Contribution of glial cells to pathology of coronavirus induced demyelination

Neural precursor cell (NPC) transplantation has emerged as a therapeutic option to treat several neurological disorders. Intracranial infection of mice with the JHM strain of mouse hepatitis virus (JHMV) results in a chronic demyelinating disease and thus has been used as a model to study multiple sclerosis (MS). The cause of MS remains unknown but viruses have been attributed to trigger the disease in genetically susceptible individuals, making it imperative to study the remyelination potential of NPCs in a virally induced demyelinating disease. We have previously shown that engraftment of fetal derived GFP-NPCs in spinal cords of JHMV-infected mice with established demyelination results in remyelination and axonal sparing. We have previously demonstrated that transplanted GFP-NPCs are susceptible to JHMV-induced cell death. From a clinical standpoint, donor-specific induced pluripotent stem cells (iPSC)-derived NPCs may be preferable to avoid the use of immunosuppressive drugs. Therefore, we sought to investigate whether iPSC derived NPCs are functionally similar to fetal-derived NPCs and whether they are susceptible to JHMV infection in a preclinical setting. iPSCNPCs are similar to GFP-NPCs in functionality as they are able to differentiate into oligodendrocytes, astrocytes, and neurons. However, iPSC-NPCs express low levels of the viral receptor CEACAM1a, making them resistant to JHMV infection and viralinduced cytopathic effects. Activated microglia can prevent maturation of oligodendrocyte precursor cells (OPCs) to myelinating oligodendrocytes and promote oxidative damage by releasing nitric oxide. In the presence of environmental insults, activated microglia can release IL- 6, IL-23, IFN-γ and TNF-α, all of which can be neurotoxic and neuroinflammatory. Microglia can also contribute to adaptive immunity by presenting antigen to CD4+ and CD8+ T cells that have entered the central nervous system (CNS) in response to infection. In order to determine the contribution of microglia to disease progression in the JHMV model, we used colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 to deplete microglia in CNS of JHMV-infected mice. Microglia depletion results in increased viral titers, morbidity and, mortality and also modulates the immunological landscape of the CNS of JHMV-infected mice.

CONTRIBUTION OF GLIAL CELLS TO PATHOLOGY OF CORONAVIRUS INDUCED DEMYELINATION by Vrushali Mangale A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Immunology Department of Pathology The University of Utah May 2019 Copyright © Vrushali Mangale 2019 All Rights Reserved The University of Utah Graduate School STATEMENT OF DISSERTATION APPROVAL The dissertation of Vrushali Mangale has been approved by the following supervisory committee members: Thomas Lane , Chair 2/15/2019 Robert Fujinami , Member 2/15/2019 Dean Tantin , Member 2/15/2019 Alex Shchleglovitov , Member 2/15/2019 June Round , Member 2/15/2019 and by Peter Jensen the Department/College/School of and by David B. Kieda, Dean of The Graduate School. Date Approved Date Approved Date Approved Date Approved Date Approved , Chair/Dean of Pathology ABSTRACT Neural precursor cell (NPC) transplantation has emerged as a therapeutic option to treat several neurological disorders. Intracranial infection of mice with the JHM strain of mouse hepatitis virus (JHMV) results in a chronic demyelinating disease and thus has been used as a model to study multiple sclerosis (MS). The cause of MS remains unknown but viruses have been attributed to trigger the disease in genetically susceptible individuals, making it imperative to study the remyelination potential of NPCs in a virally induced demyelinating disease. We have previously shown that engraftment of fetal derived GFP-NPCs in spinal cords of JHMV-infected mice with established demyelination results in remyelination and axonal sparing. We have previously demonstrated that transplanted GFP-NPCs are susceptible to JHMV-induced cell death. From a clinical standpoint, donor-specific induced pluripotent stem cells (iPSC)-derived NPCs may be preferable to avoid the use of immunosuppressive drugs. Therefore, we sought to investigate whether iPSC derived NPCs are functionally similar to fetal-derived NPCs and whether they are susceptible to JHMV infection in a preclinical setting. iPSCNPCs are similar to GFP-NPCs in functionality as they are able to differentiate into oligodendrocytes, astrocytes, and neurons. However, iPSC-NPCs express low levels of the viral receptor CEACAM1a, making them resistant to JHMV infection and viralinduced cytopathic effects. Activated microglia can prevent maturation of oligodendrocyte precursor cells (OPCs) to myelinating oligodendrocytes and promote oxidative damage by releasing nitric oxide. In the presence of environmental insults, activated microglia can release IL6, IL-23, IFN-γ and TNF-α, all of which can be neurotoxic and neuroinflammatory. Microglia can also contribute to adaptive immunity by presenting antigen to CD4+ and CD8+ T cells that have entered the central nervous system (CNS) in response to infection. In order to determine the contribution of microglia to disease progression in the JHMV model, we used colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 to deplete microglia in CNS of JHMV-infected mice. Microglia depletion results in increased viral titers, morbidity and, mortality and also modulates the immunological landscape of the CNS of JHMV-infected mice. iv TABLE OF CONTENTS ABSTRACT....................................................................................................................... iii LIST OF FIGURES .......................................................................................................... vii LIST OF ABBREVIATIONS .......................................................................................... viii ACKNOWLEDGMENTS ................................................................................................ xii Chapters 1. INTRODUCTION .......................................................................................................... 1 1.1 Multiple Sclerosis ................................................................................................ 2 1.2 Mouse Hepatitis Virus as a Preclinical Model of MS ......................................... 6 1.3 Effect of Neural Precursor Cell Engraftment in the JHMV Model ................... 10 1.4 Microglia in Neuroinflammation ....................................................................... 13 1.5 Summary ............................................................................................................ 15 1.6 References ......................................................................................................... 15 2. NEURAL PRECURSOR CELLS DERIVED FROM INDUCED PLURIPOTENT STEM CELLS EXHIBIT REDUCED SUSCEPTIBILITY TO INFECTION WITH A NEUROTROPIC CORONAVIRUS................................................................................. 27 2.1 Introduction........................................................................................................ 28 2.2 Results and Discussion ...................................................................................... 29 2.3 Materials and Methods....................................................................................... 32 2.4 Acknowledgements............................................................................................ 33 2.5 References.......................................................................................................... 33 3. MICROGLIA AID IN HOST DEFENSE AND RESTRICT THE SEVERITY OF DEMYELINATION IN RESPONSE TO INFECTION WITH A NEUROTROPIC CORONAVIRUS.............................................................................................................. 35 3.1 Abstract .............................................................................................................. 36 3.2 Introduction........................................................................................................ 37 3.3 Methods ............................................................................................................. 39 3.4 Results................................................................................................................ 44 3.5 Discussion .......................................................................................................... 53 3.6 References.......................................................................................................... 59 4. CONCLUSIONS AND FUTURE DIRECTIONS ....................................................... 76 4.1 Therapeutic Application of iPSC Derived NPCs in a Viral Model of MS ........ 77 4.2 Role of Miroglia in Host Defense and Demyelination ...................................... 80 4.3 References.......................................................................................................... 82 APPENDIX: DIFFERENTIALLY EXPRESSED GENES FROM SINGLE CELL RNA SEQUENCING ANALYSIS FROM CHAPTER 3.......................................................... 85 vi LIST OF FIGURES 2.1. Generation of mouse iPSC-NPCs .............................................................................. 30 2.2. Expression of MHC Class I and II by iPSC-NPCs .................................................... 31 2.3. Surface CEACAM1a expression is reduced in iPSC-NPCs. ..................................... 32 2.4. JHMV infection of NPCs ........................................................................................... 33 3.1. Microglia depletion causes an increase in mortality and viral load .......................... 66 3.2. Microglia depletion influences the T cell infiltrating into the CNS of JHMV infected mice ................................................................................................................................... 67 3.3. scRNASeq unveils the immunological landscape in the brains of JHMV-infected mice treated with PLX5622 or control at day 7 p.i ........................................................... 68 3.4. Figure 3.4. Microglia depletion leads to differential gene expression in T cell subsets as revealed by scRNASeq.. ............................................................................................... 69 3.5. PLX5622 treatment leads to differential gene expression in macrophages infiltrating the CNS at day 7 p.i. ......................................................................................................... 70 3.6. Microglia depletion augments JHMV-induced neuroinflammation at day 14 p.i.. ... 71 3.7. Single cell RNA sequencing reveals the dynamic immunological landscape in the spinal cords of JHMV-infected mice treated with PLX5622 or control at day 14 p.i ...... 72 3.8. Microglia depletion results in reduced expression of T cell activation markers in T cell subsets in spinal cord at day 14 p.i............................................................................. 73 3.9. Microglia depletion results in modulation of the IFN response genes and MHC class II genes in macrophages infiltrating the CNS at day 14 p.i .............................................. 74 3.10. Microglia restrict the severity of JHMV-induced demyelination ............................ 75 LIST OF ABBREVIATIONS Aβ...................................................................................................................... amyloid beta AD .......................................................................................................... alzheimer’s disease AF ........................................................................................................................ alexaFluor APC ................................................................................................... antigen presenting cell ASC ................................................................................................... antibody secreting cell APP .............................................................................................. amyloid precursor protein BBB .........................................................................................................blood brain barrier BSA .................................................................................................... bovine serum albumin CAM ........................................................................................... cellular adhesion molecule CCR .................................................................................................................. C-C receptor CD ................................................................................................... cluster of differentiation CIS ........................................................................................... clinically isolated syndrome CNS ................................................................................................... central nervous system CRISPR ....................................... clustered regularly interspaced short palindromic repeats CSF ......................................................................................................... cerebrospinal fluid CSF1R .......................................................................... colony-stimulating factor 1 receptor CXCL ...............................................................................................................C-X-C ligand CXCR............................................................................................................C-X-C receptor DC .....................................................................................................................dendritic cell EGF ................................................................................................. epidermal growth factor FDA ........................................................................................ food and drug administration GFAP ....................................................................................... glial fibrillary acidic protein GFP ................................................................................................green fluorescent protein H&E .................................................................................................. hematoxylin and eosin HBSS .................................................................................. hank’s balanced saline solution HHV ........................................................................................................ human herpesvirus HLA .............................................................................................. human leukocyte antigen HD ......................................................................................................... huntington’s disease IBA1................................................................ ionized calcium binding adaptor molecule 1 IFN ......................................................................................................................... interferon IgG .......................................................................................................... immunoglobulin G IL .......................................................................................................................... interleukin IRF1 ........................................................................................ interferon regulatory factor 1 JHMV.......... j2.2v-1 variant of the John Howard Mueller strain of Murine Hepatitis Virus KO ..........................................................................................................................knock out LFB .................................................................................................................luxol fast blue MHC ............................................................................... major histocompatibility complex MHV ...................................................................................................murine hepatitis virus MOI .................................................................................................multiplicity-of-infection MRI .......................................................................................... magnetic resonance imaging MS ............................................................................................................. multiple sclerosis NG2 ......................................................................................................neural/glial antigen 2 ix NK ..................................................................................................................... natural killer NPC ....................................................................................................... neural precursor cell NSC .............................................................................................................. neural stem cell OCB .......................................................................................................... oligoclonal bands OCT......................................................................... optimal cutting temperature compound OPC ..................................................................................... oligodendrocyte progenitor cell PAMPS ................................................................... pathogen associated molecular patterns PD .......................................................................................................... parkinson’s disease p.i .....................................................................................................................post infection PE .................................................................................................................... phycoerythrin PFA .......................................................................................................... paraformaldehyde PFU ..................................................................................................... particle forming units PLX ....................................................................................................................... Plexxikon PPMS ....................................................................... primary progressive multiple sclerosis RAG1 ................................................................................. recombination activating gene 1 ROS ..................................................................................................reactive oxygen species RRMS ........................................................................ relapsing remitting multiple sclerosis SMI-32 ......... Sternberger Monoclonal Antibody-32 for nonphosphorylated neurofilament SPMS .................................................................... secondary progressive multiple sclerosis Th ............................................................................................................................. T helper Thy1 ..................................................................... cell surface glycoprotein regulatory gene TLR ............................................................................................................toll like receptors TMEV .................................................................. Theiler’s murine encephalomyelitis virus x TNF ......................................................................................................tumor necrosis factor TREM ......................................................... triggering receptor expressed on myeloid cells WT ......................................................................................................................... wild-type YFP ..............................................................................................yellow fluorescent protein xi ACKNOWLEDGMENTS I am thankful to my mentor Dr. Thomas E. Lane for his intellectual guidance and remarkable mentorship. His constant encouragement and support through my graduate studies has helped me stay productive, and I am truly fortunate to have worked in his lab. I would like to thank my committee members, Dr. Robert Fujinami, Dr. June Round, Dr. Alex Shchleglovitov, Dr. Dean Tantin, and the Department of Pathology for their insight and feedback in these past years. I would like to acknowledge the past and present members of the Lane lab for their constant support and encouragement. I would like to thank my husband Kaushik for his love and unwavering support. Lastly, I would like to thank my mother Smita, my father Aravind, and my sister Sayali for their constant love and support. CHAPTER 1 INTRODUCTION 2 1.1 Multiple Sclerosis Multiple Sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) with no known cure and is a leading cause of disability in young adults. MS is a neuroinflammatory disease characterized by the loss of myelin (1), a lipidenriched membrane that encases axons and is critical for maintaining saltatory conduction in axons. Oligodendrocytes are resident cells of the CNS that produce myelin yet their function is rendered dysfunctional in MS patients. Histological characterization of CNS tissue from MS patients reveal lesions consisting of inflammatory infiltrates including macrophages and activated lymphocytes (2). Perivascular infiltrates including CD4+ T cells, CD8+ T cells, B cells, antibody secreting cells (ASCs), and macrophages are thought to act with the resident reactive microglia to release a milieu of proinflammatory factors, that contribute to disruption of oligodendrocyte function as well as directly damage myelin (3, 4). Multiple demyelinating lesions in the white matter tracks manifest clinically into a variety of symptoms such as vision loss, cognitive decline, and impaired motor abilities (5, 6). Clinical signs of MS vary and may include fatigue, spasticity, gait instability, urinary incontinence, and cognitive decline and might be a result of malfunctioning of motor, sensory, visual, and brainstem pathways. During the early stages of the disease, most patients may experience episodic attacks that last for weeks and are clearly separated by periods of complete recovery of symptoms, a form of MS called relapsingremitting MS (RRMS) (7). The first clinical event in these patients called clinically isolated syndrome (CIS) could be incomplete myelitis, optic neuritis or brainstem syndrome (8). The remission phase could reflect reduced neuroinflammation and partial 3 restoration of nerve conduction due to remyelination (9, 10). Remyelination could be explained by the accumulation of oligodendrocyte precursor cells (OPCs) in the subacute lesions during the early stages of MS (11). OPC maturation into myelin-producing oligodendrocytes leads to patches of remyelinated white matter with thin myelin sheaths surrounding axons (5, 12, 13). The majority of patients (~85%) are initially diagnosed with RRMS and will eventually develop secondary progressive MS (SPMS) within a decade of their initial diagnosis, while the remaining 15% are initially diagnosed with primary progressive MS (PPMS) (14). Worsening of clinical symptoms without periods of remission marks progressive stages of the disease. There is significant neurological degeneration and higher disability in progressive MS patients due to increase in cortical white matter lesions that lead to worsening of motor capabilities. Progressive stage of the disease is characterized by severe axonal damage due to axonal transections along with grey matter neuropathy (15, 16). Studies from postmortem tissue of progressive MS patients reveal reduced leukocyte infiltration in the cortical lesions but a higher number of apoptotic neurons with transected axons (17). This suggests that demyelination and axonal damage, but not the adaptive immune system majorly contribute to the neurological pathology in progressive MS patients. In addition, while resident OPCs are found highly populated near chronic lesions, they fail to proliferate and differentiate to form mature myelin producing oligodendrocytes, and hence there is a very little possibility of endogenous remyelination (11, 18). Multiple factors including genetic predisposition, location, gender, and pathogen exposure have been attributed to the etiology of MS (19-24). While some ethnicities have almost no prevalence of MS, offspring of MS patients have a higher risk of developing 4 the disease, suggesting that certain risk alleles are associated with MS (25-27). Studies have shown a significantly higher concordance in monozygotic twins as opposed to dizygotic twins (28). The human leukocyte antigen (HLA) class II region of the HLADR-2 haplotype on chromosome 6p21 was shown to be associated with MS, and HLADRB1*1501 is the major susceptibility allele in MS patients from North America or European Caucasians (19, 29-31). Like many other autoimmune diseases, women are at a higher risk of developing MS with the female-to-male ratio varying between 1.5:1 and 2.5:1 (32) , indicating that this predisposition could be hormonally related. Interestingly, location of the individual from the equator is another factor associated with MS risk. People living in regions further away from the equator have a higher risk of getting the disease and people who move from low-risk to high-risk regions tend to acquire the higher risk (33-35). Another environmental factor related to location that has been considered as a risk factor is sunlight exposure. Low vitamin D levels, due to reduced sun exposure in the northern regions, are associated with an increase in MS risk and worsening of disease symptoms (33, 34, 36-39). The differences in MS risk based on geographic location can also be explained by exposure to microbial pathogens, particularly viruses. Two viruses that have been linked to MS are Epstein-Barr virus (EBV) and human herpervirus-6 (HHV-6) (22). The risk of developing MS is ~ 15-fold higher in those infected with EBV early in life and ~ 30-fold higher with those infected with EBV in adulthood (22). Malfunctioning of the immune system due to pathogen exposure could be explained by two mechanisms- molecular mimicry and bystander activation (40-42). The incidence of MS was also higher in smokers, with duration and intensity having a higher correlation regardless of age (43). A population-based study 5 revealed that smokers are 1.5 times more likely to develop the disease and passive smokers also have a higher MS risk than unexposed populations (43). Some other environmental factors that have been suggested to play a role are gut microbiota and oral contraceptive pills (44, 45). MS diagnosis is based on comprehensive patient history and neurological examination to determine the dissemination in time (DIT) of clinical symptoms as well as excluding mimickers (46). Other supportive diagnostic criteria include tests such as magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) analysis. While an MRI can help determine presence MS lesions, CSF analysis can identify inflammatory markers such as oligoclonal bands (OCB) and an elevated immunoglobulin G (IgG) index. These inflammatory markers were found in ~85% of MS patients (46). The 2010 revised McDonald criteria is the most widely used diagnostic criteria in the MS community. MS diagnosis can be based on presence of at least two typical clinical attacks or a single typical demyelinating event along with evidence of dissemination in space (DIS) and DIT by MRI (46, 47). MS is an incurable disease of the CNS, but several disease-modifying therapies (DMTs) are available to help patients at the RRMS stage of disease. These therapies are aimed at reducing T lymphocyte infiltration into the CNS and are aimed at preventing new lesion formation. Some DMTs approved by the United States Food and Drug Administration (FDA) include interferons (IFNs), Teriflunomide, Fingolimod along with monoclonal antibodies such as Natalizumab (targets cell adhesion molecule α4-integrin), Alemtuzumab (targets CD52), and Ocrelizumab (targets CD20) (48-53). With the exception of Ocrelizumab, which was recently approved for progressive MS, most of the 6 FDA approved DMTs are for RRMS. As remyelination failure is the hallmark of progressive MS, ultimately most FDA approved therapies are less than ideal. 1.2 Mouse Hepatitis Virus as a Preclinical Model of MS Animal models that have similar clinical symptoms and histopathology as MS patients are important to better understand the nuances of the disease. Several toxin-based rodent models use toxins such as cuprizone, lysolecithin to induce demyelination by having a direct toxic effect on oligidendrocytes. However, these models may not accurately represent stages of MS patients since they lack an autoimmune component. Experimental autoimmune encephalitis (EAE) is characterized by a T and B cell-based immune response against myelin-associated proteins and shares many pathological and clinical features with MS patients. However, many therapeutic mechanisms have not been translatable from EAE model to humans, even though EAE still remains the most commonly used model to study MS. Since viruses have been implicated as a possible environmental trigger for MS, viral models of MS present excellent tools to study the pathogenesis of MS (54, 55). There are two main neurotropic viral models of MS that have provided important insights into the pathology of MS: Theiler’s murine encephalomyelitis virus (TMEV) and John Howard Muller (JHM) strain of mouse hepatitis virus (MHV) (JHMV). The neurotropic JHMV j2.2v-1 variant is a well-characterized laboratory strain of MHV that can cause severe encephalomyelitis and demyelination in adult mice (56, 57). Mouse hepatitis virus (MHV) is a single-stranded RNA virus of the group II Coronaviridae family. MHV has a wide range of strains that can cause a variety of 7 pathologies in mice depending on the age, species of the mouse as well as the route of infection. Enterotropic MHV strains are highly contagious and cause severe enteritis and can cause damaging natural outbreaks in mouse colonies (58). Some MHV strains, such as MHV-2, can cause fulminant hepatitis but are mildly neurotropic and only cause limited and resolving meningitis when infected intracranially (59). However dual i.c infection of mice with neurotropic and hepatotropic strains leads to encephalomyelitis and demyelination while intraperitoneal (i.p) infection causes hepatitis (60, 61). JHMV is a neurotropic strain of MHV that was found to cause extensive myelin destruction and was first isolated from mice that exhibited hind-limb paralysis (62). Intracranial injection (i.c) with a sublethal dose of JHMV in C57BL/6 mice results in dissemination of the virus to the brain and spinal cord (63, 64). The virus has a tropism for glial cells such as oligodendrocytes, astrocytes and microglia, yet neurons are spared (56). Within 24 hours of i.c infection, JHMV targets the ependymal layer of the lateral ventricles followed by dissemination into the parenchyma where the virus infects oligodendrocytes, microglia and astrocytes (65). The virus further disseminates into the spinal cord through the cerebral spinal fluid (CSF) where it infects the glial cells of the white matter tracts (65). Presence of virus in the glial cells and parenchyma elicits a milieu of antiviral responses, initially consisting of components of the innate immune system and later followed by the adaptive immune system. JHMV infection results in widespread white matter tract demyelination with the presence of viral RNA for up to one year post infection (p.i) (66). The spike glycoprotein and other virally encoded genes are important for neurovirulence and demyelination (67). JHMV-induced demyelination includes pathogenic immune responses against viral antigens (68-70). The innate immune 8 response consists of proinflammatory factors such as IL-1, IL-6, IL-12 and TNF-α (71, 72). Type I interferons (IFN-α and IFN-β) provide crucial host defense against the JHMV, as mice lacking IFN-α/β show elevated viral load and higher mortality and treatment of mice with external IFN-α/β shows significant reduction in viral dissemination (73, 74). Innate immune cells are recruited to the CNS at 48 hours p.i and consist of macrophages, dendritic cells (DCs), neutrophils, and natural killer (NK) cells. Matrix- metalloproteases (MMPs) along with cellular innate components such as macrophages, neutrophils, and NK cells permeate the blood brain barrier (BBB) to allow the infiltration of adaptive immune cells into the CNS (75-77); by day 5 p.i, Th1polarized T lymphocytes appear in the CNS (78). CD4+ and CD8+ T lymphocytes are essential for control of viral replication and elicit their antiviral effects by IFN-γ secretion and cytolysis (79, 80). CD8+ T cells function via cytotoxic effects on infected astrocytes and granzyme and perforin mediated lysis of infected microglia (79, 81, 82). Virusspecific CD4+ T cells support the peripheral expansion and antiviral function of CD8+ T cells and thus are critical for controlling JHMV infection in the CNS (83, 84). CD4+ T cells limit the spread of virus by secreting IFN-γ, which controls viral replication in oligodendrocytes and microglia (84, 85). CD4+ T cells are required for maintaining CD8+ T cell mediated control of viral replication (83). CD8+ T cells form the main arm of the adaptive immunity in controlling virus spread from glia and they do so through secretion of IFN-γ or, by inducing direct cytolysis (79-81). IFN-γ induces perforinmediated lysis of JHMV-infected astrocytes and microglia by inducing upregulation of MHC class I (79, 85). On the other hand, oligodendrocytes are resistant to perforinmediated lysis and clear JHMV infection by IFN-γ receptor signaling (86). 9 Histopathological examination of JHMV-infected spinal cord tissue from mice undergoing demyelination shows that myelin destruction and oligodendrocyte dysfunction is not due to apoptosis and loss of mature oligodendrocytes, but rather due to presence of inflammatory infiltrates and presentation of viral antigen by antigen presenting cells (APCs) through MHC class I and class II (87). Infectious virus particles are cleared during the chronic phase of the disease, suggesting that infection of glial cells does not promote demyelination. However, viral quasi-species are present at the later stage of the disease, which enhance inflammation and subsequently promote chronic demyelination (88, 89). Demyelination is measured by staining of spinal cord tissue of mice by a dye called luxol fast blue (LFB) that stains for fatty tissue such as myelin. LFB staining of mice persistently infected with JHMV reveals extensive spinal cord demyelination concentrated primarily in the lateral and posterior funiculus (65). Axon damage in the white matter tracts is another hallmark of JHMV-induced disease in mice. SMI-32 staining, which stains for damaged neurofilament, has indicated that axon degeneration occurs in concert with demyelination in JHMV-infected mice (90). However, some studies suggest that axon degeneration might precede oligodendrocyte dysfunction and demyelination (59). JHMV-infection of mice lacking T and B cells (RAG1-/-) shows limited demyelination and extensive viral replication and adoptive transfer of JHMV-infected splenocytes into RAG1-/- mice results in demyelination (91, 92). CD4+ T cells were demonstrated to accelerate inflammation and demyelination post JHMV infection, by trafficking macrophages into the CNS (93). These studies suggest that both CD4+ and CD8+ T cells are pivotal contributors to JHMV-induced demyelination. To summarize, numerous factors contribute to JHMV- 10 induced demyelination, making it an excellent preclinical model to study MS. 1.3 Effect of Neural Precursor Cell Engraftment in the JHMV Model MS remains an incurable chronic disease with FDA-approved DMTs aimed at reducing immune cell infiltration into the CNS in patients with RRMS(94). DMTs have very low efficacy in progressive MS patients, and the disease at this stage is accelerated primarily by reactive astrocytes, microglia, and irreversible axonal degeneration. With the exception of recently FDA approved Ocrelizumab, there are no treatment options for progressive MS patients. Therapies that could potentially help progressive MS patients should be geared toward halting the demyelination process and preventing further axonal degeneration. Remyelination could be achieved by increasing the number of mature myelinating oligodendrocytes. Cellular replacement of oligodendrocytes using neural precursor cells (NPCs) has emerged as a viable option to help progressive MS patients with severe neuronal degeneration. In the early 1990s, it was shown that cells isolated from embryos of rodents could be cultured ex-vivo and have the ability to differentiate into neurons, oligodendrocytes, and astrocytes (95, 96). Since then, scientists have been able to produce NPCs from various mammalian tissues of both embryonic and adult origin (97-99). These discoveries have opened up new therapeutic avenues as NPCs have the ability to differentiate into functional cells and also secrete immunomodulatory factors that could promote neuronal regeneration (100). Several recent studies also demonstrate the ability of culturing NPCs derived from humans in vitro and therapeutic benefits of transplanting these NPCs in preclinical models of demyelination and dysmyelination (101-103). Moreover, this work has reinforced the justification of 11 considering NPC-based therapies as a clinically viable option for treating progressive MS patients. In order to evaluate therapeutic benefits of engrafting mouse NPCs in JHMVinfected mice, MHC-mismatched NPCs were transplanted into spinal cords of infected mice with established demyelination (104). Results of NPC engraftment showed that i) NPCs survive post transplant and can migrate upto ~12mm rostral and 8mm caudal from the site of transplant, ii) NPCs preferentially differentiate into mature oligodendrocytes, and iii) NPCs can migrate to the white matter tract demyelinated areas and cause extensive axonal remyelination compared to nontransplanted controls (104). Engrafted NPCs migrate to areas of white matter damage by responding to the ligand CXCL12 through the receptor CXCR4 expressed on NPCs (105). Compared to transplantation of syngeneic NPCs, transplantation of allogeneic NPCs into spinal cords of JHMV-infected mice with demyelination resulted in immune-mediate rejection by host T lymphocytes and NK cells (106). These results demonstrate that immunosupression should be considered to promote survival, migration, and efficacy of allogeneic NPCs. In order to better understand both the migration dynamics of NPCs as well as whether oligodendrocytes derived from transplanted NPCs are capable of remyelinating demyelinated axons, GFP-expressing NPCs were transplanted into JHMV-infected Thy-1 yellow fluorescent protein (YFP) mice in which med-to large caliber axons express YFP. Through the use of two-photon microscopy, it was determined that transplanted NPCs migrate slowly in spinal cords of JHMV-infected mice compared to uninfected animals and these cells can directly remyelinate axons upon differentiation to oligodendrocytes (107). 