammation and can be considered as potential therapeutic targets for preventing CNS inflammation in MS. Keywords: NLRX1, NLRP12, NLR, multiple sclerosis, EAE. vi

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TABLE OF CONTENTS Résumé ...... iv Summary ...... vi Table of contents ...... vii List of figures ...... viii List of tables ...... ix List of abbreviations ...... 1 1. Introduction ...... 5 1.1 Immune response in the CNS ...... 5 1.1.1 Innate immune response ...... 5 1.1.2 Adaptive immune response ...... 7 1.2 Multiple sclerosis and its pathogenesis ...... 8 1.2.1 Multiple Sclerosis ...... 8 1.2.2 Risk factors of MS ...... 9 1.2.3 MS: an autoimmune or neurodegenerative disease? ...... 11 1.2.4 Immunopathogenesis of MS ...... 12 1.2.5 MS animal models ...... 15 1.2.6 MS treatments...... 16 1.3 The Regulators of Inflammation:NLRs ...... 18 1.3.1 NLRs as positive regulators of inflammation ...... 18 1.3.2 NLRs as negative regulators of inflammation ...... 22 1.3.2.1 NLRP12 ...... 23 1.3.2.2 NLRX1 ...... 25 2. Hypothesis and Objectives ...... 29 3. Articles 3.1 Article 1 ...... 30 3.2. Article 2 ...... 57 3.3. Article 3 ...... 87 4. Discussion ...... 118 4.1 NLRP12 and the inhibition of CNS inflammation ...... 118 4.2.1 NLRP12 and EAE ...... 118 4.2.2 NLRP12 and the CNS immune response ...... 119 4.2.3 NLRP12 and the peripheral immune response ...... 120 4.2. NLRX1 and the inhibition of CNS inflammation ...... 122 4.2.1 NLRX1 in EAE and MS ...... 122 4.2.2 NLRX1 and the CNS immune response ...... 125 4.2.3 NLRX1 and the peripheral immune response ...... 127 4.3 Concluding remarks and future directions ...... 128 Acknowledgments ...... 131 List of references ...... 132

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LIST OF FIGURES

THESIS

Figure 1 Outside-in and inside-out models of MS ...... 12 Figure 2 Innate and adaptive immunity in the pathogenesis of MS ...... 14 Figure 3 NLRs structure ...... 20 Figure 4 Functional Characterisation of NLRs...... 21 Figure 5 Anti-inflammatory NLRs inhibit NF-B activation ...... 25 Figure 6 NLRs balance the inflammatory response in the CNS...... 130

ARTICLE 1

Figure 1 Nlrp12 mRNA expression reaches a peak at third week post injection ...... 40 Figure 2 Nlrp12-/- mice exhibit exacerbated form of disease compared to WT mice ...... 41 Figure 3 Photomicrograph pictures of Spinal Cords stained with GFAP ...... 42 Figure 4 Photomicrograph pictures of Spinal Cords stained with Iba1 ...... 43 Figure 5 Percent level of Astrogliosis following EAE ...... 43 Figure 6 Precent level of Microgliosis following EAE ...... 44 Figure 7 The proliferation of activated T cells from WT and Nlrp12-/- mice in vitro ...... 45 Figure 8 IL-4 production by activated T cells from WT and Nlrp12-/- mice in vitro and in vivo ...... 46 Figure 9 Nlrp12 deficiency augments expression of pro-inflammatory molecules in the CNS after EAE ...... 47 Figure 10 Expression of iNOS in primary microglia cells ...... 48 Figure 11 TNF- and IL-6 concentrations following treatment with LPS in primary microglia cell ...... 49

ARTICLE 2

Figure 1 Nlrp12 inhibits Th1 response in EAE...... 67 Figure 2 Nlrp12 inhibits T cell proliferation and cytokine production by CD4+ T cells ..... 68 Figure 3 Nlrp12 does not affect the differentiation of naïve CD4+ T cells to Th1 or Th17.69 Figure 4 T cell activation increases Nlrp12 mRNA expression ...... 70 Figure 5 Nlrp12 inhibits phosphorylation of Akt and p65 in CD4+ T cells ...... 71 Figure 6 Nlrp12 inhibits IL-2 synthesis by activated T cells ...... 72 Figure 7 Nlrp12 does not modify mTOR activity ...... 72 Figure 8 SpEAE in WT 2D2 mice is associated with spinal cord inflammation and demyelination...... 74 Figure 9 Myelin-specific T cells infiltrate in the spinal cord of spEAE mice ...... 75 Figure 10 Possible mechanism of action of Nlrp12 in regulating early TCR signaling pathways, T cell activation and proliferation...... 78 Figure 11 Dual immunoregulatory function of Nlrp12 in EAE...... 81 Figure S1 Nlrp12 genotyping of a Nlrp12-/- 2D2 mouse and a WT 2D2 mouse...... 82 Figure S2 Clinical score of induced EAE in Nlrp12-/- mice compared to WT mice ...... 82 ix

ARTICLE 3

Figure 1 Nlrx1-/-2D2 mice develop spEAE ...... 89 Figure 2 Increased levels of IgG and frequency of B cells in the spinal cord of SpEAE mice ...... 90 Figure 3 CNS inflammation associated with spEAE is more severe in Nlrx1-/-2D2 compared to 2D2 mice ...... 91 Figure 4 Activation of innate immune cells induce CNS inflammation and severe paralysis in Nlrx1-/-2D2 mice ...... 93 Figure 5 Inflammation at the preclinical stages of spEAE is enhanced in the CNS of Nlrx1-/- 2D2 mice...... 94 Figure 6 NLRX1 inhibits subclinical tissue damage and the generation of neurotoxic glia in the CNS...... 95 Figure 7 NLRX1: implications for human MS...... 97 Figure 8 NLRX1 inhibits early stages of CNS inflammation, prevents toxic glia activation, and the onset of EAE ...... 101 Figure S1 Nlrx1-/-APC activate T cells as efficiently as WT APC...... 112 Figure S2 Nlrx1 inhibits T cell activation, proliferation, and differentiation to inflammatory subsets...... 112

LISTE DES TABLEAUX THESIS

Table 1 The immunopathogenesis of inflammatory and anti-inflammatory NLRs in MS patients and EAE mice...... 28

ARTICLE 2

Table 1 Nlrp12 does not prevent the development of spEAE...... 73

ARTICLE 3 Table S1 Nlrx1-/- 2D2 model compared to previous models with CD4 T cells involvement...... 114 Table S2 Demographic data of MS patients and healthy individuals. RRMS: replacing remitting MS...... 115 Table S3 NLRX1 mutations identified in MS patients ...... 115 Table S4 The primer sequences used for qPCR experiments ...... 116

LISTE DES ABRÉVIATIONS A A ADCC Antigen dependent cell cytotoxicity ALS Amyotrophic Lateral Sclerosis ASC Apoptosis-associated Speck-like protein containing CARD ATP Adenosine Triphosphate

B A BBB Blood Brain Barrier BIR Baculoviral Inhibitory Repeat

C B CARD Caspase-Recruitment Domain CATERPILLAR CARD Transcription Enhancer, R (Purine)-binding, Pyrin, Lots of Leucine Repeats CCL5 Chemokine (C-C motif) Ligand 5 CCL20 Chemokine (C-C motif) Ligand 20 CCL21 Chemokine (C-C motif) Ligand 21 CCR2 Chemokine (C-C motif) Receptor 2 CCR5 Chemokine (C-C motif) Receptor 5 CD Cluster of Differentiation CFA Complete Freund’s Adjuvant CIITA Class II Transactivator CLRs C-type Lectin Receptors CNS Central Nervous System CSF Cerebrospinal Fluid

D B DAMPs Danger Associated Molecular Patterns DCs Dendritic Cells DD Death Domain DMT Disease Modifying treatment DNA Deoxyribonucleic Acid DRP1 Dynamin related protein 1 B E EAE Experimental Autoimmune Encephalomyelitis ERK Extracellular Signal-Regulated Kinase

G B GDNF Glial-Derived Neurotrophic Factor GFAP Glial Fibrillary Acidic Protein 2

GWAS Genome-Wide Association Scan

H B HL60 Human Leukemia cell line 60 HLA Human Leukocyte Antigen HMGB1 High mobility group box 1 protein B I IAP Inhibitor of apoptosis protein IBD Inflammatory Bowel Disease ICAM-1 Intracellular Molecule-1 IFIH1 Interferon Induced with Helicase C domain 1 IFN Interferon IFN-1 Interferon-1 IFN-1 Interferon-1 IFN-1a Interferon--1a IFN-1b Interferon--1b IFN- Interferon- IGF-1 Insulin-like 1 Growth Factors IB Inhibitor of B IKK Inhibitor of B kinase IKK Inhibitor of B kinase  IL Interleukin IL-1 Interleukin-1 iNOS Interleukin-18 IP-10 Inducible Nitric Oxide Synthase IRAK Interferon gamma-induced protein 10 IL-1R Associated Kinase L B LPS Lipopolysaccharide LRRs Leucine-rich repeats

M B MAPK Mitogen-Activated Protein Kinase MBP Myelin Binding Protein/Myelin Basic Protein MCP1 Monocyte Chemoattractant Protein 1 MHC Major Histocompatibility Complex MIP1 Macrophage Inflammatory Protein 1 MMP-9 Matrix Metalloproteinase 9 MOG Myelin Oligodendrocyte Glycoprotein MS Multiple Sclerosis MRI Magnetic Resonance Imaging MyD88 Myeloid Differentiation primary-response gene 88

N NACHT NAIP, CIITA, HET-E and TP1 domain NAIP NLP family Apoptosis Inhibitor Protein NAIP1 NLP family Apoptosis Inhibitor Protein 1 NAIP4 NLP family Apoptosis Inhibitor Protein 4 NBD Nucleotide-Binding Domain NBS Nucleotide-Binding Site NF-B Nuclear Factor-Kappa B NIK NF-κB inducing kinase NK Natural Killer NLR NOD-Like Receptor NLRC4 NLR family CARD domain containing 4 NLRP12 NLR family Pyrin domain containing 12 NLRX1 NLR family X1 NO Nitric Oxide NOD Nucleotide-binding Oligomerization Domain

O OPCs Oligodendrocyte Precursor Cells

P PAMPs Pathogen Associated Molecular Patterns PLP Proteolipid Protein PML Progressive Multifocal Leukoencephalopathy PMN Polymorphonuclear PNS Peripheral Nervous System PPMS Primary-Progressive Multiple Sclerosis PRRs Pathogen Recognition Receptors PYD Pyrin domain PYPAF7 Pyrin-containing Apaf1-like protein 7

R RAG Recombination activating gene RANTES Regulated upon Activation, Normal T cell Expressed and Secreted RIG1 Retinoic Acid Inducible Gene I RLR RIG-Like Receptor RNA Ribonucleic Acid ROS Reactive Oxygen Species RRMS Relapsing-Remitting Multiple Sclerosis

S SDF-1 Stromal cell-derived factor-1α SPEAE Spontaneous EAE

T TCR T cell receptor TGF- Transforming Growth Factor  Th1 T helper 1 Th2 T helper 2 Th17 T helper 17 TIR Toll/Interleukin-1 Receptor TLR Toll-Like Receptor TNF Tumor Necrosis Factor- TRAF TNF Receptor Associated Factor 3 Treg Regulatory T cells TUFM Tu Translation Elongation Factor, Mitochondrial

V VCAM Vascular Cell Adhesion Molecule

W B WT Wild-Type

1. INTRODUCTION

1.1 Immune response in the CNS Over the past decades, it was believed that the CNS is an immune-privileged site and peripheral immune cells were not allowed to enter to the CNS under physiological condition (Carson et al., 2006). Recently, this viewpoint was dramatically changed by the discovery of the lymphatic system in the brain's meninges, where immune cells and soluble mediators drain into the deep cervical lymph nodes and communicate with peripheral immune cells under both pathological and homeostatic conditions (Louveau et al., 2015). Instead of being surrounded by physical barriers that shield the CNS from any outside contacts, the CNS is protected by dynamic biological barriers that tightly regulate the crosstalk between the CNS and the immune system (Engelhardt et al., 2017). Here I summarize the mechanisms of the CNS-immune system crosstalk in the context of innate and adaptive immunity.

1.1.1. Innate immune response The CNS innate immune compartment consists of biological barriers and the specialized resident cells that respond into the CNS infections and injuries. The dynamic biological barrier, known as the blood–brain barrier (BBB), controls the trafficking of the cells and molecules to the CNS. The endothelial cells are held together by tight junctions that control the diffusion of large (molecular weight bigger than 450 Da) and non-lipophilic molecules (Petty and Lo, 2002). The CNS-resident cells are categorized in two groups: the nerve cells and the glial cells. Neurons receive synaptic inputs and transmit electrical signals down to the axons, the long projection of neurons that are covered by a protective myelin sheath. Glia cells are 10 times more numerous than neurons and include oligodendrocytes, astrocytes, and microglia (Nolte, 2002). Oligodendrocytes derived from oligodendrocyte precursor cells/progenitors (OPCs) are the cells that tightly wrapped around axons and make myelin sheath (Jacobson & Marcus, 2008). The axonal insulation by myelin increase the speed of signal transmission and serves as a protective layer against axonal degeneration (Fitzner et al., 2006). Astrocytes are the most abundant glia in the CNS. Some of their processes, known as endfeet, make the last layer of the BBB in the brain parenchyma. Additionally, astrocytes support homeostasis and metabolism of neurons in several ways including formation and

6 maintenance of the BBB integrity; the regulation of extracellular ions and neurotransmitters; and synthesis of the neuronal metabolic substrates such as glycogen (Sofroniew, 2015). Astrocyte-mediated inflammation is associated with inflammatory responses that are characterized by robust proliferation and hypertrophy of astrocytes that is termed astrogliosis. Activated astrocytes release inflammatory cytokines such as IL-6, IL-1β, and TNF-α that affect the tight junctions of endothelial cells and control the passage of immune cells through the BBB. Moreover, astrocytes secrete different chemokines such as MCP-1, RANTES, IP- 10, SDF-1, CCL20 and IL-8, which actively recruit a variety of leukocytes such as monocytes, neutrophils, DCs, and lymphocytes from the periphery into the CNS parenchyma. Therefore, astrocytes have all the features to regulate both an inflammatory response within the CNS and the influx and activity of leucocytes. Proinflammatory cytokines and chemokines also activate the major player of the CNS innate immunity, microglia (Correale and Farez, 2015a). Microglia constantly monitor the CNS for various insults and orchestrates innate immune response within the CNS parenchyma (Herz et al., 2017). They are the only tissue- resident macrophages that origin from embryonic yolk sac at the very early stage of development, seed the brain and stay there into adulthood (Ginhoux et al., 2013). Interestingly, microglia have the ability for self-renewal and are not replenished from circulating monocytic progenitors (Lampron et al., 2013). The morphology of microglia differs from conventional macrophages by the presence of highly motile projections that constantly monitor their environment. Activated microglia increase the ability of phagocytosis and antigen presentation within the CNS (Hernandez-Pedro et al., 2013, Ransohoff and Cardona, 2010, Xanthos and Sandkuhler, 2014). In many CNS pathologies, the number of microglia often increases in a phenomenon that is called reactive microgliosis (Huber and Irani, 2015, Dendrou et al., 2015a). Furthermore, they release inflammatory mediators such as inducible nitric oxide synthase (iNOS), TNFα, IL-1β, IL-6, IL-12, which control the infections and recruit adaptive immune cells into the CNS (Gonzalez et al., 2014). The CNS also contains myeloid cells including macrophages and DCs that strategically positioned at the CNS barriers such as choroid plexus, perivascular space, and meninges (Hernandez-Pedro et al., 2013). These cells serve role of the antigen presenting cells (APC) that process the antigen and present peptide-MHC II complex to the T cells. Unlike other

7 organs such as liver, skin or intestine where dendritic cells (DCs) uptake the antigen and travel to the local lymph node, under normal conditions, there are no DCs in the CNS parenchyma and even microglia never leave the CNS (Hussain et al., 2014). Instead, there are other myeloid cells including macrophages and DCs that monitor the brain from outside of the parenchyma. These are the cells that communicate with adaptive immune cells such as T cells, which patrol the CNS from the outside of the tissue at the particular gateways; the meninges, perivascular spaces, and choroid plexus (Herz et al., 2017)

1.1.2. Adaptive immune response Naïve T cells do not enter the normal or inflamed CNS (Goverman, 2009), indicating that activated T cells at the CNS barriers are licensed to enter the CNS parenchyma (Ransohoff and Engelhardt, 2012). T cell activation is initiated within the regional lymph nodes (cervical lymph nodes), where DCs process and present the antigen in both classes I and class II MHCs to naïve T cells, leading to the specific activation of CD4+ and CD8+ T cells. Depending on the type of cytokines present in T cell environment, naïve CD4+ T cells may differentiate into different T helper (Th) subsets including Th1, Th2, Th17, and regulatory T cells (Treg) that produce various cytokines including IFN, IL-4, and IL-17 respectively. Th subsets may affect CNS inflammation in different manners, in which Th1 and Th17 responses promote CNS inflammation, while Th2 response and regulatory T cells dampen the inflammatory response of T cells and protect CNS (Luckheeram et al., 2012). Th2 cells help B cells to form plasma cells that produce high-affinity pathogen-specific antibodies (Pennock et al., 2013). As a routine immunosurveillance activity, a small number of memory T cells can access the CNS via the blood–CSF barrier at subarachnoid space (Ransohoff et al., 2003). Macrophages at perivascular space present the CNS-derived antigens to memory T cells. In pathological condition, endothelial cells of the BBB increase the expression of several adhesion molecules including Vascular Cell Adhesion Molecule (VCAM-1) that support the recruitment of T cells into the CNS (Goverman, 2009, Selewski et al., 2010). As a result, lymphocytes including T cells and B cells are found in the CNS parenchyma only after CNS injuries, infections, neurodegenerative and autoimmune diseases such as multiple sclerosis (Kamm and Zettl, 2012).

1.2 Multiple sclerosis and its immunopathogenesis

1.2.1. Multiple sclerosis Multiple sclerosis (MS) is a chronic autoimmune disease of the CNS, which is characterized by inflammation, demyelination, and axonal damage. It is the leading cause of chronic neurological disability in young adults (Dendrou et al., 2015a). Besides MS, there are rare forms of autoimmune demyelinating diseases including neuromyelitis optica (NMO), in which there is an adaptive immune response directed against the water channel aquaporin 4 (AQP4), and acute disseminated encephalomyelitis (ADEM), which is characterized by a monophasic and rapidly progressive immune mediated attack against the CNS (Hu and Lucchinetti, 2009). The name of MS was inspired by the pathological signature of the disease, the presence of multiple sclerotic plaques in the white and gray matter of the brain and spinal cord (Murray, 2009). The first description of MS dates back to 1868 when the French neurologist, Jean-Martin Charcot, described MS symptoms and plaques (McAlpine and Compston, 2005, Ntranos and Lublin, 2016). As we know today, the pathological hallmark of MS is the formation of sclerotic plaques in the white and gray matter of the CNS, which is caused by several processes including inflammation, demyelination, astrocytic glial scars, axonal damage, and neuronal loss (Yadav et al., 2015, Kutzelnigg and Lassmann, 2014). Demyelinated axons can no longer support rapid conduction of action potential, which results in a disturbed transmission of nerve impulses (Ciccarelli et al., 2014). Therefore, MS manifests in a variety of clinical symptoms such as blurred or double vision, lack of neuronal muscle coordination, loss of balance, numbness, tingling, pain, fatigue, and difficulties in moving (Compston and Coles, 2002). MS affects more than 2.5 million people worldwide (Marrie et al., 2015) and Canada has one of the highest rates of MS in the world (197/100,000 population) (Gilmour et al., 2018). It is most frequently diagnosed between the ages of 20 and 49. MS affects women 3 times more than men(Harbo et al., 2013). The clinical course of MS is different among patients. Relapsing-remitting MS (RRMS) is the common form of MS, which is found in about 70–80% of patients. In RRMS, there are clear episodes of inflammatory activity or relapses followed by remission. However, people with RRMS may develop a secondary progressive form of MS after a disease duration of about 10 years. A small percentage of

9 patients may develop primary progressive MS (About 10%), which is characterized by continuous neurodegeneration from the onset without remission (Ciccarelli et al., 2014, Wegner, 2013).

1.2.2. Risk factors of MS

➢ Genetic factors The prominent role of genetic factors in MS is shown in familial MS. For example, the monozygotic twins have a higher risk of developing MS (25-30%)(Hansen et al., 2005). Similarly, siblings of an MS individual are at least 7 times more likely to develop MS than the general population (Baranzini and Oksenberg, 2017). More than 200 loci associated with MS susceptibility have been discovered by genome-wide association scan (GWAS) performed by the International Multiple Sclerosis Genetic Consortium (Baranzini and Oksenberg, 2017). The main susceptibility locus is located within the Major Histocompatibility Complex (MHC) class II region. MHC or Human Leukocyte Antigen (HLA) genes code for cell surface molecules involved in antigen presentation to T cells and triggering the immune response (Files et al., 2015). Several studies have demonstrated that the Human Leukocyte Antigen (HLA)-DRB1*1501 has been constantly associated with MS in many populations (Alcina et al., 2012, Barcellos et al., 2006, Baranzini and Oksenberg, 2017). Moreover, many other immunologically relevant genes, particularly those involved in T cell response have been discovered, providing evidence that dysregulated T cell response is associated with MS (Sawcer et al., 2011).

➢ Gender factors It is shown that women are at higher risk of developing MS, with a sex ratio of 3:1 female to male (Reich et al., 2018). Moreover, the predisposition to many autoimmune conditions is increased amongst women (Tiniakou, Costenbader, & Kriegel, 2013), suggesting that sex hormones may play a role in higher susceptibility of women to autoimmune diseases. A growing number of studies using EAE show that male hormone testosterone and the pregnancy hormone estriol induce anti-inflammatory and neuroprotective effects, supporting the potential therapeutic effect of these hormones in MS (Gold and Voskuhl, 2009). Findings from clinical studies of testosterone therapy in male MS patients and oral estriol in female MS patients are encouraging (Voskuhl et al., 2016, Sicotte et al., 2007). However, there are

10 concerns regarding potential side effects of sex hormone therapy in MS patients, such as carcinogenesis, thrombogensis, and changes in reproductive behavior in long-term use of these agents (Houtchens and Desai, 2018). More investigations need to be done before sex hormones can be used as MS therapeutics.

➢ Environmental factors Many studies have proposed the influence of microbial infection such as viruses or bacteria on the susceptibility to MS. Among viral infections associated with MS, herpesviruses or retroviruses have been widely studied in MS (Gilden, 2005). Human herpesvirus 6 (HHV-6) and Epstein-Barr virus (EBV) are detected in MS plaques (Soldan et al., 1997). Additionally, a higher seroprevalence and higher titer of anti-EBV and anti-HHV-6 are reported in MS patients compared to age-matched controls (Virtanen and Jacobson, 2012). A recent study demonstrates fungal DNA from different species including Trichosporon mucoides in the CNS of MS patients (Alonso et al., 2018). Among bacterial agents, Mycoplasma pneumoniae, Chlamydia pneumoniae, and Staphylococcus aureus enterotoxins are associated with the development or exacerbation of MS (Libbey et al., 2014). The gut microbiome is another environmental factor that has been demonstrated to significantly influence CNS health and disease (Zhu et al., 2017). Microbiota consists of all microbes including bacteria, archaea, fungi, and viruses that exist in human body and microbiome includes genomic, protein, or metabolite content of all the microbes (Shahi et al., 2017). It is shown that gut microbiome maintains the proper functions of CNS, including brain circuitry, neurophysiology, and behavior (Sharon et al., 2016). Gut microbiome also regulates the permeability of the blood-brain barrier (BBB)(Braniste et al., 2014). In addition, gut microbiota affects the function of immune cells, directing the immune response towards homeostasis or autoimmunity. For instance, gut microbiota influences the differentiation of T cells to inflammatory subsets (Th1, Th17) or regulatory T cells (Wu and Wu, 2012). Additionally, several reports demonstrate the critical role of gut microbiota in regulating the development of APCs, in which stimulates a subset of DCs that induce the differentiation of Th17 cells (Atarashi et al., 2008). These studies support the notion that gut microbiome contributes to the development or exacerbation of CNS inflammation. In the context of MS, several studies have demonstrated gut microbial dysbiosis in MS patients with both enhancement and reduction of certain bacteria compared to healthy

11 controls (Chu et al., 2018). For example, Jangi et al. showed that Methanobrevibacter and Akkermansia are increased in MS patients, while Butyricimonas is decreased. The microbiota variations correlate with the expression of genes involved in interferon and NF-kB signaling, as well as DC maturation (Jangi et al., 2016). Interestingly, in transferring fecal content isolated from MS patients to a transgenic mouse model increased the incidence or severity of MS-like autoimmune disease. This finding suggests that gut microbiota from MS patients contain factors that exacerbate CNS inflammation and autoimmunity (Yadav et al., 2017). Global prevalence of MS shows a latitudinal gradient, where the farthest countries from the equator including the populations located at northern latitudes of Europe and North America including Canada and southern areas of Australia and New Zealand have a higher incidence of MS (Files et al., 2015; Rosati, 2001). Latitude gradient was explained by the lower levels of vitamin D in the countries located at northern latitude (O'Gorman et al., 2012). This is explained by the fact that the primary source of vitamin D in human is UVB radiation from sunlight, which varies according to latitude and season. Low vitamin D intake and its low serum level are associated with increased risk of MS (Munger et al., 2004, Munger et al., 2006). Interestingly, lower levels of vitamin D during MS relapses suggest the immunosuppressive role of vitamin D progression of MS (Alharbi, 2015). Mechanistically, vitamin D suppresses the activity of pro-inflammatory T cells including Th1 and Th17 cells and promotes the activity of regulatory T cells(da Costa et al., 2016, Zeitelhofer et al., 2017). In addition to its immunosuppressive activity, vitamin D also plays a role in the repair of damaged oligodendrocytes and induce remyelination(Shirazi et al., 2015). Therefore, vitamin D supplementation may have a therapeutic role in the treatment of MS (Alharbi, 2015). Taken together, the complex interaction between genetic factors and environmental factors contributes in the etiology of MS.

1.2.3. MS: an autoimmune or a neurodegenerative disease?

Despite the plenty of research on the immunopathology of MS, the origin of the disease is still a matter of debates. The presence of autoreactive T and B cells in the CNS strongly supports the hypothesis that MS is primarily caused by an aberrant immune response against the CNS antigens, particularly myelin, in which chronic immune response cause

12 oligodendrocyte death and progressive demyelination (outside-in model of MS) (Figure 2) (Stys et al., 2012, McAlpine and Compston, 2005, Ntranos and Lublin, 2016). It is still unknown how myelin-specific T cells are activated in the periphery. There are studies that support the activation of myelin-specific T cells by infectious agents (molecular mimicry) or non-specific T cells by superantigens (bystander activation) (Cusick et al., 2012, Libbey et al., 2014). Molecular mimicry is the term coined for the activation of autoreactive cells by cross-reactivity between self-antigens and foreign agents (Oldstone, 2005), while bystander activation assumes that autoreactive cells are activated due to nonspecific inflammatory events that occur during infections. The gut microbiome is also important to balance and regulate the immune response (Bhargava and Mowry, 2014). Inside-out model of MS supports the idea that MS is primarily a neurodegenerative disorder (Figure 1). In this model, the oligodendrocyte injury or death would be the trigger of the CNS inflammation that presumably begins in the absence of a direct immune attack (Stys et al., 2012, Patel and Balabanov, 2012). Oligodendrocytes are extremely vulnerable to oxidative stress due to their high metabolic rate, large intracellular iron stores, and low levels of anti-oxidative enzymes. Exposure to any stress reactions or metabolic disturbances leads to caspase activation and oligodendrocyte death (Patel and Balabanov, 2012). Oxidative stress also results in mitochondrial dysfunction, which causes axonal damage and oligodendrocyte apoptosis (Lassmann et al., 2012, Su et al., 2013). As a result, myelin antigens are released to the periphery (van Zwam et al., 2009, Engelhardt et al., 2016) and activate autoreactive T and B cells that cause further CNS damage (Figure 1) (Yadav et al., 2015, Kutzelnigg and Lassmann, 2014, Ciccarelli et al., 2014).

Figure 1. Outside-in and inside-out models of MS. Outside-in model supports the idea that T cells are activated in the periphery by pathogen-derived molecules (molecular mimicry) or non-specific bystander activation. The digestive-tract associated microbes are also important to balance and regulate the immune response. The activated T cells attack CNS and cause inflammation and neurodegeneration. Inside-out model argues that the CNS inflammation primarily begins in the absence of a direct immune attack, in which tissue damage or cell death within the CNS releases myelin antigen that triggers the immune response in the periphery (Gharagozloo et al., 2018a).

1.2.4. Immunopathogenesis of MS The presence of autoreactive CD4+ T cells within CNS lesions is detectable in the early stages of MS (Dendrou et al., 2015b). Th1 and Th17 cells are the main CD4+ T cell subsets implicated in disease (Legroux and Arbour, 2015). Th17 cells induce neuroinflammation by producing pro-inflammatory mediators such as IL-17, granulocyte/macrophage colony- stimulating factor (GM-CSF)(McGeachy, 2011), matrix metalloproteinases and CXCL8, a potent chemokine for recruiting neutrophils (Waisman et al., 2015). Interestingly, a recent study has identified T cells that express both IL-17 and IFN-γ cytokines in MS brain tissue, suggesting the pathological role of IL-17+ IFNγ+ T cells in human MS (Kebir et al., 2009).

