B cells going viral in the CNS: Dynamics, complexities, and functions of B cells responding to viral encephalitis.

A diverse number of DNA and RNA viruses have the potential to invade the central nervous system (CNS), causing inflammation and injury to cells that have a limited capacity for repair and regeneration. While rare, viral encephalitis in humans is often fatal and survivors commonly suffer from permanent neurological sequelae including seizures. Established treatment options are extremely limited, predominantly relying on vaccines, antivirals, or supportive care. Many viral CNS infections are characterized by the presence of antiviral antibodies in the cerebral spinal fluid (CSF), indicating local maintenance of protective antibody secreting cells. However, the mechanisms maintaining these humoral responses are poorly characterized. Furthermore, while both viral and autoimmune encephalitis are associated with the recruitment of diverse B cell subsets to the CNS, their protective and pathogenic roles aside from antibody production are just beginning to be understood. This review will focus on the relevance of B cell responses to viral CNS infections, with an emphasis on the importance of intrathecal immunity and the potential contribution to autoimmunity. Specifically, it will summarize the newest data characterizing B cell activation, differentiation, migration, and localization in clinical samples as well as experimental models of acute and persistent viral encephalitis.

Trem2 deficiency impairs recovery and phagocytosis and dysregulates myeloid gene expression during virus-induced demyelination.

BACKGROUND: Triggering receptor expressed on myeloid cells 2 (Trem2) plays a protective role in neurodegenerative diseases. By contrast, Trem2 functions can exacerbate tissue damage during respiratory viral or liver infections. We, therefore, investigated the role of Trem2 in a viral encephalomyelitis model associated with prominent Th1 mediated antiviral immunity leading to demyelination. METHODS: Wild-type (WT) and Trem2 deficient (Trem2-/-) mice were infected with a sublethal glia tropic murine coronavirus (MHV-JHM) intracranially. Disease progression and survival were monitored daily. Leukocyte accumulation and pathological features including demyelination and axonal damage in spinal cords (SC) were determined by flow cytometry and tissue section immunofluorescence analysis. Expression of select inflammatory cytokines and chemokines was measured by RT-PCR and global myeloid cell gene expression in SC-derived microglia and infiltrated bone-marrow-derived macrophages (BMDM) were determined using the Nanostring nCounter platform. RESULTS: BMDM recruited to SCs in response to infection highly upregulated Trem2 mRNA compared to microglia coincident with viral control. Trem2 deficiency did not alter disease onset or severity, but impaired clinical recovery after onset of demyelination. Disease progression in Trem2-/- mice could not be attributed to altered virus control or an elevated proinflammatory response. A prominent difference was increased degenerated myelin not associated with the myeloid cell markers IBA1 and/or CD68. Gene expression profiles of SC-derived microglia and BMDM further revealed that Trem2 deficiency resulted in impaired upregulation of phagocytosis associated genes Lpl and Cd36 in microglia, but a more complex pattern in BMDM. CONCLUSIONS: Trem2 deficiency during viral-induced demyelination dysregulates expression of other select genes regulating phagocytic pathways and lipid metabolism, with distinct effects on microglia and BMDM. The ultimate failure to remove damaged myelin is reminiscent of toxin or autoimmune cell-induced demyelination models and supports that Trem2 function is regulated by sensing tissue damage including a dysregulated lipid environment in very distinct inflammatory environments.

Neuronal Ablation of Alpha/Beta Interferon (IFN-α/ß) Signaling Exacerbates Central Nervous System Viral Dissemination and Impairs IFN-γ Responsiveness in Microglia/Macrophages.