12 CNS viruses have been considered as potential triggers of MS, especially for genetically susceptible people (108). In terms of NPC transplants, an important and clinically relevant question is whether engrafted NPCs are susceptible to infection by neurotropic viruses. There are several viruses that are capable of propagating in NPCs and cells derived from NPCs. The neonatal neurotropic Coxsackievirus can persist in the CNS and preferentially replicate in NPCs (109). Some other human pathogenic viruses that can infect and replicate in NPCs and cells derived from NPCs are human herpes simplex virus type 1 (HSV-1), Enterovirus 71, and JC virus (110-112). In terms of murine NPCs and susceptibility to JHMV infection, our lab has previously shown that glial cells derived from NPCs are susceptible to JHMV infection as seen by increased viral titers and viral antigen distributed throughout the infected monolayers (113). These results demonstrate that susceptibility to viral infection is an important factor that has to be considered for using NPCs as cell replacement therapy for neurodegenerative disorders. If immunosuppression is used to prevent rejection of allogeneic cell transplants, persistent but dormant neurotropic viruses could reactivate and can potentially infect transplanted cells, thereby mitigating any therapeutic effects of NPCs. The need to use immunosuppressive drugs can be surpassed by using NPCs derived from patient-specific induced pluripotent cells (iPSCs) for cell replacement therapy. Chapter 2 will evaluate the ability of NPCs derived from iPSCs to differentiate functionally into terminal neural cell types and also the susceptibility of iPSC-NPCs to infection with JHMV. 13 1.4 Microglia in Neuroinflammation Microglia are the resident innate immune cells of the CNS, and their density ranges from around 5% of CNS cells in the cortex and corpus callosum to 12% of cells in the substantia nigra (114). Microglia are myeloid cells originating from the embryonic mesoderm and can self-renew independent of hematopoietic cells (115). The cytokines CSF-1 and interleukin-34 (IL-34) along with transcription factors such as IRF8 are required for the survival and normal functioning of microglia (116, 117). Microglia can be recognized by the expression of markers CD11b, Iba1, Cx3Cr1, and F4/80 (118). Recent RNA-seq analysis on microglia revealed additional markers that are expressed by microglia in the healthy brain and include Tmem119, TREM2, HexB, Pry12, S100A8, S100A9, SinglecH, and Gpr34 (118, 119). Through expression of these transcripts, microglia can perform essential functions such as protection against infectious agents, sensing and polarizing towards injury and conducting essential housekeeping (120). Using their vast neural network, microglia can perform sensing functions by using products of expressing hundreds of genes (119). These scanning and sensing functions of microglia are essential to detect anomalies and provide host defense as well as housekeeping. Microglia can provide host defense against both self and non-self injurious agents. Microglia have been shown to provide defense against infectious agents such as prions, self proteins such as Aβ plaques as well as CNS tumors. Microglia are generally the first responders to injurious stimuli in the CNS and can initiate peripheral inflammation by expressing Toll-like receptors (TLRs), viral receptors, and Fc receptors (119). Finally, microglia also provide housekeeping functions such as myelin phagocytosis, phagocytosis of dead cells, and synaptic remodeling (121, 122). A recent 14 study demonstrates that microglia also dictate the activation phenotype of astrocytes depending on the microenvironment (123). Microglia and macrophages are found in active lesions with ongoing demyelination and are thought to play a pivotal role in disease progression. MS patients in the early stages of demyelination have active lesions containing neuroinflammatory microglia that expressed genes involved in antigen presentation, T cell costimulation, oxidative injury, and phagocytosis (124). Nearly 45% of monocyte-like cells in active lesions of MS patients were microglia, and there was a significant reduction of PPRY12 expression in these active lesions (124). PPRY12 is a marker for homeostatic microglia, and its loss indicates that the microglia present in active MS lesions are in an activated, proinflammatory state. In the EAE mouse model of MS, microglia release proinflammatory factors, reactive oxygen species (ROS) and recruit reactive T lymphocytes to the CNS, causing neuronal toxicity (118). However, studies also show that during the initial stages of disease, microglia promote axonal regeneration, release neurotrophic factors and clear myelin debris, suggesting an advantageous role (125). This dual nature of microglia signifies that they may be beneficial or damaging, depending on the stage of the disease and the location of microglia in the proximity of lesions. In preclinical viral models of MS such as TMEV and JHMV, microglia are required for host defense against the virus and depletion of microglia during the early stage of the disease resulted in impaired control of viral replication associated with enhanced T cell response to viral antigens (126, 127). Additionally, microglia were demonstrated to be protective in the TMEV model of encephalitis as microglia depletion increased the occurrence of seizures, 15 exacerbated hippocampal damage and caused neurodegeneration (that is not generally seen in this model) (127). These studies show that microglia have essential functions in MS and are clinically relevant as they are found in active demyelinating MS lesions (16, 124). 1.5 Summary Chapter 2 investigates whether NPCs derived from mouse iPSCs are functionally similar to fetal derived NPCs. Additionally, the susceptibility of iPSC-NPCs to infection by JHMV is evaluated. Similar to NPCs derived from striatum of day 1 postnatal GFPtransgenic mice (GFP-NPCs), iPSC derived NPCs are able to differentiate into oligodendrocytes, astrocytes and neurons. However, unlike GFP-NPCs, iPSC-NPCs express low levels of the surface receptor for JHMV and are consequently less susceptible to infection and JHMV-induced cell death. The focus of Chapter 3 is to evaluate the contribution of microglia to disease progression in JHMV-induced demyelination. Depletion of microglia leads to increased viral replication, mortality, and demyelination and modulates the immunological response to JHMV infection in the CNS. 1.6 References 1. Steinman L. Multiple sclerosis: a coordinated immunological attack against myelin in the central nervous system. Cell. 1996;85(3):299-302. 2. Lassmann H, Bruck W, Lucchinetti CF. The immunopathology of multiple sclerosis: an overview. Brain Pathol. 2007;17(2):210-8. 3. Traugott U, Reinherz EL, Raine CS. Multiple sclerosis: distribution of T cell subsets within active chronic lesions. Science. 1983;219(4582):308-10. 16 4. Traugott U, Reinherz EL, Raine CS. Multiple sclerosis. Distribution of T cells, T cell subsets and Ia-positive macrophages in lesions of different ages. J Neuroimmunol. 1983;4(3):201-21. 5. Prineas JW, Kwon EE, Goldenberg PZ, Ilyas AA, Quarles RH, Benjamins JA, et al. Multiple sclerosis. Oligodendrocyte proliferation and differentiation in fresh lesions. Lab Invest. 1989;61(5):489-503. 6. Neumann H, Medana IM, Bauer J, Lassmann H. Cytotoxic T lymphocytes in autoimmune and degenerative CNS diseases. Trends Neurosci. 2002;25(6):313-9. 7. Compston A, Coles A. Multiple sclerosis. Lancet. 2002;359(9313):1221-31. 8. Miller D, Barkhof F, Montalban X, Thompson A, Filippi M. Clinically isolated syndromes suggestive of multiple sclerosis, part 2: non-conventional MRI, recovery processes, and management. Lancet Neurol. 2005;4(6):341-8. 9. Kornek B, Storch MK, Weissert R, Wallstroem E, Stefferl A, Olsson T, et al. Multiple sclerosis and chronic autoimmune encephalomyelitis: a comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions. Am J Pathol. 2000;157(1):267-76. 10. Chang A, Tourtellotte WW, Rudick R, Trapp BD. Premyelinating oligodendrocytes in chronic lesions of multiple sclerosis. N Engl J Med. 2002;346(3):165-73. 11. Chang A, Nishiyama A, Peterson J, Prineas J, Trapp BD. NG2-positive oligodendrocyte progenitor cells in adult human brain and multiple sclerosis lesions. J Neurosci. 2000;20(17):6404-12. 12. Lucchinetti C, Bruck W, Parisi J, Scheithauer B, Rodriguez M, Lassmann H. A quantitative analysis of oligodendrocytes in multiple sclerosis lesions. A study of 113 cases. Brain. 1999;122 ( Pt 12):2279-95. 13. Roy NS, Wang S, Harrison-Restelli C, Benraiss A, Fraser RA, Gravel M, et al. Identification, isolation, and promoter-defined separation of mitotic oligodendrocyte progenitor cells from the adult human subcortical white matter. J Neurosci. 1999;19(22):9986-95. 14. Kremenchutzky M, Rice GP, Baskerville J, Wingerchuk DM, Ebers GC. The natural history of multiple sclerosis: a geographically based study 9: observations on the progressive phase of the disease. Brain. 2006;129(Pt 3):584-94. 15. Antel J, Antel S, Caramanos Z, Arnold DL, Kuhlmann T. Primary progressive multiple sclerosis: part of the MS disease spectrum or separate disease entity? Acta Neuropathol. 2012;123(5):627-38. 17 16. Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mork S, Bo L. Axonal transection in the lesions of multiple sclerosis. N Engl J Med. 1998;338(5):27885. 17. Peterson JW, Bo L, Mork S, Chang A, Trapp BD. Transected neurites, apoptotic neurons, and reduced inflammation in cortical multiple sclerosis lesions. Ann Neurol. 2001;50(3):389-400. 18. Wolswijk G. Chronic stage multiple sclerosis lesions contain a relatively quiescent population of oligodendrocyte precursor cells. J Neurosci. 1998;18(2):601-9. 19. Haines JL, Terwedow HA, Burgess K, Pericak-Vance MA, Rimmler JB, Martin ER, et al. Linkage of the MHC to familial multiple sclerosis suggests genetic heterogeneity. The Multiple Sclerosis Genetics Group. Hum Mol Genet. 1998;7(8):1229-34. 20. Barcellos LF, Sawcer S, Ramsay PP, Baranzini SE, Thomson G, Briggs F, et al. Heterogeneity at the HLA-DRB1 locus and risk for multiple sclerosis. Hum Mol Genet. 2006;15(18):2813-24. 21. International Multiple Sclerosis Genetics C. Network-based multiple sclerosis pathway analysis with GWAS data from 15,000 cases and 30,000 controls. Am J Hum Genet. 2013;92(6):854-65. 22. Ascherio A, Munger KL. Environmental risk factors for multiple sclerosis. Part I: the role of infection. Ann Neurol. 2007;61(4):288-99. 23. Ascherio A, Munger KL. Environmental risk factors for multiple sclerosis. Part II: Noninfectious factors. Ann Neurol. 2007;61(6):504-13. 24. International Multiple Sclerosis Genetics C, Wellcome Trust Case Control C, Sawcer S, Hellenthal G, Pirinen M, Spencer CC, et al. Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis. Nature. 2011;476(7359):214-9. 25. Ebers GC, Yee IM, Sadovnick AD, Duquette P. Conjugal multiple sclerosis: population-based prevalence and recurrence risks in offspring. Canadian Collaborative Study Group. Ann Neurol. 2000;48(6):927-31. 26. Sadovnick AD, Baird PA. The familial nature of multiple sclerosis: age-corrected empiric recurrence risks for children and siblings of patients. Neurology. 1988;38(6):990-1. 18 27. Flores J, Gonzalez S, Morales X, Yescas P, Ochoa A, Corona T. Absence of Multiple Sclerosis and demyelinating diseases among lacandonians, a pure amerindian ethnic group in Mexico. Mult Scler Int. 2012;2012:292631. 28. Sadovnick AD, Armstrong H, Rice GP, Bulman D, Hashimoto L, Paty DW, et al. A population-based study of multiple sclerosis in twins: update. Ann Neurol. 1993;33(3):281-5. 29. Olerup O, Hillert J. HLA class II-associated genetic susceptibility in multiple sclerosis: a critical evaluation. Tissue Antigens. 1991;38(1):1-15. 30. Haegert DG, Michaud M, Schwab C, Tansey C, Decary F, Francis G. HLA-DR beta, -DQ alpha, and -DQ beta restriction fragment length polymorphisms in multiple sclerosis. J Neurosci Res. 1989;23(1):46-54. 31. Fernandez O, Fernandez V, Alonso A, Caballero A, Luque G, Bravo M, et al. DQB1*0602 allele shows a strong association with multiple sclerosis in patients in Malaga, Spain. J Neurol. 2004;251(4):440-4. 32. Ascherio A, Munger KL. Epidemiology of Multiple Sclerosis: from risk factors to prevention-an update. Semin Neurol. 2016;36(2):103-14. 33. Dobson R, Ramagopalan S, Davis A, Giovannoni G. Cerebrospinal fluid oligoclonal bands in multiple sclerosis and clinically isolated syndromes: a metaanalysis of prevalence, prognosis and effect of latitude. J Neurol Neurosurg Psychiatry. 2013;84(8):909-14. 34. Koch-Henriksen N, Sorensen PS. The changing demographic pattern of multiple sclerosis epidemiology. Lancet Neurol. 2010;9(5):520-32. 35. Kurtzke JF. Epidemiology of multiple sclerosis. Does this really point toward an etiology? Lectio Doctoralis. Neurol Sci. 2000;21(6):383-403. 36. Ascherio A, Munger KL, White R, Kochert K, Simon KC, Polman CH, et al. Vitamin D as an early predictor of multiple sclerosis activity and progression. JAMA Neurol. 2014;71(3):306-14. 37. Jelinek GA, Marck CH, Weiland TJ, Pereira N, van der Meer DM, Hadgkiss EJ. Latitude, sun exposure and vitamin D supplementation: associations with quality of life and disease outcomes in a large international cohort of people with multiple sclerosis. BMC Neurol. 2015;15:132. 38. van der Mei IA, Ponsonby AL, Engelsen O, Pasco JA, McGrath JJ, Eyles DW, et al. The high prevalence of vitamin D insufficiency across Australian populations is only partly explained by season and latitude. Environ Health Perspect. 2007;115(8):1132-9. 19 39. van der Mei IA, Ponsonby AL, Dwyer T, Blizzard L, Simmons R, Taylor BV, et al. Past exposure to sun, skin phenotype, and risk of multiple sclerosis: casecontrol study. BMJ. 2003;327(7410):316. 40. Fujinami RS, Oldstone MB. Amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity. Science. 1985;230(4729):1043-5. 41. Fujinami RS, von Herrath MG, Christen U, Whitton JL. Molecular mimicry, bystander activation, or viral persistence: infections and autoimmune disease. Clin Microbiol Rev. 2006;19(1):80-94. 42. O'Connor KC, Bar-Or A, Hafler DA. The neuroimmunology of multiple sclerosis: possible roles of T and B lymphocytes in immunopathogenesis. J Clin Immunol. 2001;21(2):81-92. 43. James E, Dobson R, Kuhle J, Baker D, Giovannoni G, Ramagopalan SV. The effect of vitamin D-related interventions on multiple sclerosis relapses: a metaanalysis. Mult Scler. 2013;19(12):1571-9. 44. Glenn JD, Mowry EM. Emerging concepts on the gut microbiome and Multiple Sclerosis. J Interferon Cytokine Res. 2016;36(6):347-57. 45. Kotzamani D, Panou T, Mastorodemos V, Tzagournissakis M, Nikolakaki H, Spanaki C, et al. Rising incidence of multiple sclerosis in females associated with urbanization. Neurology. 2012;78(22):1728-35. 46. Yamout BI, Alroughani R. Multiple Sclerosis. Semin Neurol. 2018;38(2):212-25. 47. Polman CH, Reingold SC, Banwell B, Clanet M, Cohen JA, Filippi M, et al. Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol. 2011;69(2):292-302. 48. Jacobs LD, Cookfair DL, Rudick RA, Herndon RM, Richert JR, Salazar AM, et al. Intramuscular interferon beta-1a for disease progression in relapsing multiple sclerosis. The Multiple Sclerosis Collaborative Research Group (MSCRG). Ann Neurol. 1996;39(3):285-94. 49. Confavreux C, O'Connor P, Comi G, Freedman MS, Miller AE, Olsson TP, et al. Oral teriflunomide for patients with relapsing multiple sclerosis (TOWER): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet Neurol. 2014;13(3):247-56. 50. Cohen JA, Barkhof F, Comi G, Hartung HP, Khatri BO, Montalban X, et al. Oral fingolimod or intramuscular interferon for relapsing multiple sclerosis. N Engl J Med. 2010;362(5):402-15. 20 51. Butzkueven H, Kappos L, Pellegrini F, Trojano M, Wiendl H, Patel RN, et al. Efficacy and safety of natalizumab in multiple sclerosis: interim observational programme results. J Neurol Neurosurg Psychiatry. 2014;85(11):1190-7. 