CD8+ T cells represent the predominant T cell population in the lesions of human MS (Sinha et al., 2015). CD8 cytotoxicity is mediated through cell surface Fas ligand or release of soluble molecules such as IFNγ, TNF-α, lymphotoxin, granzyme B and perforin. During inflammation, oligodendrocytes upregulate MHC I expression, Fas, IFN-γ and TNF-α receptors, result in direct cytotoxicity of CD8+ cells (Patel and Balabanov, 2012). B cells and antibodies are also implicated in the pathogenesis of MS. Plasma cells produce specific antibodies to myelin antigens that initiate the complement cascade, leading to destruction, opsonization, and subsequent phagocytosis of the myelin sheath (von Büdingen et al., 2011). B cells also act as APCs to capture and present the antigens to autoreactive T cells. Inflammatory mediators from activated T cells such as TNFα and IFNγ stimulate macrophages and microglia to produce more proinflammatory mediators such as iNOS, TNFα, and IL-1β. Activated macrophages and microglia produce reactive oxygen species (ROS) and NO radicals that are associated with demyelination and neuronal injury in MS lesions (Luo et al., 2017). There is also evidence that astrocytes promote demyelination by regulating peripheral immune cell trafficking, modulating BBB integrity, and being a source of chemokines and cytokines such as IL-12, IL-23, and IL-15, that mediate differentiation CD4+T cells into Th1 or Th17 and activates the cytotoxicity of CD8+ T cells. Astrocyte activation induces the down-regulation of important proteins that compose the tight junctions of endothelial cells, such as claudin-5 and occludin, leading to the compromise of the BBB (Argaw et al., 2012). Astrocytes are the main source of the chemokine CCL20 or monocyte chemoattractant protein 1 (MCP1) that is a crucial chemoattractant for monocytes and T cells in EAE and MS (Correale and Farez, 2015b). The role of innate and adaptive immune cells in the immunopathogenesis of MS is summarized in Figure 2. The above immunopathological mechanisms are involved in developing MS plaques, which are the hallmark of MS pathology and histologically classified as acute and chronic lesions (Popescu and Lucchinetti, 2012). Acute lesions are more common in the early stage of MS, characterized by demyelination, reactive astrocytes, and microglia (glial scar tissue), and perivascular and parenchymal inflammation. The majority of infiltrated cells in active lesions are macrophages, followed by lymphocytes including CD4+ and CD8+ T cells, B cells and plasma cells. Chronic or inactive plaques are more common in progressive MS and are

15 associated with severe demyelination and axonal loss, oligodendrocytes death, while there are very few infiltrating leukocytes (Popescu et al., 2013).

Figure 2. Innate and adaptive immunity in the pathogenesis of MS. Myelin-reactive T cells are activated in the periphery and accumulate in the perivascular spaces, where they are reactivated by the CNS myeloid cells, such as macrophages, and enter the CNS parenchyma. CD4+ T cells are differentiated to different inflammatory subsets, such as Th1, Th17, and Th9, and once in the CNS, they promote the activation of CNS-resident innate immune cells, such as macrophages and microglia. Inflammatory mediators, such as TNFα, IL-6, IL-1, ROS, and NO, released from activated macrophages, microglia, and astrocytes damage oligodendrocytes and neurons, leading to demyelination. Activated cytotoxic CD8+ T cells directly induce apoptosis in oligodendrocytes, while plasma cells produce anti-myelin antibodies that activate the complement system and damage oligodendrocytes (Gharagozloo et al., 2018a).

1.2.5. MS animal models The understanding of MS immunopathogenesis and current drug therapies have been mainly derived from the studies in experimental autoimmune encephalomyelitis (EAE). EAE is a CD4+ T cell-mediated autoimmune disease consisting of Th1- and Th17-mediated tissue

16 damage, mononuclear cell infiltration and demyelination within the CNS (Miller, Karpus, & Davidson, 2010). Major histopathological features of EAE is the presence of multiple focal lesions within the CNS, particularly, in the spinal cord, associated with astrogliosis and microgliosis. The EAE clinical signs are scored from 0-5, depending on the severity and progression of the clinical symptoms. Clinical scores are given using the following scale: 0, no sign of disease; 1, limp tail or weakness in limbs; 2, limp tail and weakness in limb; 3, partial limb paralysis; 4, complete limb paralysis (Miller and Karpus, 2007). Depending on the research design, EAE can be induced by different protocols. Active EAE consists of two phases; the induction and the effector phase. The induction phase involves the priming of CD4+ T cells following subcutaneous immunization of mice with myelin antigens such as Myelin Oligodendrocyte Glycoprotein (MOG), emulsified in Complete Freund's Adjuvant (CFA) (Miller et al., 2010), followed by a pertussis toxin injection intraperitoneally (Miller and Karpus, 2007). Typical EAE onset is 9 to 14 days after immunization. The clinical scores reach the highest usually 20-27 days after immunization. Passive EAE is induced by adoptive transfer of myelin-specific T cells to the recipient mice. Myelin specific T cells are purified from lymph nodes and spleen of animals with active EAE and transferred intravenously or intraperitoneally into the recipient mice (Stromnes and Goverman, 2006). To study the biology of T cell response, T cells are transferred to the Rag- /- recipients, which are lymphocyte-deficient mice (Miller and Karpus, 2007). Spontaneous EAE develops in a variety of T cell receptor (TCR) transgenic mice including the MBP TCR transgenic (Lafaille et al., 1994), the 5B6 SJL/PLP TCR transgenic

(Waldner et al., 2000), and the 2D2 C57BL/6/MOG35-55 TCR transgenic (Bettelli et al., 2003). TCR transgenic animals develop a variety of symptoms ranging from conventional clinical and histological signs of EAE to optic neuritis (2D2 mice) (Bettelli et al., 2003). Since TCR transgenic mice have already high numbers of myelin-specific T cells, the mice do not need to be immunized to develop clinical signs of EAE. Therefore, this model is very useful for the study of predisposing factors of MS such as genetic and environmental factors (Ben-Nun et al., 2014).

1.2.6. MS treatments

To date, MS has no cure but there are approved medications for MS. Approved medications in this category mainly reduce the replace rates and delay the progression of the disease (Thorpe et al., 2015). They have mainly anti-inflammatory effects and are more effective in the early phases of disease development. Therefore, the majority of disease-modifying treatments (DMT) that are currently used are effective in the treatment of RRMS (Dörr and Paul, 2015). Based on their risk-benefit profile, DMT can be divided into first-line or second- line of treatment. First-line includes interferon β, glatiramer acetate, dimethyl fumarate, teriflunomide. Second-line of treatment includes fingolimod, natalizumab, alemtuzumab, and ocrelizumab. The only approved DMT for PPMS is ocrelizumab (Zimmermann et al., 2018). Interferon β is the first approved DMD for RRMS that includes interferon β-1a (Avonex®, Rebif®), interferon β-1b (Betaseron®, Extavia®), and peginterferon β-1a (Plegridy®) (Dörr and Paul, 2015). Interferon β-1a is a recombinant cytokine produced by mammalian cells, while interferon β-1b is produced in bacteria. Peginterferon β-1a is an interferon β-1a conjugated to a polyethylene glycol (PEG) molecule to increased its bioavailability(Hoy, 2015). All forms of interferon β are injected subcutaneously except Avonex® that is injected intramuscularly. The exact mechanism of interferon β therapeutic function is still unclear. However, it is shown that interferon β negatively controls a wide range of immunological processes, such as Th1 and Th17 response, production of inflammatory cytokines, and BBB permeability (Boiko et al., 2002). Glatiramer acetate (Copaxone®) is a synthetic peptide composed of 4 amino acids and structurally similar to the myelin basic protein. Its exact mechanisms of action are still unknown, however, there is evidence showing that it shifts the T cell response towards Th2, thereby suppress inflammatory Th1 response (Ziemssen and Schrempf, 2007). Dimethyl fumarate (DMF, Tecfidera®) is an oral therapy for RRMS (Montes Diaz et al., 2018). DMF exert multiple functions including immunomodulating, anti-inflammatory and anti-oxidative activities. DMF is neuroprotective and improves cell survival via activating the transcription factor nuclear factor erythroid-derived 2 (Nrf2) that induces the transcription of antioxidant genes. Moreover, DMF inhibits NF-B signaling pathway (Linker et al., 2011).

Teriflunomide (Aubagio®) is an oral therapy that inhibits pyrimidine synthesis in proliferating lymphocytes, limiting inflammatory effects on the CNS. Importantly, teriflunomide is not a DNA intercalating agent and has no effect on cell viability (Bar-Or et al., 2014). However, it is a specific blocker of the mitochondrial enzyme dihydro-orotate dehydrogenase (DHODH) that are highly expressed in proliferating lymphocytes(Bar-Or et al., 2014). Fingolimod (FTY720, Gilenya®) is the first approved oral treatment for MS. Structurally, it is the analog of sphingosine-1-phosphate (S1P) and blocks lymphocyte trafficking. When Fingolimod binds to S1P receptor, it causes receptor internalization and prevents the egress of activated lymphocytes from the lymph node (Wingerchuk & Lucchinetti, 2007). Natalizumab (Tysabri®) is the first humanized monoclonal antibody approved for MS treatment, delivered by intravenous infusion. It binds to alpha-4-integrin and blocks leukocyte adhesion to vascular cell adhesion molecule (VCAM), resulting in the prevention of T cell infiltration into the CNS (Alper & Wang, 2009; Clerico et al., 2017). The serious side effect of this drugs is its potential to reactivate latent JC virus, which can result in progressive multifocal leukoencephalopathy (PML) and death (Banwell & Anderson, 2005; Clerico et al., 2017). Alemtuzumab (Lemtrada®) is a humanized monoclonal antibody targeting CD52- antigen on the surface on leukocytes, mainly mature lymphocytes, monocytes, dendritic cells, and granulocytes. The effector mechanisms of alemtuzumab involve immunomodulation through the resetting the immune system through the depletion and repopulation of lymphocytes. Depletion of CD52 positive cells is performed via antibody-dependent cellular cytotoxicity, complement-mediated cell lysis, and apoptosis. (Willis and Robertson, 2016) Ocrelizumab (Ocrevus®) is an anti-CD20 humanized monoclonal antibody that is recently approved for the treatment of RRMS. Ocrelizumab is the only approved treatment for PPMS. It targets mature B cells by binding to CD20 and results in depletion of circulating B cells (Mulero et al., 2018). The DMT that I listed above mainly target the immune response in the periphery through depleting lymphocytes, inhibiting their proliferation or blocking their activity (Torkildsen et al., 2016). None of the DMT directly target inflammation in the CNS cells. For that reason,

19 they are inefficient in the treatment of progressive MS, which is driven mainly by the inflammation within the CNS (Mahad et al., 2015). A better understanding of the pathological mechanisms of inflammation and neurodegeneration within the CNS is needed to develop therapies that effectively treat patients with progressive MS. Next, I review the regulators of inflammation and their contributions to MS immunopathogenesis.

1.3 The Regulators of Inflammation: NLRs Neuroinflammation in MS is thought to be triggered by infection and/or tissue damage (Hernandez-Pedro et al., 2016). Pattern recognition receptors (PRRs) are a complex network of receptors that are mainly expressed in innate immune cells and can detect both triggers of infection and stress. PRRs are able to recognize unique molecular structures in the cell wall or nuclei components of microorganisms, known as pathogen-associated molecular patterns (PAMPs) as well as molecules that are released from damaged or stressed cells, known as damage-associated molecular patterns (DAMPs). There are four major families of PRRs in the cells, including Toll-like receptors (TLRs), C-type lectin receptors (CLRs), RIG- I-like receptors (RLRs), and the nucleotide-binding domain (NOD) receptors (NLRs) that are expressed either on the cell surface or inside the cells (Mogensen, 2009). The PRRs initiate an inflammatory response upon recognition of PAMPs and/or DAMPs, which leads to the secretion of cytokines and chemokines, recruitment of phagocytes, and induction of autophagy or pyroptotic cell death (Bortoluci and Medzhitov, 2010). TLRs are expressed in most cell types, either at the cell surface (TLR1, 2, 4, 5, 6, 10) or in endosomes (TLR3, 7, 8, 9). TLRs can detect a variety of molecules, including proteins, lipopeptides, and nucleic acids (single-stranded RNA, double-stranded RNA, or CpG DNA). Ligand detection by TLRs initiates intracellular signaling cascades that activate interferon regulatory factor (IRF) family members or NF-B. In macrophages, the activation of TLRs initiates phagocytosis and increases the production of ROS, which facilitates rapid clearance of microbes. Moreover, activation of NF-B signaling pathway leads to production of chemokines that recruit additional neutrophils and monocytes to the site of immunological challenge. In DCs, TLR stimulation induces the expression of co-stimulatory receptors and causes their migration to the draining lymph nodes, where they meet and activate naïve antigen-specific T cells. Depending on the type of cell that receives the stimuli, TLR

20 activation can have several outcomes that result in inflammation and activated immune response (Dowling and Mansell, 2016). CLRs are another family of PRRs. CLRs bind to carbohydrate structures, including mannose, fucose, and glucan on pathogens. They are mainly expressed by APC such as monocytes, macrophages, and DCs (Dunkelberger and Song, 2010). Binding of pathogens to CLRs leads to its internalization and degradation and subsequent antigen presentation. Mannose-binding lectin is a CLR that activates the complement system. This multipotent system of innate immunity generates three broad effector pathways: direct lysis of pathogens, generation of potent pro-inflammatory anaphylatoxins, and opsonization and clearance of target cells (Dunkelberger and Song, 2010). RLRs are cytosolic nucleic acid PRRs expressed in both immune and non-immune cells that sense cytoplasmic RNA. When cells are infected by a virus, viral dsRNA is generated in the cytoplasm during the course of replication. Infected cells sense dsRNA by RLRs and activate anti-viral signaling pathways. There are three members in RLRs family; RIG-I, melanoma differentiation associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2) (Sparrer and Gack, 2015). NLRs are the most recently discovered group of PRRs (Martinon and Tschopp, 2005, Inohara and Nunez, 2003) that sense a wide variety of PAMPs and DAMPs inside the cells. NLRs are evolutionarily conserved in many species, from sea urchins to plants and mammals, which suggests that NLRs are efficient and successful molecules in innate immunity against a wide variety of invaders. NLRs include 23 members in humans and at least 34 members in mice (Franchi et al., 2009). Structurally, NLRs consist of three highly conserved domains characterized by the C- terminal leucine-rich repeat (LRR), which is essential for ligand sensing; the central nucleotide binding ATPase domain NACHT/NBD (also known as NOD), which promotes oligomerization and activation, and the N-terminal domain, which contains either a caspase recruitment domain (CARD) or pyrin domain (PYD) and drives downstream signaling (Figure 3) (Allen, 2014). Members of the NLR family are categorized into at least 4 sub- families based on the structure of the N-terminal domain (Figure 3), including (i) NLRA or Class II transactivator (CIITA) contains acidic transactivation domain, (ii) NLRBs contains baculovirus inhibitor of apoptosis protein repeat (BIR), (iii) NLRC possesses CARD or an

21 undefined domain, (iv) and NLRP contains pyrin domain (Kawai and Akira, 2009, Di Virgilio, 2013, Barbe et al., 2014). NLRs are also categorized based on their function, either as positive or negative regulators of inflammation (Figure 4). Additionally, there are NLRs such as CIITA and NLRC5 that indirectly regulate the immune response by tuning the expression of MHC II and I on APCs (Downs et al., 2016). Since inflammation is the hallmark of MS pathology, next we discuss how NLRs regulate the inflammation in MS.

Figure 3. NLRs structure. (a) The general structure of NLRs, consists of 3 domains, including functional domain, nucleotide binding and oligomerization domain, and ligand sensing domain. (b) Classification of NLRs based on the nature of their functional domain: NLRA, an acidic transactivation (AD) domain; NLRB, a baculovirus inhibitor of apoptosis protein (IAP) repeat (BIR); NLRC, a caspase-recruitment and activation domain (CARD); and NLRP, a Pyrin domain (PYD). In NLRC subfamily, the X displays an unknown domain that has no homology with the other NLR members. Mito is the mitochondria-localization sequence that directs NLRX1 to the mitochondria (Gharagozloo et al., 2018a).

Figure 4. Functional Characterisation of NLRs. NLRs can be classified depending on their mechanism of action to inflammasome and non-inflammasome forming NLRs. The inflammasome forming NLRs assemble inflammasome that activates Caspase-1 and promotes the production of inflammatory cytokines, IL-1β and IL-18. In the group of non- inflammasome forming NLRs, some NLRs regulate MHC II expression, while other NLRs regulate NF-B signaling. The regulators of NF-B consist of NLRs that enhance (NOD-1, NOD-2) or inhibit (NLRP12, NLRX1) NF-B signaling pathway. The negative NLRs, NLRP12 and NLRX1, can inhibit both inflammasome-dependent and -independent cytokine production. NLRC5 and NLRP12 have been described to influence both inflammasome and non-inflammasome signaling pathways in a cell- and stimuli-dependent fashion (Gharagozloo et al., 2018a).

1.3.1 NLRs as positive regulators of inflammation NLRs such as NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, NLRC4, and NAIP are positive regulators of inflammation through the formation of inflammasomes (Latz et al., 2013). NLRP3 inflammasome is the best-characterized form of the inflammasome, which is activated in two steps; the first step is priming the cells by PAMPs or DAMPs via TLRs, leading to the activation of nuclear factor-kB (NF-B) signaling that triggers the expression of inflammasome-related components, including NLRP3, pro-IL-1β, and pro-IL-18. The second step is oligomerization of NLRP3 and its association with an adaptor protein ASC, and pro-caspase-1. This complex triggers the activation of caspase-1 that cleaves pro-IL-1β and pro-IL-18 into their mature and secretable forms, IL-1β and IL-18 (Shao et al., 2015). Activation of the NLRP3 inflammasome also results in pyroptosis, a caspase 1-dependent cell death, which is a highly inflammatory form of cell death. Pyroptosis results in cell lysis

23 and the release of cytosolic components into the extracellular environment (Miao et al., 2011). Previous studies showed that NLRs and their adaptors could positively influence the development and the severity of EAE (Gris et al., 2010). Deletion of Nlrp3, ASC, or the caspase-1 gene resulted in protection against EAE (Shaw et al., 2010, Gris et al., 2010). NLRP3 causes severe inflammatory symptoms in EAE by producing more IL-1β and IL-18, which stimulate the development and activation of Th1/Th17 cells and enhance their infiltration into the spinal cords (Gris et al., 2010). In an alternative pathway, NLRP3 inflammasome engages caspase-8 instead of caspase-1 (Antonopoulos et al., 2015). The importance of NLRP3-caspase-8 inflammasome was recently shown in the production of IL- 1β by T cells that support the survival of Th17 cells in EAE (Martin et al., 2016). Moreover, NLRP3 inflammasome in APCs played a critical role in upregulating chemotactic proteins, such as osteopontin, CCR2, and CXCR6 in Th1 and Th17 cells, thereby inducing T cell migration to the CNS in EAE (Inoue et al., 2012). Many reports demonstrate the role of the NLRP3 inflammasome in MS patients. The expression of caspase-1 and IL-18 are elevated in peripheral mononuclear cells from MS patients compared to cells from healthy controls (Huang et al., 2004). Moreover, the levels of IL-1β are upregulated in CSF of MS patients and correlated with the progression of MS (de Jong et al., 2002). MS treatments such as glatiramer acetate or IFNβ elevate the levels of endogenous IL-1 receptor antagonist in MS patients (Burger et al., 2009, Nicoletti et al., 1996). IFNβ treatment is also shown to attenuate the course and severity of MS by reducing the activity of NLRP3 inflammasomes via suppressing caspase-1 dependent IL-1β secretion (Malhotra et al., 2015). These findings collectively demonstrate that NLR proteins can exacerbate MS, either via formation of inflammasome or stimulation of inflammatory pathways such as NF-B and MAPK. Here, I review the biological activities of NLRs that negatively regulate inflammation. .

1.3.2 NLRs as negative regulators of inflammation Some members of NLRs family including NLRP12, NLRX1, NLRP6, and NLRC3 inhibit the inflammatory signals that are triggered by PRRs following the recognition of PAMPs and DAMPs in the cells. Here I discuss the biological features of 2 inhibitory NLRs, NLRP12 and NLRX1.

1.3.2.1 NLRP12

NLRP12 is a pyrin-containing NLR protein that initially was identified in HL60 human leukemic cell line (Shami et al., 2001). Later, two research groups simultaneously cloned the full-length sequence of human NLRP12 naming it PYPAF7(Wang et al., 2002) or Monarch- 1 (Williams et al., 2003), which eventually the HUGO Gene Nomenclature Committee (HGNC) approved the name of NLRP12 for this gene. The Nlrp12 expression is highly restricted to bone marrow and peripheral blood leukocytes, particularly polymorphonuclear cells that express much higher levels of Nlrp12 than mononuclear cells (Shami et al., 2001, Zamoshnikova et al., 2016, Lich et al., 2007). Since the discovery of NLRP12, there have been contrasting reports that demonstrate both pro-inflammatory and anti-inflammatory roles of NLRP12 in cell-type and stimuli- specific manners (Lukens et al., 2015, Allen et al., 2012, Vladimer et al., 2012, Silveira et al., 2016). Early studies showed that NLRP12 is an inflammatory NLR that interacts with ASC to form inflammasome, leading to caspase-1 activation and release of mature IL-1β. Evidence for the involvement of NLRP12 in inflammasome formation and activation are largely derived from in vitro studies where the expression of NLRP12 was significantly elevated (Wang et al., 2002). Recent studies show the role of NLRP12 in activation of inflammasome by intracellular pathogens such as Yersinia Pestis and Plasmodium infection (Vladimer et al., 2012, Ataide et al., 2014). The formation of the inflammasome by NLRP12 seems to be pathogen specific, as NLRP12 is not involved in activating the inflammasome by other pathogens such as Salmonella, Klebsiella, Escherichia, Mycobacterium, and Listeria species (Tsuchiya et al., 2010, Zaki et al., 2014, Allen et al., 2013). Taken together, these studies establish a biologically relevant role for the NLRP12 inflammasome in innate immune responses against pathogens; however, the exact molecule that triggers NLRP12 inflammasome remains unknown. On the other hand, there are studies that demonstrate NLRP12 as a negative regulator of inflammation through the inhibition of NF-B signaling in innate immune cells. It is shown that the activation of human peripheral blood granulocytes and monocytes by TLR4 or TLR2 agonists (E. coli LPS or synthetic lipopeptide Pam3Cys respectively) reduces the expression of NLRP12 (Williams et al., 2003). Moreover, the expression of NLRP12 declines significantly in myeloid cells (THP-1 human monocytic cell line) after in vitro stimulation

25 with live bacteria such as Mycobacterium or Plasmodium or cytokines such as TNFα or IFNγ (Williams et al., 2005). When NLRP12 is knocked down in THP1 cells using shRNA, the expression levels of pro-inflammatory cytokines significantly increase following LPS or M. tuberculosis treatment (Williams et al., 2005). A transcriptional repressor called B lymphocyte-induced maturation protein-1 (Blimp-1) is induced by TLR stimulation and downregulates NLRP12 expression by binding to NLRP12 promoter and recruiting histone deacetylases (Lord et al., 2009, Shi et al., 2016). These findings suggest that NLRP12 plays an anti-inflammatory role during inflammation and its expression is down regulated to induce the most effective immune response against pathogens. Mechanistically, NLRP12 was shown to suppress both canonical and non-canonical NF- κB signaling pathways. Canonical pathway is activated by a number of signals through TNFR, IL-1R, or TLR, which results in translocation of the RelA/p50 subunits to the nucleus. NLRP12 inhibits hyperphosphorylation of receptor-associated kinase (IRAK-1) that triggers IκBα degradation and p50 nuclear translocation (Zaki et al., 2011, Williams et al., 2005). The non-canonical NF-B pathway is triggered by signaling through receptors such as CD40, LT훽R, or BAFF-R. The signal activates NF-κB inducing kinase (NIK) and IKKα, which leads to p100 cleavage and nuclear translocation of p52 dimers. In non-canonical NF-κB signaling pathway, NLRP12 interacts with TRAF3 and NIK, which leads to the degradation of NIK and subsequent reduction of p100 cleavage to p52 (Figure 5) (Lich et al., 2007, Allen et al., 2012). ATP binding to NLRP12 is crucial for its inhibitory function, as the cells with an NBD mutant form of NLRP12 are not able to inhibit NF-κB activation. As a result, they produce high levels of pro-inflammatory cytokines and chemokines (Ye et al., 2008). Considering the regulatory function of NLRP12 in innate cells, Nlrp12-/- mice were shown to be highly susceptible to the inflammatory disease of intestine such as experimental colitis and colon cancer (Allen et al., 2012, Zaki et al., 2011). This is due to the increased activation of NF-κB in macrophages of Nlrp12-/- mice, which results in the production of pro- inflammatory cytokines and mediators including TNFα and IL-6.

Figure 5. Anti-inflammatory NLRs inhibit NF-B activation. Both NLRX1 and NLRP12 inhibit the activation of NF-κB canonical pathway following TLR stimulation. Nlrx1 interacts and inhibits TRAF6, while NLRP12 inhibits the phosphorylation of IRAK-1. NLRP12 can also inhibit non-canonical NF-κB signaling through regulation of TRAF3 and NIK (Gharagozloo et al., 2018a).

1.3.2.2 NLRX1

NLRX1 is the recently characterized member of NLRs that is ubiquitously expressed and uniquely localized in the mitochondria (Moore et al., 2008). Initial studies showed that NLRX1 was located in the outer membrane of mitochondria (Moore et al., 2008), however, later studies by two independent groups demonstrated that NLRX1 is located in the matrix of mitochondria (Rhee et al., 2013, Arnoult et al., 2009). The localization of NLRX1 in mitochondria is due to the presence of a functional N-terminal mitochondrial-addressing sequence (UQCRC2), which allows the targeting of NLRX1 to the mitochondrial matrix (Arnoult et al., 2009). A study by Tattoli et al., showed that NLRX1 induces the production of ROS in the cells treated by TNFα and double-stranded RNA, which result in increased activation of NF-κB inflammatory pathway (Tattoli et al., 2008). On the other hand, Xia et al. reported that NLRX1 acts as a negative regulator of NF-κB signaling. They showed that

27 in the rest cells, NLRX1 interacts with TNF receptor-associated factor 6 (TRAF6). However, after stimulation with LPS, NLRX1 rapidly dissociates from TRAF6 and binds to the IKK complex, leading to inhibition of IKKα/β phosphorylation and NF-κB activation (Figure 5) (Xia et al., 2011). Therefore, depending on the experimental condition, NLRX1 can both activate or inhibit NF-κB signaling. In the viral infection, NLRX1 acts as a negative regulator of the anti-viral signaling pathway (Moore et al., 2008, Allen et al., 2011). Once cells are infected with a virus, viral PAMPs are detected by the cytoplasmic RLRs and MDA5, which activate MAVS signaling pathway, resulting in the activation of interferon regulatory factor 3 (IRF3), NF-B, and transcription of type-1 interferon (IFN-1) (Moore et al., 2008, Allen et al., 2011). Moore et al. reported that interaction between NLRX1 and MAVS prevents the RIG-I from binding to MAVS, which results in inhibition of NF-κB activation and IFN-beta production (Moore et al., 2008). NLRX1 can induce autophagy that deletes the cytosolic viral RNA and consequently results in the inhibition of IFN-beta production. Lei et al. proposed that NLRX1 forms a multimeric complex with the cytosolic ATG proteins and a mitochondrial matrix protein, TUFM that initiate an autophagic response (Lei et al., 2012). For this reason, NLRX1 deficient cells upregulate type I IFN production and are better able to restrict the replication of a variety of viruses. Studies summarized here demonstrate the regulatory role of NLRX1 in the immune responses against viruses. NLRX1 also regulates mitochondrial dynamics and cell death. Recently, our research group showed that NLRX1 protects neuronal-like cell line (N2A cells) from necrosis (Imbeault et al., 2014). We found an increased number of mitochondria in NLRX1- overexpressed N2A cells compared to control cells, which was associated with increased phosphorylation of DRP1 and mitochondrial fission. As a result, NLRX1 switch the cell death from necrosis towards apoptosis, which inhibits neurodegeneration by preventing the release of inflammatory mediators in the tissue environment and maintaining the tissue homeostasis (Imbeault et al., 2014). Consistent with our observations, Theus et al. found that NLRX1 serves as a survival factor for neurons and significantly reduces neuronal apoptosis under hypoxic conditions (Theus et al., 2017). Regarding the mechanism of NLRX1 survival activity, a recent study shows that NLRX1 controls mitochondrial activity, thereby reduces oxygen consumption, oxidative stress and apoptosis of epithelial cells during

28 the ischemia-reperfusion injury (Stokman et al., 2017). Interestingly, a study by Girardin’s group shows that in prolonged cellular stress or glucose starvation, NLRX1 accelerates intrinsic apoptotic pathway (Soares et al., 2014). Taken together, these studies show that NLRX1 is a cell survival factor and regulator of mitochondria function. In case of severe and prolonged cellular stress, NLRX1 switch the cell death from necrosis towards apoptosis and accelerates apoptosis. Regarding sterile inflammation in autoimmune animal models, it is shown that NLRX1 regulates inflammation in EAE. A recent work by Eitas et al. demonstrates a protective role of NLRX1 in EAE by suppressing macrophage/microglial activation (Eitas et al., 2014). In this study, Nlrx1−/− mice showed increased cytokine production and enhanced tissue damage during EAE compared to the WT mice (Eitas et al., 2014). This finding is consistent with Allen et al. study, which also showed the anti-inflammatory function of NLRX1 in sterile CNS inflammation, such as traumatic brain injury. Traumatic CNS injury results in massive necrotic and apoptotic loss of neurons and glial cells. Nlrx1−/− mice exhibited significantly larger brain lesions and increased motor deficits following brain injury. Their data indicates that NLRX1 attenuates NF-κB signaling and IL-6 production in microglia (Allen et al., 2016). Interestingly, not only NLRX1 inhibits innate immune responses, but it also controls adaptive immune responses via inhibiting T cell proliferation and differentiation (Leber et al., 2017b). In a mouse model of induced colitis using dextran sodium sulfate–induced colitis, lack of NLRX1 results in increased severity of the disease and enhanced Th1 and Th17- related inflammatory cytokines such as IFN, TNFα, and IL-17. In vitro experiments revealed that Nlrx1-/- T cells have a greater ability to proliferate and differentiate into Th17 cells (Leber et al., 2017b). The role of inflammatory and anti-inflammatory NLRs in MS patients and EAE mice is summarized in Table 1.

Table 1. The immunopathogenesis of inflammatory and anti-inflammatory NLRs in MS patients and EAE mice.