Alpha/beta interferon (IFN-α/ß) signaling through the IFN-α/ß receptor (IFNAR) is essential to limit virus dissemination throughout the central nervous system (CNS) following many neurotropic virus infections. However, the distinct expression patterns of factors associated with the IFN-α/ß pathway in different CNS resident cell populations implicate complex cooperative pathways in IFN-α/ß induction and responsiveness. Here we show that mice devoid of IFNAR1 signaling in calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) expressing neurons (CaMKIIcre:IFNARfl/fl mice) infected with a mildly pathogenic neurotropic coronavirus (mouse hepatitis virus A59 strain [MHV-A59]) developed severe encephalomyelitis with hind-limb paralysis and succumbed within 7 days. Increased virus spread in CaMKIIcre:IFNARfl/fl mice compared to IFNARfl/fl mice affected neurons not only in the forebrain but also in the mid-hind brain and spinal cords but excluded the cerebellum. Infection was also increased in glia. The lack of viral control in CaMKIIcre:IFNARfl/fl relative to control mice coincided with sustained Cxcl1 and Ccl2 mRNAs but a decrease in mRNA levels of IFNα/ß pathway genes as well as Il6, Tnf, and Il1ß between days 4 and 6 postinfection (p.i.). T cell accumulation and IFN-γ production, an essential component of virus control, were not altered. However, IFN-γ responsiveness was impaired in microglia/macrophages irrespective of similar pSTAT1 nuclear translocation as in infected controls. The results reveal how perturbation of IFN-α/ß signaling in neurons can worsen disease course and disrupt complex interactions between the IFN-α/ß and IFN-γ pathways in achieving optimal antiviral responses.IMPORTANCE IFN-α/ß induction limits CNS viral spread by establishing an antiviral state, but also promotes blood brain barrier integrity, adaptive immunity, and activation of microglia/macrophages. However, the extent to which glial or neuronal signaling contributes to these diverse IFN-α/ß functions is poorly understood. Using a neurotropic mouse hepatitis virus encephalomyelitis model, this study demonstrated an essential role of IFN-α/ß receptor 1 (IFNAR1) specifically in neurons to control virus spread, regulate IFN-γ signaling, and prevent acute mortality. The results support the notion that effective neuronal IFNAR1 signaling compensates for their low basal expression of genes in the IFN-α/ß pathway compared to glia. The data further highlight the importance of tightly regulated communication between the IFN-α/ß and IFN-γ signaling pathways to optimize antiviral IFN-γ activity.

Dynamics of Virus-Specific Memory B Cells and Plasmablasts following Viral Infection of the Central Nervous System.

Humoral responses within the central nervous system (CNS) are common to many neurotropic viral infections, with antibody (Ab)-secreting cells (ASC) contributing to local protection. However, a role for virus-specific memory B cells (Bmem) within the CNS is poorly explored due to lack of robust phenotypic or functional identification in mice. This study takes advantage of the progeny of mice expressing tamoxifen-inducible Cre recombinase (Cre-ERT2) under the Aicda promoter crossed with Rosa26-loxP-tdTomato reporter mice (AIDCre-Rosa26tdTomato) to monitor B cells having undergone activation-induced cytidine deaminase (AID)-mediated somatic hypermutation (SHM) following neurotropic coronavirus infection. AID detection via tdTomato expression allowed tracking of virus-specific ASC and Bmem in priming and effector sites throughout infection. In draining lymph nodes, tdTomato-positive (tdTomato+) ASC were most prevalent prior to germinal center (GC) formation, but total tdTomato+ B cells only peaked with robust GC formation at day 14 p.i. Moreover, their proportion of Bmem dominated over the proportion of ASC throughout infection. In the CNS, tdTomato+ cells started emerging at day 14 p.i. While they initially comprised mainly Bmem, the proportions of ASC and Bmem became similar as tdTomato+ B cells increased throughout viral persistence. Delayed tamoxifen treatment demonstrated ongoing CNS recruitment of tdTomato+ B cells, mainly ASC, primed late during GC reactions. Overall, the data support the idea that virus-induced B cells exhibiting SHM require peripheral GC formation to emerge in the CNS. Ongoing GC reactions and regional signals further regulate dynamics within the CNS, with preferential maintenance of tdTomato+ B cells in spinal cords relative to that in brains during viral persistence.IMPORTANCE The prevalence and role of antigen-specific Bmem in the CNS during viral encephalomyelitis is largely undefined. A lack of reliable markers identifying murine Bmem has made it difficult to assess their contribution to local antiviral protection via antigen presentation or conversion to ASC. Using reporter mice infected with neurotropic coronavirus to track virus-specific Bmem and ASC, this report demonstrates that both subsets only emerge in the CNS following peripheral GC formation and subsequently prevail. While early GC reactions supported preferential Bmem accumulation in the CNS, late GC reactions favored ASC accumulation, although Bmem outnumbered ASC in draining lymph nodes throughout infection. Importantly, virus-specific B cells undergoing sustained GC selection were continually recruited to the persistently infected CNS. Elucidating the factors governing temporal events within GCs, as well as regional CNS cues during viral persistence, will aid intervention to modulate CNS humoral responses in the context of infection and associated autoimmune pathologies.

