Murine Encephalomyelitis Virus Research Articles

Application of Theiler's murine encephalomyelitis virus in treatment of multiple sclerosis.

Theiler's murine encephalomyelitis virus (TMEV) infected mice have been often used as an animal model for Multiple sclerosis (MS) due to their similar pathology in the central nervous system (CNS). So far, there has been no effective treatment or medicine to cure MS completely. The drugs used in the clinic can only reduce the symptoms of MS, delay its recurrence, and increase the interval between relapses. MS can be caused by many factors, and clinically MS drugs are used to treat MS regardless of what factors are caused rather than MS caused by a specific factor. This can lead to inappropriate medicine, which may be one of the reasons why MS has not been completely cured. Therefore, this review summarized the drugs investigated in the TMEV-induced disease (TMEV-IDD) model of MS, so as to provide medication guidance and theoretical basis for the treatment of virus-induced MS.

Signalling of the C-type lectin receptor CLEC12A restrains protective immunity during acute Theiler’s murine encephalomyelitis virus infection

Signalling of the C-type lectin receptor CLEC12A restrains protective immunity during acute Theiler’s murine encephalomyelitis virus infection

Exploring the Role of Platelets in Virus-Induced Inflammatory Demyelinating Disease and Myocarditis.

Theiler's murine encephalomyelitis virus (TMEV) infection has been used as a mouse model for two virus-induced organ-specific immune-mediated diseases. TMEV-induced demyelinating disease (TMEV-IDD) in the central nervous system (CNS) is a chronic inflammatory disease with viral persistence and an animal model of multiple sclerosis (MS) in humans. TMEV infection can also cause acute myocarditis with viral replication and immune cell infiltration in the heart, leading to cardiac fibrosis. Since platelets have been reported to modulate immune responses, we aimed to determine the role of platelets in TMEV infection. In transcriptome analyses of platelets, distinct sets of immune-related genes, including major histocompatibility complex (MHC) class I, were up- or downregulated in TMEV-infected mice at different time points. We depleted platelets from TMEV-infected mice by injecting them with platelet-specific antibodies. The platelet-depleted mice had significantly fewer viral antigen-positive cells in the CNS. Platelet depletion reduced the severities of TMEV-IDD and myocarditis, although the pathology scores did not reach statistical significance. Immunologically, the platelet-depleted mice had an increase in interferon (IFN)-γ production with a higher anti-TMEV IgG2a/IgG1 ratio. Thus, platelets may play roles in TMEV infection, such as gene expression, viral clearance, and anti-viral antibody isotype responses.

Diet composition and sterilization modifies intestinal microbiome diversity and burden of Theiler's virus infection-induced acute seizures.

Brain infection with Theiler's murine encephalomyelitis virus (TMEV) in C57BL/6J mice can induce acquired epileptogenesis. Diet alters acute seizure incidence in TMEV-infected mice; yet it is unclear whether intestinal dysbiosis may also impact acute or chronic behavioral comorbidities. This study thus assessed the impact of diet formulation and sterilization on acute seizure presentation, gut microbiome composition, and epilepsy-related chronic behavioral comorbidities. Baseline fecal samples were collected from male C57BL/6J mice (4- to 5-weeks-old; Jackson Labs) upon facility arrival. Mice were randomized to either autoclaved (AC) or irradiated diet (IR) (Prolab RMH 3000) or IR (Picolab 5053). Three days later, mice underwent intracerebral TMEV or phosphate-buffered saline (PBS) injection. Fecal samples were collected from a subset of mice at infection (Day 0) and Day 7 post-infection. Epilepsy-related working memory deficits and seizure threshold were assessed 6 weeks post-infection. Gut microbiome diversity was determined by 16S rRNA amplicon sequencing of fecal samples. TMEV-infected mice displayed acute handling-induced seizures, regardless of diet: 28 of 57 IR Picolab 5053 (49.1%), 30 of 41 IR Prolab RMH 3000 (73.2%), and 47 of 77 AC Prolab RMH 3000 (61%) mice displayed seizures. The number of observed seizures differed significantly by diet: IR Picolab 5053 diet-fed mice had 2.2 ± 2.8 seizures (mean ± standard deviation), IR Prolab RMH 3000 diet-fed mice had 3.5 ± 2.9 seizures, and AC Prolab RMH 3000 diet-fed mice had 4.4 ± 3.8 seizures during the 7-day monitoring period. Gut microbiome composition differed significantly in TMEV-infected mice fed the AC Prolab RMH 3000 diet, with measured differences in gram-positive bacteria. These mice also displayed worsened long-term working memory deficits. Diet-induced differences in intestinal dysbiosis in the TMEV model are associated with marked changes in acute seizure presentation, symptomatic recovery, and onset of chronic behavioral comorbidities of epilepsy. Our study reveals a novel disease-modifying impact of dietary manipulation on intestinal bacterial species after TMEV-induced acute seizures.