Subjects Major findings Reference

A homozygous missense variant in (Maver et al., MS NLRP1 (Gly587Ser) was associated 2017) NLRP1 with familial MS. Compound heterozygous mutation was (Bernales et MS observed in several MS patients. al., 2017) Nlrp3-/- mice developed ameliorated EAE, associated with a significant (Gris et al., EAE reduction of the inflammatory infiltrate 2010) to the CNS and lower production of IL- 18, IFN, and IL-17. Nlrp3−/− mice were resistant to EAE

with decreased inflammatory cell (Inoue et al., EAE infiltration to the CNS. The activation of 2012) NLRP3 inflammasome in APCs is crucial for T cell migration to the CNS. NLRP3 High dose adjuvant induced severe EAE EAE and neuronal damage in Nlrp3−/− mice, (Inoue et al., which was inflammasome-independent 2016)

Inflammatory NLRs Inflammatory and resistant to IFNβ therapy. IFNβ treatment attenuated the course (Malhotra et MS and severity of MS by reducing the al., 2015) activity of NLRP3 inflammasomes. Q705K polymorphism (rs35829419) results in overactive NLRP3 (Malhotra et MS inflammasome, which was associated al., 2015) with IFNβ response in MS patients. Nod1−/− and Nod2−/− mice were highly resistant to EAE. Reduced number of (Shaw et al., NOD EAE activated APC and activation of T cells 2011) in the CNS were observed. Nlrp12-/- mice had ameliorated EAE course with atypical symptoms, NLRP12 (Lukens et EAE including ataxia and impaired balance al., 2015) control, which was associated with increased production of IL-4. Protective role of NLRX1 in EAE. Nlrx1−/− mice showed increased NLRX1 (Eitas et al., inflammatory NLRs inflammatory

- EAE macrophage/microglial activation and 2014) cytokine production, which resulted in

Anti increased tissue damage.

2. HYPOTHESIS AND OBJECTIVES

We hypothesized that anti-inflammatory NLRs inhibit the onset and progression of MS. We tested our hypothesis using the following aims and objectives:

Aim 1: To study the anti-inflammatory role of NLRP12 in the progression of MS

Objectives:

• Evaluating the role of NLRP12 in suppressing the progression of EAE • Investigating the anti-inflammatory function of NLRP12 in microglia • Assessing the role of NLRP12 in the regulation of T cell response

Aim 2: To study the anti-inflammatory role of NLRX1 in the onset of MS

Objectives:

• Evaluating the role of NLRX1in preventing the onset of spontaneous EAE • Investigating the anti-inflammatory function of NLRX1 in innate immune cells • Assessing the anti-inflammatory role of NLRX1 in T cell response

3. ARTICLES

3.1. ARTICLE

The nod-like receptor, receptor, Nlrp12, plays an anti-inflammatory role in Experimental Autoimmune Encephalomyelitis

Marjan Gharagozloo*, Tara M. Mahvelati*, Emilie Imbeault, Pavel Gris, Echarki Zerif, Diwakar Bobbala, Subburaj Ilanguraman, Abdelaziz Amrani, Denis Gris

*Marjan Gharagozloo and Tara M. Mahvelati are first co-authors

Published in Journal of Neuroinflammation, PMID: 26521018

Résumé

Contexte La sclérose en plaques (SEP) est une maladie auto-immune déclenchée par une réaction inflammatoire anormale et caractérisée par la présence de plaque démyélinisantes dans le système nerveux central. Cette maladie affecte majoritairement les jeunes adultes entre 25 et 40 ans. Plusieurs voies de signalisation ont été identifiées comme étant des cibles thérapeutiques incluant les récepteurs TLRs (Toll-like receptors), la voie de NF-B et les récepteurs NLRs (nucleotide-binding leucine-rich repeat containing). Durant la SEP, le rôle exact des microglies reste inconnu. D’une part, elle promues l’expression de molécules pro- inflammatoires et joue le rôle de cellules présentatrices d’antigènes pour forcer les cellules T à adopter un phénotype pro-inflammatoire. D’autre part, les microglies peuvent exprimer des molécules anti-inflammatoires and inhiber la réponse inflammatoire. Ces cellules expriment une grande variété de récepteurs immunitaires tels que les récepteurs de type NLRs. Ces récepteurs sont des protéines intracellulaires régulatrices du système immunitaire inné et adaptatif. Suite à leur activation, ces protéines initient des voie pro-inflammatoire, incluant la formation de l’inflammasome et la réponse de NF-B. Parmi les NLRs, NLRP12 est une protéine intracellulaire largement exprimée dans les cellules d’origine myéloïde. Elle joue un rôle important dans les réponses inflammatoires immunes en régulant négativement la voie NF-kB. Ainsi, notre hypothèse de recherche est que NLRP12 occupe un rôle anti- inflammatoire et améliore le cours de la SEP. Méthodes Nous avons utilisé un modèle de souris bien caractérisé de la SEP, l’encéphalopathie expérimentale autoimmune (EAE). EAE a été induites chez des souris de types sauvages (WT) et des souris n’exprimant pas Nlrp12 (Nlrp12 KO) avec MOG :CFA. Les moelles épinières de ces souris ont été extraites pour l’analyse des gènes pro- inflammatoire et l’immunofluorescence. De plus, un modèle de culture primaire cellulaire primaires de microglies, d’origine corticale, a été utilisé pour l’expression de gènes pro- inflammatoire, ainsi que leurs sécrétions. Résultats Au cours de 9 semaines, dans les deux types de génotypes, la maladie était observée à son maximum autour de la 3ème semaine après immunisation. Les souris n’ayant pas le gène de Nlrp12 ont démontré un état sévère de la maladie comparativement aux souris de type sauvage. Effectivement, les souris Nlrp12 KO ont une forme exacerbée de la maladie

33 et celle-ci début plus tôt et atteint des scores cliniques plus élevés (p<0.05). Également, les symptômes corrèlent avec une augmentation de l’expression protéique de Nlrp12 dans les souris contrôles malades comparativement aux souris saines. Une augmentation significative de l’expression de Ccr5, COX-2 ainsi qu’IL-1 β était détectée dans les souris Nlrp12 KO par rapport aux souris WT. Surprenamment, aucune différence dans le pourcentage de gliose était observée dans les deux génotypes à 3 semaines post-injection. De plus, une augmentation significative de l’inflammation dépendante de NF-B a été observé dans des microglies primaire de souris Nlrp12 KO. L’essai du réactif de Griess pour l’activité d’iNOS a démontré plus de nitrate sécrété dans le milieu des souris Nlrp12 KO. Suite à un traitement au LPS, les microglies des souris Nlrp12 KO sécrètent significativement plus des cytokines TNFα et IL-6. Conclusion Ces observation suggèrent un rôle de protecteur important pour le Nlrp12 dans la suppression de l’inflammation lors du développement et la progression de la SEP. Ainsi, l’absence de Nlrp12 entraîne une augmentation de la réponse inflammatoire Keywords Nlrp12, EAE, microglie, inflammation neuronale.

Background Multiple Sclerosis (MS) is an organ-specific autoimmune disease resulting in demyelinating plaques throughout the central nervous system. In MS, the exact role of microglia remains unknown. On one hand, they can present antigens, skew T cell responses, and upregulate the expression of pro-inflammatory molecules. On the other hand, microglia may express anti-inflammatory molecules and inhibit inflammation. Microglia express a wide variety of immune receptors such as NLRs. NLRs are intracellular receptors capable of regulating both innate and adaptive immune responses. Among NLRs, Nlrp12 is largely expressed in cells of myeloid origins. It plays a role in immune inflammatory responses by negatively regulating the NF-B pathway. Thus, we hypothesize that NLRP12 suppresses inflammation and ameliorates the course of MS. Methods We used Experimental Autoimmune Encephalomyelitis (EAE), a well- characterized mouse model of MS. EAE was induced in WT and Nlrp12-/- mice with MOG:CFA. Spinal cords of healthy and immunized mice were extracted for immunofluorescence and pro-inflammatory gene analysis. Primary murine cortical microglia cell cultures of WT and Nlrp12-/- were prepared with cortices of 1-day-old pups. Cells were stimulated with LPS and analyzed for the expression of pro-inflammatory genes as well as pro-inflammatory molecule secretions. Results Over the course of 9 weeks, Nlrp12-/- mice demonstrated increased severity in the disease state, where they developed the disease earlier and reached significantly higher clinical scores compared to WT mice. Spinal cords of immunized WT mice relative to healthy WTs revealed a significant increase in Nlrp12 mRNA expression at 1, 3, and 5 weeks post injection. A significant increase in the expression of pro-inflammatory genes Ccr5, Cox2, and IL-1 was found in the spinal cords of Nlrp12-/- mice relative to WT mice (P<0.05). A significant increase in the level of gliosis was observed in the spinal cords of Nlrp12-/- mice compared to WT mice after 9 weeks of disease (P<0.05). Primary Nlrp12-/- microglia cells demonstrated significant increase in iNOS expression (P<0.05), and secreted significantly (P<0.05) more TNF-, IL-6, and NO. Conclusion Nlrp12 plays a protective role by suppressing inflammation during the development of EAE. The absence of Nlrp12 results in an increased inflammatory response.

Keywords: Nlrp12, EAE, microglia, neuroinflammation.

Background Multiple Sclerosis (MS) is among one of the most common neurodegenerative diseases affecting an estimated 2.3 million individuals worldwide [1]. This organ-specific autoimmune disease is characterized by four different types of demyelinating plaques; Type I and II which are T-cell mediated or T-cell and antibody-mediated; while Type III and IV are mediated by oligodendrocyte death [2]. In all four cases, plaques are associated with activated macrophages, microglia and astrocytes. Regardless of the type of plaque formation, inflammation plays a central role in MS pathophysiology [1, 3]. Microglia, the resident immune cells of the central nervous system (CNS), play a major role in maintaining CNS homeostasis. They’ve been shown to be associated with developing plaques and are thought to contribute to the development of MS [2, 4], as well as other chronic inflammatory neurodegenerative diseases such as Alzheimer’s [5]. During MS, activated microglia can play the role of antigen presenting cells (APCs) and therefore, skew

T cell responses towards a TH1 pro-inflammatory phenotype [1, 2, 6]. In addition, once activated, microglia upregulate the expression of pro-inflammatory molecules including but not restricted to TNFα, IL-1β, IL-6, MIP1α, iNOS, all of which have been shown to play a role in demyelination and neuronal damage [7]. There are a wide variety of immune receptors expressed by microglia that regulate its function. Pathogen-recognition receptors (PRRs) such as Nod-like Receptors (NLRs) are innate immune receptors and sensors of pathogen-associated molecular patterns (PAMPs) [8]. NLRs are a group of proteins that share a NACHT and leucine-rich repeat (LRR) domain but differ in their N-terminal effector domain. Upon recognition of their respective ligand, NLRs become activated and result in the subsequent triggering of multiple pro-inflammatory molecular pathways, such as NF-B. In addition, they are able to regulate both innate and adaptive immune responses and play a role in pathological processes [8]. Recently discovered Nlrp12 is a pyrin-containing intracellular NLR protein. It is largely expressed in the cells of myeloid origin such as monocytes and dendritic cells (DCs). The expression of Nlrp12 has been shown to play an important role in immune inflammatory responses by negatively regulating the NF-B pathway and modulatory roles, such as dendritic cell migration [9, 10]. The NF-B pathway is one of the major pathways involved in the inflammatory response. Typically, the activation of NF-B following insults results in the

36 transcription of pro-inflammatory cytokines such as TNF-, IL-1, and IL-6, chemokines such as CCL5, CCL22, and MIP1 and proteins, such as iNOS and COX2 [11, 12]. This study aims to investigate the role of NLRs in neuroinflammation, particularly to uncover the role of Nlrp12 during EAE development. In our study, results show that Nlrp12 acts to down-regulate inflammation during Experimental Autoimmune Encephalomyelitis (EAE) development. This study may have significant implications in the development of potential novel therapies to treat MS and other neuro-inflammatory degenerative diseases.

Materials & Methods Mice Nlrp12 knock-out (Nlrp12-/-) mice were kindly provided by Dr. Jenny P.-Y. Ting (Chapel Hill, NC). All of the protocols and procedures were approved by the University of Sherbrooke at University of Sherbrooke Animal Facility and Use Committee.

Experimental Autoimmune Encephalomyelitis EAE was induced in 8-10-week-old C57BL/6 female mice using a previously established protocol by Miller et al. [13]. Briefly, a 1:1 emulsion mixture of Myelin Oligodendrocyte

Glycoprotein (MOG35-55) (Genemed Synthesis Inc., San Antonio, TX) and Complete Freund’s Adjuvant (CFA) (Sigma-Aldrich, St-Louis, MO) supplemented with 100mg Mycobacterium Tuberculosis H37 RA (Difco Laboratories, Detroit, MI) was prepared using glass tuberculin syringe. The emulsion (100μL) was injected subcutaneously on each side of the midline on the lower back of each mouse for a total of 200μg MOG35-55. Pertussis toxin (200ng) (List Biological Laboratories Inc., Campbell, CA) was injected intraperitoneally on the day of and 48h following immunization. Mice were monitored every day for the development of disease. Clinical scores were given by two independent observers, using the following scale: 0: No sign of disease, 1: Limp tail or weakness in limbs, 2: Limp tail and weakness in limb, 3: Partial limb paralysis, 4: Complete limb paralysis.

Histopathology Immunized mice were anesthetized by intraperitoneal injection of Avertin® (2,2,2 tribromoethanol, approximately 240 mg/kg) (Sigma-Aldrich, St-Louis, MO) diluted in 0.9%

37 saline solution. Mice were then perfused with ice-cold PBS (Wisent, St-Bruno, QC) and spinal cords were removed and stored at -80°C immediately for RNA extraction (thoracic region) and placed in 4% paraformaldehyde (Sigma-Aldrich, St-Louis, MO) for immunofluorescence analysis (lumbar region). Spinal cord tissues were embedded in paraffin and cut into 5μm sections.

T Cell Proliferation assay T cell proliferation was performed using 3H-thymidine incorporation assay. A single cell suspension was prepared from draining lymph nodes (more precidely from inguinal and axillary lymph nodes) and spleen. CD4+ T cells were then purified using EasySep Mouse CD4+ T Cell Isolation Kit (Stem cell, Vancouver, BC), seeded in round-bottom 96-well culture plate (1x105 cells/well) and activated with plate-bound anti-CD3 (1 μg/mL) and anti- CD28 (2 μg/mL) antibodies for 3 days. During the last 18h of culture, 1 μCi of methyl-[3H]- thymidine (NEN Life Sciences, Boston, MA) was added per well. The cells were harvested onto glass fiber filter mats, and the incorporated radioactivity was measured using Top Count microplate scintillation counter (PerkinElmer, Wellesley, MA).

Intracellular IL-4 staining for flow cytometry The purified CD4+ T cells from wild-type (WT) and Nlrp12-/- mice were activated by plate bound anti-CD3 (1 μg/mL) and anti-CD28 (2 μg/mL) antibodies for 3 days. Then, the cells were stimulated with phorbol 12-myristate 13-acetate (PMA; 50ng/mL, Sigma Chemical Co., St-Louis, MO) and ionomycin (1 μg/mL, Calbiochem Corp., La Jolla, CA) for 4h at 37°C and 5% CO2 in the presence of Brefeldin A (1 μg/mL, eBioscience, San Diego, CA). After staining the cells with anti CD4+-FITC antibody (eBioscience), the cells were fixed and permeabilized using intracellular fixation and permeabilization buffer (eBioscience) and stained with anti-IL-4_PE antibody, as per the manufacturer’s instructions. Sample analysis was performed with FACS Calibur and data analysis was done using FlowJo Software (FlowJo, LLC, Ashland, OR).

RNA Extraction, cDNA synthesis, reverse transcription and real-time quantitative PCR RNA from spinal cords was extracted using TRIzol reagent (Life Technologies Inc., Burlington, ON). Tissues were homogenized with sterile beads (Qiagen, Limburg, Netherlands) at a speed of 20 Hz for 2 minutes. Chloroform (200μL) (Fisher Scientific, Ottawa, ON) was added to each tube per 1mL of TRIzol and incubated at room temperature for 15 minutes followed by centrifugation at 13000 rpm for 15 minutes at 4°C. Supernatants were collected in new tubes and 500μL Isopropanol (Fisher Scientific, Ottawa, ON) was added to each tube and incubated for 10 minutes at -80°C before spinning down at 13000 rpm for 10 minutes at 4°C. Pellets were washed with 75% Ethanol and re-suspended in 20μL RNAse-free sterile water (Wisent, St-Bruno, QC). cDNA was synthesized using Oligo(dT) primer (IDT, Coralville, IA), PCR Nucleotide Mix (GE Healthcare, Baie d’Urfe, QC), M- MuLV Reverse Transcriptase, M-MuLV Reverse Transcriptase Buffer (New England BioLabs, Whitby, ON), and RNasin Ribonuclease Inhibitor (Promega, Madison, WI). RT- PCR and RT-qPCR were used to verify the expression of Nlrp12, Mip3α, Cox2, IL-1β and Ccr5 using Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies, Santa Clara, CA). Primers (IDT, Coralville, IA) sequences: Nlrp12 F: 5'-CCT CTT TGA GCC AGA CGA AG-3', Nlrp12 R: 5'-GCC CAG TCC AAC ATC ACT TT-3', Mip3α F: 5’- CTC AGC CTA AGA GTC AAG AAG ATG-3’, Mip3α R: 5’-AAG TCC ACT GGG ACA CAA ATC-3’, Cox2 F: 5’- CCA GCA CTT CAC CCA TCA GTT-3’, Cox2 R: 5’- ACC CAG GTC CTC GCT TAT GA-3’, IL-1β F: 5’-CAT CCA GCT TCA AAT CTC GCA G-3’, IL- 1β R: 5’CAC ACA CCA GCA GGT TAT CAT C-3’, Ccr5 F: 5’-CGA AAA CAC ATG GTC AAA CG-3’, Ccr5 R: 5’-GTT CTC CTG TGG ATC GGG TA-3’, 18S F: 5'-CGG CTA CCA CAT CCA AGG AA-3', 18S R: 5'-GCT GGA ATT ACC GCG GCT-3' Samples were normalized to the internal control 18S rRNA and relative expression was calculated using the ΔΔCT method [14].

Immunofluorescence Slides were de-paraffinized in xylene (EMD Millipore, Etobicoke, ON) and hydrated in 100%, 95% and 70% ethanol gradient. Antigen unmasking was performed at sub-boiling temperature for 10 minutes in 10mM Sodium Citrate Buffer pH 6.0 (Sigma-Aldrich, St- Louis, MO). Immunofluorescence was performed in Sequenza Slide Rack and Coverplate

System (Ted Pella, Inc., Redding, CA). Slides were washed with 0.1% Triton X-100 in PBS solution, blocked in 5% FBS plus 0.1% Triton X-100 in PBS for 1 hour and incubated with primary antibody (1:1000) overnight at 4°C. Secondary antibody (1:2000) incubation was done at room temperature for 2 hours. Slides were mounted with DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL) and photomicrograph pictures were taken with Retiga SRV Mono Cooled numerical camera attached to Zeiss Axioskop 2 microscope. Pictures were stitched with Adobe Photoshop CS6 and stain density was quantified with Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD).

Antibodies: Rabbit Anti-Glial Fibrillary Acidic Protein (GFAP) antibody was purchased from Cedarlane (Burlington, ON). Rabbit Anti-Ionized calcium binding adaptor molecule 1 (Iba1) antibody was purchased from Wako (Osaka, Japan). Alexa Fluor 488 AfinniPure Goat Anti-Rabbit IgG (H+L) was purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). The percentage of microgliosis and astrogliosis in the spinal cord and grey matter were calculated as follow: 퐷𝑒𝑛𝑠𝑖𝑡푦 𝑠𝑡푎𝑖𝑛 푃𝑒𝑟𝑐𝑒𝑛𝑡푎𝑔𝑒 𝑜𝑓 퐺𝑙𝑖𝑜𝑠𝑖𝑠 (%) = × 100 푇𝑜𝑡푎𝑙 푎𝑟𝑒푎 The percentage of microgliosis and astrogliosis in the white matter were calculated as follow: 푃𝑒𝑟𝑐𝑒𝑛𝑡푎𝑔𝑒 𝑜𝑓 퐺𝑙𝑖𝑜𝑠𝑖𝑠 𝑖𝑛 𝑡ℎ𝑒 푊ℎ𝑖𝑡𝑒 푀푎𝑡𝑡𝑒𝑟 (%) 퐷𝑒𝑛𝑠𝑖𝑡푦 𝑠𝑡푎𝑖𝑛 𝑜𝑓 𝑠𝑝𝑖𝑛푎𝑙 𝑐𝑜𝑟𝑑 − 퐷𝑒𝑛𝑠𝑖𝑡푦 𝑠𝑡푎𝑖𝑛 𝑜𝑓 𝑔𝑟푎푦 𝑚푎𝑡𝑡𝑒𝑟 = ( ) 푇𝑜𝑡푎𝑙 푎𝑟𝑒푎 𝑜𝑓 𝑠𝑝𝑖𝑛푎𝑙 𝑐𝑜𝑟𝑑 − 𝑡𝑜𝑡푎𝑙 푎𝑟𝑒푎 𝑜𝑓 𝑔𝑟푎푦 𝑚푎𝑡𝑡𝑒𝑟 × 100

Primary Cell Culture Cortices from 1-day-old pups were extracted and placed onto a 100mm petri dish using aseptic techniques. Cortices were sliced with a commercial razor blade, further broken up with rigorous up-and-down motion in 10mL of medium and filtered with a 70μm filter. Cells were then plated onto a 100mm petri dish and put in an incubator of 37°C with 5% CO2. Cell culture medium DMEM/F12 (Wisent, St-Bruno, QC) was supplemented with 10% FBS (Invitrogen, Burlington, ON), 1% Penicillin-Streptomycin Solution (Wisent, St-Bruno, QC),

1% L-Glutamine Solution (Wisent, St-Bruno, QC), 0.9% Sodium Pyruvate Solution (Wisent, St-Bruno, QC), 0.9% MEM Amino Acid Solution (Wisent, St-Bruno, QC) and 0.9% Amphotericine B Solution (Wisent, St-Bruno, QC). Medium of the mixed glial culture was changed every 2 to 3 days. After 3 weeks, primary microglia cells were separated from astrocytes using EasySep CD11b positive selection kit following manufacturer’s instructions (Stem cell, Vancouver, BC).

Immunoblotting Proteins were separated on 10% polyacrylamide gels and transferred onto PVDF (Millipore, Etobicoke, ON) membranes. Membranes were blocked with PBS containing 10% nonfat milk and 0.05% Tween-20 (Sigma-Aldrich, St-Louis, MO). Membranes were washed in 1X Tris-Buffered Saline (TBS) plus 1% Tween-20 for 15 minutes and incubated with primary antibody (1:1000) overnight at 4°C and with secondary antibody (1:2000) for 2 hours at room temperature. Membranes were revealed with GE HealthCare Life Sciences Amersham ECL Plus (Baie d’Urfe, QC), viewed with Molecular Imager VersaDoc from BioRad and protein bands were quantified using NIH ImageJ software. Antibodies: β-Actin (Rabbit), iNOS (Rabbit) and Anti-Rabbit IgG HRP-linked antibodies were purchased from Cell Signaling Technology (Beverly, MA).

Cytokine measurement TNF-α and IL-6 cytokines in the supernatant of microglia culture were measured using ELISA kits purchased from BioLegend (San Diego, CA). Cerebellum and lymph node samples were homogenized in 0.5 mL of ice-cold lysis buffer (Cell Signaling Technology, Beverly, MA) supplemented with protease inhibitors (Roche Diagnosis, Mannhein, Germany) by rapid agitation for 2 min in the presence of 3-mm stainless beads. The tissue lysate was centrifuged for 10 min at 13 000xg in a cold microfuge, and the supernatant was transferred to a new tube. The concentration of proteins in the lysate was determined by Bradford protein assay. The tissue levels of IL-4 were determined using a high sensitivity IL- 4 ELISA Kit (eBioscience, San Diego, CA), and the concentration of IL-4 in serum samples was quantified using Mouse IL-4 DuoSet (R&D Systems), according to the manufacturer’s instructions.

Statistical Analysis All statistical analysis was conducted using GraphPad Prism 6 software. The results were expressed as mean ± SD. Statistical significance was determined using one-way ANOVA Kruskal-Wallis followed by Bonferroni (EAE clinical score), one-way ANOVA followed by Tukey-Kramer (Nlrp12 mRNA expression, iNOS expression in primary microglia, concentration of pro-inflammatory cytokines), two-way ANOVA followed by Tukey’s (percentage of gliosis) or one-way ANOVA followed by Dunet (pro-inflammatory mRNA expression) multiple comparison test. IL-4 results were compared between WT and Nlrp12- /- mice using Mann-Whitney U test. Statistical significance was accepted at P<0.05.

Results

Nlrp12 mRNA expression reaches a peak at the third week post injection

Following immunization with ovalbumin and MOG35-55 in CFA, spinal cords were dissected from healthy and EAE mice and analyzed for the expression of Nlrp12 mRNA (Fig.1). Nlrp12 mRNA expression in immunized mice was shown to be significantly increased relative to healthy wild-type (WT) mice at week 1 (3-fold increase), week 3 (7-fold increase), and week 5 (4-fold increase). Additionally, the level of Nlrp12 mRNA expression was increased as of the first week of EAE and reached its highest level at the third week. At 5 weeks post injection, although the expression of Nlrp12 was significantly higher in diseased mice compared to healthy mice, it was considerably lower than the third week and resembled much more the disease state of the first week. As a control, ovalbumin was injected, and the spinal cords of mice treated with ovalbumin were removed after the third week in order to keep consistency with MOG injected mice.

8 *

6 * mice 4 * 2 Fold changeover healthy 0 OVA 1w 3w 5w

Figure 1 41

Figure 1. Nlrp12 mRNA expression reaches a peak at third week post injection. Results indicate fold change in Nlrp12 mRNA expression of diseased mice over healthy mice. Mice injected with Ovalbumin as control were sacrificed 3-weeks post injection. Results are expressed as mean ± SD. Statistical significance was accepted at *P<0.05. Statistical analysis was done using one-way ANOVA followed by Tukey-Kramer multiple comparison test. N=5.

Nlrp12-/- mice exhibit exacerbated form of the disease compared to WT mice

In order to investigate the role of Nlrp12 in MS, EAE was induced in 8-10-week-old

C57BL/6 female mice. An emulsified mixture of MOG35-55 in CFA was subcutaneously injected in mice. Nlrp12-/- mice demonstrated clinical symptoms after approximately 5 days post injection whereas WT mice developed the disease roughly after 9 days. In addition, while WT mice were showing the first signs of disease, Nlrp12-/- mice already demonstrated indications of severe disease, reaching scores of 2, indicative of tail and back limb weaknesses (Fig.2). Indeed, Nlrp12-/- mice were observed to reach higher clinical scores throughout the 9-week period. More precisely they reached scores of 3-3.5, which indicates weakness in tail, back and front limbs compared to WT mice that reached scores of 2-2.5. In both genotypes, the severity of the disease outcome was observed to peak around the third week post injection and remained relatively constant throughout the 9-week period.

* * * -/- 4 Nlrp12 WT

2 Clinical Scores 1

10 20 30 40 Days after immunization Figure 2. Nlrp12-/- mice exhibit exacerbated form of disease compared to WT mice. Animals were scored daily by two independent observersFigure 2and scored based on the following scale: 0: No sign of disease, 1: Limp tail or weakness in limbs, 2: Limp tail and weakness in limbs, 3: Partial limb paralysis, 4: Complete limb paralysis. Statistical significance was accepted at *P<0.05. Statistical analysis was done by Kruskal-Wallis one-way ANOVA test followed by Bonferroni multiple comparison test. N=7.

Nlrp12-/- mice demonstrate higher percentage of reactive gliosis after EAE

In response to injury, glial cells become reactive, producing multiple pro-inflammatory proteins, as well as increasing in numbers. GFAP is an intermediate protein expressed and upregulated by astrocytes in response to CNS insults [15]. Moreover, in addition to the secretion of multiple pro-inflammatory proteins, the reactive microglial response can be measured by the extent of upregulation of Iba1 [5]. Spinal cords of healthy and immunized mice were extracted and stained for GFAP (Fig. 3) and Iba1 (Fig. 5). We observed no significant difference in the percent level of astrogliosis and microgliosis between the healthy WT and healthy Nlrp12-/- mice. Additionally, we observed no differences in the percentage of neither microgliosis nor astrogliosis between WT and Nlrp12-/- mice after 3 weeks of EAE (Fig. 4a, 6a). However, after 9 weeks of disease, Nlrp12-/- mice demonstrated a significant increase in the level of astrogliosis (30% compared to 15% in WTs) in the white matter (WM) and an observable increase within the grey matter (GM) area of the spinal cord compared to WT mice (Fig. 4b). The 10% difference between Nlrp12-/- mice and WT mice occurred within the WM. Similar results were obtained for the level of microgliosis, where Nlrp12-/- mice demonstrated increased percentage of Iba1 compared to WT mice (Fig. 6b). Indeed, the difference of 20% increase in microgliosis within the spinal cord of Nlrp12-/- mice compared to WT mice was primarily within the WM.

Figure 3. Photomicrograph pictures of Spinal Cords stained with GFAP. GFAP staining of the spinal cord, evaluating astrogliosis percentage following EAE induction. A) WT mice Healthy. B) Nlrp12-/- mice Healthy. C) WT mice 3 weeks EAE. D) Nlrp12-/- mice 3 weeks EAE. E) WT mice 9 weeks EAE. F) Nlrp12-/- mice 9 weeks EAE. Scale bar is 500m.

Figure 4. Photomicrograph pictures of Spinal Cords stained with Iba1. Iba1 staining of the spinal cord, evaluating microgliosis percentage following EAE induction. A) WT mice Healthy. B) Nlrp12-/- mice Healthy. C) WT mice 3 weeks EAE. D) Nlrp12-/- mice 3 weeks EAE. E) WT mice 9 weeks EAE. F) Nlrp12-/- mice 9 weeks EAE. Scale bar is 500m.