52. Coles AJ, Cohen JA, Fox EJ, Giovannoni G, Hartung HP, Havrdova E, et al. Alemtuzumab CARE-MS II 5-year follow-up: efficacy and safety findings. Neurology. 2017;89(11):1117-26. 53. Frampton JE. Ocrelizumab: First global approval. Drugs. 2017;77(9):1035-41. 54. Mix E, Meyer-Rienecker H, Hartung HP, Zettl UK. Animal models of multiple sclerosis--potentials and limitations. Prog Neurobiol. 2010;92(3):386-404. 55. Tsunoda I, Fujinami RS. Two models for multiple sclerosis: experimental allergic encephalomyelitis and Theiler's murine encephalomyelitis virus. J Neuropathol Exp Neurol. 1996;55(6):673-86. 56. Fleming JO, Trousdale MD, el-Zaatari FA, Stohlman SA, Weiner LP. Pathogenicity of antigenic variants of murine coronavirus JHM selected with monoclonal antibodies. J Virol. 1986;58(3):869-75. 57. Cheever FS, Daniels JB, et al. A murine virus (JHM) causing disseminated encephalomyelitis with extensive destruction of myelin. J Exp Med. 1949;90(3):181-210. 58. Barthold SW, Beck DS, Smith AL. Enterotropic coronavirus (mouse hepatitis virus) in mice: influence of host age and strain on infection and disease. Lab Anim Sci. 1993;43(4):276-84. 59. Das Sarma J, Fu L, Hingley ST, Lavi E. Mouse hepatitis virus type-2 infection in mice: an experimental model system of acute meningitis and hepatitis. Exp Mol Pathol. 2001;71(1):1-12. 60. Lavi E, Gilden DH, Wroblewska Z, Rorke LB, Weiss SR. Experimental demyelination produced by the A59 strain of mouse hepatitis virus. Neurology. 1984;34(5):597-603. 61. Lavi E, Gilden DH, Highkin MK, Weiss SR. The organ tropism of mouse hepatitis virus A59 in mice is dependent on dose and route of inoculation. Lab Anim Sci. 1986;36(2):130-5. 62. Bailey OT, Pappenheimer AM, Cheever FS, Daniels JB. A Murine Virus (Jhm) Causing Disseminated Encephalomyelitis with Extensive Destruction of Myelin : Ii. Pathology. J Exp Med. 1949;90(3):195-212. 21 63. Hosking MP, Lane TE. The Biology of Persistent Infection: Inflammation and Demyelination following Murine Coronavirus Infection of the Central Nervous System. Curr Immunol Rev. 2009;5(4):267-76. 64. Bergmann CC, Lane TE, Stohlman SA. Coronavirus infection of the central nervous system: host-virus stand-off. Nat Rev Microbiol. 2006;4(2):121-32. 65. Wang FI, Hinton DR, Gilmore W, Trousdale MD, Fleming JO. Sequential infection of glial cells by the murine hepatitis virus JHM strain (MHV-4) leads to a characteristic distribution of demyelination. Lab Invest. 1992;66(6):744-54. 66. Lane TE, Buchmeier MJ. Murine coronavirus infection: a paradigm for virusinduced demyelinating disease. Trends Microbiol. 1997;5(1):9-14. 67. Iacono KT, Kazi L, Weiss SR. Both spike and background genes contribute to murine coronavirus neurovirulence. J Virol. 2006;80(14):6834-43. 68. Glass WG, Lane TE. Functional analysis of the CC chemokine receptor 5 (CCR5) on virus-specific CD8+ T cells following coronavirus infection of the central nervous system. Virology. 2003;312(2):407-14. 69. Glass WG, Chen BP, Liu MT, Lane TE. Mouse hepatitis virus infection of the central nervous system: chemokine-mediated regulation of host defense and disease. Viral Immunol. 2002;15(2):261-72. 70. Haring JS, Perlman S. Bystander CD4 T cells do not mediate demyelination in mice infected with a neurotropic coronavirus. J Neuroimmunol. 2003;137(12):42-50. 71. Parra B, Hinton DR, Lin MT, Cua DJ, Stohlman SA. Kinetics of cytokine mRNA expression in the central nervous system following lethal and nonlethal coronavirus-induced acute encephalomyelitis. Virology. 1997;233(2):260-70. 72. Pearce BD, Hobbs MV, McGraw TS, Buchmeier MJ. Cytokine induction during T-cell-mediated clearance of mouse hepatitis virus from neurons in vivo. J Virol. 1994;68(9):5483-95. 73. Ireland DD, Stohlman SA, Hinton DR, Atkinson R, Bergmann CC. Type I interferons are essential in controlling neurotropic coronavirus infection irrespective of functional CD8 T cells. J Virol. 2008;82(1):300-10. 74. Minagawa H, Takenaka A, Mohri S, Mori R. Protective effect of recombinant murine interferon beta against mouse hepatitis virus infection. Antiviral Res. 1987;8(2):85-95. 22 75. Yong VW, Zabad RK, Agrawal S, Goncalves Dasilva A, Metz LM. Elevation of matrix metalloproteinases (MMPs) in multiple sclerosis and impact of immunomodulators. J Neurol Sci. 2007;259(1-2):79-84. 76. Hosking MP, Liu L, Ransohoff RM, Lane TE. A protective role for ELR+ chemokines during acute viral encephalomyelitis. PLoS Pathog. 2009;5(11):e1000648. 77. Savarin C, Stohlman SA, Atkinson R, Ransohoff RM, Bergmann CC. Monocytes regulate T cell migration through the glia limitans during acute viral encephalitis. J Virol. 2010;84(10):4878-88. 78. Marten NW, Stohlman SA, Zhou J, Bergmann CC. Kinetics of virus-specific CD8+ -T-cell expansion and trafficking following central nervous system infection. J Virol. 2003;77(4):2775-8. 79. Lin MT, Stohlman SA, Hinton DR. Mouse hepatitis virus is cleared from the central nervous systems of mice lacking perforin-mediated cytolysis. J Virol. 1997;71(1):383-91. 80. Parra B, Hinton DR, Marten NW, Bergmann CC, Lin MT, Yang CS, et al. IFNgamma is required for viral clearance from central nervous system oligodendroglia. J Immunol. 1999;162(3):1641-7. 81. Ramakrishna C, Stohlman SA, Atkinson RA, Hinton DR, Bergmann CC. Differential regulation of primary and secondary CD8+ T cells in the central nervous system. J Immunol. 2004;173(10):6265-73. 82. Bergmann CC, Parra B, Hinton DR, Ramakrishna C, Dowdell KC, Stohlman SA. Perforin and gamma interferon-mediated control of coronavirus central nervous system infection by CD8 T cells in the absence of CD4 T cells. J Virol. 2004;78(4):1739-50. 83. Phares TW, Stohlman SA, Hwang M, Min B, Hinton DR, Bergmann CC. CD4 T cells promote CD8 T cell immunity at the priming and effector site during viral encephalitis. J Virol. 2012;86(5):2416-27. 84. Zhou J, Hinton DR, Stohlman SA, Liu CP, Zhong L, Marten NW. Maintenance of CD8+ T cells during acute viral infection of the central nervous system requires CD4+ T cells but not interleukin-2. Viral Immunol. 2005;18(1):162-9. 85. Bergmann CC, Parra B, Hinton DR, Chandran R, Morrison M, Stohlman SA. Perforin-mediated effector function within the central nervous system requires IFN-gamma-mediated MHC up-regulation. J Immunol. 2003;170(6):3204-13. 23 86. Gonzalez JM, Bergmann CC, Ramakrishna C, Hinton DR, Atkinson R, Hoskin J, et al. Inhibition of interferon-gamma signaling in oligodendroglia delays coronavirus clearance without altering demyelination. Am J Pathol. 2006;168(3):796-804. 87. Redwine JM, Buchmeier MJ, Evans CF. In vivo expression of major histocompatibility complex molecules on oligodendrocytes and neurons during viral infection. Am J Pathol. 2001;159(4):1219-24. 88. Adami C, Pooley J, Glomb J, Stecker E, Fazal F, Fleming JO, et al. Evolution of mouse hepatitis virus (MHV) during chronic infection: quasispecies nature of the persisting MHV RNA. Virology. 1995;209(2):337-46. 89. Fleming JO, Adami C, Pooley J, Glomb J, Stecker E, Fazal F, et al. Mutations associated with viral sequences isolated from mice persistently infected with MHV-JHM. Adv Exp Med Biol. 1995;380:591-5. 90. Dandekar AA, Wu GF, Pewe L, Perlman S. Axonal damage is T cell mediated and occurs concomitantly with demyelination in mice infected with a neurotropic coronavirus. J Virol. 2001;75(13):6115-20. 91. Pewe L, Perlman S. Cutting edge: CD8 T cell-mediated demyelination is IFNgamma dependent in mice infected with a neurotropic coronavirus. J Immunol. 2002;168(4):1547-51. 92. Wu GF, Perlman S. Macrophage infiltration, but not apoptosis, is correlated with immune-mediated demyelination following murine infection with a neurotropic coronavirus. J Virol. 1999;73(10):8771-80. 93. Lane TE, Liu MT, Chen BP, Asensio VC, Samawi RM, Paoletti AD, et al. A central role for CD4(+) T cells and RANTES in virus-induced central nervous system inflammation and demyelination. J Virol. 2000;74(3):1415-24. 94. Hauser SL, Chan JR, Oksenberg JR. Multiple sclerosis: prospects and promise. Ann Neurol. 2013;74(3):317-27. 95. Temple S. Division and differentiation of isolated CNS blast cells in microculture. Nature. 1989;340(6233):471-3. 96. Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science. 1992;255(5052):1707-10. 97. Brustle O, Jones KN, Learish RD, Karram K, Choudhary K, Wiestler OD, et al. Embryonic stem cell-derived glial precursors: a source of myelinating transplants. Science. 1999;285(5428):754-6. 24 98. Onorati M, Camnasio S, Binetti M, Jung CB, Moretti A, Cattaneo E. Neuropotent self-renewing neural stem (NS) cells derived from mouse induced pluripotent stem (iPS) cells. Mol Cell Neurosci. 2010;43(3):287-95. 99. Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A, et al. Neural progenitors from human embryonic stem cells. Nat Biotechnol. 2001;19(12):1134-40. 100. Kokaia Z, Martino G, Schwartz M, Lindvall O. Cross-talk between neural stem cells and immune cells: the key to better brain repair? Nat Neurosci. 2012;15(8):1078-87. 101. Cristofanilli M, Harris VK, Zigelbaum A, Goossens AM, Lu A, Rosenthal H, et al. Mesenchymal stem cells enhance the engraftment and myelinating ability of allogeneic oligodendrocyte progenitors in dysmyelinated mice. Stem Cells Dev. 2011;20(12):2065-76. 102. Harris VK, Faroqui R, Vyshkina T, Sadiq SA. Characterization of autologous mesenchymal stem cell-derived neural progenitors as a feasible source of stem cells for central nervous system applications in multiple sclerosis. Stem Cells Transl Med. 2012;1(7):536-47. 103. Harris VK, Yan QJ, Vyshkina T, Sahabi S, Liu X, Sadiq SA. Clinical and pathological effects of intrathecal injection of mesenchymal stem cell-derived neural progenitors in an experimental model of multiple sclerosis. J Neurol Sci. 2012;313(1-2):167-77. 104. Totoiu MO, Nistor GI, Lane TE, Keirstead HS. Remyelination, axonal sparing, and locomotor recovery following transplantation of glial-committed progenitor cells into the MHV model of multiple sclerosis. Exp Neurol. 2004;187(2):254-65. 105. Carbajal KS, Schaumburg C, Strieter R, Kane J, Lane TE. Migration of engrafted neural stem cells is mediated by CXCL12 signaling through CXCR4 in a viral model of multiple sclerosis. Proc Natl Acad Sci U S A. 2010;107(24):11068-73. 106. Weinger JG, Plaisted WC, Maciejewski SM, Lanier LL, Walsh CM, Lane TE. Activating receptor NKG2D targets RAE-1-expressing allogeneic neural precursor cells in a viral model of multiple sclerosis. Stem Cells. 2014;32(10):2690-701. 107. Greenberg ML, Weinger JG, Matheu MP, Carbajal KS, Parker I, Macklin WB, et al. Two-photon imaging of remyelination of spinal cord axons by engrafted neural precursor cells in a viral model of multiple sclerosis. Proc Natl Acad Sci U S A. 2014;111(22):E2349-55. 25 108. Giovannoni G, Cutter GR, Lunemann J, Martin R, Munz C, Sriram S, et al. Infectious causes of multiple sclerosis. Lancet Neurol. 2006;5(10):887-94. 109. Tsueng G, Tabor-Godwin JM, Gopal A, Ruller CM, Deline S, An N, et al. Coxsackievirus preferentially replicates and induces cytopathic effects in undifferentiated neural progenitor cells. J Virol. 2011;85(12):5718-32. 110. Chucair-Elliott AJ, Conrady C, Zheng M, Kroll CM, Lane TE, Carr DJ. Microglia-induced IL-6 protects against neuronal loss following HSV-1 infection of neural progenitor cells. Glia. 2014;62(9):1418-34. 111. Huang HI, Lin JY, Chen HH, Yeh SB, Kuo RL, Weng KF, et al. Enterovirus 71 infects brain-derived neural progenitor cells. Virology. 2014;468-470:592-600. 112. Schaumburg C, O'Hara BA, Lane TE, Atwood WJ. Human embryonic stem cellderived oligodendrocyte progenitor cells express the serotonin receptor and are susceptible to JC virus infection. J Virol. 2008;82(17):8896-9. 113. Whitman L, Zhou H, Perlman S, Lane TE. IFN-gamma-mediated suppression of coronavirus replication in glial-committed progenitor cells. Virology. 2009;384(1):209-15. 114. Lawson LJ, Perry VH, Dri P, Gordon S. Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain. Neuroscience. 1990;39(1):151-70. 115. Tay TL, Savage JC, Hui CW, Bisht K, Tremblay ME. Microglia across the lifespan: from origin to function in brain development, plasticity and cognition. J Physiol. 2017;595(6):1929-45. 116. Kierdorf K, Erny D, Goldmann T, Sander V, Schulz C, Perdiguero EG, et al. Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways. Nat Neurosci. 2013;16(3):273-80. 117. Wang Y, Szretter KJ, Vermi W, Gilfillan S, Rossini C, Cella M, et al. IL-34 is a tissue-restricted ligand of CSF1R required for the development of Langerhans cells and microglia. Nat Immunol. 2012;13(8):753-60. 118. Ransohoff RM, El Khoury J. Microglia in health and disease. Cold Spring Harb Perspect Biol. 2015;8(1):a020560. 119. Hickman SE, Kingery ND, Ohsumi TK, Borowsky ML, Wang LC, Means TK, et al. The microglial sensome revealed by direct RNA sequencing. Nat Neurosci. 2013;16(12):1896-905. 26 120. Hickman S, Izzy S, Sen P, Morsett L, El Khoury J. Microglia in neurodegeneration. Nat Neurosci. 2018;21(10):1359-69. 121. Healy LM, Perron G, Won SY, Michell-Robinson MA, Rezk A, Ludwin SK, et al. MerTK Is a Functional regulator of myelin phagocytosis by human myeloid cells. J Immunol. 2016;196(8):3375-84. 122. Zhan Y, Paolicelli RC, Sforazzini F, Weinhard L, Bolasco G, Pagani F, et al. Deficient neuron-microglia signaling results in impaired functional brain connectivity and social behavior. Nat Neurosci. 2014;17(3):400-6. 123. Liddelow SA, Guttenplan KA, Clarke LE, Bennett FC, Bohlen CJ, Schirmer L, et al. Neurotoxic reactive astrocytes are induced by activated microglia. Nature. 2017;541(7638):481-7. 124. Zrzavy T, Hametner S, Wimmer I, Butovsky O, Weiner HL, Lassmann H. Loss of 'homeostatic' microglia and patterns of their activation in active multiple sclerosis. Brain. 2017;140(7):1900-13. 125. Yamasaki R, Lu H, Butovsky O, Ohno N, Rietsch AM, Cialic R, et al. Differential roles of microglia and monocytes in the inflamed central nervous system. J Exp Med. 2014;211(8):1533-49. 126. Wheeler DL, Sariol A, Meyerholz DK, Perlman S. Microglia are required for protection against lethal coronavirus encephalitis in mice. J Clin Invest. 2018;128(3):931-43. 127. Waltl I, Kaufer C, Gerhauser I, Chhatbar C, Ghita L, Kalinke U, et al. Microglia have a protective role in viral encephalitis-induced seizure development and hippocampal damage. Brain Behav Immun. 2018. CHAPTER 2 NEURAL PRECURSOR CELLS DERIVED FROM INDUCED PLURIPOTENT STEM CELLS EXHIBIT REDUCED SUSCEPTIBILITY TO INFECTION WITH A NEUROTROPIC CORONAVIRUS Originally published in Virology Mangale V, Marro BS, Plaisted WC, Walsh CM, Lane TE. Neural precursor cells derived from induced pluripotent stem cells exhibit reduced susceptibility to infection with a neurotropic coronavirus. Virology. 2017;511:49-55. Copyright © Elsevier 28 29 30 31 32 33 34 CHAPTER 3 MICROGLIA AID IN HOST DEFENSE AND RESTRICT THE SEVERITY OF DEMYELINATION IN RESPONSE TO INFECTION WITH A NEUROTROPIC CORONAVIRUS 3.1 Abstract Mice infected with the JHM strain of mouse hepatitis virus (JHMV) results in acute encephalomyelitis and chronic demyelination in the central nervous system (CNS). Thus, it has been used as a preclinical model to study multiple sclerosis (MS). Microglia are resident monocytes of the CNS and aid in host defense against microbes, neurogenesis and synaptic pruning. However, under chronic inflammatory conditions, activated microglia can become neurotoxic and neurodegenerative. Microglia are capable of undergoing dynamic changes in morphology as well as transcription in response to injury or infection in the CNS. In order to evaluate the specific role of microglia in JHMV-induced demyelination, colony-stimulating factor 1 receptor (CSF1R) inhibitor was used to deplete microglia. Our results demonstrate that CSF1R inhibitor PLX5622 can result in >90% depletion of microglia in JHMV-infected mice. Targeted reduction of microglia led to increased mortality associated with impaired control of viral replication as well as a dramatic increase in the severity of demyelination arguing for a protective role for microglia in host defense as well as restricting white matter damage. To better understand how microglia accomplish these tasks, we performed single cell RNA sequencing (scRNA Seq) on flow-sorted CD45-positive cells isolated from the brains and spinal cords of experimental mice at days 7 and 14 p.i., respectively. Our findings clearly show that microglia exert a broad influence on shaping the immunological landscape within the CNS of JHMV-infected mice as assessed by expression of genes involved in antigen presentation, neuroinflammation, and T cell activation. 