The Contribution of Microglia and Brain-Infiltrating Macrophages to the Pathogenesis of Neuroinflammatory and Neurodegenerative Diseases during TMEV Infection of the Central Nervous System.

The infection of the central nervous system (CNS) with neurotropic viruses induces neuroinflammation and is associated with the development of neuroinflammatory and neurodegenerative diseases, including multiple sclerosis and epilepsy. The activation of the innate and adaptive immune response, including microglial, macrophages, and T and B cells, while required for efficient viral control within the CNS, is also associated with neuropathology. Under healthy conditions, resident microglia play a pivotal role in maintaining CNS homeostasis. However, during pathological events, such as CNS viral infection, microglia become reactive, and immune cells from the periphery infiltrate into the brain, disrupting CNS homeostasis and contributing to disease development. Theiler's murine encephalomyelitis virus (TMEV), a neurotropic picornavirus, is used in two distinct mouse models: TMEV-induced demyelination disease (TMEV-IDD) and TMEV-induced seizures, representing mouse models of multiple sclerosis and epilepsy, respectively. These murine models have contributed substantially to our understanding of the pathophysiology of MS and seizures/epilepsy following viral infection, serving as critical tools for identifying pharmacological targetable pathways to modulate disease development. This review aims to discuss the host-pathogen interaction during a neurotropic picornavirus infection and to shed light on our current understanding of the multifaceted roles played by microglia and macrophages in the context of these two complexes viral-induced disease.

Understanding the link between neurotropic viruses, BBB permeability, and MS pathogenesis.

Neurotropic viruses can infiltrate the CNS by crossing the blood-brain barrier (BBB) through various mechanisms including paracellular, transcellular, and "Trojan horse" mechanisms during leukocyte diapedesis. These viruses belong to several families, including retroviruses; human immunodeficiency virus type 1 (HIV-1), flaviviruses; Japanese encephalitis (JEV); and herpesviruses; herpes simplex virus type 1 (HSV-1), Epstein-Barr virus (EBV), and mouse adenovirus 1 (MAV-1). For entering the brain, viral proteins act upon the tight junctions (TJs) between the brain microvascular endothelial cells (BMECs). For instance, HIV-1 proteins, such as glycoprotein 120, Nef, Vpr, and Tat, disrupt the BBB and generate a neurotoxic effect. Recombinant-Tat triggers amendments in the BBB by decreasing expression of the TJ proteins such as claudin-1, claudin-5, and zona occludens-1 (ZO-1). Thus, the breaching of BBB has been reported in myriad of neurological diseases including multiple sclerosis (MS). Neurotropic viruses also exhibit molecular mimicry with several myelin sheath proteins, i.e., antibodies against EBV nuclear antigen 1 (EBNA1) aa411-426 cross-react with MBP and EBNA1 aa385-420 was found to be associated with MS risk haplotype HLA-DRB1*150. Notably, myelin protein epitopes (PLP139-151, MOG35-55, and MBP87-99) are being used to generate model systems for MS such as experimental autoimmune encephalomyelitis (EAE) to understand the disease mechanism and therapeutics. Viruses like Theiler's murine encephalomyelitis virus (TMEV) are also commonly used to generate EAE. Altogether, this review provide insights into the viruses' association with BBB leakiness and MS along with possible mechanistic details which could potentially use for therapeutics.

