Sustained Infiltration of Neutrophils Into the CNS Results in Increased Demyelination in a Viral-Induced Model of Multiple Sclerosis.

Intracranial inoculation of the neuroadapted JHM strain of mouse hepatitis virus (JHMV) into susceptible strains of mice results in acute encephalomyelitis followed by a cimmune-mediated demyelination similar to the human demyelinating disease multiple sclerosis (MS). JHMV infection of transgenic mice in which expression of the neutrophil chemoattractant chemokine CXCL1 is under the control of a tetracycline-inducible promoter active within GFAP-positive cells results in sustained neutrophil infiltration in the central nervous system (CNS) that correlates with an increase in spinal cord demyelination. We used single cell RNA sequencing (scRNAseq) and flow cytometry to characterize molecular and cellular changes within the CNS associated with increased demyelination in transgenic mice compared to control animals. These approaches revealed the presence of activated neutrophils as determined by expression of mRNA transcripts associated with neutrophil effector functions, including CD63, MMP9, S100a8, S100a9, and ASPRV1, as well as altered neutrophil morphology and protein expression. Collectively, these findings reveal insight into changes in the profile of neutrophils associated with increased white matter damage in mice persistently infected with a neurotropic coronavirus.

Single-Cell RNA Sequencing Reveals the Diversity of the Immunological Landscape following Central Nervous System Infection by a Murine Coronavirus.

Intracranial (i.c.) infection of susceptible C57BL/6 mice with the neurotropic JHM strain of mouse hepatitis virus (JHMV) (a member of the Coronaviridae family) results in acute encephalomyelitis and viral persistence associated with an immune-mediated demyelinating disease. The present study was undertaken to better understand the molecular pathways evoked during innate and adaptive immune responses as well as the chronic demyelinating stage of disease in response to JHMV infection of the central nervous system (CNS). Using single-cell RNA sequencing analysis (scRNAseq) on flow-sorted CD45-positive (CD45+) cells enriched from brains and spinal cords of experimental mice, we demonstrate the heterogeneity of the immune response as determined by the presence of unique molecular signatures and pathways involved in effective antiviral host defense. Furthermore, we identify potential genes involved in contributing to demyelination as well as remyelination being expressed by both microglia and macrophages. Collectively, these findings emphasize the diversity of the immune responses and molecular networks at defined stages following viral infection of the CNS.IMPORTANCE Understanding the immunological mechanisms contributing to both host defense and disease following viral infection of the CNS is of critical importance given the increasing number of viruses that are capable of infecting and replicating within the nervous system. With this in mind, the present study was undertaken to evaluate the molecular signatures of immune cells within the CNS at defined times following infection with a neuroadapted murine coronavirus using scRNAseq. This approach has revealed that the immunological landscape is diverse, with numerous immune cell subsets expressing distinct mRNA expression profiles that are, in part, dictated by the stage of infection. In addition, these findings reveal new insight into cellular pathways contributing to control of viral replication as well as to neurologic disease.

Microglia influence host defense, disease, and repair following murine coronavirus infection of the central nervous system.

The present study examines functional contributions of microglia in host defense, demyelination, and remyelination following infection of susceptible mice with a neurotropic coronavirus. Treatment with PLX5622, an inhibitor of colony stimulating factor 1 receptor (CSF1R) that efficiently depletes microglia, prior to infection of the central nervous system (CNS) with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in increased mortality compared with control mice that correlated with impaired control of viral replication. Single cell RNA sequencing (scRNASeq) of CD45+ cells isolated from the CNS revealed that PLX5622 treatment resulted in muted CD4+ T cell activation profile that was associated with decreased expression of transcripts encoding MHC class II and CD86 in macrophages but not dendritic cells. Evaluation of spinal cord demyelination revealed a marked increase in white matter damage in PLX5622-treated mice that corresponded with elevated expression of transcripts encoding disease-associated proteins Osteopontin (Spp1), Apolipoprotein E (Apoe), and Triggering receptor expressed on myeloid cells 2 (Trem2) that were enriched within macrophages. In addition, PLX5622 treatment dampened expression of Cystatin F (Cst7), Insulin growth factor 1 (Igf1), and lipoprotein lipase (Lpl) within macrophage populations which have been implicated in promoting repair of damaged nerve tissue and this was associated with impaired remyelination. Collectively, these findings argue that microglia tailor the CNS microenvironment to enhance control of coronavirus replication as well as dampen the severity of demyelination and influence repair.

EphA2 contributes to disruption of the blood-brain barrier in cerebral malaria.

Disruption of blood-brain barrier (BBB) function is a key feature of cerebral malaria. Increased barrier permeability occurs due to disassembly of tight and adherens junctions between endothelial cells, yet the mechanisms governing junction disassembly and vascular permeability during cerebral malaria remain poorly characterized. We found that EphA2 is a principal receptor tyrosine kinase mediating BBB breakdown during Plasmodium infection. Upregulated on brain microvascular endothelial cells in response to inflammatory cytokines, EphA2 is required for the loss of junction proteins on mouse and human brain microvascular endothelial cells. Furthermore, EphA2 is necessary for CD8+ T cell brain infiltration and subsequent BBB breakdown in a mouse model of cerebral malaria. Blocking EphA2 protects against BBB breakdown highlighting EphA2 as a potential therapeutic target for cerebral malaria.