A A A A B B B B

Spinal CorSpinald CordGray MSpinalatter Gray Cord MatterWhite M GrayatterWhite Matter Matter White SMatterpinal Cord Spinal CGorday MSpinalatter G Cordray MaWttheirte MSpinal Grayatter W MatterCordhite Matter GrayWhite Matter Matter SpinWhiteal Cord Matter Gray Matter White Matter WT Healthy WT Healthy WT Healthy WT Healthy * * WT 3 weeks EAE WT*** *3 weeks EAE **** * -/- WT 3 weeks* EAE -/- **** **W* T 3 weeks EAE **** *** Nlrp12 HealthNlrp12y -/- Healthy Nlrp12 Healthy Nlrp12-/- Healthy *** *** -/- -/- Nlrp12** -/- 3 weeks EAE ** ** ** ** **25 ** ** Nlrp12 3 weekNlrp12s EA*E* 3* weeks* EAE ** 40 Nlrp12-/- 3 weeks EAE ** * 25 25 * * 25* 40 40 * * * 40 * * * * * * **** **** * * **** * *** **** *** ) ) *** *** ) 20 20 20 20 ) % %

% 30 %

( 30 ( 30 (

( 30

s s s s i i i i s s s 15 15 15 s o 15 o o o i i i i l l l l

g 20 g g A 20 20 B g 20 o o o o r r r r t

10 t t 10 10 10 t s s s s Astrogliosis (%) Astrogliosis(%) Astrogliosis (%) Astrogliosis (%) A A A A 10 10 10 10 A Spinal5 Cord5 5 Gray Matter White Matter5 B Spinal Cord Gray Matter White Matter WT Healthy 0 0 0 0 0 0 Spinal Cord Gray0 Matter White Matter 0 WT 3 weeksSpinal EAE Cord Gray**** Matter White Matter * Figure 4 Figure 4 WTF Healthyigure 4 Nlrp12-/- Healthy Figure 4 *** Nlrp12-/- 3 weeks EAE ** * 25 ** ** WT 3 weeks EAE **** 40 * * Nlrp12* -/- Healthy *** Nlrp12-/-* 3 weeks EAE ** **** 25 ** ** 40 * *** 20 * * ****30 * *** 20 44 15 30 20 15 10 20 Astrogliosis(%) Astrogliosis(%) 10 10 5 Astrogliosis (%) Astrogliosis(%) 10 5 0 0

0 Figure0 4 Figure 4 45

Figure 5. Percent level of Astrogliosis following EAE. Percentage of astrogliosis is calculated by the intensity of GFAP staining on total area of the spinal cord. A) After 3 weeks EAE. B) After 9 weeks EAE. Results are expressed as mean ± SEM. Statistical significance was accepted at *P<0.05. Statistical analysis was done by two-way ANOVA followed by Tukey’s multiple-comparison test. Each spinal cord was quantified in duplicates and/or triplicates. N=3-4.

A A A B B B A B Spinal Cord Gray Matter White Matter Spinal Cord Gray MSpinalatter CordWhite M Grayatter Matter White MatterSpinal CorSdpinal CorGdray MSpinalatteGrr aCordy MattWerhite M GrayatteWr h Matterite Matter White Matter Spinal Cord Gray Matter White Matter Spinal Cord Gray Matter White Matter WT Healthy WT Healthy WT Healthy WT 3 weeks EAE WT Healthy WT 3 weeks EAE -/- *** WT 3 weeks EAE **** * WT 3 weeks EAE * -/- Nlrp12**** Healthy *** -/- Nlrp12 Healthy -/- Nlrp12-/- Healthy *** Nlrp12 Healthy -/- Nlrp12 3 weeks EAE *** *** Nlrp12 3 weeks EAE *** Nlrp12-/- 3 weeks EAE ** ** ** *** Nlrp12-/- 3 we2e5ks EAE** ** ** *** 40 * 25 * 40 * * * * 15 *** 40 40 **** **** 15 *** * **** **** * *** ) 20 *** ) ) ** **** ** ) ** % 20 ** **** ** ** %

( 30 ( % %

(

( 30 s

i 30 i s * s 30 s s i * 15 i ** ** o s o s i 10 i

15 l 10 l o o i i g g l ** l ** 20 o o g g 20 r r o t o 10 t r r 20 20 s s t 10 t A s A A s B A A 10 5 Microgliosis(%) 5 5 Microgliosis(%) Microgliosis (%) 10 Microgliosis (%) 5 10 A Spinal Cord Gray Matter White Matter B 10 Spinal Cord Gray Matter White Matter 0 0 0 0 0 0 0 WT Healthy0 Figure 4 Spinal Cord Gray Matter White Matter Figure 4 WT 3 weeksSpinal EAE Cord Gray Matter White Matter * Figure 6 **** WT Healthy Figure Nlrp126 -/- Healthy *** WT 3 weeks EAE Nlrp12-/- 3 weeks EAE ** * 25 ** ** **** 40 * * Nlrp12* -/- Healthy *** Figure 6. Percent level of MicrogliosisNlrp12-/-* 3 weeks following EAE EAE. Percentage** of microgliosis is **** 25 ** ** 40 * *** 20 calculated *by the intensity of Iba1 staining on total area of the spinal cord. A) After 3 weeks * ****30 EAE. B) After 9* weeks EAE. Results are expressed as mean ± SEM. Statistical significance *** 20 was accepted at *P<0.05. Statistical analysis was done by two-way ANOVA followed by 15 Tukey’s multiple-comparison test. Each spinal cord30 was quantified in duplicates and/or triplicates. N=3-5. 20 15 10 20 Astrogliosis(%) Astrogliosis(%) 10 Nlrp12 negatively regulates T cell proliferation 10 5 Astrogliosis (%) Astrogliosis(%) -/- We observed higher proliferation in responses to purified10 CD4+ T cells from the Nlrp12 5 0 compared to the WT mice (Fig. 7a-c). Interestingly, while pure activation by 0anti CD3/CD28 0 antibodies resulted in the significantly higher proliferativeFigure0 responses 4 in T cells (Fig. 7c) from -/- the Nlrp12 compared to WT mice,Figure more physiological4 activation by splenocytes, although, tended to be higher in T cells from the Nlrp12-/- mice, did not result in a statistically different proliferation compared to the WT mice (Fig 7a, b).

A B

WT 47 Nlrp12-/- 42 40 42 12 12

LOG10 CFSE Figure 7 Figure 7. The proliferation of activated T cells from WT and Nlrp12-/- mice in vitro. A, B. CD4+ T cells were stained with CFSE and activated with plate-bound anti-CD3 and anti-CD28 antibodies stimulation for 3 days. The intensity of CFSE dye in the cells was analyzed by flow cytometry. No significant difference was observed. C. Purified CD4+ T cells from the WT and Nlrp12-/- mice were stimulated with anti CD3/CD28 for 72h, and the incorporation of 3H-thymidine was measured during the final 18h of cell culture. Statistical significance was accepted at *P<0.05. Statistical analysis was done by Mann-Whitney U test. N=3-6 per group.

Nlrp12 deficiency did not affect IL-4 production by activated T cells

Differences in T cells proliferation prompted us to verify the levels of IL-4 in the Nlrp12-/- mice after EAE induction. We chose to look at IL-4 in the light of recent publication by Lukens et al. that observed that Nlrp12 inhibited Th2 responses. We investigated whether Nlrp12 deficiency might affect IL-4 production by T cells in EAE mice. As shown in Fig. 8a, no significant difference was detected between the Nlrp12-/- and WT mice in the percentage of CD4+ IL4+ T cells after 3 days of activation with anti CD3/CD28 antibodies in vitro. Consistent with this finding, we did not observe any significant differences in the levels of IL-4 in lysates from the lymph nodes of the Nlrp12-/- or WT EAE mince neither by RT-PCR (Fig 8c) not by ELISA (Fig 8e). Similar observation demonstrated that there was no statistical difference between IL-4 levels in serum (Fig. 8b) and cerebellum (Fig.8d) from the Nlrp12-/- EAE mice compared to the WT EAE mice.

Figure 8. IL-4 production by activated T cells from WT and Nlrp12-/-mice in vitro and in vivo. A. CD4+ T cells were purified from the lymph nodes and spleens and stimulated with anti-CD3/CD28 antibodies. Intracellular production of IL-4 by activated CD4+ T cells was determined using flow cytometry. B. The levels of IL-4 in serum samples from WT and Nlrp12-/-EAE mice, measured by ELISA. C. The level of IL-4 mRNA in lymph nodes from WT and Nlrp12-/-EAE mice, quantified by real-time PCR. D, E. The levels of IL-4 in tissue samples from the WT and Nlrp12-/-EAE mice. Cerebellum and lymph node tissues were collected from the mice after 3 weeks of immunization with MOG:CFA. The tissues were homogenized in lysis buffer, and IL-4 levels were measured in tissue lysate by ELISA. Statistical analysis was done by Mann-Whitney U test. N=3-6 per group. No significant difference was observed.

Nlrp12 deficiency augments expression of pro-inflammatory molecules in the CNS after EAE

Looking into the mechanisms of increased inflammation in Nlrp12 mice, we analyzed mRNA expression of pro-inflammatory proteins in the spinal cords of mice 3 weeks post immunization (Fig. 9). Compared to WT mice, Nlrp12-/- mice demonstrated significantly higher levels of Cox2 (3-fold increase), IL-1β (4-fold increase), and Ccr5 (10-fold increase) mRNA expressions. Although a relative increase in the mRNA expression of Mip3α was observed, that difference was not significant. Thus, these results demonstrate that in the absence of Nlrp12, the inflammatory response is much more significant.

Figure 9. Nlrp12 deficiency augments expression of pro-inflammatory molecules in the CNS after EAE. Results indicate fold change in mRNA expression of pro-inflammatory proteins in the spinal cords of Nlrp12-/- mice relative to WT mice. A significant increase in the expression of Cox2, IL-1β, and Ccr5 mRNAs and no in the expression of Mip3α were observed. Results are expressed as mean ± SD. Statistical significance was accepted at *P<0.05. Statistical analysis was done using one-way ANOVA followed by Dunet comparison test relative to control. N=5.

Nlrp12-/- primary microglia express increased levels of reactive species and pro- inflammatory cytokines

The inflammatory response is an important feature of the innate immunity in the regulation of homeostasis. Inflammation is an innate response that occurs following the encounter of harmful bodies however, a shift towards anti-inflammatory environment occurs in order to re-establish the balance. Microglia cells play a critical role in this process. Cortices from 1- day old murine pups were removed and after 3 weeks in culture, primary microglia cells were separated from astrocytes. Stimulation with bacterial endotoxin lipopolysaccharide (LPS) revealed a significant increase (2-fold increase) in the expression of inducible nitric oxide synthase (iNOS), the enzyme responsible for the production of nitric oxide (NO), in Nlrp12- /- mice microglia compared to WT microglia (Fig. 10a and 10b). Supernatants following LPS stimulation were further analyzed by Griess reagent assay and we observed significantly

49 more (2.5-fold increase) nitrates secreted in the media from the microglia of Nlrp12-/- mice compared to WT. We additionally observed a dose response effect (Fig. 10c).

Figure 10. Expression of iNOS in primary microglia cells. Microglia cells (1×105) from Nlrp12-/- mice and WT were stimulated with 1g/mL LPS for 12hrs. A) Western blot analysis. B) Densitometric analysis of iNOS. C) Concentration of Nitrates using Griess reagent assay. Results are expressed as mean ± SD. Statistical significance was accepted at *P<0.05. Statistical analysis was done using one-way ANOVA followed by Tukey-Kramer multiple comparison test. N=5.

To further characterize microglial response, purified microglia from both genotypes were incubated with 500ng/mL LPS for 12h and supernatants were analyzed for the presence of pro-inflammatory cytokines TNF-α and IL-6. At basal level, we observed no differences between WT and Nlrp12-/- microglia. However, after treatment with LPS, microglia from Nlrp12-/- mice secreted more than 2-fold increase in TNF-α (Fig. 11a) and IL-6 (Fig. 11b) concentrations compared to WT microglia. Once again demonstrating that in the absence of Nlrp12, the cellular environment is more inflammatory.

Figure 11. TNF-α and IL-6 concentrations following treatment with LPS in primary microglia cells. Microglia cells (1×105) from Nlrp12-/- mice and WT were stimulated with 500 ng/mL LPS for 12hrs. A) ELISA for TNF-α concentration. B) ELISA for IL-6 concentration. Results are expressed as mean ± SD. Statistical significance was accepted at *P<0.05. Statistical analysis was done using one-way ANOVA followed by Tukey-Kramer multiple comparison test. N=5.

Discussion The process of inflammation is a fundamental response aimed at protecting the body from foreign and detrimental causes. Neuroinflammation can become harmful if it is unregulated and prolonged. A continuous and persistent response will eventually lead to a chronic state of inflammation, a prominent feature of many neurodegenerative diseases, including MS. NLRP12 is of interest for the study of MS notably due to its restricted expression in cells derived from hematopoietic origins such as monocytes, dendritic cells, and granulocytic cells and most recently, T cells [16] and its the role in attenuating the inflammatory response by interfering in both branches of the NF-B pathway [9, 17]. To investigate the implication of Nlrp12 in MS, EAE was induced in WT and in Nlrp12- /- mice. Our results demonstrated that in mice lacking the Nlrp12 gene, EAE developed earlier compared to WT mice, and Nlrp12-/- mice showed increased severity throughout the course of the disease. Interestingly, after EAE induction, Nlrp12 mRNA expression was significantly increased in WT mice compared to healthy WTs. These results suggest that Nlrp12 plays an important role in maintaining the level of inflammation and ensuring that a hyper-inflammatory state does not occur. In fact, the expression profile of Nlrp12 over the course of the disease is suggestive of this regulatory role. Indeed, previous studies have shown that in response to live bacteria such as Mycobacterium Tuberculosis, TNF-, and

INF, a reduction in Nlrp12’s expression is in accordance with an increase in the inflammatory response [18, 19]. Moreover, Nlrp12’s overexpression has been previously shown to attenuate the inflammatory response by negatively regulating the NF-B pathways [17]. A study conducted by Shami PJ. et al. demonstrated that the expression of Nlrp12 is increased in response to nitric oxide [20]. NO is a reactive molecule that is produced at sites of inflammation and in MS, it is involved in lesion development [21]. As T cell responses play a crucial role in the development of EAE [12], we evaluated purified CD4 T cell proliferative response after CD3/CD28 activation and we saw significantly elevated proliferation of T cells from the Nlrp12-/- mice. We then evaluated T cells proliferation using recall response 10 days after EAE induction by stimulating purified CD4 T cells with MOG peptide in the presence of splenocytes. We observed the tendency of T cells from the Nlrp12- /- mice for a higher proliferation rate; however, these differences never reached a statistical significance. These results are similar to those published by Lukens et al. [16], where authors observed that pure anti-CD3/CD28 activation resulted in significantly higher proliferation in T cells from Nlrp12-/- mice, while in the presence of splenocytes, differenced between T cell proliferation of different genotypes were greatly reduced. In this work, the authors propose that the cell autonomous effect of Nlrp12 T cells shifts T cell differentiation to TH2-IL4 producing phenotype [16]. We measured the concentration of IL-4 in the serum, lymph nodes, and brain samples from WT and from Nlrp12-/- mice at 3 weeks after EAE and did not find any differences in the expression of IL-4. These results are consistent with our observation that there are no differences in the percentage of IL-4 producing cells and that results from experiments in complex cellular interaction at the tissue level in vivo can be different from results of clean anti CD3/CD28 activation in vitro. Till today, no exact mechanism has been described that explains Nlrp12 activity in different cell types. Nlrp12 has been shown to inhibit classical and alternative pathways of NF-kB in different cell types and different stimulations; for extensive review, please read Tuncer et al. [9]. In the light of these controversies, the different KO strategies to remove Nlrp12 may have produced an uncontrolled variable that resulted in different phenotypes [22, 23]. Future studies should address these differences. In our studies, we observed that Nlrp12-/- mice demonstrated more severe course of EAE according to classical evaluation of clinical scores, while in the work by Lukens and co-

52 workers, the authors noted appearances of the atypical EAE. These results are intriguing, as overall effect of Nlrp12 on the EAE pathology was similar to our observations. Furthermore, EAE is a well-characterized and the most widely used mouse model to study MS [13]. It exhibits the main features of MS pathology such as inflammation, destruction of myelin, and reactive gliosis. Moreover, many of the current therapies for MS, such as Tysabri were developed following EAE studies [24]. However, it is important to note that the evaluations of clinical scores are subjective. In our studies, we did not measure the degree of atypical EAE as there is no quantifiable scale to evaluate this pathology. Observing video clips published by Lukens et al. (supplemental materials), we can tell that Nlrp12 mice was severely compromised and had impaired righting reflex, which suggests severe weakness/paralysis of the hind limbs as well as paralysis of the trunk muscles. To further elucidate how Nlrp12 is playing a protective role in the disease, spinal cords of both WT and Nlrp12-/- mice were analyzed for the expression of genes implicated in EAE as well as in MS. Our results demonstrated a significant increase in the mRNA expression of Cox-2, IL-1, and Ccr5 genes in Nlrp12-/- mice compared to WT mice, suggesting a protective role played by Nlrp12 in EAE at the level of proinflammatory gene expression. The increase in expression of proinflammatory molecules in Nlrp12-deficient phenotype has been demonstrated by multiple studies [22, 25]. Next, we demonstrated that Nlrp12 inhibits inflammation during EAE at the level of microglia. We showed that Nlrp12 deficiency augments pro-inflammatory microglial phenotypes by using purified primary microglia cells from WT and Nlrp12-/- mice. Consistent with our in vivo observation, stimulation of microglia with LPS resulted in a significant increase of iNOS expression, NO, TNF, and IL-6 secretion from Nlrp12-/- microglia cells compared to WT microglia. These results are consistent with the suppressive role of Nlrp12 in cells of myeloid origin [26]. A report by Lukens et al. also found increased inflammatory response in the CNS tissue of Nlrp12-/- mice compared to WT controls, although, microglia responses per se were not verified. Furthermore, the notion of inhibitory NLRs is not new. Similar to our results, stimulation of primary Nlrx1-/- microglia cells revealed a significant increase in the pro-inflammatory response, thus, showing a suppressive role for Nlrx1 in microglial activation [27].

The roles of microglia and astrocytes are well defined in the pathology of MS. Previous studies on Nlrp3 have demonstrated that the absence of this receptor results in better disease outcome and reduced gliosis following EAE [28]. Spinal cord of Nlrp12-/- mice and WT mice were stained with Iba1 and GFAP in order to assess the extent of microgliosis and astrogliosis, respectively. Surprisingly, no differences in the percentage of gliosis were observed between the two genotypes at the third week, however after 9 weeks, Nlrp12-/- mice demonstrated significantly increased gliosis compared to WT mice. Additionally, in both genotypes the majority of gliosis occurs within the white matter area of the spinal cord. Although a quantitative difference was not observed at the third week, in vitro study suggests qualitative changes in microglia activation. Indeed, upon LPS stimulation, microglia from Nlp12-/- mice released significantly more pro-inflammatory mediators. Furthermore, the remarkable increase in Ccr5 mRNA expression observed in Nlrp12-/- mice suggests that Nlrp12 may be playing a crucial role in the influx of inflammatory infiltrates. CCR5 is a chemokine receptor that is expressed primarily by monocytes, macrophages, effector T cells, immature dendritic cells and NK cells [29]. Moreover, previous studies in both animal and in MS patients have demonstrated the upregulation of CCR5 in inflammatory lesions [30- 32]. Also, a chronic over-expression of IL-1 has been shown to result in the disruption of the blood-brain barrier (BBB) and in the infiltration of leukocytes such as macrophages, DCs and neutrophils [33, 34]. Thus, the increase of Ccr5 and IL-1 mRNA in the spinal cords of Nlrp12-/- mice compared to WT mice during EAE supports the notion of an increased influx of inflammatory cells in these mice. In fact, the entry of pro-inflammatory leukocytes into the CNS is an early phenomenon capable of initiating events that result in BBB disruption and neuroinflammation [35]. Interestingly, previous studies have demonstrated a reduction in inflammatory infiltrates within the CNS in EAE-induced Nlrp3-/- mice, where Nlrp3 was shown to play an inflammatory role by inducing immune cell migration whereas, our results suggest that Nlrp12 plays a protective role by maintaining the level of inflammatory influx [36, 37]. Thus, future studies should focus on evaluating in details the presence of inflammatory infiltrates in order to clarify the driving force responsible for the differences observed between WT and Nlrp12-/- mice.

Conclusion The study of NLRs and their functions has been mainly studied in the context of host and pathogen interactions. Their role in mediating the inflammatory response is well recognized, while their role in other diseases is an emerging field. Recent reports suggest that Nlrs may play detrimental, as well as a beneficial role in the progression of EAE. For example, Nod1, Nod2, and Nlrp3 augment inflammation and T cell responses that lead to increased EAE severity. On the other hand, the expression of Nlrp12 and Nlrx1 inhibits the expression of pro-inflammatory genes, suppressing inflammation and reducing the severity of EAE [27]. In many neurodegenerative diseases, the regulation of neuro-inflammatory responses is a key target for therapeutic interventions. Numerous studies have focused on the role and contribution of T- and B-lymphocytic responses in MS and much of the pathophysiology of MS has gravitated around the adaptive branch of the immune system. The implication of the adaptive immune response is undeniable in this disorder, given that the primary cause of damages in the nervous system of MS patients is due to CNS inflammation, where CD4+ autoreactive T cells primarily react to myelin epitope, enter the CNS and result in the destruction of myelin [3]. At this stage, we are not excluding the role of Nlrp12 in T cell responses during EAE. However, it is vital to understand the underlying cause of the inflammatory process in MS. Thus, it is important to focus on the innate immune response, since in essence, inflammation is a response of innate immunity [38], [46]. Thus, our findings that Nlrp12 plays a role in microglia activation during EAE may help find the mechanism that regulates CNS specific inflammation.

Competing interest The authors declare that they have no competing interests. Acknowledgments We thank Multiple Sclerosis Society of Canada and Fonds de recherche du Québec - Santé for financial support.

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3.2. ARTICLE

The Dual Immunoregulatory function of Nlrp12 in T Cell-Mediated Immune Response: Lessons from Experimental Autoimmune Encephalomyelitis

Marjan Gharagozloo, Shaimaa Mahmoud, Camille Simard, Tara M. Mahvelati, Abdelaziz Amrani, and Denis Gris

Program of Immunology, Department of Pediatrics, CR-CHUS, Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke, Quebec, Canada

Published in Cells, PMID: 30150571

Résumé Bien que l'étiologie de la sclérose en plaques (SEP) reste énigmatique, le rôle des cellules T est incontestablement central dans cette pathologie. Les cellules immunitaires répondent aux agents pathogènes et aux signaux de danger via les récepteurs de reconnaissance de formes (PRR). Plusieurs rapports impliquent Nlrp12, un PRR intracellulaire, dans le développement d'une maladie semblable à la SEP chez les souris, appelée encéphalomyélite auto-immune expérimentale (EAE). Dans cette étude, nous avons utilisé la modèle EAE pour tester l'hypothèse selon laquelle Nlrp12 inhibe la réponse Th1 et empêche l'auto-immunité médiée par les lymphocytes T. Nous avons constaté que Nlrp12 joue un rôle protecteur dans l'EAE en réduisant le ratio IFNγ/IL-4 dans les ganglions lymphatiques, alors qu'il potentialise le développement d'EAE spontanée (spEAE) chez des souris transgéniques 2D2 exprimant un récepteur de cellules T (TCR). En examinant le mécanisme d’action de Nlrp12 dans la réponse des cellules T, nous avons constaté qu'il inhibe la prolifération des cellules T et supprime la réponse Th1 en réduisant la production d'IFNγ et d'IL-2. Suite à l'activation du TCR, Nlrp12 inhibe la phosphorylation de Akt et de NF-κB, alors qu'il n'a aucun effet sur la phosphorylation de S6 dans la voie mTOR. En conclusion, nous proposons un modèle pouvant expliquer la double fonction immunorégulatrice de Nlrp12 dans l'EAE. Nous proposons également un modèle expliquant le mécanisme moléculaire de la régulation de la réponse des lymphocytes T dépendant de Nlrp12.

Abstract Although the etiology of multiple sclerosis (MS) remains enigmatic, the role of T cells is unquestionably central in this pathology. Immune cells respond to pathogens and danger signals via pattern-recognition receptors (PRR). Several reports implicate Nlrp12, an intracellular PRR, in the development of a mouse MS-like disease, called Experimental Autoimmune Encephalomyelitis (EAE). In this study, we used induced and spontaneous models of EAE, as well as in vitro T cell assays, to test the hypothesis that Nlrp12 inhibits Th1 response and prevents T-cell mediated autoimmunity. We found that Nlrp12 plays a protective role in induced EAE by reducing IFN/IL-4 ratio in lymph nodes, whereas it potentiates the development of spontaneous EAE in 2D2 T cell receptor (TCR) transgenic mice. Looking into the mechanism of Nlrp12 activity in T cell response, we found that it inhibits T cell proliferation and suppresses Th1 response by reducing IFN and IL-2 production. Following TCR activation, Nlrp12 inhibits Akt and NF-B phosphorylation, while it has no effect on S6 phosphorylation in mTOR pathway. In conclusion, we propose a model that can explain the dual immunoregulatory function of Nlrp12 in EAE. We also propose a model explaining the molecular mechanism of Nlrp12-dependent regulation of T cell response.

1. Introduction Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS), where autoreactive immune responses are involved in demyelination and CNS damage. The etiology and pathogenesis of the disease remain elusive. However, several lines of evidence demonstrate that adaptive immune response plays a key role in the pathogenesis of MS and experimental autoimmune encephalomyelitis (EAE), the mouse model of MS[1- 3]. The major components of the adaptive immunity, T cells, are initially activated by antigen presenting cells (APCs) in lymph nodes. Activated T cells migrate into the CNS across the blood brain barrier (BBB) and reactivated again in perivascular space, where CNS-resident cells including microglia and macrophages present myelin antigens to T cells [4]. Thus, those activated CD4+ T cells orchestrate the functions of other adaptive immune cells, such as CD8+ T cells and B cells, as well as innate immune cells in the CNS and periphery [5]. Depending on the composition of the cytokine milieu, naïve CD4+ T cells may differentiate into different T helper (Th) subsets including Th1, Th2, and Th17 that produce signature cytokines such as IFN, IL-4, and IL-17 respectively. The differentiation of naïve CD4+ T cells into Th1, Th2, or Th17 types are governed by transcription factors, known as Tbet, GATA3, and RORt respectively[6]. Th subsets affect the CNS inflammation in different ways. Th1 and Th17 responses potentiate the CNS inflammation, while Th2 response dampens inflammatory response and protects CNS damage[6]. These findings highlight the importance of T cell-mediated immunity in MS pathology. APCs including dendritic cells, macrophages, and microglia are innate immune cells that trigger T cell activation [7]. These cells create the first line of response by recognizing pathogens and/or danger signals via pattern-recognition receptors (PRR)[8]. NOD-like receptors (NLRs) are intracellular PRR that are mainly expressed by cells of hematopoietic origin and regulate both innate and adaptive immune responses [9]. Recently, NLRs have gained attention since 3 members of the family including CIITA, Nlrc5, and Nlrp3 regulate transcription of molecules that shape adaptive immune responses. CIITA [10], and Nlrc5 [11,12] show transcriptional activities for MHC II and MHC I molecules respectively, while Nlrp3 acts as a Th2 transcription factor and promotes IL-4 production [13]. In addition, activation of NLRs often leads to production and secretion of proinflammatory cytokines such as IL-1 and IL-18 that in turn potentiate differentiation of Th1 and Th17 subsets [9,14].

These findings highlight the key role of NLR proteins in shaping T cell response and adaptive immunity. Not all NLRs are proinflammatory. Nlrp12 is a recently discovered member of NLRs that is shown to be a negative regulator of both canonical and non-canonical nuclear factor-κB (NF-κB) signaling pathways [15]. Previous studies showed that Nlrp12-/- mice are highly vulnerable to inflammatory diseases such as experimental colitis and colorectal tumor development [16-19]. In the context of CNS inflammation, the lack of Nlrp12 resulted in increased CNS inflammation and exacerbated course of EAE [19]. Nlrp12-/- mice developed earlier and more severe form of EAE than wild-type (WT) mice. This phenotype parallel with significant increases in the expression of pro-inflammatory genes in the spinal cords of Nlrp12-/- mice relative to WT mice. Experiments using mouse primary microglia cultures demonstrated that Nlrp12 significantly inhibits production of the inflammatory mediators such as inducible nitric oxide synthase (iNOS), Tumor Necrosis Factor (TNF)α, IL-6, and nitric oxide (NO) [19]. However, the ability of Nlrp12 to modulate T cell responses remains poorly defined. A recent article by Lukens et al. revealed that Nlrp12 is expressed not only by myeloid cells but also by T cells. It negatively regulates NF-κB signaling, T cells proliferation, and the secretion of Th1/Th2/Th17 cytokines [20]. Non-surprisingly, Nlrp12 deficient mice developed enhanced inflammatory symptoms in T-cell-mediated autoimmune diseases such as colitis and atopic dermatitis [20]. However, in EAE model, lack of Nlrp12 promotes Th2 response and IL-4 secretion, which results in a milder form of EAE with atypical symptoms, including ataxia and impaired balance control [20]. Collectively, current findings and controversies indicate that the exact immunoregulatory functions of Nlrp12 in T cell activation and T cell-mediated autoimmunity are poorly understood. In this study, we investigated the immunoregulatory role of Nlrp12 in T cell responses using classical induced-EAE and spontaneous EAE (spEAE) models. We further characterized the role of Nlrp12 in regulating T cell receptor (TCR) signaling pathways and IL-2 production.

2. Materials and methods

2.1. Mice

All the protocols and procedures were approved by the University of Sherbrooke Animal Facility and Use Committee (Protocols #280-15, April 04, 2017; #335-17B, February 22, 2018). Nlrp12 knock-out (Nlrp12−/−) mice on C57BL/6J background were kindly provided by Dr. Jenny P. Y. Ting (Chapel Hill, NC). Mice were backcrossed for at least 15 generation.

The 2D2 transgenic mice expressing a TCR specific for the myelin oligodendrocyte (MOG35– −/− 55) peptide were purchased from Jackson Laboratory. Nlrp12 and WT mice were crossed with 2D2 mice to generate Nlrp12-/- 2D2 mice. We genotyped all the animals for Nlrp12 and 2D2 (supplementary protocol) and only those animals that were Nlrp12-/- and 2D2+ were included in the study (supplementary Figure 1). Moreover, the expression of V11 receptor was verified with flow cytometry. The mice were maintained under specific pathogen-free conditions in the animal facility of the faculty of medicine, at the University of Sherbrooke.