Increased expression of chondroitin sulfate proteoglycans in dentate gyrus and amygdala causes postinfectious seizures.

Alterations in the extracellular matrix are common in patients with epilepsy and animal models of epilepsy, yet whether they are the cause or consequence of seizures and epilepsy development is unknown. Using Theiler's murine encephalomyelitis virus (TMEV) infection-induced model of acquired epilepsy, we found de novo expression of chondroitin sulfate proteoglycans (CSPGs), a major extracellular matrix component, in dentate gyrus (DG) and amygdala exclusively in mice with acute seizures. Preventing the synthesis of CSPGs specifically in DG and amygdala by deletion of the major CSPG aggrecan reduced seizure burden. Patch-clamp recordings from dentate granule cells revealed enhanced intrinsic and synaptic excitability in seizing mice that was significantly ameliorated by aggrecan deletion. In situ experiments suggested that dentate granule cell hyperexcitability results from negatively charged CSPGs increasing stationary cations on the membrane, thereby depolarizing neurons, increasing their intrinsic and synaptic excitability. These results show increased expression of CSPGs in the DG and amygdala as one of the causal factors for TMEV-induced acute seizures. We also show identical changes in CSPGs in pilocarpine-induced epilepsy, suggesting that enhanced CSPGs in the DG and amygdala may be a common ictogenic factor and potential therapeutic target.

High throughput method for detecting murine brain atrophy using a clinical 3T MRI

BackgroundThere is a lack of understanding of the mechanisms by which the CNS is injured in multiple sclerosis (MS). Since Theiler’s murine encephalomyelitis virus (TMEV) infection in SJL/J mice is an established model of progressive disability in MS, and CNS atrophy correlates with progressive disability in MS, we used in vivo MRI to quantify total ventricular volume in TMEV infection. We then sought to identify immunological and virological biomarkers that correlated with increased ventricular size.MethodsMice, both infected and control, were followed for 6 months. Cerebral ventricular volumes were determined by MRI, and disability was assessed by Rotarod. A range of immunological and virological measures was obtained using standard techniques.ResultsDisability was present in infected mice with enlarged ventricles, while infected mice without enlarged ventricles had Rotarod performance similar to sham mice. Ventricular enlargement was detected as soon as 1 month after infection. None of the immunological and virological measures correlated with the development of ventricular enlargement.ConclusionsThese results support TMEV infection with brain MRI monitoring as a useful model for exploring the biology of disability progression in MS, but they did not identify an immunological or virological correlate with ventricular enlargement.

Theiler's Murine Encephalomyelitis Virus Replicates in Primary Neuron Cultures and Impairs Spine Density Formation.

In this study, we examined infection with the highly neurovirulent GDVII, the less neurovirulent DA strains, and with a mutant DA, which lacks the L* protein (L*-1) involved in viral persistence and demyelinating disease, to analyze the direct effects of Theiler's murine encephalomyelitis virus (TMEV) replication using primary cultures of mouse brain hippocampal neurons. All viruses replicate in cultured neurons, with GDVII having the highest titers and L*-1 the lowest. Accordingly, all were positive for viral antigen staining 3 days postinfection (dpi), and DA and L*-1 were also positive after 12 dpi. NeuN+immunostaining showed an early and almost complete absence of positive cells in cultures infected with GDVII, an approximately 50% reduction in cultures infected with DA, and fewer changes in L*-1 strains at 3 dpi. Accordingly, staining with chloromethyltetramethylrosamine orange (Mitotracker OrangeTM) as a parameter for cell viability showed similar results. Moreover, at 1 dpi, the strain DA induced higher transcript levels of neuroprotective genes such as IFN-Iβ, IRF7, and IRF8. At 3 dpi, strains GDVII and DA, but not the L*-1 mutant, showed lower PKR expression. In addition, confocal analysis showed that L*-1-infected neurons exhibited a decrease in spine density. Treatment with poly (I:C), which is structurally related to dsRNA and is known to trigger IFN type I synthesis, reduced spine density even more. These results confirmed the use of mouse hippocampal neuron cultures as a model to study neuronal responses after TMEV infection, particularly in the formation of spine density.