Alpha/Beta Interferon (IFN-α/ß) Signaling in Astrocytes Mediates Protection against Viral Encephalomyelitis and Regulates IFN-γ-Dependent Responses.

The contribution of distinct central nervous system (CNS) resident cells to protective alpha/beta interferon (IFN-α/ß) function following viral infections is poorly understood. Based on numerous immune regulatory functions of astrocytes, we evaluated the contribution of astrocyte IFN-α/ß signaling toward protection against the nonlethal glia- and neuronotropic mouse hepatitis virus (MHV) strain A59. Analysis of gene expression associated with IFN-α/ß function, e.g., pattern recognition receptors (PRRs) and interferon-stimulated genes (ISGs), revealed lower basal mRNA levels in brain-derived astrocytes than in microglia. Although astrocytes poorly induced Ifnß mRNA following infection, they upregulated various mRNAs in the IFN-α/ß pathway to a higher extent than microglia, supporting effective IFN-α/ß responsiveness. Ablation of the IFN-α/ß receptor (IFNAR) in astrocytes using mGFAPcre IFNARfl/fl mice resulted in severe encephalomyelitis and mortality, coincident with uncontrolled virus replication. Further, virus spread was not restricted to astrocytes but also affected microglia and neurons, despite increased and sustained Ifnα/ß and ISG mRNA levels within the CNS. IFN-γ, a crucial mediator for MHV control, was not impaired in infected mGFAPcre IFNARfl/fl mice despite reduced T cell CNS infiltration. Unexpectedly however, poor induction of IFN-γ-dependent major histocompatibility complex (MHC) class II expression on microglia supported that defective IFN-γ signaling contributes to uncontrolled virus replication. A link between sustained elevated IFN-α/ß and impaired responsiveness to IFN-γ supports the novel concept that temporally limited early IFN-α/ß responses are critical for effective antiviral IFN-γ function. Overall, our results imply that IFN-α/ß signaling in astrocytes is not only critical in limiting early CNS viral spread but also promotes protective antiviral IFN-γ function.IMPORTANCE An antiviral state established by IFN-α/ß contains initial viral spread as adaptive immunity develops. While it is apparent that the CNS lacks professional IFN-α/ß producers and that resident cells have distinct abilities to elicit innate IFN-α/ß responses, protective interactions between inducer and responder cells require further investigation. Infection with a glia- and neuronotropic coronavirus demonstrates that astrocytes mount a delayed but more robust response to infection than microglia, despite their lower basal mRNA levels of IFN-α/ß-inducing components. Lethal, uncontrolled viral dissemination following ablation of astrocyte IFN-α/ß signaling revealed the importance of IFN-α/ß responses in a single cell type for protection. Sustained global IFN-α/ß expression associated with uncontrolled virus did not suffice to protect neurons and further impaired responsiveness to protective IFN-γ. The results support astrocytes as critical contributors to innate immunity and the concept that limited IFN-α/ß responses are critical for effective subsequent antiviral IFN-γ function.

Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication.

Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I). This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU) activity is key to prevent early induction of double-stranded RNA (dsRNA) host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis-within the replicase complex-suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses.

Blockade of sustained tumor necrosis factor in a transgenic model of progressive autoimmune encephalomyelitis limits oligodendrocyte apoptosis and promotes oligodendrocyte maturation.