2.2. Induction of EAE and Tissue Collection

EAE was induced in 8-10-week old WT or Nlrp12−/− female mice as previously described [19]. An emulsion mixture of MOG35−55 (Genemed Synthesis Inc., San Antonio, TX), complete Freund’s Adjuvant (CFA) (Sigma-Aldrich, St. Louis, MO), and Mycobacterium tuberculosis H37 RA (Difco Laboratories, Detroit, MI) was prepared and injected subcutaneously in the flank with a total of 200 μg MOG35–55 and 500 μg Mycobacterium. Mice were also injected intraperitoneally on days 0 and 2 with 200 ng Pertussis toxin (List Biological Laboratories Inc., Campbell, CA). After 3 weeks of immunization, mice were sacrificed, perfused with ice-cold phosphate-buffered saline (PBS) (Wisent, St. Bruno, QC), and the CNS tissues were collected.

2.3. Intracellular Staining and Flow Cytometry

CD4+ T cells were purified from lymph nodes and spleens using Mouse CD4+ T Cell Isolation Kit (eBioscience) and activated with plate-bound anti-CD3 (eBioscience, clone:145-2C11, 1 μg/mL) and anti-CD28 (eBioscience, clone:37.51, 2 μg/mL) antibodies for indicated times. T cell proliferation was assessed by Ki67 intranuclear staining following

64 fixation and permeabilization in the Foxp3/Transcription Factor staining kit purchased from eBioscience. For intracellular staining of cytokines, the cells were stimulated with phorbol 12-myristate 13-acetate (PMA; 500 ng/mL, Sigma Chemical Co., St. Louis, MO) and inomycin (1 μg/mL, Calbiochem Corp., La Jolla, CA) for 5 h at 37°C in the presence of Brefeldin A (10 μg/mL, eBioscience, San Diego, CA). Cells were stained with anti-CD4- FITC antibody (eBioscience), fixed, and permeabilized using intracellular fixation and permeabilization buffer (eBioscience). Then, cells were stained with anti-IFN-PE, anti-IL- 4-PE, IL-17-PerCP-Cy5.5, Tbet-PE, or RORt-PE antibody, as per the manufacturer’s instructions (eBioscience). Sample acquisition was performed with Beckman Coulter CytoFlex and data were analyzed using CytExpert 2 software (Beckman Coulter). Plots were prepared using CytExpert 2 and FlowJo (San Carlos, California, USA) software.

2.4. Quantitative RT-PCR

RNA was extracted from CD4+ T cells using TRIzol reagent (Life Technologies Inc., Burlington, ON), and cDNA was synthesized as previously described [19]. Reverse transcription PCR (RT-PCR) was used to verify the expression of Nlrp12 in activated T cell using KiCqStart™ SYBR® Green qPCR ReadyMix (Sigma Aldrich, St. Louis, MO). Primers (IDT, Coralville, IA) sequences were as follows: Nlrp12F: 5′-CCT CTT TGA GCC AGA CGA AG-3′, Nlrp12R: 5′-GCC CAG TCC AAC ATC ACT TT-3′, 18SF: 5′-CGG CTA CCA CAT CCA AGG AA-3′, and 18SR: 5′-GCT GGA ATT ACC GCG GCT-3′. The relative expression was calculated using the ΔΔCT method [21].

2.5. Cytokine Measurement

IFN and IL-4 cytokines in the supernatant of activated CD4+ T cell culture and tissue lysates were measured using ELISA kits as previously described[19]. Briefly, lymph node, spinal cord, and cerebellum tissues were homogenized in lysis buffer supplemented with protease inhibitors (Cell Signaling Technology) by rapid agitation using 3-mm stainless beads and a TissueLyser II (Qiagen) homogenizer for 2 min. The levels of IL-4 in tissue lysates were determined using a high sensitivity IL-4 ELISA Kit (eBioscience, San Diego, CA) according to the manufacturer’s instruction. The amount of IFN was determined using IFN kit purchased from PeproTech (Rocky Hill, NJ).

2.6. Differentiation of 2D2 T cells Toward Th1 or Th17 in vitro

Naïve CD4+ T cells were purified from the spleens and lymph nodes of Nlrp12-/- 2D2 and WT 2D2 mice using MagniSort Naïve CD4 T cell Enrichment Kit (eBiosciences). Purified CD4+ T cells were stimulated with MOG (50µg/ml) in the presence of WT splenocytes at 1:1 ratio and Th1-, Th2- or Th17- polarizing condition (Th1: IL-12 (10 ng/ml), anti-IL-4 (10 μg/ml), IL-2 (10 ng/ml); Th2: IL-4 (10 μg/ml), hTGF-β1 (10 ng/ml), IL-2 (10 ng/ml) and Th17: anti-IL-12 (10 μg/ml), anti-IL-4 (10 μg/ml), anti-IFN-γ (10 μg/ml), mIL-6 (10 ng/ml), hTGF-β1 (10 ng/ml)). Recombinant cytokines and antibodies were purchased from Biolegend (San Diego, CA) and eBioscience (San Diego, CA) respectively. After 72h of culture, cells were stained for MOG-TCR transgenic surface marker, V11, and Th1- or Th17- associated markers (intracellular cytokine and transcription factor). The percentage of Th1 or Th17 cells were evaluated by Flow cytometry.

2.7. T cell Activation and Signaling Pathways

To analyze p65 phosphorylation upon CD3 cross-linking, CD4+ T cells were purified from the lymph nodes of Nlrp12-/- and WT mice and incubated with 10 μg/ml anti-CD3 (eBioscience, San Diego, CA) for 30 min on ice, followed by washing and resuspending the cells in serum-free medium. T cells were incubated with 10 μg/ml cross-linking anti-mouse IgG antibody (R&D systems) for 20 min at 37oC. Akt phosphorylation was quantified after activation of CD4+ T cells with PMA/Iono for 10 min at 37oC, while the phosphorylation of S6 was analyzed after 24h activation of CD4+ T cells with plate-bound anti-CD3/CD28 antibodies. Following stimulation in a defined period of time, T cells were washed and lysed in the lysis buffer plus proteinase and phosphatase inhibitor (Cell Signaling Technology). Proteins were then separated by on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membrane. Transfers were blocked for 1h at room temperature with 5% nonfat milk in TBS/0.1% Tween 20 (TBST) and then incubated overnight at 4 °C in the primary antibodies (Cell Signaling Technology) diluted in TBST. The membranes were washed 3 times with TBST and incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Cell Signaling Technology) diluted 1/1000 in TBST for 1h at room temperature. The immunoblots were developed with Lumigen ECL ultra reagent, imaged with ChemiDocTM (Bio-Rad) and analyzed using Image Lab software.

2.8. Phosphoflow Cytometry

Phosphorylation of S6 ribosomal protein was confirmed by flow cytometry. Following 24h stimulation of CD4+ T cells with plate-bound anti-CD3/CD28, cells were immediately fixed and permeabilized using Fix and Perm Kit (eBioscience). After washing with perm buffer (eBioscience) and blocking with 5% Fetal Bovine Serum, cells were incubated with Phospho-S6 antibody (Cell Signaling Technology) for 1h at room temperature, followed by washing with perm buffer and incubation with Alexa Fluor® 555 (Cell Signaling Technology) conjugated antibody. Samples were acquired by Beckman Coulter CytoFlex and data was analyzed using CytExpert 2 software (Beckman Coulter).

2.9. SpEAE and Histological Analysis

WT 2D2 or Nlrp12-/- 2D2 mice were monitored for the development of spEAE. Animals were sacrificed after 4 months of monitoring or after the development of EAE at the peak score of 4. The immunized mice were sacrificed as described in section 2.2. and the spinal cords were removed and fixed in 4% formaldehyde for 24h. The spinal cord tissues were embedded in paraffin and cut into 5-μm sections and stained with haematoxylin and eosin (H&E) stain and immunofluorescence for astrocyte marker (GFAP), microglia marker (Iba1), and myelin basic protein (MBP). All slides were scanned using a digital slide scanner NanoZoomer-XR C12000 (Hamamatsu, Hamamatsu City, Japan) and viewed using NDPview2 software (Hamamatsu).

2.10. Analysis of CNS-infiltrating Mononuclear Cells by Flow Cytometry

Dissected spinal cords were filtered through a 70 mm nylon sieve (BD Pharmingen) with a syringe plunger to get a uniform tissue homogenate that was digested with collagenase D (2.5 mg/ml, Roche Diagnostics) and DNase I (1 mg/ml, Sigma-Aldrich) with agitation at 37°C for 45 minutes. Mononuclear cells were isolated by percoll (Sigma-Aldrich) centrifugation, in which 300µl percoll was mixed with 1 ml cell suspension overlaid with 700µl Hank's Balanced Salt Solution (HBSS). The samples were centrifuged at 12000×g for 15 min without break. Myelin and cell debris were aspirated, and the mononuclear pellet was resuspended and washed with HBSS before staining for surface markers and analysis by flow cytometry.

2.11. Statistical Analysis

All statistical analyses were conducted using GraphPad Prism 7 software. Results were expressed as the mean ± standard deviation. Statistical differences between WT and Nlrp12−/− samples were assessed by Student’s T-test. Level of Nlrp12 expression was assessed by one-way ANOVA, and cellular signaling by two way ANOVA. The significance level was set at P < 0.05.

3. Results

3.1. Nlrp12 Modulates Th1/Th2 Balance in EAE mice

Our previous study demonstrated that Nlrp12-/- mice develop earlier and more severe EAE compared to WT mice [19]. Since Th1 cells play a crucial role in the pathogenesis of EAE, we examined whether the imbalance of Th1/Th2 response might lead to exacerbated EAE in Nlrp12-/- mice. We collected lymph nodes and the CNS tissues including spinal cord and cerebellum from WT and Nlrp12-/- EAE mice at day 21 after MOG-CFA immunization and measured the level of IFN and IL-4 as the designated cytokines for Th1 and Th2 cells respectively. As shown in Fig. 1, we found significantly higher levels of IFN in lymph node extracts from Nlrp12-/- EAE mice, while IL-4 levels were significantly lower than WT EAE mice. In addition, the ratio of IFN/IL-4 was higher in lymph node extracts of Nlrp12-/- EAE mice, suggesting a higher Th1/Th2 ratio in Nlrp12-/- EAE (Fig. 1A). No difference was observed in the levels of IFN and IL-4 in the extracts of CNS tissues (spinal cord and cerebellum), however, IFN/IL-4 ratio was significantly lower in spinal cords from Nlrp12- /- EAE mice compared to WT EAE mice (Fig. 1B). We then evaluated the production of cytokines in ex vivo activated CD4+ T cells.

3.2. Nlrp12 Inhibits the Production of IFN by CD4+ T cells in vitro

To determine whether Nlrp12 could inhibit IFN production by T cells, we purified CD4+ T cells from WT and Nlrp12-/- mice and stimulated them with anti-CD3/CD28 antibodies to activate T cells with TCR and costimulatory signals simultaneously [22]. After 72h, cell culture supernatants were collected and the levels of IFN and IL-4 were measured by ELISA. As shown in Fig. 2A, activated CD4+ T cells from Nlrp12-/- mice secreted

68 significantly higher levels of IFN and IL-4 compared to WT T cells. We then tested whether the increased levels of both Th1- and Th2-associated cytokines were related to the increased proliferation of Nlrp12-/- T cells compared to WT T cells. Ki67 is a nuclear protein that is expressed by the cells in all cell cycle phases except G0 phase [23]. Following activation by anti-CD3/CD28 antibody for 24h, the CD4+ T cells were stained for Ki67 and analyzed by flow cytometry. Results showed significantly higher numbers of Nlrp12-/- T cells in the cell cycle compared to WT T cells (Fig. 2B). However, no significant difference was detected between Nlrp12-/- and WT T cells in the percentage of IL-4 or IL-17 producing T cells after 3 days activation with anti-CD3/CD28 antibodies in vitro (Fig. 2C). We then examined whether the exacerbated EAE in Nlrp12-/- mice was associated with the increased differentiation of myelin-specific T cells to Th1 or Th17 cells.

Figure 1. Nlrp12 inhibits Th1 response in EAE. Th1- and Th2- related cytokines in tissue lysates from EAE mice. CNS tissues and lymph nodes were collected from EAE mice after 3 weeks of immunization with MOG/CFA. The tissues were homogenized in lysis buffer and cytokine levels were measured in tissue lysate by ELISA. (A) The levels of IFN, IL-4 and IFN/IL-4 ratio in lymph node extracts from WT and Nlrp12 −/− EAE mice; (B) The levels of IFN, IL-4, and their ratios in the spinal cord and (C) cerebellum of EAE mice, n = 5–6 per group, *P<0.05.

Figure 2. Nlrp12 inhibits T cell proliferation and cytokine production by CD4+ T cells. (A) Purified CD4+ T cells from WT and Nlrp12-/- mice were stimulated with anti-CD3/CD28 for 72 h. Cell culture supernatants were collected and the levels of IFN and IL-4 were measured by ELISA, n=4; (B) Purified CD4+ T cells from WT and Nlrp12-/- mice were stimulated with anti-CD3/CD28 for 24 h and the proliferation of CD4+ T cells was evaluated using Ki67 staining and flow cytometry (C) CD4+ T cells were activated with anti-CD3/CD28 for 48h and the expression of intracellular cytokines was measured using flow cytometry. Representative flow cytometric plots show the expression of IFN, IL-4, and IL-17 in CD4+ T cells, n=4; *P < 0.05.

3.3. Nlrp12 Has no Effect on T cell Differentiation toward Th1 and Th17

We evaluated whether Nlrp12 would favor differentiation of naïve T cells towards inflammatory Th subsets. We purified naïve CD4+ T cells from lymph nodes and spleens of Nlrp12-/- 2D2 and WT 2D2 mice and cultured with MOG-pulsed WT splenocytes in the presence or absence of Th1 or Th17 polarizing cytokines. After 72h of incubation, cells were harvested and stained for CD4 surface marker and Th1 (IFN and Tbet) or Th17 (IL-17 and RORt) intracellular markers. Flow cytometry results show that Nlrp12 did not affect the differentiation of CD4+ T cells toward Th1 or Th17 (Fig. 3).

Figure 3. Nlrp12 does not affect the differentiation of naïve CD4+ T cells to Th1 or Th17. Naïve CD4+ T cells from WT and Nlrp12-/- mice were purified and stimulated with MOG- splenocytes in the presence of Th1 or Th17 polarizing cytokines for 3 days. After 5h stimulation with PMA/ionomycin, cells were stained for V11 and Th1- or Th17- associated cytokine/transcription factor and analyzed by flow cytometry, n=3; Representative flow cytometric plots show the expression of Th1- and Th17-related markers in V11+ T cells.

3.4. Nlrp12 Expression Is Increased in Activated T cells

To test whether TCR activation modulated the expression of Nlrp12, we activated CD4+ T cells with either anti-CD3 or anti-CD3/CD28 antibodies for 24h and measured the expression of Nlrp12 gene by qPCR. As shown in Fig. 4A, the mRNA expression of Nlrp12 was significantly increased in T cells following activation by anti-CD3 or anti-CD3/CD28 antibodies. The level of Nlrp12 expression remains high even after 48h stimulation of T cells by anti-CD3/CD28 (Fig.4B). Since we observed change of Nlrp12 expression soon after T cell activation, we asked the question whether Nlrp12 could modify early TCR signaling events before commitment of T cells to a certain Th subset.

3.5. Nlrp12 Inhibits Phosphorylation of Akt and NF-κB Signaling in Activated T cells

Akt can be activated by TCR signaling in T cell. Accordingly, we studied the early TCR signaling and Akt phosphorylation in T cells downstream of TCR, where Akt is activated by protein kinase C (PKC). For mitogenic stimulation that bypasses the TCR, T cells were treated with the combination of PMA, a permeable specific activator of PKC and calcium ionophore that synergizes with PMA effect in enhancing the activation of PKC [24]. We activated CD4+ T cells by PMA/ionomycin or CD3 cross-linking antibody and evaluated the phosphorylation of Akt and p65 NF-κB subunit by Western blot. As shown in Fig. 5, a significant increase in the phosphorylation of Akt and p65 NF-κB subunit were found in Nlrp12-/- CD4+ T cells compared to WT CD4+ T cells.

Figure 4. T cell activation increases Nlrp12 mRNA expression. (A) Increased expression of Nlrp12 mRNA in CD4+ T cells activated with anti-CD3 or anti-CD3/CD28 antibodies for 24h. The higher expression was found in T cells activated with anti-CD3/CD28 antibody 71

72 compared to T cell treatment with anti-CD3 or PBS; (B) The expression of Nlrp12 is increased in activated CD4+ T cells after 24 and 48h activation with anti(α)-CD3/CD28 antibody; results are presented relative to the expression of Nlrp12 in inactive cells; n=4, *P < 0.05.

Figure 5. Nlrp12 inhibits phosphorylation of Akt and p65 in CD4+ T cells. The cells were activated either with PMA/ionomycin for 10 min or with cross-linking anti-CD3 antibody (anti-CD3/IgG) for 20 min at 37oC. (A) Samples were analyzed by SDS-PAGE and immunoblotting for phospho-Akt (S473) and phospho-p65. Total Akt and p65 expression in the same lysates were used to normalize the expression of phosphorylated molecule between samples; (B) The ratio of phosphorylated to total molecules were compared between WT and Nlrp12-/- cells, n=3, *P < 0.05.

3.6. Nlrp12 Inhibits IL-2 Synthesis but Does Not Modify Ca2+/calmodulin-Dependent T cell activation Since the phosphorylation of NF-κB promotes IL-2 synthesis, we tested whether Nlrp12 inhibit IL-2 production in MOG-specific transgenic CD4+ T cells. CD4+ T cells from Nlrp12- /- 2D2 and WT 2D2 mice were activated with MOG-pulsed splenocytes and IL-2 expression by CD4+ T cells were quantified using flow cytometry. Furthermore, to test whether Nlrp12 modifies Ca2+/calmodulin-dependent T cell activation, we incubated the T cells with and Ca2+/calmodulin inhibitor (cyclosporin) for 24h and quantified the percentage of CD4+ IL- 2+ T cells by flow cytometry. As shown in Fig. 6, Nlrp12 inhibits IL-2 production by MOG- activated CD4+ T cells; however, it does not interfere with the immunosuppressive activities of cyclosporine, since cyclosporin inhibits IL-2 production in both Nlrp12-/- and WT T cells.

Figure 6. Nlrp12 inhibits IL-2 synthesis by activated T cells. CD4+ T cells from WT and Nlrp12-/- mice were activated with anti-CD3/CD28 for 24h in the presence of cyclosporin or its vehicle (DMSO) for 24h and intracellular expression of IL-2 were quantified using flow cytometry; (B) Representative flow cytometric histograms showing the population of IL-2+ T cells in vehicle or cyclosporin-treated group, n=3, *P < 0.05.

3.7. Nlrp12 Does not Affect the Phosphorylation of S6 ribosomal Protein in mTOR pathway

IL-2 is known to activate mTOR pathway and to promote cellular growth and clonal expansion of effector T cells [25]. Since we found an increased production of IL-2 and enhanced proliferation of Nlrp12-/- CD4+ T cells, we investigate whether Nlrp12 affects mTOR pathway in T cells. We evaluated the phosphorylation of S6 ribosomal protein, which is a critical component of 40 S ribosomal subunit and sits at the downstream of mTOR pathway (Fig. 10). Following 24h activation by plate-coated anti-CD3/CD28 antibodies, no significant difference was found between Nlrp12-/- and WT CD4+ T cells in the levels of phospho-S6, determined by Western blotting and flow cytometry (Fig. 7A, B, C).

Figure 7. Nlrp12 does not modify mTOR activity. (A) The phosphorylation of S6 ribosomal protein (pS6) as an indicator of mTOR activity was assessed by Western blotting; (B) The ratio of pS6 to β-Tubulin in WT and Nlrp12-/- CD4+ T cells activated with anti-CD3/CD28 for 24h, as quantified by Western blot, n=3; (C) Phospho-flow analysis of pS6 in CD4+ T cells activated with anti-CD3/CD28 for 24h, n=3.

3.8. Nlrp12-/- 2D2 mice Are Resistant to the Development of spEAE

Given the anti-inflammatory role of Nlrp12 in EAE [19,20], we investigated whether Nlrp12-/- 2D2 mice would develop spEAE. As expected, 6% of WT 2D2 mice developed EAE spontaneously (Table 1). However, surprisingly, none of Nlrp12-/- 2D2 mice developed spEAE (Table 1). Pathological examination of WT 2D2 spEAE mice revealed marked inflammation associated with infiltration of mononuclear cells to the spinal cord, increased expression of microglia and astrocyte markers, Iba1 and GFAP respectively (Fig. 8). The inflammation was associated with demyelination, as shown in by immunofluorescence staining of myelin basic protein (MBP) (Fig. 8). Using flow cytometry, we found a similar percentage of myelin-specific T cells (V11+) in the spleens of Nlrp12-/- 2D2 mice and WT 2D2 mice (Fig. 9). However, a high percentage of leukocytes (CD45high) including V11+ T cells infiltrated to the spinal cord of WT 2D2 spEAE compared to healthy mice (Fig. 9).

Genotype Total (n) SpEAE (%) Age of onset (weeks) EAE score WT 2D2 101 6 10.1 ± 4.7 3.6 ± 0.5

Nlrp12-/- 2D2 30 0 - -

Table 1. Nlrp12 does not prevent the development of spEAE. None of Nlrp12-/- 2D2 mice developed spEAE, while 6 WT 2D2 mice out of 101 developed spEAE, manifested with ascending paralysis. Nlrp12-/- 2D2 mice were monitored for 16 weeks and no sign of classical EAE were observed.

Figure 8. SpEAE in WT 2D2 mice is associated with spinal cord inflammation and demyelination. Representative histopathological examination of the spinal cords from healthy Nlrp12-/- 2D2 mice and spEAE WT 2D2 mice. H&E staining reveals an extensive mononuclear cell infiltration in spinal cord of spEAE mice (shown by black arrows). Immunofluorescent staining of spinal cords from WT 2D2 SpEAE shows an increased expression of GFAP (astrocyte marker) and Iba1 (macrophage/microglia marker) mice compared to Nlrp12-/- 2D2 healthy mice. Focal demyelinating lesions (shown by white arrows) are observed in spEAE spinal cords using MBP staining. Similar histopathological features to healthy Nlrp12-/- 2D2 mice were found in healthy WT 2D2 mice (images not shown).

Figure 9. Myelin-specific T cells infiltrate in the spinal cord of spEAE mice. (A) The percentage of T cells expressing myelin-specific transgenic TCR (V11+) were comparable in the spleen of healthy and spEAE mice; however, the percentage of V11+ T cells were increased in the spinal cord of spEAE mice compared to WT healthy and Nlrp12-/- healthy mice (n=6), *P < 0.05; (B) Representative flow cytometry plots of CD45+ V11+ T cells in the spinal cord of Nlrp12-/- 2D2 healthy and WT 2D2 spEAE mice.

4. Discussion

Early reports showed the expression and anti-inflammatory function of Nlrp12 in innate immune cells of myeloid origin such as DC and macrophages [18,26]. However, a very recent report by Lukens et al. revealed the expression of Nlrp12 in T cells [20]. The current study aimed to investigate the immunoregulatory function of Nlrp12 in T cell-mediated immune response in EAE. Our results suggest that Nlrp12 plays pivotal role in Th1/Th2 balance by inhibiting Th1 peripheral responses in the favor of Th2. We demonstrated that in in lymph nodes of Nlrp12-/- mice, Th1 to Th2 ratio is increased compared to WT mice. This shift, in part, can be explained by significant increases in the production of IFN by Nlrp12- /- T cells. Interestingly, Nlrp12 does not play a role in the differentiation of naïve T cells but upregulates IL-2 production and proliferation of CD4+ T cells. The effect of Nlrp12 is associated with its increased expression and inhibition of major molecular pathways including Akt and NF-B in activated T cells. 76

Previously, we published that Nlrp12-/- mice develop more severe form of EAE than WT mice, which is associated with exacerbated spinal cord inflammation and increased activation of microglia in Nlrp12-/- mice compared to WT mice [19]. In the present study, we found an increased Th1 dominant response in lymph nodes of Nlrp12-/- EAE mice, suggesting that Nlrp12 suppresses Th1 activation in the periphery. Interestingly, we did not find any change in the levels of IFN and IL-4 in the CNS from Nlrp12-/- EAE mice compared to WT EAE mice. However, the IFN/IL-4 ratio significantly increased in the spinal cord of Nlrp12-/- EAE mice compared to WT EAE mice, which is consistent with Lukens et al. observation of enhanced Th2 response and increased IL-4 production in the CNS of Nlrp12- /- EAE mice [20]. In contrast to Luckens’ study where Nlrp12-/- mice developed atypical EAE signs, we found severe classical EAE signs in Nlrp12-/- mice compared to WT mice [19]. Several plausible explanations of these discrepancies were proposed in our recent review [9]. One possibility might be related to the difference in MOG-adjuvant immunization protocols between various labs. In our report, WT and Nlrp12-/- animals were immunized with a total dose of 200µg MOG [19], which is two-fold higher than the immunization dose used by Lukens et al. [20]. Interestingly, a recent study showed that immunization with low or high MOG concentration can modify the patterns of inflammatory cytokines [27]. Their study demonstrates that anti-inflammatory cytokines such as IL-10 and TGF significantly increase in the CNS of EAE animal immunized with 100µg MOG compared to 300µg MOG immunization. Therefore, it is possible that the severe EAE signs in Nlrp12-/- mice in our study were driven by lower levels of IL-10 and TGF anti-inflammatory cytokines or by higher levels of other inflammatory cytokines such as Granulocyte-macrophage colony- stimulating factor (GM-CSF) in the CNS [28,29]. Another possible explanation for observing different EAE profiles is the difference in the environmental conditions and different knockout strategies. It was shown that some C57BL/6 colonies have acquired a missense mutation in the Nlrp12 gene that can affect neutrophil responses [30]. In another study, genetic ablation of Nlrp12 was found to cause significant changes in microbiota [17]. Nevertheless, these variabilities highlight the complex immunoregulatory nature of Nlrp12 that warrants further investigation. Given the important role of T cells in the pathogenesis of EAE, we further investigated whether Nlrp12 controls T cell proliferation and activation in a T cell-intrinsic manner. We

78 found a significant increase of IFN and IL-4 levels in the supernatant of Nlrp12-/- compared to WT T cells. However, when we measured the levels of intracellular cytokines by flow cytometry, higher percentage of CD4+ IFN+ T cells were found in Nlrp12-/- group, while the percentages of CD4+ IL-4+ T cells or CD4+ IL-17+ T cells did not change between both groups. We addressed this discrepancy with Ki67 staining and our flow cytometry results revealed that activated Nlrp12-/- T cells proliferate significantly more than WT T cells, which explains why we found increased production of both IFN and IL-4 in the supernatant of activated Nlrp12-/- T cells. Consistent with our findings, Lukens et al., observed the higher expression of activation markers, enhanced proliferation, and elevated secretion of Th1/Th2/Th17 cytokines by Nlrp12-/- T cells compared to WT T cells in vitro [20]. Collectively, these results show that Nlrp12 inhibits the activation of inflammatory T cell subsets including Th1 and Th17. We found no difference between Nlrp12-/- or WT T cells in the expression of Th1- or Th17- associated molecules, suggesting that Nlrp12 does not affect the differentiation of T cells to Th1 or Th17. In a recent study by Cai et al., purified CD4+ T cells from Nlrp12-/- or WT mice were activated with anti-CD3 and polarizing cytokines for 6 days. They showed that the differentiation of Nlrp12-/- T cells to Th1 or Th17 cells were significantly lower than WT T cells. However, no difference was found between WT and Nlrp12-/- T cells in Th2 differentiation [31]. Taken together, it appears that Nlrp12-/- T cells respond differently to the environmental stimuli, depending on the type of activating signals, incubation period, and polarizing conditions. Our results, in agreement with Lukens’ study [20], showed that Nlrp12 inhibits T cell activation and proliferation. We found that the expression of Nlrp12 was significantly increased in T cells following activation by anti-CD3 or anti-CD3/CD28 antibodies. The increased level of Nlrp12 expression remained high even after 48h stimulation of T cells, suggesting that Nlrp12 modulate T cell signaling pathways upon TCR stimulation. Since multiple signaling pathways are involved in T cell activation and proliferation, we hypothesized that signaling pathways were inhibited by Nlrp12 in activated T cells. It is well- established that TCR activation triggers calcineurin (a Ca2+/calmodulin-dependent phosphatase), MAPK, and NF-B signaling pathways, leading to the activation of nuclear factor of activated T cell (NFAT), AP1, and NF-κB transcription factors that initiate IL-2

79 transcription [22]. In this regard, we evaluated the phosphorylation of p65 (NF-B subunit) and Akt in activated CD4+ T cells. Western blot results demonstrated a significant increase in the phosphorylation of p65 in activated Nlrp12-/- T cells compared to WT T cells, highlighting the inhibitory effect of Nlrp12 on NF-B signaling pathway (Fig. 10). These results are in agreement with previous studies that show Nlrp12 suppresses canonical and non-canonical NF-κB pathways [16,20,32,33]. The NF-κB signaling cascade interacts with several parallel pathways including the signaling cascades initiated by phosphatidylinositol 3-kinase (PI3K) and Akt. We found a significant increase of Akt(Ser 473) phosphorylation in Nlrp12-/- T cells compared to WT T cells. These results are corroborated by study that demonstrated that Nlrp12 negatively regulated Akt signaling pathway in affected tumor tissues in colitis model[16]. Interestingly, Akt acts upstream of NF-κB, where the activation of PI3K/Akt signaling pathway leads to phosphorylation and degradation of IκB protein, resulting in nuclear translocation and transcriptional activation of NF-κB[34]. Taken together, our results demonstrate that Nlrp12 inhibits Akt signaling pathway, which, subsequently, affects downstream pathways including NF-κB pathway. Moreover, phosphorylated Akt blocks the activity of two G1- checkpoint inhibitors, p21 and p27, and promotes cell cycle progression [35]. Collectively, our results suggest that Nlrp12 controls T cell proliferation and cytokine production by inhibiting the activation of Akt, that, in turn, affects several downstream effectors (Fig. 10).