Chronic encephalomyelitis virus exhibits cellular tropism and evades pDCs by binding to sialylated integrins as the cell surface receptors.

Theiler's murine encephalomyelitis virus (TMEV) causes a chronic demyelinating disease similar to multiple sclerosis in mice. Although sialic acids have been shown to be essential for TMEV attachment to the host, the surface receptor has not been identified. While type I interferons play a pivotal role in the elimination of the chronic infectious Daniel (DA) strain, the role of plasmacytoid dendritic cells (pDCs) is controversial. We herein found that TMEV binds to conventional DCs but not to pDCs. A glycomics analysis showed that the sialylated N-glycan fractions were lower in pDCs than in conventional DCs, indicating that pDCs are not susceptible to TMEV infection due to the low levels of sialic acid. TMEV capsid proteins contain an integrin recognition motif, and dot blot assays showed that the integrin proteins bind to TMEV and that the viral binding was reduced in the desialylated αX β2 . αX β2 protein suppressed TMEV replication in vivo, and TMEV co-localized with integrin αM at the cell membrane and TLR 3 in the cytoplasm, suggesting that αM serves as the viral attachment and entry. These results show that the chronic encephalomyelitis virus utilizes sialylated integrins as cell surface receptors, leading to cellular tropism to evade pDC activation.

Effect of Siponimod on Brain and Spinal Cord Imaging Markers of Neurodegeneration in the Theiler's Murine Encephalomyelitis Virus Model of Demyelination.

Siponimod (Sp) is a Sphingosine 1-phosphate (S1P) receptor modulator, and it suppresses S1P- mediated autoimmune lymphocyte transport and inflammation. Theiler's murine encephalomyelitis virus (TMEV) infection mouse model of multiple sclerosis (MS) exhibits inflammation-driven acute and chronic phases, spinal cord lesions, brain and spinal cord atrophy, and white matter injury. The objective of the study was to investigate whether Sp treatment could attenuate inflammation-induced pathology in the TMEV model by inhibiting microglial activation and preventing the atrophy of central nervous tissue associated with neurodegeneration. Clinical disability score (CDS), body weight (BW), and rotarod retention time measures were used to assess Sp's impact on neurodegeneration and disease progression in 4 study groups of 102 animals, including 44 Sp-treated (SpT), 44 vehicle-treated, 6 saline-injected, and 8 age-matched healthy controls (HC). Next, 58 (22 SpT, 22 vehicle, 6 saline injected, and 8 HC) out of the 102 animals were further evaluated to assess the effect of Sp on brain region-specific and spinal cord volume changes, as well as microglial activation. Sp increased CDS and decreased BW and rotarod retention time in TMEV mice, but did not significantly affect most brain region volumes, except for lateral ventricle volume. Sp suppressed ventricular enlargement, suggesting reduced TMEV-induced inflammation in LV. No significant differences in spine volume changes were observed between Sp- and vehicle-treated animals, but there were differences between HC and TMEV groups, indicating TMEV-induced inflammation contributed to increased spine volume. Spine histology revealed no significant microglial density differences between groups in gray matter, but HC animals had higher type 1 morphology and lower type 2 morphology percentages in gray and white matter regions. This suggests that Sp did not significantly affect microglial density but may have modulated neuroinflammation in the spinal cord. Sp may have some effects on neuroinflammation and ventricular enlargement. However, it did not demonstrate a significant impact on neurodegeneration, spinal volume, or lesion volume in the TMEV mouse model. Further investigation is required to fully understand Sp's effect on microglial activation and its relevance to the pathophysiology of MS. The differences between the current study and previous research using other MS models, such as EAE, highlight the differences in pathological processes in these two disease models.

Anti-Glycolipid Antibody Examination in Five EAE Models and Theiler's Virus Model of Multiple Sclerosis: Detection of Anti-GM1, GM3, GM4, and Sulfatide Antibodies in Relapsing-Remitting EAE.