37 3.2 Introduction MS was traditionally thought to be predominantly a T cell mediated disease, supported by studies on the EAE model. However, recent studies have shown that the resident monocytes of the brain called microglia are important players in MS disease progression (1). Microglia are resident monocytes of the CNS and are often considered as the first line of defense against pathogenic infections in the CNS (2). In addition, they maintain homeostasis in the CNS by aiding neurogenesis, neuronal function, and synaptic pruning (3-5). Microglia reside in the CNS parenchyma, and although they are considered as the resident brain macrophages, they differ from macrophages in their location, morphology and function. Microglia have a characteristic ramified morphology and are functionally distinct to other brain macrophages such as perivascular and meningeal macrophages (6, 7). Although often deemed as weak antigen presenting cells (APCs), microglia are capable of rapidly transforming into an activated macrophage phenotype upon stimulation (8). Ramified microglia express MHC Class II constitutively on their surface but have the ability to rapidly change their morphology to an activated amoeboid form in response to environmental insults such as microbial infections. In addition to morphologic changes, activated microglia increase surface expression of receptors associated with activation as well as secrete a milieu of cytokines/chemokines such as IL6, IL-23, IFN-γ, and TNF-α that are thought to provide host defense but may also prove to be neurotoxic, neurodegenerative or neuroinflammatory (6, 9, 10). Activated microglia can express toll like receptors (TLRs) and mannose receptors, and these receptors can recognize pathogen associated molecular patterns (PAMPs) such as lipopolysaccharide and peptidoglycan of bacterial cell wall (11). Microglia can also contribute to adaptive 38 immunity by presenting antigen to CD4+ and CD8+ T lymphocytes, which enter the CNS in response to infection (11). In several neurodegenerative diseases, microglia are thought to undergo reactive microgliosis. Reactive microglia were found surrounding amyloid-β plaques in postmortem specimens from people with Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease (12-14). In addition, activated MHC class II expressing microglia and macrophages were found in the proximity of damaged axons in MS postmortem tissue, suggesting these cells contribute to axonopathy (15). A genetic dysfunction of microglia is observed in human diseases such as Nasu-Hakola and hereditary diffuse leukoencephalopathy with spheroids, suggesting that microglia play a crucial role in neurodegeneration in this disease (16, 17). Activated microglia can hinder brain repair by interfering with the differentiation of OPCs into myelinating oligodendrocytes by producing heat shock protein 60, inducing TNF-α signaling and promoting oxidative damage by releasing nitric oxide synthase (18, 19). Intracranial infection with neurotropic JMHV leads to an acute encephalomyelitis and followed by chronic immune-mediated demyelinating disease in surviving mice (20, 21). JHMV infection in the CNS results in the release of inflammatory cytokines and chemokines that in turn recruit innate and adaptive immune cells into the CNS (22-24). One of the early cellular responders of viral infection from the periphery are macrophages, and systemic depletion of macrophages increases mortality but does not influence viral clearance nor does dramatically affect demyelination (25). Previously, it has been difficult to define the distinct roles of microglia and macrophages due to the inability of differentiating the two cell types in the CNS. However, recent studies using 39 the experimental autoimmune encephalomyelitis (EAE) model have indicated a detrimental role for infiltrating macrophages, and a protective role for microglia in disease progression (26-28). In order to understand the role of microglia in neurological disorders, recent studies have used an inhibitor of CSF1R to deplete microglia. CSF1R is expressed on microglia, macrophages and osteoclasts and has two ligands- CSF1 and IL34 (29). CSF1R knockout mice are devoid of microglia and it has been shown that microglia are the only cell types in the brain that express CSF1R (30, 31). Upon withdrawal of CSF1R inhibitor, microglia have been shown to repopulate from nestinpositive cells found in the CNS (32). A recent study by Perlman and colleagues (33) evaluated the role of microglia in response to infection by JHMV by using a CSF1R inhibitor and demonstrated that microglia are required for protection from viral encephalomyelitis during acute stages of infection. However, the impact of microglial depletion tailoring antiviral immune responses within following JHMV infection as well as on demyelination has not yet been well defined. The focus of this chapter is to explore the effect of microglia depletion on immune infiltration, demyelination, and remyelination in JHMV-infected mice. 3.3 Methods 3.3.1 Mice and viral infection Five-week old C57/BL6 male mice were purchased from The Jackson Laboratory, and all experimental mice were maintained in a specific pathogen free facility at the University of Utah. Mice were infected intracranially with 250 plaque forming units (PFU) of JHMV strain J2.2v-1 in 30µL of sterile Hanks balanced sterile solution (HBSS), 40 and animals were euthanized at days 7,14 or 21 post infection (p.i), using approved institutional animal care guidelines. Clinical disease in JHMV-infected mice was evaluated using a previously described scale (34). To determine viral titers within brains, experimental animals were sacrificed at defined times p.i, brains isolated, homogenized, and plaque assay were performed on the DBT astrocytoma cell line, as described previously (35). 3.3.2 Microglia depletion by PLX5622 treatment AIN-76A (Research Diets, NJ) rodent chow formulated with CSF1R inhibitorPLX5622 at a dose of 1,200 mg/kg of chow was kindly provided by Plexxikon, Inc (Berkeley, CA). Mice were fed with either PLX5622 chow or control chow 7 days prior to viral infection, and feed was continued until the mice were sacrificed to harvest tissues at defined times p.i. 3.3.3 Flow cytometry Flow cytometry was performed to phenotype immune cells infiltrating into the CNS post JHMV i.c infection, using previously established protocols (34, 36). Briefly, brain tissue was homogenized by using frosted microscope slides to make a single cell suspension; subsequently infiltrating leukocytes were isolated by using a two-step Percoll gradient separation. Isolated immune cells were then treated with anti-CD16/32 Fc Block (BD Biosciences, San Jose, CA) at a dilution of 1:200 for 20 minutes and immunostaining was performed using the following rat antimouse antibodies: CD45, CD3, CD4, CD8, CD11b, F4/80, MHV S510 tetramer (NIH), and MHV M133 tetramer 41 (NIH). The following gating schemes were used to identify immune populations using FloJo software: CD4+ T cells (CD45hi CD3+ CD4+), CD8+ T cells (CD45hi CD3+ CD8+), virus specific CD4+ T cells (CD45hi CD3+ M133-tet+ CD4+), virus specific CD8+ T cells (CD45hi CD3+ S510-tet+ CD8+), macrophages (CD45hi Cd11b+ or CD45hi F4/80+), and microglia (CD45int Cd11b+ or CD45int F4/80+). 3.3.4 Histopathology Following sacrifice, mice were perfused with 20 ml of 4% paraformaldehyde, at which point spinal cords were isolated and fixed overnight with 4% paraformaldehyde at 4o C. Effaced spinal cords were then placed in 30% sucrose for 5 days and subsequently cut into 2 mm sections and embedded in optimal cutting temperature compound (OCT) and frozen for sectioning. Frozen blocks of spinal cords were cryosectioned (coronal or sagittal) on slides at a thickness of 8µm and desiccated for 3 hours before histological analysis. Coronal spinal cord sections were stained with hematoxylin and eosin (H&E) and counter stained with luxol fast blue (LFB) in order to evaluate the severity of demyelination using ImageJ software, as previously described. 3.3.5 Immunofluorescence Spinal cord sections were processed as described above. Slides were desiccated for at least 3 hours and blocked with 10% normal goat serum or normal donkey serum for 1 hour at room temperature. Primary antibodies were incubated overnight at 4o C or for 1 hour at room temperature and include rabbit anti-GFAP 1:500 (Invitrogen, Carlsbad CA), rabbit anti-Iba1 1:500 (Wako Chemicals, Richmond VA), rabbit anti-MBP (Abcam, 42 Cambridge UK), and chicken anti-Neurofilament (Abcam, Cambridge UK). Following incubation with primary antibody, slides were washed three times in 1X PBS and were then incubated for 1 hour in the appropriate AlexaFluor (AF)-conjugated secondary antibodies (Invitrogen, Carlsbad CA): goat antirabbit AF594 (1:500), goat antirabbit AF488 (1:500) and goat antichicken AF594. Following incubation with secondary antibodies, slides were washed three times in 1X PBS and mounted with Fluoromount-G with DAPI (Invitrogen, Carlsbad CA). Mounted slides were then dried overnight in the dark and imaged using a Nikon A1 confocal microscope at the University of Utah Cell Imaging Core. 3.3.6 Single cell RNA sequencing (scRNA Seq) Immune cells were isolated as described above from brain (day 7 p.i) and spinal cord (day 14 p.i) and stained with DAPI and APC conjugated anti-CD45 for 20 minutes on ice in 1X PBS containing 0.5% bovine serum albumin (BSA). Live CD45+ cells were enriched through the use of BD FACSAria flow sorter (University of Utah HSC core) and washed with 0.04% BSA once. Samples were then processed for single cell RNA sequencing via the 10X Genomics platform following the manufacturer’s protocol. RNA sequencing was performed via Aligent Hiseq next generation sequencer. Sequencing data were processed by using 10X Genomics CellRanger pipeline and analyzed using the Seurat R package. Gene expression signatures defining cell clusters were analyzed after combining day 7 PLX5622 and day 7 PLX control samples together and separately combining day 14 samples- day 14 PLX5622 and day 14 PLX control samples together. Cells from each aggregated sample dataset were clustered into corresponding immune 43 cell populations by a shared nearest neighbor modularity optimization-based clustering algorithm using the Seurat package. The resulting clusters were defined using an immune-cell scoring algorithm (https://aekiz.shinyapps.io/Cell_identity_predictor/) that compares the gene signatures of each cluster in the experimental dataset with the microarray data available in the Immunological Genome (ImmGen) Project Database (37). Expression levels and distribution of population-specific immune cell markers were then analyzed to further refine the identified clusters and expose any subpopulations that should be manually separated as independent clusters. Once the clusters were established and identified, plots were generated using Seurat, ggpubr and fgsea R packages (Appendix). 3.3.7 Electron microscopy For EM analysis, mice were sacrificed and perfused with 0.1M cacodylate buffer containing 2.5% glutaraldehyde and 2% paraformaldehyde. Spinal cords were isolated and embedded in Epon (Danbury,CT) epoxy resin. Spinal cords (from thoracic vertebrate 7-9) were then cut into ultrathin sections and stained with uranyl acetate and analyzed, as described previously (38, 39). In experimental adult animals, the relation between axon circumference and myelin sheath thickness (number of lamellae) is defined by g-ratio (axon diameter/total fiber diameter). During remyelination myelin sheaths are abnormally thin for the axons they surround. Analysis was performed in ventral white matter columns of spinal cords. For PLX control treated animals a total of 145 axons were scored (n=2) from 17 randomly selected fields, and for PLX5622-treated animals, a total of 208 axons were scored (n=2) from 31 randomly selected fields. 44 3.3.8 Statistical analysis GraphPad Prism was used to perform statistical analyses. Data for each experiment are presented as mean + standard error of mean (SEM). For flow cytometry analysis unpaired student’s t test was used to determine significance and a p value of < 0.05 was considered statistically significant. Wilcoxon test was used for analyzing gene expression in scRNASeq clusters, and the resulting p values were corrected for multiple comparisons by Holm-Sidak method (*p ≤0.05; **p ≤0.01; ***p ≤0.001; ****p ≤0.0001). 3.4 Results 3.4.1 Depletion of microglia in JHMV-infected mice leads to an increase in morbidity, mortality, and viral load To evaluate the contribution of microglia to disease progression in JHMVinfected mice, CSF1R inhibitor PLX5622 was used to deplete microglia in JHMVinfected animals. When fed to C57BL/6 mice, PLX5622 has been reported to cross the blood brain barrier (BBB) and target microglia in a dose-dependent manner (31, 40). At 7 days post-JHMV infection (14 days of drug treatment), PLX5622 treatment resulted in a significant decrease in the number of microglia (CD45mid F4/80+) compared to PLX control treated animal brains (Fig. 3.1A,B). PLX5622-mediated microglia depletion did not affect the number of macrophages (CD45hi F4/80+) that infiltrated into the brains of JHMV-infected mice (Fig. 3.1A,B). In order to further evaluate microglial depletion in PLX5622-treated mice, spinal cord sections were stained with an anti-Iba1 antibody (Fig. 3.1C). Confocal microscopy revealed reduced Iba1+ cells in spinal cord sections, 45 indicating that PLX5622 effectively reduces microglia in the spinal cord (Fig. 3.1C). Confocal microscopy revealed reduced iba1+ cells in spinal cord sections, indicating that PLX5622 can deplete microglia in the spinal cord (Fig. 3.1C). Upon microglia depletion, JHMV-infected mice experienced a significant increase in clinical scores (p<0.0001) compared to control treated animals (Fig. 3.1D). Additionally, mortality was significantly (p<0.05) increased in PLX5622-fed, JHMV-infected mice, with only a 60% survival rate by day 21 p.i (Fig. 3.1E). Depletion of microglia in JHMV-infected mice also correlated with a significant (p<0.0005) increase in viral titers at 7 days p.i. (Fig 3.1F); however, virus was undetectable by plaque assay at day 14 p.i in PLX5622 or control treated animals. These findings indicate that control of viral replication within the CNS of PLX5622-treated mice was impaired during acute disease, and this ultimately was not sustained out to later stages of infection. 3.4.2 Microglia depletion influences T cell infiltration into CNS of JHMV-infected mice The increase in CNS viral titers in PLX5622-treated mice argues that microglia are required for controlling viral replication during the acute stages of infection and support recent findings by Perlman and colleagues (33). To evaluate the effect of microglial depletion on the influx of virus-specific T cells into the CNS, JHMV-infected animals fed either PLX5622 or control chow were sacrificed at 7 days p.i, and brains were harvested to evaluate the number of T cells via flow cytometric analysis. PLX5622treated mice resulted in a significant increase (p<0.05) in the total number of CD8+ T cells in brains of compared to control animals (Fig. 3.2A,B). In contrast, no significant 46 differences in total numbers of CD4+ T cells were observed between PLX5622 and control animals at day 7 p.i. (Fig. 3.2A,B). Moreover, microglia depletion did not significantly modulate numbers of virus-specific CD4+ and CD8+ T cell response at 7 days p.i in JHMV-infected animals compared to control mice as determined by staining for MHC class II and MHC class I virus-specific tetramers, respectively (Fig. 3.2C,D). 3.4.3 scRNA Seq unveils the immunological environment in the D7 JHMV-infected brain post PLX5622 treatment Since microglia depletion resulted in higher infiltration of T cells and macrophages at day 7 following JHMV infection, we decided to further evaluate the phenotypes of infiltrating immune cells at the single cell transcriptome level. To this end, we utilized the 10X Genomics single cell RNA sequencing technology on CD45+enriched cells isolated from the brains of JHMV-infected brain treated with either PLX5622 or control. C57 BL/6 mice were either fed PLX5622 or control chow for 7 days followed by intracranial infection with 250 PFU of JHMV. C57 BL/6 mice were either fed PLX5622 or control chow for 7 days, followed by intracranial infection with 250 PFU of JHMV. Mice were sacrificed at D7 p.i, and CD45+ cells were sorted from isolated brains (Fig. 3.3A). Cells from PLX5622 treated and untreated samples were aggregated, and an unsupervised clustering analysis based on similarity of gene expression signatures was performed, using the Seurat single cell genomics R package (37, 41). This analysis revealed 15 distinct clusters of lymphoid and myeloid lineage (Fig. 3.3B). Identification of gene clusters was based on an algorithm that compares gene expression signatures of scRNA Seq clusters with the publicly available Immunological 47 Genome Project (ImmGen) database. Furthermore, differential expression analysis was performed to identify distinguishing markers within clusters in cases where the algorithm could not make a clear distinction. Fifteen distinct clusters were identified consisting of CD4+ T cells, CD8+ T cell subsets, monocytes, NK cells, neutrophil subsets, macrophage subsets, and dendritic cell (DC) subsets (Fig. 3.3B). The algorithm-assisted identification of clusters was further verified by examining the expression of known cell markers in our dataset (Fig. 3.3C). The expression of known cellular markers rightly corresponded with the respective identified cell clusters (Fig. 3.3C). Differential expression analysis revealed distinct gene signatures of top 5 highly expressed genes in support of unique molecular signatures of each cluster (Fig. 3.3D). This allowed us to compare the average expression of the top 5 highly expressed genes in each cluster between the PLX5622 and control treated animals (Fig. 3.3D). An overlay of t-SNE plots of control treated (Fig. 3.3E, blue dots) and PLX5622 treated (Fig. 3.3E, amber dots) shows the dynamics of immune cells infiltrating at day 7 post-JHMV infection. PLX5622 treatment led to a reduced expression of the microglia cluster in the PLX5622-treated group, supporting our flow data and emphasizing that PLX5622 selectively depletes microglia (Fig. 3.3E). PLX5622 treatment also resulted in enrichment of some T cell populations and a pronounced difference in clustering of B cells (Fig. 3.3E). A slight increase in the frequency of naïve CD8+ T cells, effector CD8+ T cells, CD4+ T cells, and B cells was observed upon PLX5622 treatment (Fig. 3.3F). These data reveal the dynamics of immune cells infiltrating the CNS of mice infected with JHMV upon microglia depletion. 48 3.4.4 Microglia depletion leads to differential gene expression in T cell subsets at day 7 p.i The effector CD8+ and CD4+ T cell clusters from day 7 scRNA Seq analysis (Fig 3.3) were further examined for differences in gene expression of T cell activation markers and effector molecules. Microglia depletion leads to an increase in expression of T cell activation markers such as CD27 and PD-1 (Pdcd1) in the effector CD8+ T cell cluster (Fig 3.4A). Furthermore, the gene expression of T cell effector molecules required for the cytotoxic activity of effector T cells and control of JHMV infection in glial cells e.g., granzyme B (Gzmb) and perforin (Prf1) were significantly up regulated in CD8+ effector T cells of microglia depleted mice (Fig. 3.4A). The anti-inflammatory marker IL10rα was significantly reduced in CD8+ effector T cells isolated from microglia-depleted mice (Fig. 3.4A) suggesting that CD8+ effector T cells of microglia-depleted mice express elevated levels of activation markers compared to controls. Evaluation of the CD4+ T cell cluster showed an increase in gene expression of T cell activation marker CD27 yet a significant decrease in other activation markers such as CD44, Il2rα (CD25), Il2rβ, and IL10rα (Fig. 3.4B). 