Figure 10. Possible mechanism of action of Nlrp12 in regulating early TCR signaling pathways, T cell activation and proliferation. Activation signals from TCR activates multiple downstream signaling pathways in T cells, presented as (1) NFAT, (2) NF-B, (3) MAPK, and (4) mTOR pathways. These pathways lead to nuclear translocation of NFAT, NF-B and AP1 transcription factors and IL-2 transcription. IL-2 binds to its receptor on the cell surface and initiates mTOR pathway. TCR activating signals induces the expression of Nlrp12 that consequently inhibits NF-κB and MAPK signaling pathways, which consequently suppress IL-2 production. Upstream of NF-B pathway, Nlrp12 inhibits Akt phosphorylation that controls NF-B signaling on one side and mTOR activity and cell cycle progression on the other side. Taken together, Nlrp12 has a broad range of regulatory activity that controls hyper-proliferation and activation of T cells. The pathways shown in dashed lines are not affected by Nlrp12 including NFAT signaling and mTOR/S6 phosphorylation.

One of the signaling pathways downstream of Akt phosphorylation is mTOR pathway, which induces protein synthesis and cell growth by regulating ribosomal p70S6 kinase 1 (S6K1) and eukaryotic translation factor 4E-binding protein 1 (4EBP1). S6K1 phosphorylates and activates S6, a ribosomal subunit involved in initiating protein synthesis machinery. Using western blotting and flow cytometry, we found no difference between Nlrp12-/- T cells and WT T cells in the level of S6 phosphorylation. Therefore, it is possible that Nlrp12 regulates protein synthesis and cell growth via modulating the activity of 4EBP1 (Fig. 10). Further investigation is warranted to uncover the regulatory mechanism of Nlrp12 on mTOR signaling pathway. The results of this study and previous publications suggest that Nlrp12 inhibits NF-κB and MAPK signaling pathways in activated T cells [20,36]. The transcription of IL-2 gene is regulated by NF-κB, MAPK, and Ca2+/calmodulin-dependent pathways [37,38]. Accordingly, we hypothesized that Nlrp12 inhibits IL-2 production by activated CD4+ T cells. Flow cytometry data revealed a higher percentage of CD4+ IL-2+ T cells in activated Nlrp12-/- CD4+cells compared to WT CD4+ T cells, suggesting that Nlrp12 suppresses IL-2 production by activated T cells. Incubation with cyclosporin inhibited IL-2 production by both Nlrp12-/- and WT T cells, indicating that lack of Nlrp12 does not affect Ca2+/calmodulin signaling pathway in T cells. Our in vitro findings, together with results obtained from in vivo EAE model, support the idea that Nlrp12 inhibits T cell responses. In 2D2 mice, due to the presence of many MOG- reactive T cells, about 4-14% of mice developed spEAE [39,40]. In 2D2 mice, the percentage of V11+ T cells in the spleen were the same between healthy and spEAE animals, showing

81 that our affected and non-affected animals had similar percentage of MOG-reactive T cells. However, a high percentage of MOG-reactive T cells infiltrated to the spinal cord of WT 2D2 spEAE mice, which confirms the presence of autoreactive T cells in inflamed spinal cord. The healthy Nlrp12-/- 2D2 animals have only a few V11+ T cells in the spinal cord and do not contain pathology. The fact that none of Nlrp12-/- 2D2 mice develop disease suggest that Nlrp12 does not inhibit the development of spEAE and even can serve as contributing factor in the pathology of EAE. Due to slow rate of breeding of Nlrp12-/- 2D2 mice (unpublished observation), these conclusions are based on the observation of thirty Nlrp12-/- 2D2 mice. Interestingly, previous studies report that in induced EAE, Nlrp12 can prevent [19] or promote [20] CNS inflammation. To explain the observed controversy, we propose a dual immunoregulatory function for Nlrp12, in which Nlrp12 can act as an inflammatory or anti-inflammatory molecule, depending on the type and severity of immunological challenge. The hypothetical model of Nlrp12 immunoregulation is shown in Fig. 11. The bifunctional nature of Nlrp12 has been previously reported in several studies [16,19,20,31,36,41,42]. Early in vitro studies suggest that Nlrp12 is an inflammatory NLR that interacts with ASC to form inflammasome [43]. Recent reports also support this idea and show that Nlrp12 activates inflammasome in Yersinia Pestis and Plasmodium infections [42,44]. Moreover, several behavioral outcomes are similar between ASC-/- and Nlrp12-/- mice [45]. On the other hand, there are studies that classify Nlrp12 as an anti-inflammatory molecule and the inhibitor of NF-B signaling pathway [16,18,32,46]. In this regard, Nlrp12- /- mice were shown to be highly susceptible to inflammatory diseases of intestine such as experimental colitis and colon cancer [16,18]. Taken together, these findings support the dual immunoregulatory nature of Nlrp12, that may vary in a cell-specific or stimulus-specific manner. In conclusion, the present study provides new insight into the immunoregulatory role of Nlrp12 in T cell-mediated CNS inflammation, triggered by different modes of immunological challenges (induced EAE and spEAE). We demonstrated for the first time that Nlrp12 inhibits early TCR signaling pathways. In this way, Nlrp12 plays critical roles in balancing T cell response to control overt activation and maintain cellular homeostasis. Indeed, the fine-tuned triggering of Nlrp12 by different immunological challenges determine

82 whether the outcome of the challenge would be an inflammatory or anti-inflammatory response. Factors determining the immunoregulatory function of Nlrp12 remain to be determined.

Figure 11. Dual immunoregulatory function of Nlrp12 in EAE. The severity of immunological challenge, ranging from low to high, can influence the regulatory function of Nlrp12 in EAE. In this model, when there is no MOG/CFA immunization, myelin-specific T cells face a low level of challenge. In this case, Nlrp12 plays an inflammatory role, which explains why WT 2D2 mice developed spEAE and Nlrp12-/- 2D2 mice were resistant to spEAE. On the other hand, with MOG/CFA immunization, T cells encounter immunological challenge that may vary from medium to high levels. In mild challenge, perhaps with lower dose of MOG-CFA immunization, Nlrp12 is a bifunctional molecule, acting as an inflammatory or anti-inflammatory molecule. In this condition, EAE mice developed mild EAE signs, mostly atypical symptoms such as ataxia [20]. When MOG/CFA immunization provides high levels of immunological challenge, Nlrp12 acts as an anti-inflammatory molecule, inhibiting severe signs of classical EAE [19]. Given the fact that Nlrp12 ligand is still unknown, what exactly tunes Nlrp12 response as a proinflammatory or anti- inflammatory molecules, needs further investigation.

Author Contributions: MG, AA, and DG designed the study and the experiments; MG performed the experiments, statistical analysis, and wrote the manuscript. SM and CS monitored the animals, genotyped the mice, dissected the tissues, and reviewed the manuscript. TAM immunized mice for EAE and collected the tissues. MG, AA, and DG contributed to the conceptual reading and critical editing of the manuscript. All authors read and approved the manuscript. Acknowledgments: This work was supported by the scholarships from MS Society of Canada, Fonds de recherche du Québec-Santé (FRQS), and Association de la sclérose en plaques de l’Estrie (ASPE) to MG. Conflicts of Interest: The authors declare that they have no conflict of interest.

Supplementary Information:

Figure S1. Nlrp12 genotyping of a Nlrp12-/- 2D2 mouse and a WT 2D2 mouse. DNA was obtained from ear samples.

Genotyping of Nlrp12-/- 2D2 mice Genotyping of Nlrp12-/- mice was performed as previously described[47]. Molecular weights for Nlrp12-/- and WT bands are 390 and 318 base pairs (bp) respectively. Genotyping of 2D2 mice was performed based on Jackson laboratory protocol (Tg(Tcra2D2)1Kuch). Molecular weight for 2D2 band is 675 bp.

Figure S2: Clinical score of induced EAE in Nlrp12-/- mice compared to WT mice, published by Gharagozloo et al. in J Neuroinflammation, 2015. Nlrp12-/- mice developed earlier and exacerbated EAE compared to WT mice after MOG-CFA immunization. Animals were scored daily based on the following scale: 0, no sign of disease; 1, limp tail or weakness in limbs; 2, limp tail and weakness in limbs; 3, partial limb paralysis; and 4, complete limb paralysis. Statistical analysis was done by Kruskal- Wallis one-way ANOVA test followed by Bonferroni multiple comparison test. n = 7, *P < 0.05.

References

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3.3. ARTICLE

NLRX1 inhibits the early stages of CNS inflammation and prevents the onset of spontaneous autoimmunity

Marjan Gharagozloo1, Shaima Mahmoud1, Camille Simard1, Kenzo Yamamoto2, Diwakar Bobbala3, Subburaj Ilangumaran3, Albert Lamontagne4, Samir Jarjoura4, Jean-Bernard Denault1, Véronique Blais1, Louis Gendron1, Carles Vilariño-Güell5, A Dessa Sadovnick5, Jenny P. Ting6, Abdelaziz Amrani7, and Denis Gris1*

1Department of Pharmacology and Physiology, Faculty of Medicine, Université de Sherbrooke, QC, Canada; 2Department of Chemical Engineering and Biotechnological Engineering, Université de Sherbrooke, Sherbrooke, QC, Canada 3Department of Anatomy and Cell biology, Faculty of Medicine, Université de Sherbrooke, QC, Canada; 4Department of Neurology, Faculty of Medicine, MS Clinic, Université de Sherbrooke, QC, Canada 5Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada 6Departemnt of Microbiology and Immunology, Lineberger Cancer Center, University of North Carolina at Chapel Hill, NC, USA. 7Department of Pediatrics, Faculty of Medicine, Université de Sherbrooke, QC, Canada

Corresponding author* Denis Gris [email protected]

Submitted to PLOS Biology# PBIOLOGY-D-19-00332R1, Feb 2019

Résumé

Un récepteur de type NOD, NLRX1, appartient à la famille de récepteur immunitaire inné. NLRX1 est situé dans la mitochondrie et inhibe les principales voies pro-inflammatoires telles que l’interféron de type I et le facteur nucléaire-κB (NF-κB). Nous avons généré un nouveau modèle de sclérose en plaques (SEP), spontané avec une évolution rapide, en croisant des souris transgéniques portant des cellules T spécifiques de la myéline à des souris Nlrx1- / -. La maladie affecte 54% de la descendance et les symptômes cliniques sont en corrélation avec une démyélinisation sévère et une inflammation du système nerveux central. En utilisant des souris déficientes en lymphocytes et une série d'expériences de transfert adoptif, nous avons démontré que la susceptibilité génétique à la SEP se situe dans le compartiment immunitaire inné. Nous montrons que NLRX1 inhibe les astrocytes neurotoxiques et réduit la mort des neurones et des oligodendrocytes in vitro. De plus, nous avons découvert plusieurs mutations au sein de NLRX1 dans les familles touchées par la SEP. En résumé, nos résultats soulignent l’importance de la réponse immunitaire innée du SNC et fournissent de nouvelles preuves pour le modèle inversé de la SEP.

Abstract: The nucleotide-binding, leucine-rich repeat containing protein X1 (NLRX1) is a mitochondria-located innate immune sensor that inhibits major proinflammatory pathways such as type I interferon and nuclear factor-κB. We generated a novel, spontaneous, and rapidly progressing mouse model of multiple sclerosis (MS) by crossing transgenic mice that have myelin-specific T cells with Nlrx1-/- mice. The disease affects 54% of the progeny and the clinical symptoms correlate with severe demyelination and inflammation in the central nervous system. Using lymphocyte-deficient mice and series of adoptive transfer experiments, we demonstrate that genetic susceptibility to MS lies within the innate immune compartment. We show that NLRX1 inhibits neurotoxic astrocytes and reduces the death of neurons and oligodendrocytes in vitro. Moreover, we discovered several mutations within NLRX1 that run in MS affected families. In summary, our findings highlight the importance of the CNS innate immune response and provide new evidence for the inside-out model of MS.

Keywords: NLRX1, multiple sclerosis, Spontaneous EAE, inflammation, astrocytes, microglia, T cells

Number of Figures: 8 90

Introduction

Multiple sclerosis (MS) is a neurological disease that affects young population, leading to long-term disabilities and bringing staggering cost to society1. It is associated with inflammation-driven demyelination and neurodegeneration within the central nervous system (CNS)1,2.

Over the last century, multiple studies have confirmed the autoimmune nature of MS3. Infiltration of various subsets of myelin-specific T and B cells precedes the development of inflammatory foci, demyelinating plaques, and axonal damage2. Surprisingly, all people have myelin autoreactive lymphocytes4 and despite the extensive efforts to define MS immunopathology, the origin of the disease is still a matter of debate. Two main models have been proposed to explain the etiology of MS: outside-in and inside- out. According to the first model, MS is primarily caused by aberrant peripheral immune responses outside of the CNS in the secondary lymphoid organs such as spleen and lymph nodes. Following the overactivation and infiltration of the autoreactive T and B cells into the CNS, hyperinflammatory response leads to oligodendrocyte death and progressive demyelination5. This model gave rise to the majority of contemporary disease-modifying therapies6. Second, the inside-out model presents the idea that MS is primarily initiated by neurodegenerative processes, in which oligodendrocyte and/or neuronal injury or death triggers the CNS inflammation in the absence of direct immune attack5. This inflammation leads to the drainage of CNS antigens into secondary lymphoid organs and consequent activation of autoreactive T and B cells7. After this activation, the scenarios of MS development are similar between outside-in and inside-out models. In both models, inflammation is present at all stages of the disease. It is triggered either by the infiltration of peripheral immune cells into the CNS or by the activation of CNS-resident cells including microglia and astrocytes. Both innate and adaptive immune responses are involved in potentiating demyelinating neuroinflammatory disease in MS1. In our research, we looked into the details of activation of glial cells in the CNS including microglia and astrocytes. These cells create the first line of immune response by recognizing pathogens and/or danger signals via pattern-recognition receptors (PRR), such

91 as nucleotide-binding oligomerization domain, leucine-rich repeat containing proteins (NLRs)8. NLRs regulate both innate and adaptive immune responses5. In the context of neuroinflammation, some NLRs such as NLRP1 and NLRP3 promote the development of autoimmune encephalomyelitis (EAE), a mouse model of MS9,10 , whereas anti-inflammatory NLRs such as NLRX1 and NLRP12 inhibit CNS inflammation11,12. NLRX1 is a recently characterized member of the NLR family that is ubiquitously expressed and uniquely localized to the mitochondria. Since its discovery, NLRX1 has been implicated in multiple pathophysiological processes including oxidative damage, mitochondrial dynamics, and cell death13-17. In addition, NLRX1 acts as a negative regulator of TLR-mediated NF-κB signaling18. Eitas et al. observed more severe EAE and enhanced tissue damage in Nlrx1-/- mice compared to wild type (WT) mice11. They reported that microglia from Nlrx1-/- mice released more pro-inflammatory cytokines and chemokines11, suggesting the protective role of NLRX1 in the progression of MS. Several T cell receptor (TCR) transgenic mouse models have been developed to study the pathogenic role of T cells in MS19. Surprisingly, the high numbers of myelin-specific T cells are not sufficient to induce spontaneous EAE (spEAE) in 2D2 mice20, suggesting that other mechanisms are necessary to break the immunological tolerance and induce CNS autoimmunity. In the current study, we demonstrate that the severe spEAE in the absence of Nlrx1 in 2D2 mice is due to the enhanced activation of innate immune cells within the CNS. We provide evidence of subclinical CNS inflammation in asymptomatic Nlrx1-/- 2D2 mice that contributes to the generation of neurotoxic glia and the death of neurons and oligodendrocytes.

Results

Nlrx1-/- 2D2 mice develop spEAE

We found spEAE in 54% (21 out of 39) of Nlrx1-/- 2D2 mice, while only 6% (6 out of 95) of 2D2 mice developed spEAE (Fig.1a). The spEAE frequency was similar between males and females (Fig. 1b) and the age of onset for the majority of Nlrx1-/- 2D2 mice was between 6-9 weeks (n=16, 76%), while just 24% (n=5) of Nlrx1-/- 2D2 mice developed spEAE at the later 91

92 age. The clinical scores rapidly increased from 1.5 to 4 within one week with no signs of recovery (Fig. 1c). The onset of spEAE followed a seasonal pattern. Out of all mice the most frequent incidence of the disease was in summer (n=11, 53%), followed by spring (n=4, 19%) and the least frequency was observed in fall and winter (n=3, 14%). We then assessed the presence of inflammation and demyelination in the CNS of Nlrx1-/- 2D2 spEAE mice.

SpEAE is associated with CNS inflammation and demyelination

The level of expression of spinal microglial and astrocytic markers, mRNA expression of inflammatory mediators including Tnf, Il-1b, and Ccr5 were significantly increased, while the expression of myelin basic protein (MBP) was significantly decreased in Nlrx1-/-2D2 compared to healthy 2D2 mice. (Fig.1d-f). Additionally, hematoxylin staining demonstrated a massive infiltration of inflammatory cells into the spinal cord of Nlrx1-/-2D2 spEAE animals (Fig. 1g), in which nuclear localization of NF-B p65 subunit was observed (Fig. 1h). Furthermore, flow cytometry analysis showed significant increases in accumulation of peripheral immune cells (CD45high) including a greater percentage of activated CD11b+ MHCII+ myeloid cells in the spinal cord of Nlrx1-/-2D2 spEAE mice compared to 2D2 mice (Fig.1i, j). Immunofluorescence analysis of focal lesions revealed the nuclear localization of NF-B p65 subunit in CD68+ cells (Fig. 1k). Moreover, the spinal cords of Nlrx1-/-2D2 spEAE mice showed a marked infiltration of myelin-specific transgenic TCR (V11+) T cells into the CNS (Fig. 1l-n), and enhanced expression of T helper (Th)1-associated transcription factor, Tbet, in the spinal cords of Nlrx1-/- 2D2 spEAE mice (Fig. 1o). Immunoblotting and immunofluorescence tests revealed significant increases of IgG in the spinal cord of Nlrx1-/-2D2 spEAE mice compared to control (Fig. 2a-c). Flow cytometry analysis of inflamed spinal cord tissue demonstrated significant increases in the number of CD19+ B cells and levels of serum anti- myelin oligodendrocyte glycoprotein (MOG) antibody in Nlrx1-/-2D2 spEAE animals (Fig. 2d-f). Although we did not observe any differences between the capability of WT and Nlrx1-/- splenocytes antigen presenting cells (APC) in activating T cells (Supplementary Fig. S1a-e), we found significant increases in the proliferation of T cells from Nlrx1-/-2D2 mice compared to 2D2 confirmed by 3H-thymidine incorporation assay and Ki67 staining (Supplementary Fig. S2a-c). We also observed

93 elevated production of IFN by activated Nlrx1-/- 2D2 T cells (Supplementary Fig. S2f, g) and differentiation bias to Th1 and Th17 cells (Supplementary Fig. S2h, i).

Figure 1. Nlrx1-/- 2D2 mice develop spEAE, which is associated with the CNS inflammation and demyelination. (a) The frequency of spEAE in 2D2 and Nlrx1-/- 2D2 mice; (b) The frequency of spEAE in male and female Nlrx1-/-2D2 mice; (c) Clinical score of Nlrx1-/- 2D2 mice, showing a progressive EAE from the onset; (d) A representative immunofluorescent staining of spinal cords from healthy 2D2 and spEAE Nlrx1-/-2D2 mice for GFAP, Iba1, and MBP markers. White arrows show astrogliosis (GFAP), microgliosis (Iba1) and focal demyelinating lesions (MBP); (e) The quantification of fluorescence intensity of stained markers (n=5); (f) The mRNA expression levels of Tnf, I1-1b, and Ccr5, quantified by qPCR (n=5); (g) A representative H&E staining of the spinal cords from Nlrx1-/-2D2 spEAE and healthy 2D2 mice; (h) Nuclear localization of NF-kB p65 subunit in the focal lesions of a spEAE spinal cord, shown by white arrows; (i) The percentage of CD45high CD11b+ MHCII+ cells in the spinal cord of spEAE mice (n=7); (j) Representative flow cytometry plots showing the expression of CD11b+MHCII+ myeloid cells in CD45high cell population; (k) Nuclear localization of NF-kB p65 subunit in CD68+ macrophages (white arrows) in the spEAE spinal cord lesions; (l) An immunofluorescent image of CD3+ T cells (shown by white arrows) in the spinal cords of Nlrx1-/-2D2 mice; (m) The percentage of V11+ T cells in the spinal cord and brain from Nlrx1-/-2D2 mice (n=8); (n) Flow cytometric analysis of CD45+ V11+ T cells in the brain and spinal cord of spEAE and healthy animals; (o) The expression of T cell-associated transcription factors in the spinal cord of Nlrx1-/-2D2 spEAE mice compared to heathy mice (n=4); *P ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, as determined by the Student's t test.

Figure 2. Increased levels of IgG and frequency of B cells in the spinal cord of SpEAE mice. (a) Representative western blot of IgG in the spinal cord of spEAE mice and healthy mice; (b) Quantitative analysis of IgG/Tubulin ratio in healthy and Nlrx1-/-2D2 spEAE spinal cords (n=6); (c) Representative images of immunofluorescence staining for IgG leakage into the spinal cords of Nlrx1-/-2D2 spEAE mice and healthy spinal cord sections; (d) The percentage of CD19+ B cells in the spinal cord and brain of spEAE mice compared to healthy mice (n=8); (e) Flow cytometry analysis of CD45+CD19+ B cells in the spinal cord of healthy and spEAE mice; (f) Serum levels of anti-MOG IgG in spEAE and healthy mice (n=4), measured by ELISA, mean absorbance at OD 450 nm is shown. Error bars indicate standard deviations. *P ≤ 0.05, determined by the Student's t test.

Nlrx1-/- 2D2 mice develop more severe EAE than 2D2 mice

By comparing rare cases of spEAE in 2D2 mice to Nlrx1-/- 2D2, we noted significantly higher clinical scores, increased numbers of focal lesions, higher percentage of CD45high CD11b+ cells, and activated CD11b+ MHCII+ cells in Nlrx1-/- 2D2 spEAE mice. Among CD45high cells, no difference was found between the percentage of lymphoid cells including myelin- specific V11+ T cells and CD19+ B cells in the spinal cords of spEAE mice (Fig 3a-g). These findings led us to investigate the immunoregulatory role of NLRX1 in innate immune cells beyond antigen presentation and T cell activation using lymphocyte-deficient Rag-/- mice.

Figure 3. CNS inflammation associated with spEAE is more severe in Nlrx1-/-2D2 compared to 2D2 mice. (a) Representative images of H&E staining of lumbar spinal cords from spEAE mice with Nlrx1-/-2D2 or 2D2 genotype. The circles show focal lesions; (b) The number of focal lesions counted in spinal cords from spEAE mice (n=5 in each group); (c) EAE clinical score in Nlrx1-/- 2D2 or 2D2 mice at the time of sacrifice (n=6 in 2D2 group vs n=15 in Nlrx1- /- 2D2 group); (d) The percentage of CD45high cells in the spinal cord of Nlrx1-/-2D2 spEAE mice compared to 2D2 spEAE mice, quantified by flow cytometry as shown in the representative plots; (e) Similar percentages of V11+ T cells and CD19+ B cells in the spinal cords of spEAE mice (n=6); (f) Enhanced numbers of activated CD11b+MHCII+

96 monocyte/macrophage in CD45high cell population, quantified in spinal cords samples from Nlrx1-/-2D2 or 2D2 spEAE mice; (g) The quantification of CD11b+MHCII+ cells in CD45low or CD45high gates. Data are presented in mean and standard deviation. *P ≤ 0.05 determined by the Student's t test.

NLRX1 inhibits innate immune response and prevents CNS inflammation

Fourteen days post-immunization of Rag-/- and Nlrx1-/-Rag-/-, flow cytometry data showed significant increases in the percentages of CD45high leukocytes and activated CD11b+MHCII+ myeloid cells in the brain of Nlrx1-/-Rag-/- mice compared to Rag-/- mice (Fig.4a, b). We transferred MOG-activated 2D2 T cells into Rag-/- and Nlrx1-/- Rag-/- mice and following 21 days of immunization with MOG-Complete Freund's Adjuvant (CFA) emulsion plus pertussis toxin (PTX), we observed several statistically significant changes. We found more severe hind limb paralysis, higher EAE clinical score (3.5±0.7 vs 1.5±0.7), and increased accumulation of CD45high cells in Nlrx1-/-Rag-/- compared to Rag-/- mice (Fig. 4d-f). Moreover, we found demyelinating lesions, inflammatory foci, and elevated Iba1 expression in the spinal cords of Nlrx1-/-Rag-/- mice (Fig. 4g-h). Additionally, the percentages of CD11b+ MHCII+ myeloid cells and myelin-specific T cells were significantly increased in Nlrx1-/-Rag- /- mice compared to Rag-/- mice (Fig. 4i, j) At the preclinical stage, we observed significantly higher percentages of CD45low microglia CD11b+ MHCII populations in the spinal cord of Nlrx1-/-2D2 mice, while the percentage of CD45high cells was significantly increased in both brain and spinal cord tissues from Nlrx1-/- 2D2 mice compared to 2D2 mice (Fig 5a-c). Although, the percentages of T cells and B cells in the CNS tissues from both strains of mice were comparable (Fig. 5d), we found a marked increase in the expression of Tnf, Il-1b, Ccl20, Ccr5, and NOS2 mRNA in the brains of Nlrx1-/- 2D2 mice (Fig. 5e).

Figure 4. Activation of innate immune cells induce CNS inflammation and severe paralysis in Nlrx1-/-Rag-/- mice. (a) The infiltration of CD45high leukocytes to the brain of Nlrx1-/-Rag-/- mice compared to Rag-/- mice 14 days after immunization with MOG-CFA emulsion plus pertussis toxin (PTX), quantified by flowcytometry as shown in representative plots; (b) The percentage of activated CD11b+MHCII+ microglia/macrophage in CD45+ cells, quantified by flowcytometry as shown in representative plots; (c) Adoptive transfer of 2D2 T cells followed by MOG-CFA/PTX immunization caused hind limb paralysis in Nlrx1-/-Rag-/- mice, while Rag-/- mice never got paralyzed; (d) The clinical score of mice 3 weeks after adoptive transfer and immunization (n=4); (e) The ratio of CD45high cells (myeloid cells) to CD45Low cells (microglia) in the spinal cord of Nlrx1-/-Rag-/- mice compared to Rag-/- mice following adoptive T cell transfer and MOG-CFA/PTX immunization (n=4); (f) The H&E staining of spinal cords from Nlrx1-/-Rag-/- mice and Rag-/- mice, black arrows show the infiltration of mononuclear cells; (g) The expression of GFAP, Iba1 and MBP in the spinal cord of Nlrx1- /-Rag-/- mice compared to Rag-/- mice; (h) Quantification of stained markers (Iba1: microglia, GFAP: astrocyte, MBP: myelin basic protein) using image J, (n=4); (i) The percentage of V11+ T cells in the spinal cord and brain of Nlrx1-/-Rag-/- mice compared to Rag-/- mice (n=4); (j) The percentage of activated CD11b+MHCII+ microglia/macrophage in the spinal

98 cord and brain of Nlrx1-/-Rag-/- mice compared to Rag-/- mice (n=4). Data are presented in mean and standard deviation. *P value<0.05 determined by the Student's t test.

Figure 5. Inflammation at the preclinical stages of spEAE is enhanced in the CNS of Nlrx1- /-2D2 mice. (a) The percentages of CD45low microglia and CD45high myeloid cells to the spinal cord and brain of Nlrx1-/-2D2 mice compared to 2D2 mice, quantified by flow cytometry; (b) Representative plots showing CD45low and CD45high gates in spinal cord and brain samples from Nlrx1-/-2D2 and 2D2 mice; (c) The percentage of activated myeloid cells (CD11b+MHCII+) in the spinal cord and brain of Nlrx1-/-2D2 and 2D2 mice (n=7); (d) the percentage of V11+ T cells and CD19+ B cells in the spinal cords and brains of Nlrx1-/-2D2 and 2D2 mice, quantified by flow cytometry (n=7); (e) The mRNA levels of inflammatory mediators in Nlrx1-/-2D2 brains compared to 2D2 brains, quantified by qPCR. *P ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, as determined by the Student's t test.

NLRX1 inhibits tissue damage and the generation of neurotoxic astrocytes

In the brain of Nlrx1-/- 2D2 mice, we found a significant increase in tissue injury defined by the levels of High mobility group box 1 (HMGB1) that was associated with significant increases in the expression of A1-related reactive astrocyte genes, while A2-related astrocyte genes were significantly decreased compared to 2D2 mice (Fig. 6a,b). We observed similar pattern of increased expression of A1-related genes in Nlrx1-/- brains compared to WT brains (Fig. 6c). To better understand the role of NLRX1 in astrocyte phenotype, we stimulated standard glial cultures from Nlrx1-/- and WT mice with Lipopolysaccharides (LPS)/IFN and found that Nlrx1-/- glia express significantly more A1-related transcripts, increased microglial activation, and TNFα production than WT glia (Fig. 6d, e). We further investigated the effect of conditioned medium collected from LPS/IFN-treated glial cells on the cell death of neurons and oligodendrocytes. The results show a significantly lower cell survival and higher cell death in both N2A and MO3.13 cells treated with Nlrx1-/- glia compared to WT glia conditioned medium (Fig. 6f-h).

Figure 6. NLRX1 inhibits subclinical tissue damage and the generation of neurotoxic glia in the CNS. (a) The level of HMGB1 in the spinal cord of asymptomatic Nlrx1-/-2D2 mice compared to 2D2 mice, determined using western blot and quantified by the ratio of HMGB1

100 to -Tubulin (n=5); (b) The levels of A1-related gene expression in brain from Nlrx1-/-2D2 mice compared to 2D2 mice, while A2-related gene expression is diminished (n=7); (c) The expression of A1-related genes in the brains of Nlrx1-/- mice compared to WT mice (n=7); (d) A heatmap diagram showing the fold change of pan-, A1- or A2-transcript levels in Nlrx1- /- glia after 24h stimulation with LPS (500ng/ml) or LPS+IFN (10ng/ml) compared to the corresponding WT controls (n=5 in each group); (e) TNFα level in the conditioned medium (CM), collected 24h after LPS/IFN treatment of glia culture and measured by ELISA; (f) The toxicity of MO3.13 and N2A cells after 24h incubation with Nlrx1-/- or WT glia CM, measured by MTT assay; (g) Expression of apoptosis (annexin V) and necrosis (propidium iodide: PI) markers on MO3.13 and N2A cells incubated with Nlrx1-/-glia CM compared to WT glia CM for 24h (n=4); (h) Representative flow cytometry plots used for the quantification of annexin V and PI expressions on MO3.13 and N2A cells. *P ≤ 0.05, ** ≤ 0.01, *** ≤ 0.001, as determined by the two-tailed unpaired Student's t test.