The microbiota protects from viral-induced neurologic damage through microglia-intrinsic TLR signaling.

Symbiotic microbes impact the function and development of the central nervous system (CNS); however, little is known about the contribution of the microbiota during viral-induced neurologic damage. We identify that commensals aid in host defense following infection with a neurotropic virus through enhancing microglia function. Germfree mice or animals that receive antibiotics are unable to control viral replication within the brain leading to increased paralysis. Microglia derived from germfree or antibiotic-treated animals cannot stimulate viral-specific immunity and microglia depletion leads to worsened demyelination. Oral administration of toll-like receptor (TLR) ligands to virally infected germfree mice limits neurologic damage. Homeostatic activation of microglia is dependent on intrinsic signaling through TLR4, as disruption of TLR4 within microglia, but not the entire CNS (excluding microglia), leads to increased viral-induced clinical disease. This work demonstrates that gut immune-stimulatory products can influence microglia function to prevent CNS damage following viral infection.

T cell-selective deletion of Oct1 protects animals from autoimmune neuroinflammation while maintaining neurotropic pathogen response.

BACKGROUND: Treatments for autoimmune diseases aim to dampen autoreactivity while preserving normal immune function. In CD4+ T cells, the transcription factor Oct1/Pou2f1 is a dispensable transcription factor for T cell development and response to primary infection, but promotes expression of target genes, including Il2 and Ifng, under conditions of antigen reencounter. As a result, they are more strongly expressed upon secondary stimulation. Such repeated antigen encounters occur in memory recall responses, in autoimmunity where self-antigen can be recognized multiple times, and in chronic infection where foreign antigen is persistent. Based on these previous findings, we hypothesized that Oct1 loss would protect animals from autoimmunity but maintain normal responses to pathogens in the CNS. OBJECTIVE: We used a conditional mouse Oct1 (Pou2f1) allele and a CD4-Cre driver to determine the effect of T cell-specific Oct1 loss on autoimmune- and viral-induced neuroinflammation using an autoantigen-driven EAE model of autoimmunity and a JHMV model of viral infection. RESULTS: Oct1 conditional deletion mitigated clinical scores and reduced infiltrating T cells and cytokine production in the EAE model. Consistently, Oct1-deficient CD4+ T cells stimulated in vitro showed increased expression of markers associated with T cell anergy, particularly in the absence of co-stimulatory signals. In contrast, anti-viral T cell effector functions are intact in the absence of Oct1, with no changes in neuroinflammation, infiltrating T cells or cytokine production. CONCLUSION: Our findings uncover a significant difference between the effect of Oct1 loss on autoimmune and anti-pathogen responses, which potentially could be exploited for therapeutic benefit.

Disrupted CXCR2 Signaling in Oligodendroglia Lineage Cells Enhances Myelin Repair in a Viral Model of Multiple Sclerosis.

CXCR2 is a chemokine receptor expressed on oligodendroglia that has been implicated in the pathogenesis of neuroinflammatory demyelinating diseases as well as enhancement of the migration, proliferation, and myelin production by oligodendroglia. Using an inducible proteolipid protein (Plp) promoter-driven Cre-loxP recombination system, we were able to assess how timed ablation of Cxcr2 in oligodendroglia affected disease following intracranial infection with the neurotropic JHM strain of mouse hepatitis virus (JHMV). Generation of Plp-Cre-ER(T)::Cxcr2flox/flox transgenic mice (termed Cxcr2-CKO mice) allows for Cxcr2 to be silenced in oligodendrocytes in adult mice following treatment with tamoxifen. Ablation of oligodendroglia Cxcr2 did not influence clinical severity in response to intracranial infection with JHMV. Infiltration of activated T cells or myeloid cells into the central nervous system (CNS) was not affected, nor was the ability to control viral infection. In addition, the severity of demyelination was similar between tamoxifen-treated mice and vehicle-treated controls. Notably, deletion of Cxcr2 resulted in increased remyelination, as assessed by g-ratio (the ratio of the inner axonal diameter to the total outer fiber diameter) calculation, compared to that in vehicle-treated control mice. Collectively, our findings argue that CXCR2 signaling in oligodendroglia is dispensable with regard to contributing to neuroinflammation, but its deletion enhances remyelination in a preclinical model of the human demyelinating disease multiple sclerosis (MS).IMPORTANCE Signaling through the chemokine receptor CXCR2 in oligodendroglia is important for developmental myelination in rodents, while chemical inhibition or nonspecific genetic deletion of CXCR2 appears to augment myelin repair in animal models of the human demyelinating disease multiple sclerosis (MS). To better understand the biology of CXCR2 signaling on oligodendroglia, we generated transgenic mice in which Cxcr2 is selectively ablated in oligodendroglia upon treatment with tamoxifen. Using a viral model of neuroinflammation and demyelination, we demonstrate that genetic silencing of CXCR2 on oligodendroglia did not affect clinical disease, neuroinflammation, or demyelination, yet there was increased remyelination. These findings support and extend previous findings suggesting that targeting CXCR2 may offer a therapeutic avenue for enhancing remyelination in patients with demyelinating diseases.