Anti-glycolipid antibodies have been reported to play pathogenic roles in peripheral inflammatory neuropathies, such as Guillain-Barré syndrome. On the other hand, the role in multiple sclerosis (MS), inflammatory demyelinating disease in the central nervous system (CNS), is largely unknown, although the presence of anti-glycolipid antibodies was reported to differ among MS patients with relapsing-remitting (RR), primary progressive (PP), and secondary progressive (SP) disease courses. We investigated whether the induction of anti-glycolipid antibodies could differ among experimental MS models with distinct clinical courses, depending on induction methods. Using three mouse strains, SJL/J, C57BL/6, and A.SW mice, we induced five distinct experimental autoimmune encephalomyelitis (EAE) models with myelin oligodendrocyte glycoprotein (MOG)35-55, MOG92-106, or myelin proteolipid protein (PLP)139-151, with or without an additional adjuvant curdlan injection. We also induced a viral model of MS, using Theiler's murine encephalomyelitis virus (TMEV). Each MS model had an RR, SP, PP, hyperacute, or chronic clinical course. Using the sera from the MS models, we quantified antibodies against 11 glycolipids: GM1, GM2, GM3, GM4, GD3, galactocerebroside, GD1a, GD1b, GT1b, GQ1b, and sulfatide. Among the MS models, we detected significant increases in four anti-glycolipid antibodies, GM1, GM3, GM4, and sulfatide, in PLP139-151-induced EAE with an RR disease course. We also tested cellular immune responses to the glycolipids and found CD1d-independent lymphoproliferative responses only to sulfatide with decreased interleukin (IL)-10 production. Although these results implied that anti-glycolipid antibodies might play a role in remissions or relapses in RR-EAE, their functional roles need to be determined by mechanistic experiments, such as injections of monoclonal anti-glycolipid antibodies.

C57BL/6J mice exposed to perfluorooctanoic acid demonstrate altered immune responses and increased seizures after Theiler's murine encephalomyelitis virus infection.

Neurological diseases can stem from environmental influences such as antecedent viral infections or exposure to potential toxicants, some of which can trigger immune responses leading to neurological symptoms. Theiler's murine encephalomyelitis virus (TMEV) is used to model human neurological conditions associated with prior viral infections, with outcomes partly attributable to improper induction and regulation of the immune response. Perfluorooctanoic acid (PFOA) can alter pathologies known to influence neurological disease such as inflammatory responses, cytokine expression, and glial activation. Co-exposure to TMEV and PFOA was used to test the hypothesis that early life exposure to the potential immunotoxicant PFOA would affect immune responses so as to render TMEV-resistant C57BL/6J (B6) mice susceptible to viral-induced neurological disease. Neonate B6 mice were exposed to different treatments: non-injected, sham-infected with PBS, and TMEV-infected, with the drinking water of each group including either 70 ppt PFOA or filtered water. The effects of PFOA were evaluated by comparing neurological symptoms and changes in immune-related cytokine and chemokine production induced by viral infection. Immune responses of 23 cytokines and chemokines were measured before and after infection to determine the effects of PFOA exposure on immune response. Prior to infection, an imbalance between Th1, Th2, and Treg cytokines was observed in PFOA-exposed mice, suppressing IL-4 and IL-13 production. However, the balance was restored and characterized by an increase in pro-inflammatory cytokines in the non-infected group, and a decrease in IL-10 in the PFOA + TMEV group. Furthermore, the PFOA + TMEV group experienced an increase in seizure frequency and severity. Overall, these findings provide insight into the complex roles of immune responses in the pathogenesis of virus-associated neurological diseases influenced by co-exposures to viruses and immunotoxic compounds.