BACKGROUND: Tumor necrosis factor (TNF) is associated with several neurodegenerative disorders including multiple sclerosis (MS). Although TNF-targeted therapies have been largely unsuccessful in MS, recent preclinical data suggests selective soluble TNF inhibition can promote remyelination. This has renewed interest in regulation of TNF signaling in demyelinating disease, especially given the limited treatment options for progressive MS. Using a mouse model of progressive MS, this study evaluates the effects of sustained TNF on oligodendrocyte (OLG) apoptosis and OLG precursor cell (OPC) differentiation. METHODS: Induction of experimental autoimmune encephalomyelitis (EAE) in transgenic mice expressing a dominant-negative interferon-γ receptor under the human glial fibrillary acidic protein promoter (GFAPγR1Δ) causes severe non-remitting disease associated with sustained TNF. Therapeutic effects in GFAPγR1Δ mice treated with anti-TNF compared to control antibody during acute EAE were evaluated by assessing demyelinating lesion size, remyelination, OLG apoptosis, and OPC differentiation. RESULTS: More severe and enlarged demyelinating lesions in GFAPγR1Δ compared to wild-type (WT) mice were associated with increased OLG apoptosis and reduced differentiated CC1+Olig2+ OLG within lesions, as well as impaired upregulation of TNF receptor-2, suggesting impaired OPC differentiation. TNF blockade during acute EAE in GFAPγR1Δ both limited OLG apoptosis and enhanced OPC differentiation consistent with reduced lesion size and clinical recovery. TNF neutralization further limited increasing endothelin-1 (ET-1) expression in astrocytes and myeloid cells noted in lesions during disease progression in GFAPγR1Δ mice, supporting inhibitory effects of ET-1 on OPC maturation. CONCLUSION: Our data implicate that IFNγ signaling to astrocytes is essential to limit a detrimental positive feedback loop of TNF and ET-1 production, which increases OLG apoptosis and impairs OPC differentiation. Interference of this cycle by TNF blockade promotes repair independent of TNFR2 and supports selective TNF targeting to mitigate progressive forms of MS.

Protective Humoral Immunity in the Central Nervous System Requires Peripheral CD19-Dependent Germinal Center Formation following Coronavirus Encephalomyelitis.

B cell subsets with phenotypes characteristic of naive, non-isotype-switched, memory (Bmem) cells and antibody-secreting cells (ASC) accumulate in various models of central nervous system (CNS) inflammation, including viral encephalomyelitis. During neurotropic coronavirus JHMV infection, infiltration of protective ASC occurs after T cell-mediated viral control and is preceded by accumulation of non-isotype-switched IgD+ and IgM+ B cells. However, the contribution of peripheral activation events in cervical lymph nodes (CLN) to driving humoral immune responses in the infected CNS is poorly defined. CD19, a signaling component of the B cell receptor complex, is one of multiple regulators driving B cell differentiation and germinal center (GC) formation by lowering the threshold of antigen-driven activation. JHMV-infected CD19-/- mice were thus used to determine how CD19 affects CNS recruitment of B cell subsets. Early polyclonal ASC expansion, GC formation, and virus-specific ASC were all significantly impaired in CLN of CD19-/- mice compared to wild-type (WT) mice, consistent with lower and unsustained virus-specific serum antibody (Ab). ASC were also significantly reduced in the CNS, resulting in increased infectious virus during persistence. Nevertheless, CD19 deficiency did not affect early CNS IgD+ B cell accumulation. The results support the notion that CD19-independent factors drive early B cell mobilization and recruitment to the infected CNS, while delayed accumulation of virus-specific, isotype-switched ASC requires CD19-dependent GC formation in CLN. CD19 is thus essential for both sustained serum Ab and protective local Ab within the CNS following JHMV encephalomyelitis.IMPORTANCE CD19 activation is known to promote GC formation and to sustain serum Ab responses following antigen immunization and viral infections. However, the contribution of CD19 in the context of CNS infections has not been evaluated. This study demonstrates that antiviral protective ASC in the CNS are dependent on CD19 activation and peripheral GC formation, while accumulation of early-recruited IgD+ B cells is CD19 independent. This indicates that IgD+ B cells commonly found early in the CNS do not give rise to local ASC differentiation and that only antigen-primed, peripheral GC-derived ASC infiltrate the CNS, thereby limiting potentially harmful nonspecific Ab secretion. Expanding our understanding of activation signals driving CNS migration of distinct B cell subsets during neuroinflammatory insults is critical for preventing and managing acute encephalitic infections, as well as preempting reactivation of persistent viruses during immune-suppressive therapies targeting B cells in multiple sclerosis (MS), such as rituximab and ocrelizumab.

Activated GL7+ B cells are maintained within the inflamed CNS in the absence of follicle formation during viral encephalomyelitis.

