Multiple sclerosis (MS) leaves millions of people worldwide with impaired central nervous system (CNS) function. To make matters worse, a great deal is still unknown about the physiological landscape of MS pathogenesis and of what factors might exacerbate or ameliorate the severity of disease outcomes. As the resident immune cell of the CNS, microglia are considered active members of this pathogenic landscape, but whether their contributions are more destructive or protective remains controversial. Using the microglia-depleting drug PLX5622, an inhibitor of colony stimulating factor 1 receptor (CSF1R), it was demonstrated that upon depletion of microglia in mice infected with the neurotropic JHM strain of the mouse hepatitis virus (JHMV) results in an increase in viral load and severity of demyelination with a hindered capacity to remyelinate. Additionally, single-cell RNA sequencing (scRNAseq) revealed that microglia depletion prior to JHMV infection results in increased expression of transcripts associated with disease, including Apoe, Trem2, and Spp1 (osteopontin), and reduced expression of remyelination associated transcripts Igf1 (insulin-like growth factor-1), Lpl (lipoprotein lipase), and Cst7 (cystatin F) in macrophage populations and remaining microglia. However, the mechanisms by which microglia reduce the severity of demyelination and overall disease outcome is not yet understood. ScRNAseq was employed to apprehend the heterogeneous populations of cells at defined times post-infection in the pathogenic landscape following CNS infection with JHMV. Results exposed a subpopulation of microglia that have elevated expression of remyelination markers Igf1, Lpl, and Cst7, that are present at day 21 p.i., in chronic disease, when remyelination efforts are occurring. Cystatin F expression drastically increases in microglia when demyelination occurs and has been associated with remyelination in various pre-clinical models of demyelination and is even found overlapping with remyelinating areas of MS plaques in human post-mortem tissue. As microglia phagocytosis of compact myelin results in increased expression of Cst7, cystatin F may act as a signaling mediator to negatively regulate the inflammatory environment that causes demyelination. Therefore, we chose to investigate whether the effects of microglia on reducing demyelination and promoting remyelination were mediated through microglia expression and secretion of cystatin F. Using a cystatin F germline KO mouse (Cst7 KO), we demonstrated that JHMV infection of the CNS results in more severe clinical disease in mice lacking Cst7 when compared with infected wild-type littermates (WT), which correlated with a significant increase in demyelination in Cst7 KO mice at both day 14 and 21 p.i as well as impaired remyelination at day 21 p.i. Genetic ablation of cystatin F did not impact acute disease with no impairment in control of virus replication or effect on immune cell infiltration into the CNS at day 7 p.i. relative to WT littermates. However, at day 21 p.i., we found increased infiltration of CD8+, CD4+, and virus specific CD4+ T cells into spinal cords. Consistently, scRNAseq on CD45+ cells from spinal cords of uninfected and infected Cst7 KO and WT littermates revealed increased expression of T cell chemoattractant Cxcl9 by microglia and macrophages and increased expression of activation markers for CD4+ T cells (Icos, Ifng, Tnfrsf4) and CD8+ T cells (Gzmb, Prf1, Ifng). Furthermore, expression of disease-associated transcripts Apoe and Trem2 were elevated in microglia and monocyte/macrophage populations. Together, these findings indicate that cystatin F plays a role in chronic JHMV-induced disease by restricting the severity of demyelination and promoting remyelination, potentially through negative regulation of T cell recruitment an activation. Results from this research provide further insight into the roles of microglia in disease and repair in a pre-clinical model of chronic MS and illuminate a potential mechanism for microglia in contributing to repair.

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Multiple sclerosis (MS) is a complex neurological disease that causes permanent disability in young adults. The cause of MS is still unknown, although both genetic and environmental factors are believed to play an important role in onset of disease. FTY720 is the first oral drug approved by the FDA to treat relapsing-remitting forms of MS presumably by inhibiting the egress of autoreactive T cells from the lymph nodes thereby preventing infiltration into the central nervous system (CNS). Treated-patients display a reduction in relapses, a decrease in new lesion formation and diminished brain volume loss shown by MRI.