ASC- and caspase-1-deficient C57BL/6 mice do not develop demyelinating disease after infection with Theiler’s murine encephalomyelitis virus

Theiler's murine encephalomyelitis virus (TMEV) induces an acute polioencephalomyelitis and a chronic demyelinating leukomyelitis in SJL mice. C57BL/6 (B6) mice generally do not develop TMEV-induced demyelinating disease (TMEV-IDD) due to virus elimination. However, TMEV can persist in specific immunodeficient B6 mice such as IFNβ−/− mice and induce a demyelinating process. The proinflammatory cytokines IL-1β and IL-18 are activated by the inflammasome pathway, which consists of a pattern recognition receptor molecule sensing microbial pathogens, the adaptor molecule Apoptosis-associated speck-like protein containing a CARD (ASC), and the executioner caspase-1. To analyze the contribution of the inflammasome pathway to the resistance of B6 mice to TMEV-IDD, ASC- and caspase-1-deficient mice and wild type littermates were infected with TMEV and investigated using histology, immunohistochemistry, RT-qPCR, and Western Blot. Despite the antiviral activity of the inflammasome pathway, ASC- and caspase-1-deficient mice eliminated the virus and did not develop TMEV-IDD. Moreover, a similar IFNβ and cytokine gene expression was found in the brain of immunodeficient mice and their wild type littermates. Most importantly, Western Blot showed cleavage of IL-1β and IL-18 in all investigated mice. Consequently, inflammasome-dependent activation of IL-1β and IL-18 does not play a major role in the resistance of B6 mice to TMEV-IDD.

Ovalbumin-specific CD4+ and CD8+ T cells contribute to different susceptibility for Theiler's murine encephalomyelitis virus persistence.

Theiler's murine encephalomyelitis virus (TMEV) is the causative agent of TMEV-induced demyelinating disease (TMEV-IDD); a well-established animal model for the chronic progressive form of human multiple sclerosis (MS). In susceptible mice with an inadequate immune response, TMEV-IDD is triggered by virus persistence and maintained by a T cell mediated immunopathology. OT-mice are bred on a TMEV-resistant C57BL/6 background and own predominantly chicken ovalbumin (OVA)-specific populations of CD8+ T cells (OT-I) or CD4+ T cells (OT-II), respectively. It is hypothesized that the lack of antigen specific T cell populations increases susceptibility for a TMEV-infection in OT-mice on a TMEV-resistant C57BL/6 background. OT-I, OT-II, and C57BL/6 control mice were infected intracerebrally with the TMEV-BeAn strain. Mice were scored weekly for clinical disease and after necropsy, histological and immunohistochemical evaluation was performed. OT-I mice started to develop progressive motor dysfunction between 7 and 21 days post infection (dpi), leading up to hind limb paresis and critical weight loss, which resulted in euthanasia for humane reasons between 14 and 35 dpi. OT-I mice displayed a high cerebral virus load, an almost complete absence of CD8+ T cells from the central nervous system (CNS) and a significantly diminished CD4+ T cell response. Contrarily, only 60% (12 of 20) of infected OT-II mice developed clinical disease characterized by mild ataxia. 25% of clinically affected OT-II mice (3 of 12) made a full recovery. 5 of 12 OT-II mice with clinical disease developed severe motor dysfunction similar to OT-I mice and were euthanized for humane reasons between 13 and 37 dpi. OT-II mice displayed only low virus-immunoreactivity, but clinical disease correlated well with severely reduced infiltration of CD8+ T cells and the increased presence of CD4+ T cells in the brains of OT-II mice. Though further studies are needed to reveal the underlying pathomechanisms following TMEV infection in OT mice, findings indicate an immunopathological process as a main contributor to clinical disease in OT-II mice, while a direct virus-associated pathology may be the main contributor to clinical disease in TMEV-infected OT-I mice.

Dissecting the role of H-2D bclass I molecule in the development of brain atrophy during Theiler’s murine encephalomyelitis virus (TMEV) infection