Central nervous system (CNS) inflammation associated with viral infection and autoimmune disease results in the accumulation of B cells in various differentiation stages. However, the contribution between peripheral and CNS activation remains unclear. During gliatropic coronavirus induced encephalomyelitis, accumulation of protective antibody secreting cells is preceded by infiltration of B cells with a naïve and early differentiation phenotype (Phares et al., 2014). Investigation of the temporal dynamics of B cell activation in draining cervical lymph nodes (CLN) and the CNS revealed that peak CNS infiltration of early activated, unswitched IgD+ and IgM+ B cells coincided with polyclonal activation in CLN. By contrast, isotype-switched IgG+ B cells did not accumulate until peripheral germinal center formation. In the CNS, unswitched B cells were confined to the perivascular space and meninges, with only rare B cell clusters, while isotype-switched B cells localized to parenchymal areas. Although ectopic follicle formation was not observed, more differentiated B cell subsets within the CNS expressed the germinal center marker GL7, albeit at lower levels than CLN counterparts. During chronic infection, CNS IgDint and IgD- B cell subsets further displayed sustained markers of proliferation and CD4 T cell help, which were only transiently expressed in the CLN. A contribution of local CD4 T cell help to sustain B cell activation was supported by occasional B cells adjacent to T cells. The results suggest that accumulation of differentiated B cell subsets within the CNS is largely dictated by peripheral activation, but that local events contribute to their sustained activation independent of ectopic follicle formation.

Sustained TNF production by central nervous system infiltrating macrophages promotes progressive autoimmune encephalomyelitis.

BACKGROUND: Tumor necrosis factor (TNF) has pleiotropic functions during both the demyelinating autoimmune disease multiple sclerosis (MS) and its murine model experimental autoimmune encephalomyelitis (EAE). How TNF regulates disability during progressive disease remains unresolved. Using a progressive EAE model characterized by sustained TNF and increasing morbidity, this study evaluates the role of unregulated TNF in exacerbating central nervous system (CNS) pathology and inflammation. METHODS: Progressive MS was mimicked by myelin oligodendrocyte glycoprotein (MOG) peptide immunization of mice expressing a dominant negative IFN-γ receptor alpha chain under the human glial fibrillary acidic protein promoter (GFAPγR1∆). Diseased GFAPγR1∆ mice were treated with anti-TNF or control monoclonal antibody during acute disease to monitor therapeutic effects on sustained disability, demyelination, CNS inflammation, and blood brain barrier (BBB) permeability. RESULTS: TNF was specifically sustained in infiltrating macrophages. Anti-TNF treatment decreased established clinical disability and mortality rate within 7 days. Control of disease progression was associated with a decline in myelin loss and leukocyte infiltration, as well as macrophage activation. In addition to mitigating CNS inflammation, TNF neutralization restored BBB integrity and enhanced CNS anti-inflammatory responses. CONCLUSIONS: Sustained TNF production by infiltrating macrophages associated with progressive EAE exacerbates disease severity by promoting inflammation and disruption of BBB integrity, thereby counteracting establishment of an anti-inflammatory environment required for disease remission.

Myd88 Initiates Early Innate Immune Responses and Promotes CD4 T Cells during Coronavirus Encephalomyelitis.

UNLABELLED: Myd88 signaling is critical to the control of numerous central nervous system (CNS) infections by promoting both innate and adaptive immune responses. Nevertheless, the extent to which Myd88 regulates type I interferon (IFN) versus proinflammatory factors and T cell function, as well as the anatomical site of action, varies extensively with the pathogen. CNS infection by neurotropic coronavirus with replication confined to the brain and spinal cord induces protective IFN-α/ß via Myd88-independent activation of melanoma differentiation-associated gene 5 (MDA5). However, a contribution of Myd88-dependent signals to CNS pathogenesis has not been assessed. Infected Myd88(-/-) mice failed to control virus, exhibited enhanced clinical disease coincident with increased demyelination, and succumbed to infection within 3 weeks. The induction of IFN-α/ß, as well as of proinflammatory cytokines and chemokines, was impaired early during infection. However, defects in both IFN-α/ß and select proinflammatory factors were rapidly overcome prior to T cell recruitment. Myd88 deficiency also specifically blunted myeloid and CD4 T cell recruitment into the CNS without affecting CD8 T cells. Moreover, CD4 T cells but not CD8 T cells were impaired in IFN-γ production. Ineffective virus control indeed correlated most prominently with reduced antiviral IFN-γ in the CNS of Myd88(-/-) mice. The results demonstrate a crucial role for Myd88 both in early induction of innate immune responses during coronavirus-induced encephalomyelitis and in specifically promoting protective CD4 T cell activation. In the absence of these responses, functional CD8 T cells are insufficient to control viral spread within the CNS, resulting in severe demyelination. IMPORTANCE: During central nervous system (CNS) infections, signaling through the adaptor protein Myd88 promotes both innate and adaptive immune responses. The extent to which Myd88 regulates antiviral type I IFN, proinflammatory factors, adaptive immunity, and pathology is pathogen dependent. These results reveal that Myd88 protects from lethal neurotropic coronavirus-induced encephalomyelitis by accelerating but not enhancing the induction of IFN-α/ß, as well as by promoting peripheral activation and CNS accumulation of virus-specific CD4 T cells secreting IFN-γ. By controlling both early innate immune responses and CD4 T cell-mediated antiviral IFN-γ, Myd88 signaling limits the initial viral dissemination and is vital for T cell-mediated control of viral loads. Uncontrolled viral replication in the absence of Myd88 leads to severe demyelination and pathology despite overall reduced inflammatory responses. These data support a vital role of Myd88 signaling in protective antimicrobial functions in the CNS by promoting proinflammatory mediators and T cell-mediated IFN-γ production.