We employ a viral model of demyelination to study various aspects related to the pathogenesis of MS. Intracerebral infection with the neurotropic JHM strain of mouse hepatitis virus (JHMV) results in an acute encephalomyelitis followed by chronic immune-mediated demyelination, that is similar both clinically and histologically to MS. The primary focus of research of this dissertation is to investigate whether FTY720 affects the host response and/or disease progression in response to JHMV infection of the CNS. Administration of FTY720 to JHMV-infected mice resulted in increased mortality associated impaired ability to control CNS viral replication that correlated with dampened accumulation of virus-specific CD4+ and CD8+ T cells. Treatment with FTY720 did not affect proliferation, cytokine secretion, or cytolytic activity by virus-specific T cells. Administration of FTY720 resulted in diminished severity of JHMV-induced demyelination associated with reduced T cell accumulation within the CNS. These findings indicate that FTY720 mutes effective antiviral immune responses by limiting infiltration of virus-specific T cells within the CNS following infection with a neurotropic virus.

Engraftment of mouse neural progenitor cells (NPCs) into the CNS of JHMV-infected mice with established demyelination results in extensive remyelination and axonal sparing. FTY720 readily penetrates the CNS. Therefore, the effect of FTY720 treatment on the biology of NPC's following CNS transplantation into JHMV-infected mice was examined. FTY720 increased NPC migration and proliferation within the spinal cord but did not diminish neuroinflammation, decrease the severity of demyelination nor increase remyelination. Therefore, FTY720 does not enhance the therapeutic potential of NPCs in a model of chronic-induced demyelination.

Evaluating the functional contributions of microglia in host defense and disease in a model of mouse coronavirus-induced neurologic disease

By

Yuting Cheng

Doctor of Philosophy in Biological Science

University of California, Irvine, 2022

Professor Thomas E. Lane, Chair

An important unmet clinical need for multiple sclerosis (MS) patients is an effective method for promoting axonal sparing and remyelination that can ameliorate clinical symptoms associated with demyelination and restore motor function. Previous studies from our laboratory demonstrating an important role for microglia in restricting the severity of demyelination and promoting remyelination in a viral model of demyelination. In brief, intracranial instillation of the neuroadapted JHM strain of mouse hepatitis virus (JHMV, a member of the Coronaviridae family) results in an acute encephalomyelitis followed by a chronic immune-mediated demyelinating disease similar both clinically and histologically with MS. Targeted depletion of microglia via administration of PLX5622, a brain penetrant small molecule antagonist specific for colony-stimulating factor 1 receptor (CSF1R) that efficiently depletes microglia, prior to infection with JHMV resulted in a dramatic increase in the severity of spinal cord demyelination and markedly impaired remyelination. To better understand how microglia impact disease progression in mice persistently infected with JHMV that have established demyelination, PLX5622 was administered to mice at day 14 post-infection (p.i.) and animals remained on drug for 14 days at which point animals were sacrificed and the severity of spinal cord demyelination and remyelination evaluated. Our findings indicate that PLX5622-mediated ablation of microglia for 2 weeks beginning at day 14 p.i. did not lead to an increase in mortality but resulted in a significant (p<0.05) increase in the severity of spinal cord demyelination at day 28 p.i. when compared to control mice. Infiltration of both total and virus-specific CD4+ and CD8+ T cells into the CNS was not affected in response to PLX5622-mediated microglia ablation yet control of viral replication was impaired that correlated with muted expression of MHC class II. Finally, we observed decreased expression of mRNA transcripts encoding remyelination-associated proteins Cystatin F and Lipoprotein lipase. BulkRNAseq analysis of spinal cords indicated that targeting microglia resulted in a dramatic reduction in gene expression profiles associated with cell-mediated immune responses and phagocytosis. These findings indicate that microglia continue to impact control of viral replication within the CNS post-acute phase of disease as well as restrict ongoing demyelination and remyelination in mice persistently infected with JHMV.