3.4.5 PLX5622 treatment leads to differential gene expression in macrophages infiltrating the CNS at D7 p.i As PLX5622 treatment led to a trending increase in CD45hi F4/80+ macrophages infiltrating the brain at D7 post JHMV-infection (Fig. 3.1B), we next evaluated the differential gene expression profiles in the D7 macrophage cluster of PLX5622 or control treated animals (Fig. 3.3B). Gene set enrichment analysis (GSEA) of the macrophage 49 population at D7 p.i showed a significantly higher enrichment of genes involved in the IFNα response pathway in the PLX5622 treated macrophages compared to the control samples (Fig. 3.5A). Similarly, microglia depletion led to a significantly higher enrichment of genes involved in the IFNγ response pathway (Fig. 3.5B). Differential gene expression analysis by scRNA Seq showed that PLX5622 treatment led to a decrease in expression of MHC class II genes- H2Eb1, H2DMa, and H2Eb1, suggesting an impaired ability to present antigen to CD4+ T cells (Fig. 3.5C). In contrast, microglia depletion led to an increase in gene expression of MHC class I genes- H2K1, H2Q7, H2M3 and H2T22 in the macrophage cluster (Fig. 3.5D). 3.4.6 Microglia depletion modulates JHMV-induced neuroinflammation at day 14 p.i. By day 14 p.i., microglia numbers remained significantly (p<0.05) depleted in PLX5622-treated mice compared to control animals whereas such treatment did not affect macrophage numbers (Fig. 3.6A). PLX5622 treatment led to increased numbers (p<0.05) of CD8+ T cells and a trending increase in CD4+ T cells at day14 p.i (Fig. 3.6B). However, there is a significant (p<0.05) increase in virus-specific CD4+ T cells (Fig. 3.6C) and CD8+ T cells (p<0.05) (Fig. 3.6D) in PLX5622-treated mice compared to the control-treated mice. 50 3.4.7 Microglia influence gene expression profiles of immune cells infiltrating into the CNS of JHMV-infected mice We next evaluated the phenotypes of immune cells infiltrating into the spinal cords of JHMV-infected mice treated with either PLX5622 or control at 14 p.i by scRNA Seq analysis. In brief, CD45+ cells were isolated from spinal cords via flow sorting and processed for scRNASeq using the 10X Genomics platform (Fig. 3.7A). Samples from PLX5622-treated and control mice were aggregated, and an unsupervised clustering analysis based on similarity of gene expression signatures was performed, using the Seurat single cell genomics R package (37, 41). Clustering analysis revealed 17 distinct clusters of lymphoid and myeloid lineage (Fig. 3.7B). Gene clusters were analyzed based on an algorithm that compares gene expression signatures of scRNASeq clusters with the publicly available Immunological Genome Project (ImmGen) database (37). Furthermore, differential expression analysis was performed to identify distinguishing markers within clusters in cases where the algorithm could not make a clear distinction. The 17 clusters that were identified consisted of CD8+ T cell subsets, CD4+ T cells, monocytes, NK cells, neutrophil subsets, macrophage subsets, and DC subsets (Fig. 3.7B). The algorithm-assisted clustering was further verified by examining the expression of known cell markers in our dataset (Fig. 3.7C). As expected, the expression of known cellular markers corresponded with the respective identified cell clusters (Fig. 3.7C). Differential expression analysis revealed distinct gene signatures of top 5 highly expressed genes in support of unique molecular signatures of each cluster and allowed us to compare the average expression of the top 5 highly expressed genes between clusters (Fig. 3.7D). Combined t-SNE plots of control treated (Fig. 3.7E, green dots) and 51 PLX5622 treated (Fig. 3.7E, grey dots) depict the dynamics of immune cells infiltrating the spinal cord at day 14 post-JHMV infection. As predicted, PLX5622 treatment led to a reduced expression of the microglia cluster in the PLX5622 treated group, supporting our flow and immunostaining data (Fig. 3.7E). PLX5622 treatment led to a slight decrease in the frequency of CD8+ T cell subsets and CD4+ T cells but an increase in the macrophage population (Mac 1) (Fig. 3.7F). 3.4.8 Targeting microglia results in reduced expression of T cell activation markers in T cell subsets in spinal cord at D14 p.i The activated CD8+ T cell and CD4+ T cell clusters from day 14 scRNA Seq clustering analysis were evaluated to determine differences in T cell activation markers and effector molecules between cells from PLX5622 treated and control treated cells. In contrast to cells at D7 p.i, we see a significantly lower gene expression of the T cell cytotoxic effector molecules granzyme B and perforin upon PLX5622 treatment (Fig. 3.8A). PLX5622 causes a significant reduction in gene expression of the T cell activation markers Il2rβ, Il2rγ, CCR5, and CD27 (Fig. 3.8A). Microglia depletion led to a reduction in gene expression of activation markers such as Pdcd1 and CTLA-4 but a significant decrease in the anti-inflammatory marker Il10rα (Fig. 3.8A). Examination of the CD4+ T cell cluster revealed reduced expression of the activation marker Il21r, the proinflammatory marker Il27rα and the anti-inflammatory marker Il10rα (Fig. 3.8B). 52 3.4.9 Microglia depletion results in modulation of the IFN response genes and MHC class II and class I genes in macrophages infiltrating the CNS at day 14 p.i scRNA Seq clustering analysis revealed that microglia depletion results in an increase in frequency of macrophages in the Mac1 population at D14 p.i (Fig. 3.7E,F). To further characterize these macrophages, we conducted a GSEA analysis of genes enriched in macrophages in response to IFNα and IFNγ. We observed a significantly higher enrichment of IFNα (Fig. 3.9A) and IFNγ (Fig. 3.9B) responsive genes in macrophages of the control treated group compared to the PLX5622 treated group. Differential gene expression analysis of macrophage cluster showed that PLX5622 treatment led to a significantly low expression of the MHC class II genes H2Ab1, H2DMa, and H2Eb1, suggesting a defect in antigen presentation by these cells (Fig. 3.9C). Microglia depletion resulted in a significantly higher expression of MHC class I genes H2Ke6, H2Q4, and H2T23 in the macrophage cluster at D14 p.i (Fig. 3.9D). 3.4.10 Microglia restrict the severity of JHMV-induced demyelination Luxol fast blue (LFB) staining of spinal cord sections revealed that the severity of demyelination was significantly (p<0.005) increased in PLX5622-treated mice compared to control treated animals at days14 and 21 p.i. (Fig. 3.10A,B). To determine the effect of microglia depletion on remyelination, EM analysis was performed on spinal cord sections isolated at day 21 p.i. g-ratio’s, a well-accepted metric for remyelination (42), were calculated. High magnification (1200X) images of spinal cords in the ventral white matter columns within the thoracic vertebrae 6-10 of control and PLX5622-treated mice 53 were used to calculate g-ratio’s. Remyelinated axons are characteristically analyzed by the appearance thin myelin sheaths compared to thicker myelin sheaths of a myelinated axon (Fig 3.10C). In the PLX5622 treated mice, there was an overall increase of demyelinated axons but fewer remyelinated axons were observed (Fig 3.10C). To accurately compare these observations, we quantified the g-ratios and myelin thickness of PLX control and PLX5622-treated mice. PLX5622 treatment of JHMV-infected mice resulted in significantly higher g-ratios compared to control treated mice (Fig 3.10D,E) and a significant (p<0.00001) decrease in myelin thickness compared to control treated animals (Fig 3.10F,G). These findings demonstrate that microglia depletion results in enhanced demyelination and lowers the endogenous remyelination capability following JHMV-induced demyelination. 3.5 Discussion MS is a multifaceted disease that involves complex interactions between various cells such as glia, neurons, and immune cells culminating in focal demyelinating lesions and axonal damage. Although MS was traditionally thought to be predominantly a CD4+ T cell mediated disease, other immune cells including CD8+ T cells, B cells, neutrophils, and macrophages are now recognized as potentially important contributors to disease progression. Furthermore, resident cells of the CNS including astrocytes and microglia are now recognized as being involved in different aspects related to either protection and/or disease progression (1, 43, 44). For example, microglia are capable of undergoing dynamic transformation in response to environmental stimuli and secrete a variety of cytokines/chemokines and recruit peripheral immune cells into the CNS (45). 54 Pathological hallmarks of MS include areas of active demyelination and astrogliosis (46), and active demyelinating lesions are consistent with inflammation and reactive microgliosis (47). Microglia and macrophages were found to be the most abundant immune cell types in active MS lesions, with activated microglia present through both early and late MS stages (48, 49). Until recently, it was difficult to distinguish between microglia and macrophages phenotypically, as microglia are capable of transforming into a macrophage phenotype (8). However, the marker TMEM119 is now recognized as a bona fide microglia marker and has been used to distinguish between microglia and macrophages (50, 51). A majority of IBA-1- positive cells in new active lesions express TMEM 119, suggesting that the initial tissue damage seen in MS lesions is associated with microglia (49, 52). The role of microglia in neurodegenerative diseases has been not been well defined due to difficulties in separating microglia function from either resident or inflammatory macrophages. However, certain genes expressed exclusively in parenchymal microglia such as TREM2, CD33, and CR1 have been identified as risk genes for AD (53, 54). Microglia can be protective in AD by recognizing and clearing amyloid beta (Aβ)- plaques, but persistent Aβ- microglia interaction can exacerbate amyloid plaque deposition (55). In Parkinson’s disease (PD), microglia can accumulate near α-synuclein deposits and become proinflammatory (56). In MS, microglia can be detrimental or beneficial depending on the stage of disease progression. In EAE, microglia release reactive oxygen and nitrogen species (ROS and RNS), proinflammatory cytokines, and recruit reactive T lymphocytes, all of which are neurotoxic and harmful to oligodendrocytes (57). In contrast, at disease onset, microglia promote remyelination, 55 clearance of myelin debris and release neurotrophic factors (58). In addition to potentially contributing to disease, how microglia function in either host defense and/or disease following viral infection was also not well understood. Recent studies employing inhibitors of CSF1R that reduce microglia within the CNS has proven to be extremely helpful in delineating roles for these cells following infection. For example, a recent study using the Theiler’s murine encephalomyelitis virus (TMEV) showed that PLX5622- mediated inhibition of microglia led to reduced immune response to infection, resulting in increased viral proliferation and enhanced seizure development (59). Similar to Wheeler et al. (33), we found that treatment of mice with PLX5622 led to a >90% depletion of microglia in the CNS, and i.c. inoculation with JHMV resulted in worsening of clinical disease and an increase in mortality of JHMV-infected mice (Fig. 3.1A-E). Additionally, PLX5622-mediated microglia depletion impaired control of viral replication at day 7 compared to control mice yet by day 14 virus was not detected in either PLX5622-treated on control animals, suggesting that microglia were critical in aiding in host defense during the acute phase of disease (Fig. 3.1F). We next evaluated whether the delayed viral replication had an effect on the number of T cells and macrophages infiltrating the brain at day 7 p.i. Unlike reports from a similar study (33), microglia depletion in our study did not result in a decrease in CD4+ T cells infiltrating the CNS. In contrast, there was a significant increase in the density of CD8+ T cells infiltrating the CNS upon microglia depletion (Fig. 3.2B). Although PLX5622 affects the receptor CSF1R, which is also expressed on peripheral macrophages, we observed a trending increase in macrophages in the CNS of PLX5622 treated mice (Fig. 3.1B). The influx of macrophages and CD8+ T cells could be a 56 compensatory effect to provide additional host defense to control the delayed viral replication in the absence of microglia. In order to obtain better insight into how targeted reduction in microglia impacted the immune response to JHMV infection of the CNS, we performed scRNA Seq on CD45+ cells infiltrating into the CNS of PLX5622-treated and control mice at day 7 p.i. Clustering analysis revealed 15 different clusters of different immune cells infiltrating the brain at day 7 p.i (Fig. 3.3). scRNA Seq analysis revealed that microglia depletion resulted in discrete changes in the immune cell populations at the single cell RNA level. For example in the CD8+ effector cells from microglia depleted samples had increased gene expression of the effector molecules granzyme B and perforin (Fig. 3.4A), suggesting a higher cytotoxic CD8+ T cell activity. In contrast, microglia depletion resulted in low expression of the activation markers CD44 and CD25 (Il2ra) on CD4+ T cells (Fig. 3.4B), indicating that microglia are required to activate these cells, either directly or indirectly. Interestingly, macrophages from microglia-depleted mice expressed high levels of the MHC class I genes- H2K1, H2Q7, H2M3, and H2T22 but had low expression of MHC class II genes- H2Ab1, H2DMa, and H2Eb1 (Fig 3.5B,C). Additionally, we observe a significant enrichment of IFNα and IFNγ response genes in macrophages in PLX5622-treated animals, and this may reflect that in the absence of microglia, these cells are targets for viral replication leading to enhanced response to IFN-a as well as IFN-g expressed by infiltrating activated T cells (Fig 3.5A). Microglia depletion also resulted in an increase in numbers of both CD4+and CD8+ T cells as well as virus-specific CD4+ and CD8+ T cells in the CNS at day 14 p.i. However, in contrast to day 7 p.i, CD8+ T cells from microglia-depleted mice have reduced gene expression of 57 the effector molecules granzyme B and perforin at day 14 p.i., and this may reflect the reduction in viral titers within the CNS (Fig. 3.8A). Additionally, at day 14 p.i, macrophages from control-treated mice had significantly higher enrichment of IFN-α and IFN-γ response genes compared to macrophages from microglia-depleted mice. Moreover, macrophages from PLX5622 mice had comparatively low expression of MHC class II genes yet expression of MHC class I was enhanced (Fig. 3.9B,C). Wheeler et.al., (33) show that microglia depletion results in an impaired CD4+ T cell response and infiltrating macrophages have defects in MHC class II antigen presentation following JHMV infection. In contrast, our study shows that microglia depletion results in an increase in infiltrating T cells and higher expression of genes involved in MHC class I expression on macrophages. Similar to Wheeler et. al., we observe that macrophages from microglia depleted mice may have defects in MHC class II antigen presentation. The different results from the two studies could be attributed to the lab strain of JHMV as well as dose of JHMV used for each study. Next, we evaluated the effect of microglia depletion on demyelination in JHMVinfected mice. Oxidative stress has been suggested to play a major role in pathogenesis of demyelinating diseases (60). Oligodendrocyte precursor cells (OPCs) are sensitive to oxidative stress as they contain low levels of antioxidant enzymes and high levels of proapoptotic proteins (61, 62) and activated microglia, and macrophages are considered the major sources of ROS in MS (63). Indeed, ablation of microglia resulted in protection of grey and white matter in EAE mice (64). Additionally, microglia depletion by CSF1R inhibition in the EAE model led to reduced immune activation and demyelination (65). Treatment with PLX5622 during the symptomatic phase of EAE led to a dramatic 58 improvement in clinical disease (65). The reduced inflammation upon PLX5622 treatment led to preservation of mature, myelinating-oligodendrocytes and thus provided an environment for enhanced remyelination (65). In our study, we show that microglia depletion results in an increase in severity of JHMV-induced demyelination (Fig. 3.10A,B). T cells and macrophages form the major components of the immune system that contribute to demyelination in the JHMV model. Demyelination is reduced in mice that lack CD4+ or CD8+ T cells (34). Additionally, JHMV-induced demyelination is also reduced in chronically infected mice that have defects in macrophage trafficking (66-68). The increase in severity of demyelination observed in this study upon PLX5622 treatment could be due to the higher influx of CD4+, CD8+ T cells, and macrophages. Remyelination is the process of regeneration of myelin around axons occurring during and/ or following demyelination (60). Key endogenous processes that aid in remyelination are clearance of myelin debris and recruitment and proliferation of OPCs that become myelinating oligodendrocytes (69). Phagocytosis of myelin debris by microglia is an important first step for initiating remyelination and insufficient clearance of myelin debris is associated with an inadequate regenerative response (70). Additionally, microglia support oligodendrocyte proliferation by secreting factors such as IGF-1, FGF-2, and TNF-α (71). Oligodendrocyte differentiation was enhanced when cerebellar slices were cultured with conditioned media from immunoregulatory microglia (M2) in vitro and decreased in vivo after depletion of M2 microglia, suggesting an important role for microglia in oligodendrocyte differentiation and subsequently remyelination (71). Since these studies demonstrate the importance of microglia in remyelination, we evaluated whether microglia depletion affected remyelination during 59 JHMV-induced demyelination by conducting EM analysis on spinal cord sections at day 21 p.i. We found that PLX5622 mediated microglia depletion leads to significantly higher g-ratios (Fig. 3.10C,D,E) and lower myelin thickness (Fig. 3.10C,F,G). This suggests that microglia depletion resulted in lowering the endogenous remyelination capacity, potentially due to reduced myelin debris clearance and/ or inability of OPCs to differentiate into myelin competent oligodendrocytes. In conclusion, our study demonstrates that microglia aid in viral clearance, modulate the immune response to infection, restrict the severity of demyelination and promote remyelination in a viral model of MS. 3.6 References 1. Jack C, Ruffini F, Bar-Or A, Antel JP. Microglia and multiple sclerosis. J Neurosci Res. 2005;81(3):363-73. 