NLRX1 expression analysis in MS patients and EAE mice

Given the fact that peripheral immune cells play one of the key roles in the pathogenesis of relapsing remitting MS (RRMS), we quantified the expression of NLRX1 in peripheral blood mononuclear cells (PBMCs) from MS patients (Table S2) and found that PBMCs from RRMS patients express significantly higher levels of NLRX1 mRNA than healthy controls (Fig. 7a). Although we did not find any differences between the two groups in the expression of TNF, there was a significant positive correlation between the mRNA levels of NLRX1 and TNF in the PBMCs from RRMS patients (Fig. 7b, c). Similarly, in mice, we found a significant increase in the mRNA levels of Nlrx1 in spleen and brain tissues from EAE mice compared to healthy mice (Fig. 7d).

Assessment of NLRX1 genetic variants in MS patients

To determine whether NLRX1 genetic variants are implicated in the onset of MS in humans, we mined exome sequencing data from 326 MS patients and 123 healthy controls. Variants identified exclusively in MS patients, with a minor allele frequency below 1% in publicly available databases (GnomAD)21, and resulting in missense or nonsense substitutions, were considered potentially disease relevant. This analysis identified five missense mutations (p.Lys172Asn, p.Pro189Leu, p.Leu237His, p.Arg471Trp, and p.Arg860Trp), each in one patient; and one nonsense mutation (p.Glu192Ter) in three MS patients (Table S3). Genotyping NLRX1 variants in a case-control series from Canada only identified p.Arg471Trp in two additional patients and two controls, and p.Glu192Ter in two additional

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101 patients and one control. Segregation analysis within families did not support co-segregation with MS for p.Pro189Leu, p.Leu237His, and p.Arg471Trp, with less than 75% of affected family members harboring the mutations (Fig 7f). NLRX1 p.Lys172Asn was only observed in two affected individuals and two obligate carriers from one family. Similarly, p.Arg860Trp was only present in a mother and daughter, both diagnosed with MS. The nonsense mutation, p.Glu192Ter was identified in four multi-incident families and one patient without family history of MS. In these families, the majority of individuals diagnosed with MS were found to carry the p.Glu192Ter mutation (9/10); however, 14 healthy individuals, including three obligate carriers, were also found to harbour this mutation, suggesting reduced penetrance.

Figure 7. NLRX1: implications for human MS. (a) Expression of NLRX1 in the peripheral blood mononuclear cells (PBMC) from MS patient (n=18) compared to healthy donors (n=31), quantified using qPCR. The groups demographic and clinical data are shown in supplementary Table S2; (b) The mRNA levels of TNFα expression in PBMC from MS 101

102 patients (n=12) and healthy individuals (n=28), measured by qPCR; (c) Correlation between mRNA levels of NLRX1 and TNFα in the PBMC from MS samples (n=12), statistically determined using Pearson correlation; (d) Expression of Nlrx1 in the spleen and brain of EAE mice compared to healthy mice. *P ≤ 0.05, *** ≤ 0.001, as determined by the Student's t test; (e) NLRX1 conservation in orthologs. Evolutionarily conserved positions for the identified mutations are highlighted in black. Organism and RefSeq accession numbers are provided; (f) Pedigrees for families identified with NLRX1 mutations. Black filled symbol, MS; gray filled, unaffected obligate carrier. Heterozygote mutation carriers (MT) and wild-type (WT) genotypes are indicated.

Discussion

Here we propose a novel mechanism that emphasises the inside-out model of MS. Our study demonstrates the crucial role of the inflammatory status of the CNS innate immune compartment in the development of EAE spontaneously. This work provides the first evidence of the potential role of an innate immune receptor, NLRX1, in predisposition to EAE. We generated Nlrx1-/- 2D2 mice that are genetically more susceptible to EAE due to high numbers of autoreactive T cells and increased CNS tissue inflammation. We found that Nlrx1-/- 2D2 mice are ten times more likely to develop spEAE than 2D2 mice, indicating that Nlrx1 prevents the onset of EAE. Although NLRX1 inhibits both innate and adaptive immune responses, the expression of Nlrx1 in the innate immune compartment is necessary for the development of spontaneous disease. Mechanistically, we demonstrate that under inflammatory conditions, NLRX1 inhibits A1 in the favor of A2, a less toxic glial phenotype. In the absence of NLRX1, the stimulation of astrocytes and microglia results in the environment that is toxic to neurons and oligodendrocytes. Importantly, we found that the pattern of NLRX1 expression is similar between mice and human, suggesting the relevance of our findings in designing new therapies for MS patients. This is further supported by the identification of six rare NLRX1 mutations in MS patients, including a p.Glu192Ter truncation in 10 patients. In agreement with work by Eitas et al., that showed NLRX1 inhibits the progression of EAE following immunization with MOG-CFA11, we demonstrated that NLRX1 reduces the severity of spEAE. Furthermore, our results suggest that Nlrx1 plays role in the tissue inflammation before the appearance of clinical symptoms. Thus Nlrx1, both, inhibits triggers of EAE and inhibits its severity, which explains why Nlrx1-/-2D2 animals develop a rapidly progressing EAE with no recovery. Similar acute progressive EAE course has been

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103 previously reported in TCR transgenic mice in the SJL background and Tnfr2-/-2D2 female mice 22,23. Although there are many TCR transgenic models of EAE (Table S1), our study is the first to demonstrate the crucial role of a PRR in the etiology of spEAE. We provide evidence that expression of Nlrx1 in innate immune compartment is sufficient to suppress T cell- mediated autoimmunity. Furthermore, we observed a seasonal pattern in the onset of spEAE, as we found the highest frequency of Nlrx1-/- 2D2 spEAE in the summer, suggesting that environmental factors influence the disease onset. Although the nature of such fluctuations is unknown, these results are in agreement with studies that found seasonal changes in disease activity in MS patients24 and in animal models25. To our knowledge, this is the first time that seasonality is reported in a TCR transgenic mouse model of MS. Pathophysiology of the spEAE in Nlrx1-/-2D2 mice is consistent with the autoimmune nature of the disease. Histopathological examination revealed the massive inflammation and demyelination in the spinal cord of Nlrx1-/-2D2 mice. Activated inflammatory macrophages, myelin-specific Tbet+ T cells, and IgG deposit in the spinal cord of Nlrx1-/-2D2 mice suggest Th1 mediated autoimmune bias. These findings parallel other mouse models of spEAE26,27. For example, it is shown that Tbet-/-2D2 mice are protected from the development of EAE26. Comparing to occasional cases of spEAE in 2D2 mice to Nlrx1-/-2D2, we found higher EAE clinical score in Nlrx1-/-2D2 spEAE mice, which was associated with increased numbers of focal lesions. Although the percentages of myelin-specific T cells in the spinal cord of spEAE animals from both genotypes are similar, the differentiation of CD4+ T cells toward the inflammatory Th1 or Th17 cells is enhanced in Nlrx1-/- 2D2 spEAE mice. Interestingly, this effect is T cell autonomous since it does not depend on the context of APCs. Consistent with our findings, a previous study by Leber et al., reported the greater proliferation rates and the higher ability of Nlrx1-/- T cells to differentiate into Th17 phenotype, when the cells were activated by a non-specific stimulator, anti-CD3 and anti-CD28, in vitro28. However, NLRX1 has no effect on the differentiation of T cells to Treg cells28. Our findings and previous research11 suggest that NLRX1 inhibits initiation and progression of EAE on at least two levels. At the level of innate immunity, it inhibits activation of microglia and macrophages and at the level of adaptive immunity, NLRX1 inhibits activation and proliferation of encephalitogenic T cells phenotypes.

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To differentiate between the role of NLRX1 in innate and adaptive immune responses, we transferred experimental paradigm onto Rag-/- background and found that, after adoptive transfer of activated T cells, Nlrx1-/- Rag-/- mice developed significantly more severe EAE compared to Rag-/- mice. Interestingly, even in the absence of T cells, after immunization with CFA, we found greater infiltration of CD45 and CD11b myeloid cells in the CNS of Nlrx1-/- Rag-/- mice. Consistent with our findings, Soulika et al. reported that the mRNA levels of CD45, CD11b, and Il-1b in the spinal cords of Rag-/- mice were significantly induced after 7 days of MOG-CFA injection29, indicating that the activation of CNS innate cells begins prior to substantial accumulation of peripheral immune cells in the CNS. Furthermore, we found an increased percentage of CD45low microglia and CD45high macrophages in the CNS of asymptomatic Nlrx1-/-2D2 mice compared to healthy 2D2 mice. We found significant increases in the levels of multiple proinflammatory mediators including Tnf, NOS2, Ccr5, and Ccl20 at the subclinical stage in Nlrx1-/- 2D2. It is shown that CCR5 is widely expressed on APC such as macrophages and microglia, and effector T cells30, whereas CCL20 mainly derived from TNFα-activated astrocytes and functions as a chemoattractant for recruiting CCR6-expressing Th17 cells to the brain31. In vitro studies show that IL-1β induces the production of CCL20 in astrocytes, leading to BBB disruption and ultimately promoting the influx of inflammatory cells32,33. Also, we found an enhanced level of HMGB1 in the spinal cord of Nlrx1-/- 2D2 mice, suggesting the presence of a subclinical tissue damage in the CNS. Furthermore, our study demonstrates that NLRX1 inhibits innate immune response at the level of the neurotoxic glia favoring A2 astrocytes. We observed the similar expression pattern of A1 astrocyte genes in the brains from Nlrx1-/- mice regardless of genetic background, which indicates that the generation of A1-astrocytes in Nlrx1-/- brains is not dependent on myelin-specific T cells, but is related to overactivated microglia. A recent study showed that neurotoxic glia, known as A1 astrocytes, are induced by microglial inflammatory mediators such as IL-1α, TNFα, and C1q and induce the death of neurons and oligodendrocytes34. Consistent with these findings, we found that after LPS/IFN treatment, Nlrx1-/- glia had an increased expression of A1-genes transcripts associated with higher production of TNFα compared to WT glia. This resulted in increased cytotoxicity of Nlrx1-/-

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105 glia as conditioned medium from Nlrx1-/- culture induced significantly more cell death in N2A neuroblastoma and MO3.13 oligodendrocyte cell lines. Taken together, we found that NLRX1 plays a key role in guarding the CNS and preventing the onset of inflammation. On one hand, NLRX1 intrinsically inhibits the autoreactive T cell response and on the other hand, it prevents the function of innate immune response, particularly in the CNS. NLRX1 suppresses the generation of inflammatory microglia and neurotoxic astrocytes that play a destructive role in the CNS by inducing death in neurons and oligodendrocytes. Additionally, in human MS, NLRX1 control the level of inflammatory cytokines, keeping the inflammatory status of the PBMCs in check. The identification of rare NLRX1 mutations in MS patients, particularly p.Lys172Asn, p.Glu192Ter and p.Arg860Trp, supports this hypothesis. However, given their low frequency further analysis in larger cohorts of MS patients is needed to confirm their role in the onset of disease. Based on the above findings, we propose that NLRX1 plays role of a guardian protein that keeps in check the CNS inflammatory status and preventing the onset of MS (Fig. 8). Our model supports the inside-out model of MS but putting inflammation upstream of the first neurodegenerative event5.

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Figure 8. NLRX1 inhibits early stages of CNS inflammation, prevents toxic glia activation, and the onset of EAE. Failure to maintain proper inflammatory balance in Nlrx1-/- microglia results in milieu that promotes toxic A1 astrocyte phenotype (A1). This results in damage to neurons and oligodendrocytes and increase T cell chemoattractant gradient. Upon activation and differentiation in secondary lymphoid organs Th1 biased T cells in Nlrx1-/- mice migrate to CNS and induce autoimmune attack. Subclinical stage, phase 1: Unknown factors trigger the inflammatory pathways particularly NF-B in microglia, leading to the production of inflammatory cytokines such as TNFα and IL-1 that promote generation of neurotoxic A1 astrocytes. At this moment we cannot exclude that astrocytes from Nlrx1-/- mice themselves are prone to differentiation to A1 phenotype. As a result, a limited number of oligodendrocytes dies and myelin antigen is drained to the deep cervical lymph nodes. At the same time A1 astrocytes increase expression of T cell chemokine such as CCL20. Subclinical stage, phase 2: In the lymph nodes, autoreactive T cells proliferate and differentiate into encephalitogenic T cells subsets (Th1, Th17). Clinical stage, phase 3: Clonal expansion of autoreactive encephalitogenic T cells, activation of myeloid cells such as monocytes and macrophages, and production of inflammatory mediators leads to their excavations into CNS. Clinical stage, phase 4: activated T cells and monocyte/macrophages infiltrate to the CNS, interact with CNS-resident cells and boost the inflammation, resulting in reactive gliosis, progressive inflammatory demyelination, neurodegeneration, and eventually the appearances of neurological symptoms. NLRX1 has a broad range of regulatory activity, preventing the onset of clinical signs at the levels of CNS and periphery.

Acknowledgment

The authors would like to thank the healthy volunteers and the patients at the MS Clinic, CHUS, Sherbrooke, QC, Canada for participating in our study. We are grateful to MS nurse, Geneviève Morin, at the Department of Neurology, CHUS, for taking patients’ blood samples.

Author Contributions

M.G., A.A. and D.G. designed the study and the experiments; M.G. performed the experiments, statistical analysis and wrote the manuscript. S.M. and C.S. monitored the animals, genotyped the mice, and dissected the tissues. K.Y. helped in genotyping and dissecting the animals. D.B. and S. I. helped for 3H thymidine assay. A.L. and S.J. visited the patients in MS clinic, collected the information, took the patients consents, and referred them to the nurse for blood sampling. J.B.D. and V.B., helped for MTT assay. C.V. and A.D.S. provided the human genetic data. M.G., S.J., L.G., C.V., J.T., J.B.D., A.A., and D.G. contributed to the conceptual reading and critical editing of the manuscript. All authors read and approved the manuscript.

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Funding

This research was funded by scholarships from the MS Society of Canada, Fonds de Recherche du Québec-Santé (FRQS) and Association de la sclérose en plaques de l’Estrie (ASPE) to M.G. Research grant from FMMS Universite des Sherbrooke to D.G. Additional funds were provided by the Michael Smith Foundation for Health Research (16827) and Canadian Institutes of Health Research (MOP-137051) to C.V.G.

Conflicts of Interest

The authors declare no conflict of interest.

Methods

Immunization and EAE induction

All the protocols and procedures were approved by the University of Sherbrooke Animal Facility and Use Committee (Protocols #280-15, April 04, 2017; #335-17B, February 22, 2018). All mice were on a C57Bl/6J background and were backcrossed for at least 15 generation. The 2D2 TCR transgenic mice specific for the myelin oligodendrocyte -/- (MOG35–55) peptide were purchased from Jackson Laboratory. Nlrx1 mice were crossed with 2D2 mice or Rag-/-mice to generate Nlrx1-/- 2D2 or Nlrx1-/- Rag-/- mice respectively. EAE was induced in 8-10-week old WT or Nlrx1-/- female mice as previously described35.

Histological Analysis

Mice were sacrificed, perfused with ice-cold phosphate-buffered saline (PBS) (Wisent, St. Bruno, QC), and the spinal cords were removed and fixed in 4% formaldehyde for 24h. 5- μm sections were used for haematoxylin and eosin (H&E) or immunofluorescence staining for the markers including CD3, GFAP, Iba1, MBP, or IgG. All slides were scanned using a digital slide scanner NanoZoomer-XR C12000 (Hamamatsu, Hamamatsu City, Japan) and viewed using NDPview2 software (Hamamatsu). Fluorescence intensities of the stained markers were quantified using Fiji (ImageJ) software (NIH).

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Flow cytometric analysis of CNS-infiltrating Mononuclear Cells

CNS tissue was digested with collagenase D (2.5 mg/ml, Roche Diagnostics) and DNase I (1 mg/ml, Sigma-Aldrich) and filtered through a 70 mm nylon sieve as described previously12. Mononuclear cells were isolated by percoll (Sigma-Aldrich) centrifugation. The samples were centrifuged at 12000×g for 15 min without break washed stained surface markers and analysis by flow cytometry. Myeloid markers included anti- CD45-FITC, anti-CD11b-PE, anti-MHCII-PE-Cy5 and lymphoid markers included anti- CD4-FITC, anti-V11-APC, and anti-CD19-PE. All antibodies were purchased from eBioscience, San Diego, CA.

T cell activation and T cell differentiation in vitro

CD4+ T cells were purified from the single cell suspension prepared from lymph nodes and spleens using MagniSort CD4 T cell Enrichment Kit (eBiosciences) and activated with MOG-pulsed splenocytes for indicated times. T cell proliferation was quantified using 3H- thymidine incorporation assay and Ki67 intranuclear staining following fixation and permeabilization using Foxp3/Transcription Factor staining kit (eBioscience). Intracellular staining of cytokines was performed as previously described12. Sample acquisition was performed with Beckman Coulter CytoFlex and data were analyzed using CytExpert 2 software (Beckman Coulter).

Quantitative RT-PCR

RNA was extracted from cells using TRIzol protocol (Life Technologies Inc., Burlington, ON), and cDNA was synthesized as previously described35. Primers sequences are 36 presented in Table S4. The relative expression was calculated using the ΔΔCT method .

Cytokine Measurement and Western blotting

The level of IFN or TNFα in the cell culture supernatants were measured using ELISA kits as described by the manufacturer (PeproTech, Rocky Hill, NJ). The Serum level of anti- MOG IgG were quantified using ELISA assay as described by Mantegazza et al.37 For western blotting, tissues were homogennized in the lysis buffer plus proteinase and

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109 phosphatase inhibitor (Cell Signaling Technology). Proteins were measured and separated on SDS-polyacrylamide gels and transferred to nitrocellulose membrane. The immunoblots were developed with Lumigen ECL ultra reagent, imaged with ChemiDocTM (Bio-Rad) and analyzed using Image Lab software.

Adoptive transfer

CD4+ T cells were activated with MOG for 48h and then harvested, washed and resuspended for a total of 3 × 106 CD4+ T cells in 500µl of PBS. T cells were transferred intraperitoneally into Rag-/- or Nlrx1-/-Rag-/- mice followed by MOG-CFA immunization35 and singe pertussis toxin (500 ng) i.p. injection.

Cell Cytotoxicity Assay

Homogenized brains from 1-day-old pups were passed through a 70-μm filter. The cells were cultured in DMEM/F12 medium and 10 % FBS (Invitrogen, Burlington, ON) as previously escribed35.Glial cells were treated with LPS (100 ng/ml) and/or IFN (10 ng/ml) for 24h and the conditioned medium were collected and stored at -80 oC. Effect of glia- conditioned medium on the viability of N2A and MO3.13 cells (kindly provided by Dr. Nathalie Arbour, CRCHUM, Montreal, Canada) was determined by MTT assay38. N2A or MO3.13 cells (1×104) were grown in 96-well plates in DMEM/F12 medium for 24 h. Thereafter, medium was replaced with astrocyte-conditioned medium and the incubation was continued for an additional 24 h. Flow cytometric analysis of cell death was done with Annexin V Apoptosis Detection Kit (eBioscience) per manufacturer's instructions.

MS subjects and NLRX1 expression

Patients diagnosed with relapsing remitting MS (n=18), based on the revised McDonald Diagnostic Criteria, were recruited from the Multiple Sclerosis Clinic at the University of Sherbrooke by a board-certified neurologist. On the day of blood sampling, all subjects were afebrile and had no signs and symptoms of infection based on history, physical examination, and responses to a survey. The age-matched control group consisted of 23 healthy volunteers. The study was Institutional Review Boards of the University of Sherbrooke.

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Genetic Methods

Biological samples from 2,480 MS patients and 1,024 healthy controls were collected through the longitudinal Canadian Collaborative Project on the Genetic Susceptibility to Multiple Sclerosis (CCPGSMS)39 with informed consent, and approval from the ethical review board at the University of British Columbia. All patients were diagnosed with MS according to Poser criteria prior to 200140, or McDonald criteria thereafter41. Cohort demographics have been described elsewhere42. Exome sequencing data from 326 MS patients and 123 healthy controls was generated as previously described43, and variants of interest were genotyped using TaqMan probes. Sanger sequencing was used to confirm non- reference genotype calls and to assess segregation within families as previously described44.

Statistical Analysis

Statistical analyses were conducted using GraphPad Prism 7 software. Results were expressed as the mean ± standard deviation. Statistical differences between WT and Nlrx1-/- samples were assessed by two-tailed unpaired Student's t test. The significance level was set at P ≤ 0.05.

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SUPPLEMENTARY MATERIAL

Figure S1. Nlrx1-/-APC activate T cells as efficiently as WT APC. (a) The expression of T cell activation markers (CD69 and CD25) by 2D2 T cells, activated with MOG in the presence of WT APC or Nlrx1-/-APC for 24h, quantified by flow cytometry (n=4); (b) The expression of proliferation marker, Ki67, by MOG-activated 2D2 T cells in the presence of MOG-pulsed WT APC or Nlrx1-/-APC for 48h (n=4); (c) A representative flow cytometry plot showing the pick of proliferating CD4+Ki67+ T cells activated by MOG-pulsed WT splenocytes (blue line) or Nlrx1-/-splenocytes (red line) for 24h; (d) The differentiation of T cells to inflammatory T cell subtypes (Th1 and Th17) by MOG-activated 2D2 T cells in the presence of WT APC or Nlrx1-/-APC and polarizing cytokines for 72h, quantified by flow cytometry (n=4).

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Figure S2. Nlrx1 inhibits T cell activation, proliferation, and differentiation to inflammatory subsets. (a) The expression of early activation marker, CD69, in Nlrx1-/- or 2D2 T cells after 24h activation with MOG (n=5); (b) The kinetics of CD25 (IL-2R) expression on Nlrx1-/- or 2D2 T cells after 24, 48, and 72h activation with MOG (n=5); (c) Higher proliferation of Nlrx1-/- 2D2 compared to 2D2 T cells after 48h activation with MOG-pulsed splenocytes in vitro quantified by [3H]-thymidine assay; (d) Enhanced proliferation of Nlrx1-/-2D2 T cells compared to 2D2 T cells after 24h activation with MOG-pulsed splenocytes, quantified using Ki67 staining and flow cytometry; (e) A representative flow cytometry plot showing the higher pick of proliferating CD4+Ki67+ Nlrx1-/- T cells (red line) compared to CD4+Ki67+ WT T cells (blue line) after 24h activation; (f) Increased production of IFN by activated Nlrx1-/-2D2 T cells compared to 2D2 T cells quantified by ELISA; (g) Flow cytometric analysis of IFN+CD4+T cells in Nlrx1-/-2D2 T cells or 2D2 T cells after 48h activation with MOG-pulsed splenocytes (n=6); (h) Flow cytometric quantification of Nlrx1-/-2D2 or 2D2 T cells differentiation to Th1 (IFN+CD4+T cells) or Th17 (RORt+CD4+T cells) activated with MOG-pulsed splenocytes for 72h in the presence of Th1 or Th17 polarizing cytokines (n=4). *P ≤ 0.05 determined by the Student's t test.

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Table S1. Nlrx1-/- 2D2 model compared to previous models with CD4 T cells involvement.

Clinical Genotype Antigen Strain Incidence Reference sign Models with CD4 T cells involvement MBP B10.PL 14-44% MBP TCR transgenic Paralysis 45 Ac1-11 mouse/I-Au MBP TCR MBP B10.PL 100% Paralysis 46 transgenic/Rag-/- Ac1-9 mouse/I-Au Humanized MBP TCR MBP Human/HLA 100% Paralysis 47 transgenic /Rag-/- 84-102 DR2, C57BL/6 PLP SJL/J mouse/ 83%, PLP TCR transgenic Paralysis 23 139-151 I-As 45% Humanized MBP TCR MBP Human/HLA- Severe 86.4% 48 transgenic /Rag-/- 85–99 DR2b Paralysis Optic MOG NOD mouse/ NOD TCR Transgenic 64% neuritis; 49 35-55 I-Ag7 Paralysis Optic MOG TCR Transgenic MOG C57BL/6 4.0% neuritis; 20 (2D2) 35-55 mouse/I-Ab Paralysis MOG C57BL/6 Tob1-/-2D2 44% Paralysis 50 35-55 mouse/I-Ab MOG C57BL/6 Tbet-/-2D2 0.0% Paralysis 26 35-55 mouse/I-Ab Models with CD4 T cells and B cells involvement Optic MOG C57BL/6 IgHMOG 2D2 51.1% neuritis; 51 35-55 mouse/I-Ab Paralysis Optic MOG C57BL/6 Il-21R-/- IgHMOG 2D2 25.0% neuritis; 52 35-55 mouse/I-Ab Paralysis Models with CD4 T cells and cytokine involvement 92% MOG C57BL/6 Tnfr-/-2D2 female, Paralysis 22 35-55 mouse/I-Ab 8% male MOG C57BL/6 Tnf-/-2D2 7.9% Paralysis 22 35-55 mouse/I-Ab Model with CD4 T cells and innate receptor involvement MOG Current Nlrx1-/-2D2 C57BL/6J 54% Paralysis 35-55 study

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Table S2. Demographic data of MS patients and healthy individuals. RRMS: replacing remitting MS. Characteristics Healthy (n=32) MS (n=18) Sex (female/male) 18/14 13/5 Age (year) 30.15± 11.74 40.11± 11.68 Duration of MS (year) - 9.27± 6.80 Type of MS - RRMS Treated with MS disease modifying drugs* (n) - 14 *MS disease modifying drugs including Teriflumonide, n=4; Dimethyl fumarate, n=3; Fingolimod, n=1; Natalizumab, n=1; Glatiramer acetate, n=1; interferon beta-1a, n=4.

Table S3. NLRX1 mutations identified in MS patients. Genomic coordinates from NCBI Build 37.1 (hg19) and dbSNP refSNP (rs) identifiers from build 150 are provided. Estimated effect on protein function was assessed with the Combined Annotation Dependent Depletion (CADD) phred-scale scores. Sample counts and/or minor allele frequency (MAF) for the Genome Aggregation Database (gnomAD), MS patients, and healthy controls are given. NA, not available.

Chromosome: Reference: cDNA Amino acid CADD GnomAD MS Controls Position alternate change change dbSNP ID phred MAF MAF (n) MAF (n) 11:119044474 G:C c.516G>C p.K172N NA 25.7 4×10-6 0.0002 (1) 0 (0) 11:119044524 C:T c.566C>T p.P189L rs375942096 24.8 4×10-5 0.0002 (1) 0 (0) 11:119044532 G:T c.574G>T p.E192X rs146286979 35.0 0.0002 0.0010 (5) 0.0005 (1) 11:119044668 T:A c.710T>A p.L237H NA 23.4 NA 0.0002 (1) 0 (0) 11:119045723 C:T c.1411C>T p.R471W rs145673513 29.8 0.0002 0.0006 (3) 0.0010 (2) 11:119053026 C:T c.2578C>T p.R860W NA 22.2 4×10-6 0.0002 (1) 0 (0)

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Table S4. The primer sequences used for qPCR experiments

Mouse genes Primer Sequences F: 5′-CCT CTT TGA GCC AGA CGA AG-3′ Nlrx1 R: 5′-GCC CAG TCC AAC ATC ACT TT-3′ Inflammatory mediators F: 5'-GGC ATG GAT CTC AAA GAC AAC C-3' Tnf R: 5-'CAG GTA TAT GGG CTC ATA CCA G-3' F: 5'-CAT CCA GCT TCA AAT CTC GCA G-3' Il-1 R: 5'-CAC ACA CCA GCA GGT TAT CAT C-3' F: 5'-CAT TGG AAG TGA AGC GTT TCG-3' NOS2 R: 5'-CAG CTG GGC TGT ACA AAC CTT C-3' F: 5'-CGA AAA CAC ATG GTC AAA CG-3' Ccr5 R: 5'-GTT CTC CTG TGG ATC GGG TA-3' F: 5'-AAG TCC ACT GGG ACA CAA ATC-3' Ccl20 R: 5'-CTC AGC CTA AGA GTC AAG AAG ATG-3' T cells F: 5'-GCC AGG GAA CCG CTT ATA-3' Tbet R: 5'-CCT TGT TGT TGG TGA GCT TTA-3' F: 5'-ACG GCC CTG GTT CTC ATC A-3' Rorc R: 5'-CCA AAT TGT ATT GCA GAT GTT CAA C-3' F: 5'-GAA AGC GGA TAC CAA ATG A-3' Foxp3 R: 5'-CTG TGA GGA CTA CCG AGC C-3' Astrocytes F: 5′-GAG GCG ACC ATA ATG TCG TT-3′ Amigo2 R: 5′-GCA TCC AAC AGT CCG ATT CT-3′ F: 5′-CAC CGC CAA GAA TCG CTA C-3' C3 R: 5′-GAT CAG GTG TTT CAG CCG C-3' F: 5′-GGG GTC ACT GTC TGA CCA CT-3′ Gbp2 R: 5′-GGG AAA CCT GGG ATG AGA TT-3′ F: 5′-GGG GCA ATA GCT CAT TGG TA-3′ Ligp1 R: 5′-ACC TCG AAG ACA TCC CCT TT-3′ F: 5′-CTT CAG ATG CAA GCA ACA A-3′ Fbln5 R: 5′-AGG CAG TGT CAG AGG CCT TA-3′ F: 5′-GTG AAC AGC ATG AGG GGT TT-3′ Ggta R: 5′-GTT TTG TTG CCT CTG GGT GT-3′ F: 5′-CCT CTG GCT GTG GAC AAA AT-3′ S100a10 R: 5′-CTG CTC ACA AGA AGC AGT GG-3′ F: 5′-AAC AAG CTC TGT TGC CCA TT-3′ Ptx3 R: 5′-TCC CAA ATG GAA CAT TGG AT-3′ F: 5′-CTT CAA TCC TCC TCG ACT GG-3′ Clcf1 R: 5′-TAC GTC GGA GTT CAG CTG TG-3′ Slc10 F: 5′-GCT TCG GTG GTA TGA TGC TT-3′ 120

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R: 5′-CCA CAG GCT TTT CTG GTG AT-3′ F: 5′-TCC TGG AAC AGC AAA ACA AG-3' Gfap R: 5′-CAG CCT CAG GTT GGT TTC AT-3' F: 5′-GAG ATC ACT GGC AAG CAC GA-3' Hsbp1 R: 5′-ATT GTG TGA CTG CTT TGG GC-3' F: 5′-CAA ACG CCG AGT ACC TTG CT-3' Steap4 R: 5′-CAG ACA AAC ACC TGC CGA CT-3' F: 5′- CGC TAG AGC AGA TAC CAC GA-3' Timp1 R: 5′- CCA GGT CCG AGT TGC AGA AA-3' Housekeeping gene F: 5′-CGG CTA CCA CAT CCA AGG AA-3′ 18S R: 5′-GCT GGA ATT ACC GCG GCT-3′. Human genes F: 5'-CCT CTG CTC TTC AAC CTG ATC-3' NLRX1 R: 5'-CCT CTC GAA ACA TCT CCA GC-3' F: 5'-ACT TTG GAG TGA TCG GCC-3' TNF R: 5'-GCT TGA GGG TTT GCT ACA AC-3' F: 5'-GAT TCC ACC CAT GGC AAA TTC-3' GAPDH R: 5'-AGC ATC GCC CCA CTT GAT T-3'

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4. DISCUSSION

MS is a chronic inflammatory disease of the CNS, manifested by relapses in the early phase followed by progressive disability in the later phase (Reich et al., 2018). Innate immune response plays a critical role in driving both phases, however, the current MS treatments mainly target peripheral immune response with limited effect on CNS inflammation (Torkildsen et al., 2016). Since the trigger of inflammation is still unknown, developing novel treatments to prevent CNS inflammation seems like an impossible approach in MS. However, natural endogenous inhibitors of inflammation can potentially serve as therapeutic targets within the CNS to prevent the onset and progression of MS. In this study, we uncovered the cellular and molecular mechanisms of anti-inflammatory NLRs in the onset and progression of MS. Specifically, we studied the anti-inflammatory functions of NLRP12 and NLRX1 in regulating innate and adaptive immune responses during CNS inflammation. We used in vitro assays and several EAE models including, active, passive, and spontaneous EAE. Here I discuss our findings in two main sections consist of NLRP12 and NLRX1 studies.