Abstract Brain atrophy is a common feature of many neurological diseases as diverse as Alzheimer’s disease, cerebral palsy, Huntington’s disease, multiple sclerosis, epilepsy, encephalitis, neurosyphilis, neuroAIDS, and Covid-19 infection. We have developed a murine model of brain atrophy using the Theiler’s murine encephalomyelitis virus (TMEV) infection mouse model of multiple sclerosis. In this study, we investigated the contribution of the MHC class I molecule, H-2Db, in generating an immune response associated with the onset of brain atrophy using mice with a C57BL/6 background. The H-2D bClass I molecule not only contributed to significantly more ventricular enlargement in a T2-weighted MRI at 45 d.p.i., but this ventricular enlargement correlated to CD8 T cell infiltration. To further define the cellular and molecular mechanisms of MHC class I molecules in TMEV induced brain atrophy we investigated the cell specific contribution of brain resident antigen presenting cells (APCs) in ventricular atrophy. We developed novel bi-transgenic mouse lines with tamoxifen induced conditional ablation of the H-2Db class I molecule in Cx3CR1+ brain resident myeloid cells. Cx3CR1cre+/Db Lox P mice presented with significantly less ventricular atrophy at 45 d.p.i., compared to cre- littermates along with a decrease in CD8 T cell infiltration. These results strongly imply that antigen presentation by H-2Db on myeloid cells, at least in part is responsible for the development of brain atrophy and provided insight into the molecular mechanism of this poorly understood neuropathology. NS103212, NS122174

Viral infection in the brain induces priming and maintenance of a novel antigen specific CD8 T cell population within the bone marrow that dysregulates the stem cell niche

Abstract Bone marrow is a primary immune organ responsible for stem cell maintenance and hematopoiesis. While the bone marrow niche is mainly studied in the context of hematopoiesis, aging, and mechanisms underlying stem cell and plasma cell survival, changes of the niche during brain viral infections and primary T cell responses is unknown. Intracranial infection with the neurotropic Theiler’s Murine Encephalomyelitis Virus (TMEV) results in an acute infection in the C57BL/6 mouse which is cleared 30 days post infection due to generation of an antigen specific CD8 T cell response raised against the immunodominant epitope VP2 121–130. We determined that 7–10% of CD8 T cells present in the sternal and femoral bone marrows and 15–20% in the cranial bone marrow were antigen specific 7 days post intracranial TMEV infection. Over 70% of these CD8 T cells were located outside of the vasculature as determined by IV labeling. Importantly, continuous treatment with FTY720, which sequesters T cells outside of the blood, did not eliminate antigen specific CD8 T cells in any bone marrow compartment. CD8 T cells in the bone marrow established durable memory following viral clearance and were reactivated upon cognate antigen reencounter. Both acute TMEV infection and antigen specific reactivation caused an increase in lineage −, Sca-1 +, ckit +(LSK) cells in the bone marrow. This LSK dysregulation was abrogated upon CD8 T cell depletion. We conclude that brain viral infections induce in situ effector and memory T cell responses within the bone marrow compartment. Both acute and memory recall CD8 T cell responses cause stem cell dysregulation. Our data hence paves the way for crucial studies of bone marrow resident antigen specific CD8 T cells in health and disease. Supported by grants from NIH (K99NS117799-01A1 (KA), R01NS103212 (AJJ), and RF1NS122174(AJJ)

Antigen-specific reactivation of brain tissue resident memory CD8 T cells directly induces severe neuroinflammation and blood-brain-barrier disruption

Abstract Intracranial infection with neurotropic Theiler’s Murine Encephalomyelitis Virus induces robust generation of brain TRMs against the immunodominant antigen, VP2 121–130, following viral clearance in C57BL/6 mice. While the presence of TRMs in the brain is documented, neurological consequences of TRM reactivation and their causal role in neuropathology remain understudied. We disabled peripheral CD8 T cells via anti CD8 depleting strategies and FTY720 treatment to determine the causal link between brain TRM reactivation and neuropathology. We depleted peripheral T cells and then administrated VP2 peptide IV to reactivate TRMs 60–200 days post infection as this elegant technique isolates responses of brain TRMs away from other branches of memory. We used state-of-the-art flow cytometry and MRI analysis to evaluate neurological consequences of TRM reactivation. TRM reactivation induced severe neuroinflammation in the brain as evident by infiltration of myeloid cells and blasting of T cells. MRI detected gadolinium leakage marking severe blood-brain-barrier (BBB) disruption when brain TRMs were reactivated. Depletion of peripheral CD8 T cells with low-dose anti CD8 antibodies or their sequestration via FTY720 did not alter neuroinflammation indicating brain TRM reactivation causally initiates neuropathology. Confirming this, tissue depletion of all TRMs in the brain using high-dose anti CD8 antibodies prevented both neuroinflammation and neuropathology. Hence, TRM reactivation causes severe neuroinflammation and BBB disruption from within the brain independently of peripheral T cells. Targeting brain TRM reactivation is a novel potential therapeutic for patients with many acute and chronic neurological diseases. Supported by grants from NIH (K99NS117799-01A1 (KA), R01NS103212 (AJJ), and RF1NS122174(AJJ)