2. Rock RB, Gekker G, Hu S, Sheng WS, Cheeran M, Lokensgard JR, et al. Role of microglia in central nervous system infections. Clin Microbiol Rev. 2004;17(4):942-64, table of contents. 3. Prinz M, Priller J, Sisodia SS, Ransohoff RM. Heterogeneity of CNS myeloid cells and their roles in neurodegeneration. Nat Neurosci. 2011;14(10):1227-35. 4. Paolicelli RC, Bolasco G, Pagani F, Maggi L, Scianni M, Panzanelli P, et al. Synaptic pruning by microglia is necessary for normal brain development. Science. 2011;333(6048):1456-8. 5. Schafer DP, Lehrman EK, Kautzman AG, Koyama R, Mardinly AR, Yamasaki R, et al. Microglia sculpt postnatal neural circuits in an activity and complementdependent manner. Neuron. 2012;74(4):691-705. 6. Nguyen MD, Julien JP, Rivest S. Innate immunity: the missing link in neuroprotection and neurodegeneration? Nat Rev Neurosci. 2002;3(3):216-27. 7. Polfliet MM, van de Veerdonk F, Dopp EA, van Kesteren-Hendrikx EM, van Rooijen N, Dijkstra CD, et al. The role of perivascular and meningeal macrophages in experimental allergic encephalomyelitis. J Neuroimmunol. 2002;122(1-2):1-8. 60 8. Kettenmann H, Hanisch UK, Noda M, Verkhratsky A. Physiology of microglia. Physiol Rev. 2011;91(2):461-553. 9. Wierzba-Bobrowicz T, Gwiazda E, Kosno-Kruszewska E, Lewandowska E, Lechowicz W, Bertrand E, et al. Morphological analysis of active microglia--rod and ramified microglia in human brains affected by some neurological diseases (SSPE, Alzheimer's disease and Wilson's disease). Folia Neuropathol. 2002;40(3):125-31. 10. Smith JA, Das A, Ray SK, Banik NL. Role of pro-inflammatory cytokines released from microglia in neurodegenerative diseases. Brain Res Bull. 2012;87(1):10-20. 11. Bsibsi M, Ravid R, Gveric D, van Noort JM. Broad expression of Toll-like receptors in the human central nervous system. J Neuropathol Exp Neurol. 2002;61(11):1013-21. 12. Itagaki S, McGeer PL, Akiyama H, Zhu S, Selkoe D. Relationship of microglia and astrocytes to amyloid deposits of Alzheimer disease. J Neuroimmunol. 1989;24(3):173-82. 13. McGeer PL, Itagaki S, Boyes BE, McGeer EG. Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains. Neurology. 1988;38(8):1285-91. 14. Sapp E, Kegel KB, Aronin N, Hashikawa T, Uchiyama Y, Tohyama K, et al. Early and progressive accumulation of reactive microglia in the Huntington disease brain. J Neuropathol Exp Neurol. 2001;60(2):161-72. 15. Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mork S, Bo L. Axonal transection in the lesions of multiple sclerosis. N Engl J Med. 1998;338(5):27885. 16. Rademakers R, Baker M, Nicholson AM, Rutherford NJ, Finch N, Soto-Ortolaza A, et al. Mutations in the colony stimulating factor 1 receptor (CSF1R) gene cause hereditary diffuse leukoencephalopathy with spheroids. Nat Genet. 2011;44(2):200-5. 17. Paloneva J, Manninen T, Christman G, Hovanes K, Mandelin J, Adolfsson R, et al. Mutations in two genes encoding different subunits of a receptor signaling complex result in an identical disease phenotype. Am J Hum Genet. 2002;71(3):656-62. 61 18. Li Y, Zhang R, Hou X, Zhang Y, Ding F, Li F, et al. Microglia activation triggers oligodendrocyte precursor cells apoptosis via HSP60. Mol Med Rep. 2017;16(1):603-8. 19. Pang Y, Campbell L, Zheng B, Fan L, Cai Z, Rhodes P. Lipopolysaccharideactivated microglia induce death of oligodendrocyte progenitor cells and impede their development. Neuroscience. 2010;166(2):464-75. 20. Cheever FS, Daniels JB, Pappenheimer AM, Bailey OT. A murine virus (JHM) causing disseminated encephalomyelitis with extensive destruction of myelin. J Exp Med. 1949;90(3):181-210. 21. Fleming JO, Trousdale MD, el-Zaatari FA, Stohlman SA, Weiner LP. Pathogenicity of antigenic variants of murine coronavirus JHM selected with monoclonal antibodies. J Virol. 1986;58(3):869-75. 22. Parra B, Hinton DR, Lin MT, Cua DJ, Stohlman SA. Kinetics of cytokine mRNA expression in the central nervous system following lethal and nonlethal coronavirus-induced acute encephalomyelitis. Virology. 1997;233(2):260-70. 23. Parra B, Hinton DR, Marten NW, Bergmann CC, Lin MT, Yang CS, et al. IFNgamma is required for viral clearance from central nervous system oligodendroglia. J Immunol. 1999;162(3):1641-7. 24. Pearce BD, Hobbs MV, McGraw TS, Buchmeier MJ. Cytokine induction during T-cell-mediated clearance of mouse hepatitis virus from neurons in vivo. J Virol. 1994;68(9):5483-95. 25. Xue S, Sun N, Van Rooijen N, Perlman S. Depletion of blood-borne macrophages does not reduce demyelination in mice infected with a neurotropic coronavirus. J Virol. 1999;73(8):6327-34. 26. Moreno MA, Burns T, Yao P, Miers L, Pleasure D, Soulika AM. Therapeutic depletion of monocyte-derived cells protects from long-term axonal loss in experimental autoimmune encephalomyelitis. J Neuroimmunol. 2016;290:36-46. 27. Chuang TY, Guo Y, Seki SM, Rosen AM, Johanson DM, Mandell JW, et al. LRP1 expression in microglia is protective during CNS autoimmunity. Acta Neuropathol Commun. 2016;4(1):68. 28. Vainchtein ID, Vinet J, Brouwer N, Brendecke S, Biagini G, Biber K, et al. In acute experimental autoimmune encephalomyelitis, infiltrating macrophages are immune activated, whereas microglia remain immune suppressed. Glia. 2014;62(10):1724-35. 62 29. Patel S, Player MR. Colony-stimulating factor-1 receptor inhibitors for the treatment of cancer and inflammatory disease. Curr Top Med Chem. 2009;9(7):599-610. 30. Ginhoux F, Greter M, Leboeuf M, Nandi S, See P, Gokhan S, et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science. 2010;330(6005):841-5. 31. Erblich B, Zhu L, Etgen AM, Dobrenis K, Pollard JW. Absence of colony stimulation factor-1 receptor results in loss of microglia, disrupted brain development and olfactory deficits. PLoS One. 2011;6(10):e26317. 32. Elmore MR, Najafi AR, Koike MA, Dagher NN, Spangenberg EE, Rice RA, et al. Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. Neuron. 2014;82(2):38097. 33. Wheeler DL, Sariol A, Meyerholz DK, Perlman S. Microglia are required for protection against lethal coronavirus encephalitis in mice. J Clin Invest. 2018;128(3):931-43. 34. Lane TE, Liu MT, Chen BP, Asensio VC, Samawi RM, Paoletti AD, et al. A central role for CD4(+) T cells and RANTES in virus-induced central nervous system inflammation and demyelination. J Virol. 2000;74(3):1415-24. 35. Hirano N, Murakami T, Fujiwara K, Matsumoto M. Utility of mouse cell line DBT for propagation and assay of mouse hepatitis virus. Jpn J Exp Med. 1978;48(1):71-5. 36. Blanc CA, Rosen H, Lane TE. FTY720 (fingolimod) modulates the severity of viral-induced encephalomyelitis and demyelination. J Neuroinflammation. 2014;11:138. 37. Ekiz HA, Huffaker TB, Grossmann AH, Stephens WZ, Williams MA, Round JL, et al. MicroRNA-155 coordinates the immunological landscape within murine melanoma and correlates with immunity in human cancers. JCI Insight. 2019. 38. Carbajal KS, Miranda JL, Tsukamoto MR, Lane TE. CXCR4 signaling regulates remyelination by endogenous oligodendrocyte progenitor cells in a viral model of demyelination. Glia. 2011;59(12):1813-21. 39. Blanc CA, Grist JJ, Rosen H, Sears-Kraxberger I, Steward O, Lane TE. Sphingosine-1-phosphate receptor antagonism enhances proliferation and migration of engrafted neural progenitor cells in a model of viral-induced demyelination. Am J Pathol. 2015;185(10):2819-32. 63 40. Dagher NN, Najafi AR, Kayala KM, Elmore MR, White TE, Medeiros R, et al. Colony-stimulating factor 1 receptor inhibition prevents microglial plaque association and improves cognition in 3xTg-AD mice. J Neuroinflammation. 2015;12:139. 41. Butler A, Hoffman P, Smibert P, Papalexi E, Satija R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol. 2018;36(5):411-20. 42. Franklin RJ, Goldman SA. Glia Disease and Repair-Remyelination. Cold Spring Harb Perspect Biol. 2015;7(7):a020594. 43. Ponath G, Ramanan S, Mubarak M, Housley W, Lee S, Sahinkaya FR, et al. Myelin phagocytosis by astrocytes after myelin damage promotes lesion pathology. Brain. 2017;140(2):399-413. 44. Ransohoff RM, Brown MA. Innate immunity in the central nervous system. J Clin Invest. 2012;122(4):1164-71. 45. Lampron A, Pimentel-Coelho PM, Rivest S. Migration of bone marrow-derived cells into the central nervous system in models of neurodegeneration. J Comp Neurol. 2013;521(17):3863-76. 46. Popescu BF, Lucchinetti CF. Pathology of demyelinating diseases. Annu Rev Pathol. 2012;7:185-217. 47. Lassmann H. Mechanisms of white matter damage in multiple sclerosis. Glia. 2014;62(11):1816-30. 48. Bogie JF, Stinissen P, Hendriks JJ. Macrophage subsets and microglia in multiple sclerosis. Acta Neuropathol. 2014;128(2):191-213. 49. van der Valk P, Amor S. Preactive lesions in multiple sclerosis. Curr Opin Neurol. 2009;22(3):207-13. 50. Bennett ML, Bennett FC, Liddelow SA, Ajami B, Zamanian JL, Fernhoff NB, et al. New tools for studying microglia in the mouse and human CNS. Proc Natl Acad Sci U S A. 2016;113(12):E1738-46. 51. Satoh J, Kino Y, Asahina N, Takitani M, Miyoshi J, Ishida T, et al. TMEM119 marks a subset of microglia in the human brain. Neuropathology. 2016;36(1):3949. 52. O'Loughlin E, Madore C, Lassmann H, Butovsky O. Microglial phenotypes and functions in Multiple Sclerosis. Cold Spring Harb Perspect Med. 2018;8(2). 64 53. Sims R, van der Lee SJ, Naj AC, Bellenguez C, Badarinarayan N, Jakobsdottir J, et al. Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglialmediated innate immunity in Alzheimer's disease. Nat Genet. 2017;49(9):137384. 54. Hu X, Pickering EH, Hall SK, Naik S, Liu YC, Soares H, et al. Genome-wide association study identifies multiple novel loci associated with disease progression in subjects with mild cognitive impairment. Transl Psychiatry. 2011;1:e54. 55. Gold M, El Khoury J. beta-amyloid, microglia, and the inflammasome in Alzheimer's disease. Semin Immunopathol. 2015;37(6):607-11. 56. Croisier E, Moran LB, Dexter DT, Pearce RK, Graeber MB. Microglial inflammation in the parkinsonian substantia nigra: relationship to alpha-synuclein deposition. J Neuroinflammation. 2005;2:14. 57. Ransohoff RM, El Khoury J. Microglia in health and disease. Cold Spring Harb Perspect Biol. 2015;8(1):a020560. 58. Yamasaki R, Lu H, Butovsky O, Ohno N, Rietsch AM, Cialic R, et al. Differential roles of microglia and monocytes in the inflamed central nervous system. J Exp Med. 2014;211(8):1533-49. 59. Waltl I, Kaufer C, Gerhauser I, Chhatbar C, Ghita L, Kalinke U, et al. Microglia have a protective role in viral encephalitis-induced seizure development and hippocampal damage. Brain Behav Immun. 2018;74:186-204. 60. Luo C, Jian C, Liao Y, Huang Q, Wu Y, Liu X, et al. The role of microglia in multiple sclerosis. Neuropsychiatr Dis Treat. 2017;13:1661-7. 61. Blomgren K, Hagberg H. Free radicals, mitochondria, and hypoxia-ischemia in the developing brain. Free Radic Biol Med. 2006;40(3):388-97. 62. Butts BD, Houde C, Mehmet H. Maturation-dependent sensitivity of oligodendrocyte lineage cells to apoptosis: implications for normal development and disease. Cell Death Differ. 2008;15(7):1178-86. 63. Miller E, Wachowicz B, Majsterek I. Advances in antioxidative therapy of multiple sclerosis. Curr Med Chem. 2013;20(37):4720-30. 64. Heppner FL, Greter M, Marino D, Falsig J, Raivich G, Hovelmeyer N, et al. Experimental autoimmune encephalomyelitis repressed by microglial paralysis. Nat Med. 2005;11(2):146-52. 65 65. Nissen JC, Thompson KK, West BL, Tsirka SE. Csf1R inhibition attenuates experimental autoimmune encephalomyelitis and promotes recovery. Exp Neurol. 2018;307:24-36. 66. Wu GF, Perlman S. Macrophage infiltration, but not apoptosis, is correlated with immune-mediated demyelination following murine infection with a neurotropic coronavirus. J Virol. 1999;73(10):8771-80. 67. Glass WG, Hickey MJ, Hardison JL, Liu MT, Manning JE, Lane TE. Antibody targeting of the CC chemokine ligand 5 results in diminished leukocyte infiltration into the central nervous system and reduced neurologic disease in a viral model of multiple sclerosis. J Immunol. 2004;172(7):4018-25. 68. Glass WG, Liu MT, Kuziel WA, Lane TE. Reduced macrophage infiltration and demyelination in mice lacking the chemokine receptor CCR5 following infection with a neurotropic coronavirus. Virology. 2001;288(1):8-17. 69. Domingues HS, Portugal CC, Socodato R, Relvas JB. Oligodendrocyte, astrocyte, and microglia crosstalk in myelin development, damage, and repair. Front Cell Dev Biol. 2016;4:71. 70. Neumann H, Kotter MR, Franklin RJ. Debris clearance by microglia: an essential link between degeneration and regeneration. Brain. 2009;132(Pt 2):288-95. 71. Voss EV, Skuljec J, Gudi V, Skripuletz T, Pul R, Trebst C, et al. Characterisation of microglia during de- and remyelination: can they create a repair promoting environment? Neurobiol Dis. 2012;45(1):519-28. 66 Figure 3.1. Microglia depletion causes an increase in mortality and viral load. (A) Representative flow cytometry plots showing PLX5622 mediated depletion of microglia (gated as CD45int F4/80+ cells) in brains of JHMV infected mice. (B) Quantification of flow cytometry data show that PLX5622 causes a significant reduction in the total number of microglia but not macrophages (gated as CD45hi F4/80+ cells) (****p<0.0005) (n=6/group). (C) Representative images showing depletion of microglia in spinal cords of PLX5622 treated mice compared to control mice as determined by fluorescent Iba-1 staining. (D) PLX5622 treatment leads to higher (****p<0.0001) clinical scores in JHMV infected mice compared to control-treated mice, correlating with increased mortality (n=10/group)(E). The overall increase in morbidity/morality in PLX5622treated mice was associated with significantly (****p<0.0005) higher viral titers in the brain at day 7 p.i. compared to control mice (n=5/group). Data from (A) and (B) are representative of 2 independent experiments, and all data are presented as mean+SEM. 67 Figure 3.2. Microglia depletion influences the T cell infiltration into CNS of JHMVinfected mice (A) Representative flow plots showing an increase in CD4+ and CD8+ T cells in PLX5622 treated mice compared to control mice. (B) Quantification shows a significant increase in CD8+ T cells and a trending increase in CD4+ T cells in brains of PLX5622 treated mice. (*p<0.05) compared to control mice (n=6/group). (C) Microglia depletion causes a trending increase in virus-specific CD4+ T cells (C) and CD8+ T cells. (D) When compared to control mice, data from (A) and (B) are representative of 2 independent experiments, and all data are presented as mean+SEM. 68 Figure 3.3. sc RNASeq unveils the immunological landscape in the brains of JHMVinfected mice treated with PLX5622 or control at day 7 p.i (A) Schematic showing the experimental outline used for scRNASeq of CD45+ cells isolated from spinal cords of PLX5622-treated or control mice at 7 days post-JHMV infection. (B) T-distributed stochastic neighbor embedding (t-SNE) plots of scRNASeq data revealing 15 distinct cell clusters (aggregate data from PLX5622 and control treated animals at 7 days p.i). (C) Dot plot presenting expression of selected genes in the 15 cell clusters. Size of the dots is representative of the frequency of cells within a particular cluster expressing the gene of interest, while the color intensity of the dot is indicative of levels of expression of a particular gene. (D) Heat map showing the top 5 differentially expressed genes in the 15 clusters. Different columns represent clusters and rows are indicative of genes. (E) t-SNE plot showing the immune landscape in brains of control (blue) and PLX5622 (amber) treated animals at 7 days p.i. (F) Frequency of cell clusters in brains of control and PLX5622 treated animals at 7 days post-JHMV infection. 69 Figure 3.4. Microglia depletion leads to differential gene expression in T cell subsets as revealed by scRNASeq. (A) Expression levels of T cell activation markers and effector molecules within the CD8+ T cell cluster are shown. (B) Expression levels of T cell activation markers within the CD4+ T cell cluster are shown. Each black dot in the plot represents a single cell; normalized expression values were used. The box plot shows interquartile range; the bold horizontal line represents median, and average expression per sample is represented by the red dot. Wilcoxon test was used for statistical analysis (*p≤ 0.05; ** p≤ 0.01; ***p≤ 0.001; ****p≤ 0.0001) 70 Figure 3.5. PLX5622 treatment leads to differential gene expression in macrophages infiltrating the CNS at day 7 p.i. (A) GSEA of macrophages in scRNASeq data at day 7 p.i. Macrophages from PLX5622 treated animals show an enrichment of genes involved in the IFNα response pathway and (B) IFNγ response pathways. (C) Expression levels of the MHC class II genes H2Ab1, H2DMa, and H2Eb1 were significantly lower in macrophages from PLX5622 treated animals. (D) Gene expression levels of MHC class I genes H2K1, H2Q7, H2M3 and H2T22 were significantly higher in macrophages from PLX5622 treated animals. Each box plot shows interquartile range; the bold horizontal line represents median, and average expression per sample is represented by the red dot. Wilcoxon test was used for statistical analysis (*p≤ 0.05; ** p≤ 0.01; ***p≤ 0.001; ****p≤ 0.0001). 