4.1. NLRP12 and the inhibition of CNS inflammation

4.1.1. NLRP12 and EAE

NLRP12 is an inhibitor of NF-B pathway and its expression is restricted to immune cells. Several studies have reported the bifunctional nature of NLRP12 in a variety of experimental designs (Allen et al., 2012, Arthur et al., 2010, Vladimer et al., 2012). On one hand, in non- sterile inflammation such as inflammation induced by bacteria including Yersinia Pestis and Plasmodium infections, NLRP12 interacts with ASC to form inflammasome and induce inflammation (Wang et al., 2002, Vladimer et al., 2012, Ataide et al., 2014). On the other hand, NLRP12 p1ays an anti-inflammatory role in sterile-inflammation such as experimental model of colitis and colon cancer (Allen et al., 2012, Zaki et al., 2011). Despite the large number of studies, the role of NLRP12 remains unclear. The conflicting results about the functional role of NLRP12 are also observed in active EAE, in which the CNS inflammation is induced in mice by the antigen-adjuvant (MOG- CFA) immunization. We found that NLRP12 inhibits the progression and severity of active EAE (Gharagozloo M et al., 2015). In contrast to our finding, Lukens et al. observation

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123 supports the inflammatory function of NLRP12 to provoke CNS inflammation (Lukens et al., 2015). These findings support the dual immunoregulatory nature of NLRP12 that may vary in the different experimental systems in a cell-specific or stimulus-specific manner. Beyond the role of NLRP12 in the progression of EAE, we further investigated whether NLRP12 could inhibit the onset of EAE. We used myelin-specific TCR transgenic 2D2 mice that have high numbers of myelin-specific CD4+ T cells. Very low frequency of these animals (4%) develop spEAE. Since NLRP12 is an anti-inflammatory NLRs and suppress NF-B in immune cells, we wondered whether Nlrp12-/- 2D2 mice would develop spEAE more than 2D2 mice. Surprisingly, none of Nlrp12-/- 2D2 mice developed spEAE, while a very low rate of spEAE (6%) was found in 2D2 mice. This observation shows that Nlrp12 plays an opposite role in active EAE and spEAE. Our finding in spEAE model is similar to Lukens’ observation in active EAE (Lukens et al., 2015), showing the role of NLRP12 in potentiating CNS inflammation. These findings highlighting the bifunctional nature of NLRP12. Given the fact that NLRP12 expression is restricted to immune cells, its immunoregulatory function varies depending on the modes of immunological challenge (Gharagozloo et al., 2018b). For instance, NLRP12 plays an anti-inflammatory role in active EAE, where MOG-CFA immunization strongly activates peripheral immune cells, particularly CD4+ T cells and macrophages, resulting in infiltration of peripheral immune cells into the CNS. In spEAE, however, there is no immunization, and compared to active EAE, the immunological challenge on the side of periphery is very mild. In this case, NLRP12 serves as an inflammatory molecule. Given the fact that Nlrp12 ligand is still unknown, what exactly tunes NLRP12 response as a proinflammatory or anti-inflammatory molecule, needs further investigation.

4.1.2. NLRP12 and the CNS immune response

In response to the CNS insults such as infection or tissue injury, glial cells become reactive and increase in numbers. The reactive glial response can be measured by the expression of astrocyte or microglia marker (GFAP and Iba1 respectively). We found higher extent of astrogliosis and microgliosis in the spinal cords of Nlrp12-/- mice associated with increased expression of Il-1b and Ccr5 in Nlrp12-/- spinal cords compared to WT, which supports the notion of an increased influx of inflammatory cells into the CNS of these mice. CCR5 is

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124 primarily expressed by DC, monocytes, macrophages, effector and memory T cells (Barmania and Pepper, 2013). Also, the over-expression of IL-1β results in the permeability of the BBB, leading to the infiltration of leukocytes into the CNS (Argaw et al., 2006). These results demonstrate that lack of NLRP12 increases the infiltration of leukocytes into the CNS, which eventually enhances the CNS inflammation. Our in vitro results demonstrate that NLRP12 suppress the inflammatory response of microglia, supported by increased production of inflammatory mediators such as iNOS, NO, TNF, and IL-6 from Nlrp12-/- microglia cells following stimulation with LPS (Gharagozloo et al., 2015). Similarly, a report by Lukens et al. also found increased inflammatory response in the CNS tissue of Nlrp12-/- mice although, microglia responses per se were not determined (Lukens et al., 2015).

4.1.3. NLRP12 and the peripheral immune response

NLRs have recently gained more attention in T cell immunology, since 3 members of the family including CIITA, NLRC5, and NLRP3 regulate the transcription of molecules that shape T cell immune response. CIITA (Reith et al., 2005), and NLRC5 (Neerincx et al., 2014, Meissner et al., 2012) show transcriptional activities for MHC II and MHC I molecules respectively, while NLRP3 (Ting and Harton, 2015, Bruchard et al., 2015) acts as a transcription factor and promote Th2 differentiation and IL-4 production. These novel findings highlight the key role of NLR molecules in shaping T cell response and adaptive immunity. Past reports demonstrated the expression of NLRP12 in myeloid cells. However, the expression of NLRP12 in T cells has been verified recently (Lukens et al., 2015), which opens the possibility that NLRP12 controls T cell response. Several publications demonstrate the importance of Th1 and Th2 balance in the pathogenesis of MS, where a shift from a Th1 towards Th2 response has a beneficial effect on the clinical course of the disease. Additionally, it is shown that the therapeutic effect of glatiramer acetate, an MS disease modifying-drug, is associated with promoting Th2 response (Valenzuela et al., 2007). We studied the immunomodulatory role of NLRP12 in balancing Th1 and Th2 responses in active EAE and we found that NLRP12 favors Th2 response in the lymph nodes. We found increased ratio of Th1/Th2-related cytokines (IFNγ/IL-4) in the lymph nodes from

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Nlrp12-/- EAE mice. Strikingly, the regulatory effect of NLRP12 on balancing T cell response in lymph nodes was completely opposite of the CNS, where we found decreased ratio of Th1/Th2-related cytokine in the spinal cord of Nlrp12-/- EAE mice. Our finding is consistent with Lukens’ observation of the increased production of IL-4 in the CNS of Nlrp12-/- EAE mice (Lukens et al., 2015). These findings suggest the prominent inhibitory effect of NLRP12 on Th1/Th2 balance in lymph nodes, where T cells are primarily activated. Using in vitro assays, we found that NLRP12 inhibits T cell proliferation, activation, and IFNγ production in a T cell-intrinsic manner (Gharagozloo et al., 2018b, Gharagozloo et al., 2015). Our data are consistent with Lukens’ observation of elevated secretion of Th1/Th2/Th17 cytokines by Nlrp12-/- T cells compared to WT T cells (Lukens et al., 2015). Additionally, we found that NLRP12 does not play a role in the differentiation of naïve T cells to Th1 and Th17 subsets (Gharagozloo et al., 2018b). Our finding, however, is in contrast to Cai et al., who reported a reduced Th1 and Th17 differentiation in Nlrp12-/- T cells, while Th2 differentiation remained similar to WT T cells (Cai et al., 2016). Another study by Silveira et al. showed that Nlrp12-/- T cells produced more IFNγ compared to WT controls in response to Brucella abortus in vivo (Silveira et al., 2016). They reported the increased IL-12 production by macrophages upon B. abortus infection that skewed T cell differentiation to Th1 (Silveira et al., 2016). Overall, it appears that the type of stimulus and the condition of cells in vitro influence the regulatory role of NLRP12 in T cells, which give rise to variable outcome. Collectively, the consistent findings of NLRP12 inhibitory effect on T cell activation and proliferation support the idea that NLRP12 regulate the early TCR signaling pathways that are associated with T cell priming. Notably, we found increased expression of Nlrp12 in T cells upon TCR stimulation, indicating the association of NLRP12 with TCR activation. Early TCR signaling pathways include 3 main pathways: (i) calcineurin (a Ca2+/calmodulin-dependent phosphatase), (ii) MAPK, and (iii) NF-B signaling pathways. TCR activation triggers these 3 pathways, which lead to the activation of nuclear factor of activated T cell (NFAT), AP1, and NF-κB transcription factors. The 3 transcription factors including NFAT, AP1, and NF-κB bind to the promoter of IL-2 gene and initiate IL-2 transcription (Smith-Garvin et al., 2009a).

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We found increased production of IL-2 by Nlrp12-/- T cells, which led us to evaluated whether NLRP12 inhibits the phosphorylation of p65 (NF-B subunit) in T cells. Lukens et al. showed that NLRP12 inhibits canonical and non-canonical NF-B pathways in purified T cells activated with anti-CD3 plus anti-CD28 in vitro (Lukens et al., 2015). However, in our study, we tested NLRP12 regulatory function in early TCR signaling pathways, right downstream of TCR without CD28 co-stimulation. This is a very important event of T cell activation, which demonstrates the T-cell intrinsic regulatory effect of NLRP12 in immune response, even before T cells interact with co-stimulatory molecules on APC and commitment of the cells to various T helper subsets (Smith-Garvin et al., 2009b). We further investigated Akt activation upstream of NF-B in activated Nlrp12-/- and WT T cells and we found that NLRP12 inhibits Akt phosphorylation in activated T cells, which subsequently affects downstream pathways including NF-κB pathway. However, NLRP12 does not affect Ca2+/calmodulin signaling pathway in T cells. (Gharagozloo et al., 2018b).

Taken together, using in vitro and in vivo assays, our study uncovers the T cell-intrinsic regulatory role of NLRP12 in the context of CNS inflammation and provides a detailed picture of NLRP12 activity in regulating TCR signaling pathways. Although the inhibitory role of NLRP12 in microglia and T cells can inhibit the progression and severity of active EAE, its anti-inflammatory activity seems to be insufficient to prevent the onset of spEAE. It might be due to the fact that NLRP12 expression is restricted to immune cells, thereby its regulatory function is more dominant in peripheral immune response compared to the CNS. In the second aim of our study, we investigated the inhibitory role of NLRX1 in the regulation of CNS inflammation and MS immunopathogenesis.

4.2. NLRX1 and the inhibition of CNS inflammation

4.2.1. NLRX1 in EAE and MS

NLRX1 is a mitochondrial innate immune sensor that ubiquitously expressed and inhibits inflammation via suppressing NF-κB signaling pathway. A previous study by Eitas et al. showed the protective role of NLRX1 in the progression of active EAE, in which Nlrx1-/- mice developed more severe EAE compared to WT mice following immunization with MOG-CFA (Eitas et al., 2014). We hypothesized that NLRX1 inhibits the very early stages of the CNS inflammation and prevents the onset of spontaneous EAE (spEAE). To address

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127 our hypothesis, we generated a new mouse model of MS by crossing Nlrx1-/- mice with myelin-specific TCR transgenic mice (2D2). The resulting progeny are genetically susceptible to spEAE due to high numbers of autoreactive T cells and absence of Nlrx1 protection. Nlrx1-/- 2D2 mice develop spEAE, about 10 times more than 2D2 mice, indicating that NLRX1 prevents the onset of EAE. Interestingly, spEAE frequency was similar between males and females and the classical EAE symptoms appeared in the highest frequency at young age between 6-9 weeks. Several mechanisms are proposed in the literature that regulate the onset and severity of spEAE in different models of TCR transgenic mice. In TCR transgenic SJL mice, Zhang et al. showed that the age-associated development of spEAE correlates with a decline in regulatory function of T cells and the production of anti-inflammatory cytokine, IL-10, following stimulation with myelin antigen (Zhang et al., 2008). They suggest that regulatory T cells control the onset of spEAE. Another study showed that the induction of gut dysbiosis triggers the development spEAE in young adult mice, by upregulating the expression of C3 and downregulating Foxp3 gene in the spleen of young adult mice. Consequently, they suggest that gut dysbiosis breaks peripheral tolerance and trigger the activation of encephalitogenic T cells (Yadav et al., 2017). We also found that Nlrx1-/-2D2 spEAE animals develop an acute EAE with a progressive course and no recovery that resembled acute progressive course of EAE. Similar acute progressive EAE course has been previously reported in PLP-TCR transgenic mice in SJL background and Tnfr2-/-2D2 female mice only (Miller et al., 2015, Waldner et al., 2000). Interestingly, we observed a seasonal pattern in the onset of the disease, in which spEAE was most frequent in the summer and the least frequency was observed in fall and winter. To best of our knowledge, this is the first report of the seasonal pattern in the onset of spEAE. Similarly, it is shown that the season at immunization significantly influence the susceptibility to EAE, in which the chances of being affected are 90% higher for those mice immunized in the summer compared to those injected in the winter (Teuscher et al., 2004). Several studies show that seasonal variation is a complex combination of endogenous circannual rhythm driven and synchronized by light and melatonin (Chemineau et al., 2008). The fact that the seasonal variation was detected under controlled environmental conditions suggests that an endogenous circannual rhythm may control the onset. In MS patients, there

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128 are studies that show seasonal changes in disease activity (Farez et al., 2015, Watad et al., 2017). Comparing to rare cases of spEAE in 2D2, we found higher EAE clinical score and increased spinal cord lesions in Nlrx1-/-2D2 spEAE mice, indicating the anti-inflammatory role of NLRX1 in the severity and progression of the disease. This finding is consistent with the inhibitory effect of NLRX1 in the progression of active EAE (Eitas et al., 2014). Taken together, our findings support the notion that NLRX1 is an effective inhibitor of the CNS inflammation, which is able to prevent both onset and progression of EAE. The unique wide-range inhibitory function of NLRX1 is associated with its global expression in all cells at the periphery and the CNS. To the best of our knowledge, our report is the first to show a natural endogenous inhibitor of inflammation implicated in the pathogenesis of MS. However, the key role of NLRX1 as an endogenous inhibitor of inflammation is poorly understood in human MS. Plenty of research demonstrate the contribution of inflammatory PBMCs including lymphocytes and APC in the pathogenesis of MS (Weissert, 2013). We quantified the expression of NLRX1 in PBMCs of patients with RRMS and healthy controls and we found higher levels of NLRX1 expression in PBMCs from RRMS than healthy controls. Measuring mRNA levels of TNFα revealed no difference in the expression of TNFα between MS and control groups. However, a significant positive correlation between the mRNA levels of NLRX1 and TNFα was found in the PBMCs from RRMS patients. Similarly, we found a significant increase in the mRNA levels of Nlrx1 in both brain and spinal cord tissues of EAE mice compared to healthy mice. This finding suggests that NLRX1 expression varies depends on the inflammatory status of the cells, in which increased expression of NLRX1 keeps the increased level of inflammatory marker in check. What exactly tunes NLRX1 expression and function according to the endogenous inflammatory signals needs further investigation. Several reports demonstrate the genetic variants of NLRs that increase the susceptibility to inflammatory and autoimmune diseases in human. They are mainly polymorphisms of inflammatory NLRs including NLRP1 and NLRP3 (Magitta et al., 2009, Alkhateeb et al., 2013, Malhotra et al., 2015, Bernales et al., 2017). Regarding anti-inflammatory NLRs, mutations have been found in NLRP12 that are associated with autoinflammatory syndromes, including some forms of familial cold auto-inflammatory syndromes, atopic dermatitis and

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129 hereditary periodic fever (I Jéru et al., 2008; Isabelle Jéru et al., 2011; Kanazawa, 2014). A recent study provides evidence of a single nucleotide polymorphism in NLRX1 (rs4245191) that increases the risk of type 2 diabetes mellitus and diabetic cerebral infarction (Zeng et al., 2017). However, the genetic association between NLRX1 and MS is poorly understood. In collaboration with Dr. Carles Vilariño-Güell at the University of British Colombia, we studied mined exome sequencing data from MS patients and healthy controls. His group identified five missense mutations and one nonsense mutation in MS patients. They genotyped these six variants in a large cohort of MS patients and healthy controls. They identified families affected with MS carrying the NLRX1 nonsense mutation. This finding suggests that NLRX1 can be a genetic risk factor associated with MS. All missense mutations are located in NBD domain of NLRX1 (p.Lys172Asn, p.Pro189Leu, p.Leu237His, and p.Arg471Trp) except one of them that is found in the C- terminal domain (p.Arg860Trp). NBD domain promotes oligomerization and activation of the NLRX1, while C-terminal domain is a ligand-sensing domain (Gharagozloo et al., 2018a). The nonsense mutation results in the change of Glutamic acid to the stop codon (p.E192X) and production of a truncated NLRX1 without full-length NBD and ligand- sensing domains. How NLRX1 mutations affect protein function is currently under investigation in our lab. Next, I explain the mechanism of NLRX1 anti-inflammatory function at the levels of CNS and periphery.

4.2.2. NLRX1 and the CNS immune response

Innate immune response provides the first line of defense in the CNS. Cell death and tissue damage in the CNS trigger innate immune response that leads to the activation and recruitment of activated macrophages and T cells from the periphery (Lampron et al., 2013). In this regard, we investigated the anti-inflammatory activity of NLRX1 in the onset of spEAE. We found severe inflammation and demyelination in the spinal cords of Nlrx1-/-2D2 spEAE mice, associated with massive infiltration of inflammatory macrophages, enhanced gliosis and elevated expression of inflammatory mediators including Tnf, Il-1b, and, Ccr5. Similarly, in active and passive models of EAE, Eitas et al. showed that NLRX1 attenuates microglia inflammatory activities (Eitas et al., 2014).

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In addition to their inflammatory response, microglia and macrophages are APC that can interact with T cells via antigen processing and presentation. We next investigated whether NLRX1 affects T cell activation via modulating the antigen presenting capacity of APC. We found a comparable efficacy of WT APC and Nlrx1-/-APC in activating T cells, indicating that the immunoregulatory role of NLRX1 in innate immune response is beyond antigen presentation and T cell activation. We performed a series of adoptive transfer experiment in lymphocyte-deficient Rag-/- and Nlrx1-/-Rag-/- mice to understand the anti- inflammatory function of NLRX1 in a complex interaction of cells and cytokines in vivo. We observed a severe hind limb paralysis and higher EAE clinical score in Nlrx1-/-Rag- /- mice, which was correlated with focal demyelination and inflammation in the spinal cord. Elevated expression of Iba1 revealed a significant increase in macrophage/microglia activation in Nlrx1-/-Rag-/- mice compared to Rag-/- mice. All together, these findings demonstrate the inhibitory function of NLRX1 in innate immune cells. We next evaluated the activation of innate immune cells and the expression of inflammatory mediators in the brains of asymptomatic animals and we found early signs of inflammation in the brains of Nlrx1-/-2D2 mice compared to 2D2. Interestingly, we found no myelin-specific T cells in the CNS of Nlrx1-/-2D2 mice compared to 2D2 mice. These data provide the evidence that NLRX1 suppresses subclinical and early stages of inflammation in the CNS, which involves local cells and is independent of T and B cells. In addition to inflammatory signs, we found a significant increase in the levels of danger signal, HMGB1, in the spinal cords of Nlrx1-/-2D2 mice, indicating the status of cell death and tissue damage at the subclinical stage. A group of cells that may be involved in inducing cell death and CNS damage are neurotoxic astrocytes (Liddelow et al., 2017). Ben Barres’ group discovered two different phenotypes of reactive astrocytes, A1 or A2 astrocytes, that are induced in CNS inflammation (Liddelow et al., 2017, Zamanian et al., 2012). A1 astrocytes up-regulate many classical complement cascade genes and are shown to be damaging to neurons and oligodendrocytes, while A2 astrocytes up-regulate many neurotrophic factors and are protective to the CNS. A1 astrocytes are induced by microglial inflammatory mediators such as IL-1α, TNFα, and C1q and induce the death of neurons and oligodendrocyte. The presence of A1 astrocytes is shown in many human neurodegenerative diseases including MS (Liddelow et al., 2017). In the brains of asymptomatic Nlrx1-/-2D2

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131 mice, we found a significant increase in the expression of A1-related astrocyte genes, while A2-related astrocyte genes were significantly decreased compared to 2D2 mice. We also found that Nlrx1-/- glial cells produce more TNFα upon stimulation with LPS/IFN in vitro, which possibly contributes to the increased expression of A1-related genes in Nlrx1-/- astrocyte. Liddelow et al. showed that A1 astrocytes could cause neurodegeneration by secreting an unknown soluble neurotoxin that rapidly kills neurons and mature oligodendrocytes (Liddelow et al., 2017). The culture medium collected from LPS/IFN-treated Nlrx1-/- glial cells induce an enhanced level of cell death in both N2A (neuroblastoma) and MO3.13 (oligodendrocytes) cell lines, suggesting that NLRX1 inhibit the production of neurotoxin by A1 astrocyte. Given the fact that NLRX1 is a NF-B inhibitor, it is possible that NF-B signaling pathway is involved in the production of mysterious neurotoxin by astrocytes. The nature and the identity of that soluble neurotoxin await further investigation. Taken together, our results demonstrate that NLRX1 plays a key role in guarding the CNS and preventing the onset of inflammation. NLRX1 prevents the function of innate immune response and the generation of inflammatory microglia and neurotoxic astrocytes. We propose a model that supports the inside-out model of MS. NLRX1 inhibits the CNS inflammation in the subclinical stage, which primarily begins in the absence of myelin- specific T cells. A neuronal/oligodendrocyte injury releases myelin antigen that triggers the immune response in the periphery and recruits autoreactive T cells to the CNS, which cause inflammation and demyelination in the clinical stage of the disease. The mechanism of NLRX1 action in inhibiting the CNS inflammation and preventing the onset of MS is summarized in a model published in our third article.

4.2.3. NLRX1 and the peripheral immune response

Activation of myelin-specific T cells in the periphery and infiltration into the CNS is an important step in the immunopathology of MS (Dendrou et al., 2015a). We found a marked infiltration of myelin-specific T cells, mainly Th1 subset, to the spinal cord of Nlrx1-/-2D2 spEAE mice. These findings parallel other mouse models of spEAE (Bettelli et al., 2004, Pöllinger et al., 2009) and demonstrate the importance of Th1 cells in the development of

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132 spEAE. For example, it is shown that Tbet-/-2D2 mice are protected from the development of EAE(Bettelli et al., 2004). Our in vitro experiments show the inhibitory effect of Nlrx1 on T cell activation and proliferation. Additionally, we found that Nlrx1 significantly inhibits the differentiation of naïve T cells to myelin-specific Th1 or Th17 cells in vitro. Consistent with our findings, a previous study by Leber et al., reported the greater proliferation rates and the higher ability of Nlrx1-/- T cells to differentiate into Th17 phenotype (Leber et al., 2017a). However, Nlrx1 has no effect on the differentiation of T cells to Treg cells (Leber et al., 2017a). Taken together, NLRX1 inhibits encephalitogenic T cell response. The molecular mechanisms of NLRX1 regulation of T cell function and TCR signaling are poorly understood and need further investigation.

4.3. Conclusion and future perspectives

The healthy immune system has a homeostatic balance between cells and molecules, which are maintained with a variety of regulatory mechanism. In the context of MS, the mechanism of homeostatic balance in T cell response have been extensively investigated. For instance, the classical Th1/Th2 paradigm provides a useful model for understanding the pathogenesis of MS (Romagnani, 1997). The latest progress in the study of Th subsets has opened a new venue to reconsider the etiology of immune-mediated inflammatory diseases beyond the Th1/Th2 balance. The opposing immune functions of Treg and Th17 lymphocytes and the plasticity of Treg/Th17 differentiation introduced the Treg/Th17 paradigm in the pathology of MS (Jamshidian et al., 2013). Similar to the balance of T cell response, several studies show the disruption of immune balance between different types of innate immune cells including M1/M2 macrophage/microglia (Martinez and Gordon, 2014, Amici et al., 2017) or A1/A2 astrocytes (Liddelow et al., 2017) in MS. Taken together, a healthy immune system maintains the balance between inflammatory and anti-inflammatory response. Considering NLRs as regulators of inflammation, I propose a model that explains how NLRs maintain the balance of immune response. According to the model, infection and tissue injury trigger inflammatory NLRs that initiate inflammatory response. The increased activation of inflammatory NLRs and the impaired function of anti-inflammatory NLRs lead to CNS inflammation and MS (Figure 6).

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In MS, the CNS is damaged by a complex inflammatory process that is present at all stages of the disease. The damage starts by the infiltration of T cells, B cells, and monocytes to the CNS. Then it becomes less inflammatory and more neurodegenerative in progressive phase, in which microglia are involved in ongoing CNS inflammation (Hemmer et al., 2015). Therefore, the dysregulated unbalanced inflammatory response in innate and adaptive immune subsets cause the onset and progression of MS. Numerous disease-modifying therapies have been developed over the past 3 decades, which the majority of them are only effective in RRMS but not in progressive MS (Dargahi et al., 2017). These pieces of evidence highlight the need for the development of new therapies that stop the triggers of inflammation at the initial phase and prevent new CNS damage. Over the past decades, many experimental drugs have been developed to inhibit inflammatory function of NLRs (Gharagozloo et al., 2018a). Targeting anti-inflammatory NLRs, however, is a novel approach in regulation of inflammation, which is poorly addressed so far. The current study provides a detailed picture of immunoregulatory mechanisms of NLRX1 and NLRP12 in MS and suggest them as the potential candidates for developing novel treatments for MS. The ligands for NLRX1 and NLRP12 is still unknown and the nature of their biological trigger remains poorly described. However, a recent study reported a number of NLRX1 binding molecules that are found in our food including punicic acid and docosahexaenoic acid (Lu et al., 2015). These molecules suppress the NF-κB activity in macrophages in vitro and in the dextran sulfate sodium (DSS) model of colitis in a NLRX1- dependent manner. This study shows a great potential of NLRX1 in the treatment of inflammatory diseases such as MS. NLRX1 is ubiquitously expressed and its global anti-inflammatory affects different cell types in the periphery and the CNS. This unique property makes NLRX1 an exceptional candidate for treatment of MS, particularly progressive MS. NLRX1 can also be considered as a potential risk factor for MS.

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Figure 6. NLRs balance the inflammatory response in the CNS. Pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) trigger the immune response via NLRs. Upon sensing ligands, inflammatory NLRs not only initiate inflammatory response in innate immune cells, such as macrophages and microglia, but also bridge the immune response from innate to adaptive immune response via instructing T cell response by dendritic cells (DCs) to generate different subsets of pathogenic T helper subsets (e.g., Th1, Th17). On the other hand, anti-inflammatory NLRs inhibit the production of inflammatory mediators by macrophages and microglia, suppress the differentiation of T cells to inflammatory subsets, and protect neurons from necrosis. Thereby, the increased activation of inflammatory NLRs and the impaired function of anti-inflammatory NLRs lead to central nervous system inflammation and demyelination in MS (Gharagozloo et al., 2018a).

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ACKNOWLEDGMENTS

First of all, I would like to express my deep appreciation and gratitude to my wonderful supervisor, Dr. Denis Gris, for his wonderful mentorship, encouragement, thoughtfulness, patience, and continuous support. I am also grateful to Dr. Abdelaziz Amrani, for his support, constructive comments and constant encouragement. I would like to thank my lovely friends in the lab, Camille, Shaimaa, and Kenzo, for their kindness and helpfulness. They maintain a relaxed atmosphere in the lab, which made it not only stimulating to work but also fun. My sincere thanks to the Jury Committee, Dr Abdelaziz Amrani, Dr Xavier Rouncou and Dr Samuel David, for the evaluation of this work and their constructive comments. I would like to thank the healthy volunteers and the patients at the MS Clinic, CHUS, 400 Sherbrooke, QC, Canada for participating in our study. We are grateful to MS nurse, Geneviève Morin, at the Department of Neurology, CHUS, for taking patients’ blood samples. I’d like to acknowledge MS Society of Canada, Fonds de Recherche 413 du Québec-Santé (FRQS) and Association de la sclérose en plaques de l’Estrie (ASPE) for my PhD scholarship. Last, but not least, I thank my family. No words can describe the care, guidance, love, understanding, and support they tirelessly gave me.

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