Critical role of TLR activation in viral replication, persistence, and pathogenicity of Theiler's virus.

Theiler's murine encephalomyelitis virus (TMEV) establishes persistent viral infections in the central nervous system and induces chronic inflammatory demyelinating disease in susceptible mice. TMEV infects dendritic cells, macrophages, B cells, and glial cells. The state of TLR activation in the host plays a critical role in initial viral replication and persistence. The further activation of TLRs enhances viral replication and persistence, leading to the pathogenicity of TMEV-induced demyelinating disease. Various cytokines are produced via TLRs, and MDA-5 signals linked with NF-κB activation following TMEV infection. In turn, these signals further amplify TMEV replication and the persistence of virus-infected cells. The signals further elevate cytokine production, promoting the development of Th17 responses and preventing cellular apoptosis, which enables viral persistence. Excessive levels of cytokines, particularly IL-6 and IL-1β, facilitate the generation of pathogenic Th17 immune responses to viral antigens and autoantigens, leading to TMEV-induced demyelinating disease. These cytokines, together with TLR2 may prematurely generate functionally deficient CD25-FoxP3+ CD4+ T cells, which are subsequently converted to Th17 cells. Furthermore, IL-6 and IL-17 synergistically inhibit the apoptosis of virus-infected cells and the cytolytic function of CD8+ T lymphocytes, prolonging the survival of virus-infected cells. The inhibition of apoptosis leads to the persistent activation of NF-κB and TLRs, which continuously provides an environment of excessive cytokines and consequently promotes autoimmune responses. Persistent or repeated infections of other viruses such as COVID-19 may result in similar continuous TLR activation and cytokine production, leading to autoimmune diseases.

CD28-signaling can be partially compensated in CD28-knockout mice but is essential for virus elimination in a murine model of multiple sclerosis.

The intracerebral infection of mice with Theiler's murine encephalomyelitis virus (TMEV) represents a well-established animal model for multiple sclerosis (MS). Because CD28 is the main co-stimulatory molecule for the activation of T cells, we wanted to investigate its impact on the course of the virus infection as well as on a potential development of autoimmunity as seen in susceptible mouse strains for TMEV. In the present study, 5 weeks old mice on a C57BL/6 background with conventional or tamoxifen-induced, conditional CD28-knockout were infected intracerebrally with TMEV-BeAn. In the acute phase at 14 days post TMEV-infection (dpi), both CD28-knockout strains showed virus spread within the central nervous system (CNS) as an uncommon finding in C57BL/6 mice, accompanied by histopathological changes such as reduced microglial activation. In addition, the conditional, tamoxifen-induced CD28-knockout was associated with acute clinical deterioration and weight loss, which limited the observation period for this mouse strain to 14 dpi. In the chronic phase (42 and 147 dpi) of TMEV-infection, surprisingly only 33% of conventional CD28-knockout mice showed chronic TMEV-infection with loss of motor function concomitant with increased spinal cord inflammation, characterized by T- and B cell infiltration, microglial activation and astrogliosis at 33-42 dpi. Therefore, the clinical outcome largely depends on the time point of the CD28-knockout during development of the immune system. Whereas a fatal clinical outcome can already be observed in the early phase during TMEV-infection for conditional, tamoxifen-induced CD28-knockout mice, only one third of conventional CD28-knockout mice develop clinical symptoms later, accompanied by ongoing inflammation and an inability to clear the virus. However, the development of autoimmunity could not be observed in this C57BL/6 TMEV model irrespective of the time point of CD28 deletion.