Alzheimer’s disease (AD) is the most prevalent form of dementia among elderly individuals, and although our understanding of some of the genetic causes of AD has improved, other risk factors contributing to the progression of AD require further evaluation. Accumulating evidence indicates that infections significantly exacerbate AD-like pathological changes, suggesting that infection-mediated inflammation may increase the susceptibility of developing AD later in life. This is further highlighted in the face of the ongoing COVID-19 pandemic, as COVID-19 patients exhibit a spectrum of neurologic disease symptoms. COVID-19 appears to impact brain structure and function, and while definitive mechanisms between COVID-19 and neurodegeneration remain poorly understood, several suggest that COVID-19 may induce or accelerate neurodegenerative processes. Microglia play a role not only in host defense to infection, but contribute to disease pathogenesis in AD. They are the resident immune cells in the CNS, monitor homeostasis and respond to injury and inflammation and can release proinflammatory chemokines and cytokines, triggering both innate and adaptive immune responses. Therefore, understanding the influence of SARS-CoV-2 on the CNS and AD pathology is crucial, and necessitates further research. To address this, experiments were carried out to evaluate the immune response in SARS-CoV-2 susceptible mice (k18-hACE2 ) and the contributions of microglia in host defense and neuroinflammation in this context. Intranasal inoculation of k18-hACE2 mice with SARS-CoV-2 resulted in significant weight loss, viral replication within the lungs, and severe disease. Examination of the brains of infected mice revealed extensive viral replication and spread within neurons, with evidence of microglial activation, contributing to the neuroinflammatory environment. We showed that pharmacological depletion of microglia with CSF1R inhibitor in chow (PLX5622) prior to SARS-CoV-2 infection, did not affect viral replication, but resulted in a marked reduction in proinflammatory chemokine and cytokine expression within the brains compared to infected control chow animals. These findings support the notion that microglia do contribute to the neuroinflammatory response, in part, through influencing expression of chemokines/cytokines in response in the k18-hACE2 mouse model of SARS-CoV-2 infection. The ability of the virus to efficiently infect and replicate within the CNS in this model contrasts with clinical findings in COVID-19 patients, where extensive CNS infection is rare, and so we found the k18-hACE2 mouse to not be the best model to accurately recapitulate COVID-19 disease presentation and pathogenesis. To better model COVID-19 clinical symptoms observed in human patients, we utilized a mouse-adapted SARS-CoV-2 -MA10 (MA10) to infect mice. This virus is able to recognize and bind to mouse ACE2, therefore it does not rely on the use of genetically-modified mice expressing human ACE2 for infection. Intranasal inoculation of C57BL/6 mice with MA10 results in lung infection associated inflammation and respiratory disease similar to COVID-19. Importantly, MA10 does not readily infect the CNS of infected mice which makes use of this virus a more reliable and accurate model of COVID-19. We utilized MA10 to evaluate the long-term effect of infection on neurodegenerative processes in laboratory mice and an established preclinical transgenic mouse models of AD. Intranasal inoculation of WT and 5xFAD mice with SARS-CoV-2- MA10 resulted in a dramatic increase in expression of gene transcripts associated with modulation of the immune system and other inflammatory pathways in the lungs of infected animals during initial phases of infection that eventually returned to baseline levels of expression after infection had resolved. In the brains, we observed lasting gene expression changes in the absence of viral RNA. These changes were associated with neuronal and synaptic dysfunction, and lasted well beyond viral clearance. These lasting changes in gene expression did not translate to detectable changes in neuropathology associated with neuroinflammation (astrocytes, microglia, and inflammatory monocytes) or other AD-associated pathology indicative or neuronal dysfunction (Aβ plaques or dystrophic neurites) in the brains of 5xFAD mice. No evidence of overt neuronal degeneration was found; moreover, no overt changes in synapse number were noted. These findings indicate that experimental infection of mice with MA10 is associated with detectable and enduring changes in the brain associated with neuronal dysfunction, even though gene expression changes do not directly translate into altered neuropathological changes. However, these long-lasting changes in gene expression provide some mechanistic insights that aid in our understanding how cognitive dysfunction may result from COVID-19. Moreover, this study provides evidence that viral infection that results from a lower viral dose or less severe variants of SARS-CoV-2 may not necessarily lead to permanent or long-term deleterious consequences in the brain.