71 Figure 3.6. Microglia depletion augments JHMV-induced neuroinflammation at day 14 p.i. (A) Representative flow cytometry plots and quantified bar graphs show PLX5622-mediated depletion of microglia at day 14 following JHMV infection compared to control mice (* p<0.05) (n=8/group). (B) Microglia depletion causes a significant increase in CD8+ T cells and a trending increase in CD4+ T cells (*p<0.05)(n=8/group). Lack of microglia causes a significant increase in (C) virusspecific CD4+ T cells (* p<0.05) (n=5/group) and (D) CD8+ T cells (** p<0.005) (n=5/group). All data are representative of two independent experiments and are presented as mean+SEM. 72 Figure 3.7. Single cell RNA sequencing reveals the dynamic immunological landscape in the spinal cords of JHMV-infected mice treated with PLX5622 or control at day 14 p.i (A) Schematic showing the experimental outline used for scRNASeq of CD45+ cells isolated from spinal cords of mice JHMV-infected mice treated with either PLX5622 or control chow at day 14 p.i. (B) T-distributed stochastic neighbor embedding (t-SNE) plots of scRNASeq data revealing 17 distinct cell clusters (aggregate data from PLX5622 and control treated animals at 14 days p.i). (C) Dot plot presenting expression of selected genes in the 17 cell clusters. (D) Heat map showing the top 5 differentially expressed genes in the 17 clusters. (E) t-SNE plot showing the immune landscape in spinal cords of control (green) and PLX5622 (grey) treated animals at D14 p.i. (F) Frequency of cell clusters in spinal cords of control and PLX5622 treated animals at D14 p.i. 73 Figure 3.8. Microglia depletion results in reduced expression of T cell activation markers in T cell subsets in spinal cord at day 14 p.i (A) Expression levels of T cell activation and effector molecules within the activated CD8+ T cell cluster are shown. (B) Expression of T cell activation markers in the CD4+ T cell cluster in spinal cords at D14 p.i. Each black dot in the plot represents a single cell. Normalized expression values were used. Each box plot shows interquartile range; the bold horizontal line represents median, and average expression per sample is represented by the red dot. Wilcoxon test was used for statistical analysis (*p≤ 0.05; ** p≤ 0.01; ***p≤ 0.001; ****p≤ 0.0001) 74 Figure 3.9. Microglia depletion results in modulation of the IFN response genes and MHC class II and class I genes in macrophages infiltrating the CNS at day 14 p.i. GSEA plot depicting the (A) IFN-α and (B) IFN-γ responsive genes in macrophage populations isolated from the spinal cords of JHMV-infected mice treated with either PLX5622 or control at day 14 p.i. (C) Differential gene expression analysis showing expression of MHC class II genes in the spinal cord macrophage cluster of PLX5622 and control treated animals at day 14 p.i. (D) Differential gene expression of MHC class I genes in spinal cord macrophage cluster of PLX5622 and control treated animals at day 14 p.i. Each box plot shows interquartile range; the bold horizontal line represents median and average expression per sample is represented by the red dot. Wilcoxon test was used for statistical analysis (*p≤ 0.05; ** p≤ 0.01; ***p≤ 0.001; ****p≤ 0.0001) 75 Figure 3.10. Microglia restrict the severity of JHMV-induced demyelination (A) Representative images of H&E/LFB-stained spinal cord sections showing an increase in severity of white matter demyelination (dashed blue lines) in JHMV-infected mice treated with PLX5622 compared to control treated mice at day 21 p.i. (B) Quantification of percentage demyelination at day 14 and 21 p.i show significantly higher demyelination after PLX5622 treatment (*p<0.05; **p<0.005) (n=12-15/group, day 14; n=5/6 group for day 21 p.i, (C) Representative EM images (1200X) from PLX5622 and control spinal cords showing normal myelinated axons (white arrowheads), demyelinated axons (black arrows) and remyelinated axons (blue arrows) at D21 p.i. (D) Calculation of g-ratio of PLX control (145 axons from a total of 17 fields) and PLX5622 (208 axons from a total of 31 fields) (****p<0.00001) (n=2/group). (E) Scatter plot depicting individual axons from lateral white matter columns of control (black) and PLX5622 (red) treated mice as a function of axon diameter. (F) Quantification of myelin thickness shows significantly (****p<0.00001) thinner myelin sheaths in PLX5622 treated mice compared to mice. (G) Scatter plot depicting myelin thickness of individual axons from PLX control (black) and PLX5622 (red) treated mice as a function of axon diameter. Data are representative of two independent experiments and are presented as mean+SEM. CHAPTER 4 CONCLUSIONS AND FUTURE DIRECTIONS 77 4.1 Therapeutic Application of iPSC Derived NPCs in a Viral Model of MS NPC transplantation has been suggested as a potential therapeutic option for neuroinflammatory and demyelinating diseases as they represent attractive sources for generating myelin competent oligodendrocytes (1, 2). In rodent autoimmune models of MS, NPC transplantation has resulted in migration of NPCs to the demyelinated white matter tracts and an improvement in clinical sequelae (3, 4). However, it is imperative to study the remyelination potential of NPCs in a viral model of demyelination, as this will give important information into whether cell replacement therapies are effective within the CNS where persistent neurotropic viruses may be prevalent. In order to address this, we have previously shown that engraftment of postnatal-derived NPCs in spinal cords of JHMV-infected mice resulted in an improvement of clinical outcome, remyelination, and axonal sparing (5-7). In a clinical setting, donor specific NPCs derived from iPSCs may be advantageous in order to potentially bypass the need for immunosuppressive drugs, which might leave the patient susceptible to opportunistic infections. In the present work, NPCs were derived ex vivo from mouse iPSCs by supplying epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Upon withdrawal of EGF and bFGF NPCs differentiated into the three neural subtypes, e.g., oligodendrocytes, astrocytes, and neurons, similar to the postnatal GFP-NPCs. This finding shows that NPCs derived from iPSCs have similar functional properties as GFP-NPCs. However, iPSC-NPCs are unique with respect to expression of MHC Class I on their cell surface and susceptibility to JHMV infection. Under normal physiologic conditions, GFP-NPCs have undetectable levels MHC Class I and Class II but IFN-γ treatment can stimulate expression of MHC 78 on NPCs (8). iPSC-NPCs on the other hand, constitutively express MHC class I on their surface and neither JHMV-infection nor IFN-γ treatment modulates MHC expression levels. Quantitative PCR and flow cytometry revealed that iPSC-NPCs express low levels of the JHMV receptor CEACAM1a. Reduced expression of the viral receptor lead to reduced susceptibility to JHMV infection and limited viral replication. Due to the impaired ability of the virus to enter the cell, iPSC-NPCs were resistant to viral-induced cytopathic effects. In contrast, GFP-NPCs are susceptible to JHMV-infection in vitro and prone to viral-induced cell death (8). Furthermore, engrafted GFP-NPCs are susceptible to JHMV infection in vivo (9). These results suggest that transplanting cells susceptible to neurotropic viruses might be counteractive in a clinical setting, as viral infection might cause cell death and prevent differentiation into myelin competent oligodendrocytes. In the context of the JHMV model, our results indicate that iPSC-NPCs might be a better cell replacement option to GFP-NPCs as they are resistant to infection and have an increased potential of differentiating into oligodendrocytes and remyelination. In order to potentially use NPCs for remyelination, it is important to consider the possibility of engrafted cells to be targeted by resident neurotropic viruses. Indeed there are several persistent neurotropic viruses known to preferentially target and replicate in NPCs (1012). In a clinical setting, transplant recipients might be administered immunosuppressive drugs to prevent rejection of transplanted cells. This might lead to recrudescence of latent neurotropic viruses, which would otherwise be kept in check by immune surveillance. Subsequently, the therapeutic benefit of engrafted NPCs might be diminished as reactivated neurotropic viruses may infect them and cause cell death. This study suggests that engraftment of NPCs lacking receptors of known neurotropic viruses might protect 79 these cells from viral induced cell death and facilitate better clinical recovery. An immediate next step for this project would be to evaluate the remyelination potential of iPSC-NPCs in vivo in the JHMV model of demyelination. Since these cells show the potential of differentiating into oligodendrocytes in vitro, it is important to evaluate whether surgical transplantation of iPSC-NPCs in spinal cords of mice inflicted by JHMV-induced demyelination leads to migration to areas of demyelination and whether they can effectively remyelinate axons. Since we have demonstrated that iPSCNPCs are resistant to infection in vitro, another important experiment would be geared toward determining whether iPSC-NPCs are resistant to JHMV infection in vivo and whether these cells can survive and remyelinate axons in presence of a persistent JHMV infection. In order to address these questions, we will be evaluating whether depletion of the viral receptor CEACAM1a on postnatal GFP-NPCs make them resistant to JHMV infection and viral induced cell death in vitro. As GFP-NPCs can be detectable by fluorescence microscopy, we will be able to detect whether cells lacking the viral receptor can migrate to areas of demyelination and promote recovery in the presence of persistent viral infection and ongoing demyelination. To this end, we will utilized the CRISPR-Cas9 system to target CEACAM1a in GFP-NPCs evaluate whether CEACAM1a knockout GFP-NPCs survive when transplanted in spinal cords of JHMVinfected mice and whether these mice have better clinical outcomes compared to the CEACAM1a+ GFP-NPCs. 80 4.2 Role of Microglia in Host Defense and Demyelination The exact mechanism of MS pathology is not yet defined, but it is known that a variety of complex interactions between cell types including glia, neurons, and immune cells are involved. Although MS was initially thought to be predominantly a T cell mediated disease, recent studies show microglia as key players in disease progression (13). Microglia are essential cells in CNS inflammation, and they are the first line of defense against pathogenic infections in the CNS (14). In response to environmental cues, microglia can become highly activated and secrete a milieu of cytokines/chemokines such as IL-6 and IFNγ to provide host defense but can also become neurotoxic or neuroinflammatory (15-17). Microglia are thought to undergo reactive microgliosis and play a detrimental role in Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD) (18-20). Reactive microglia can hinder repair by interfering with differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes (21, 22). In the present work, we have explored the role of microglia in host defense, neuroinflammation and demyelination in the context of JHMV-induced demyelinating disease. We show that targeted reduction of microglia led to an increase in mortality associated with an impaired control of viral replication. Since microglia are the first responders to pathogenic infection in the CNS, depletion resulted in an increase in viral titers at day 7 p.i. However, viral replication was controlled by day 14 p.i, indicating that microglia are most crucial during the initial stages of infection. Microglia depletion augmented T cells and macrophages infiltrating the CNS. The infiltrating macrophages expressed low levels of genes involved in MHC class II expression, indicating a defect in 81 antigen presentation. Single cell RNA sequencing showed that microglia are capable of modulating the adaptive as well as innate immune response to JHMV infection, as assessed by expression of genes involved in antigen presentation, T cell activation, and neuroinflammation. Microglia depletion led to an increase in severity of demyelination, potentially due to the higher influx of CD4+ and CD8+ T cells infiltrating the brain upon PLX5622 treatment. Microglia have non redundant roles in the remyelination process as they help clear myelin debris as well as support OPC differentiation (23, 24). We observe a dampened endogenous remyelination capacity at day 21 p.i in JHMV-infected mice treated with PLX5622. These results suggest that microglia have a protective role in host defense as well as in preventing white matter damage and supporting remyelination by oligodendrocytes. Overall, this study highlights the importance of microglia in host defense as well as immune modulation. In a viral model of MS, our study is the first of its kind to evaluate the effect of microglia depletion on immune cells infiltrating the brain at a single cell RNA level. These results help us examine the role of microglia in modulating functions of other immune populations such as dendritic cells and the subsequent effect on antigen presentation. The next step in this study is to delineate the effect of microglia depletion on immune cell types such as dendritic cells, NK cells, monocytes, and B cells in response to JHMV infection. To this end, we will analyze the differences in gene expression profiles of these cells in the presence and absence of microglia and how these changes ultimately influence disease progression in JHMV-induced demyelination. Additionally, we will deplete microglia after the virus is cleared and determine the effect on modulation of T cell phenotypes and also on demyelination and remyelination. Lastly, we will analyze the 82 effects of microglia depletion on astrocyte phenotype. Emerging evidence shows that reactive microglia can modulate astrocytosis and determine the fate of astrocytes under diverse pathological conditions (25-27). We will analyze the effect of microglia depletion on astrocytosis in JHMV-induced demyelinating disease. 4.3 References 1. Ben-Hur T, Rogister B, Murray K, Rougon G, Dubois-Dalcq M. Growth and fate of PSA-NCAM+ precursors of the postnatal brain. J Neurosci. 1998;18(15):577788. 2. Brustle O, Jones KN, Learish RD, Karram K, Choudhary K, Wiestler OD, et al. Embryonic stem cell-derived glial precursors: a source of myelinating transplants. Science. 1999;285(5428):754-6. 3. Ben-Hur T, Einstein O, Mizrachi-Kol R, Ben-Menachem O, Reinhartz E, Karussis D, et al. Transplanted multipotential neural precursor cells migrate into the inflamed white matter in response to experimental autoimmune encephalomyelitis. Glia. 2003;41(1):73-80. 4. Pluchino S, Quattrini A, Brambilla E, Gritti A, Salani G, Dina G, et al. Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature. 2003;422(6933):688-94. 5. Carbajal KS, Schaumburg C, Strieter R, Kane J, Lane TE. Migration of engrafted neural stem cells is mediated by CXCL12 signaling through CXCR4 in a viral model of multiple sclerosis. Proc Natl Acad Sci U S A. 2010;107(24):11068-73. 6. Greenberg ML, Weinger JG, Matheu MP, Carbajal KS, Parker I, Macklin WB, et al. Two-photon imaging of remyelination of spinal cord axons by engrafted neural precursor cells in a viral model of multiple sclerosis. Proc Natl Acad Sci U S A. 2014;111(22):E2349-55. 7. Totoiu MO, Nistor GI, Lane TE, Keirstead HS. Remyelination, axonal sparing, and locomotor recovery following transplantation of glial-committed progenitor cells into the MHV model of multiple sclerosis. Exp Neurol. 2004;187(2):254-65. 8. Plaisted WC, Weinger JG, Walsh CM, Lane TE. T cell mediated suppression of neurotropic coronavirus replication in neural precursor cells. Virology. 2014;449:235-43. 9. Weinger JG, Plaisted WC, Maciejewski SM, Lanier LL, Walsh CM, Lane TE. Activating receptor NKG2D targets RAE-1-expressing allogeneic neural 83 precursor cells in a viral model of multiple sclerosis. Stem Cells. 2014;32(10):2690-701. 10. Chucair-Elliott AJ, Conrady C, Zheng M, Kroll CM, Lane TE, Carr DJ. Microglia-induced IL-6 protects against neuronal loss following HSV-1 infection of neural progenitor cells. Glia. 2014;62(9):1418-34. 11. Ruller CM, Tabor-Godwin JM, Van Deren DA, Jr., Robinson SM, Maciejewski S, Gluhm S, et al. Neural stem cell depletion and CNS developmental defects after enteroviral infection. Am J Pathol. 2012;180(3):1107-20. 12. Schaumburg C, O'Hara BA, Lane TE, Atwood WJ. Human embryonic stem cellderived oligodendrocyte progenitor cells express the serotonin receptor and are susceptible to JC virus infection. J Virol. 2008;82(17):8896-9. 13. Jack C, Ruffini F, Bar-Or A, Antel JP. Microglia and multiple sclerosis. J Neurosci Res. 2005;81(3):363-73. 14. Rock RB, Gekker G, Hu S, Sheng WS, Cheeran M, Lokensgard JR, et al. Role of microglia in central nervous system infections. Clin Microbiol Rev. 2004;17(4):942-64, table of contents. 15. Nguyen MD, Julien JP, Rivest S. Innate immunity: the missing link in neuroprotection and neurodegeneration? Nat Rev Neurosci. 2002;3(3):216-27. 16. Kettenmann H, Hanisch UK, Noda M, Verkhratsky A. Physiology of microglia. Physiol Rev. 2011;91(2):461-553. 17. Smith JA, Das A, Ray SK, Banik NL. Role of pro-inflammatory cytokines released from microglia in neurodegenerative diseases. Brain Res Bull. 2012;87(1):10-20. 18. Itagaki S, McGeer PL, Akiyama H, Zhu S, Selkoe D. Relationship of microglia and astrocytes to amyloid deposits of Alzheimer disease. J Neuroimmunol. 1989;24(3):173-82. 19. McGeer PL, Itagaki S, Boyes BE, McGeer EG. Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains. Neurology. 1988;38(8):1285-91. 20. Sapp E, Kegel KB, Aronin N, Hashikawa T, Uchiyama Y, Tohyama K, et al. Early and progressive accumulation of reactive microglia in the Huntington disease brain. J Neuropathol Exp Neurol. 2001;60(2):161-72. 21. Li Y, Zhang R, Hou X, Zhang Y, Ding F, Li F, et al. Microglia activation triggers oligodendrocyte precursor cells apoptosis via HSP60. Mol Med Rep. 2017;16(1):603-8. 84 22. Pang Y, Campbell L, Zheng B, Fan L, Cai Z, Rhodes P. Lipopolysaccharideactivated microglia induce death of oligodendrocyte progenitor cells and impede their development. Neuroscience. 2010;166(2):464-75. 23. Neumann H, Kotter MR, Franklin RJ. Debris clearance by microglia: an essential link between degeneration and regeneration. Brain. 2009;132(Pt 2):288-95. 24. Voss EV, Skuljec J, Gudi V, Skripuletz T, Pul R, Trebst C, et al. Characterisation of microglia during de- and remyelination: can they create a repair promoting environment? Neurobiol Dis. 2012;45(1):519-28. 25. Liddelow SA, Guttenplan KA, Clarke LE, Bennett FC, Bohlen CJ, Schirmer L, et al. Neurotoxic reactive astrocytes are induced by activated microglia. Nature. 2017;541(7638):481-7. 26. Jha MK, Kim JH, Song GJ, Lee WH, Lee IK, Lee HW, et al. Functional dissection of astrocyte-secreted proteins: implications in brain health and diseases. Prog Neurobiol. 2018;162:37-69. 27. Shinozaki Y, Shibata K, Yoshida K, Shigetomi E, Gachet C, Ikenaka K, et al. Transformation of astrocytes to a neuroprotective phenotype by microglia via P2Y1 receptor downregulation. Cell Rep. 2017;19(6):1151-64. APPENDIX DIFFERENTIALLY EXPRESSED GENES FROM SINGLE CELL RNA SEQUENCING ANALYSIS OF CHAPTER 3 86 A.1 Differential Gene Expression of Clusters at Day 7 p.i 87 88 89 90 91 92 93 94 95 96 97 98 99 A.2 Differential Gene Expression of Clusters at Day 14 p.i 100 101 102 Mac1 103 104