CXCL13 promotes isotype-switched B cell accumulation to the central nervous system during viral encephalomyelitis.

Elevated CXCL13 within the central nervous system (CNS) correlates with humoral responses in several neuroinflammatory diseases, yet its role is controversial. During coronavirus encephalomyelitis CXCL13 deficiency impaired CNS accumulation of memory B cells and antibody-secreting cells (ASC) but not naïve/early-activated B cells. However, despite diminished germinal center B cells and follicular helper T cells in draining lymph nodes, ASC in bone marrow and antiviral serum antibody were intact in the absence of CXCL13. The data demonstrate that CXCL13 is not essential in mounting effective peripheral humoral responses, but specifically promotes CNS accumulation of differentiated B cells.

IL-27 limits central nervous system viral clearance by promoting IL-10 and enhances demyelination.

IL-27 is a pleiotropic member of the IL-6 and IL-12 cytokine family composed of the IL-27p28 and the EBV-induced gene 3. IL-27 and its receptor mRNA are both upregulated in the CNS during acute encephalomyelitis induced by the JHM strain of mouse hepatitis virus (JHMV) and sustained during viral persistence. Contributions of IL-27 to viral pathogenesis were evaluated by infection of IL-27Rα-chain-deficient (IL-27Rα(-/-)) mice. The absence of IL-27 signaling accelerated virus control within the CNS associated with increased IFN-γ secreting virus-specific CD4+ and CD8+ T cells. Abrogation of IL-27 signaling did not affect virus-specific CD8+ T cell-mediated IL-10 production or cytolytic activity or Foxp3+ regulatory T cell populations. However, IL-10 production by virus-specific CD4+ T cells was reduced significantly. Despite increased T cell-mediated antiviral function in IL-27Rα(-/-) mice, the virus persisted in the CNS at similar levels as in wild-type mice. Nevertheless, IL-27Rα(-/-) mice exhibited decreased clinical disease during persistence, coincident with less severe demyelination, the hallmark tissue damage associated with JHMV infection. Overall, these data demonstrate that in contrast to viral infections at other sites, IL-27 does not play a proinflammatory role during JHMV-induced encephalomyelitis. Rather, it limits CNS inflammation and impairs control of CNS virus replication via induction of IL-10 in virus-specific CD4+ T cells. Furthermore, in contrast to its protective role in limiting CNS autoimmunity and preventing immunopathology, these data define a detrimental role of IL-27 in promoting demyelination by delaying viral control.

Interleukin-10 is a critical regulator of white matter lesion containment following viral induced demyelination.

Neurotropic coronavirus induces an acute encephalomyelitis accompanied by focal areas of demyelination distributed randomly along the spinal column. The initial areas of demyelination increase only slightly after the control of infection. These circumscribed focal lesions are characterized by axonal sparing, myelin ingestion by macrophage/microglia, and glial scars associated with hypertrophic astrocytes, which proliferate at the lesion border. Accelerated virus control in mice lacking the anti-inflammatory cytokine IL-10 was associated with limited initial demyelination, but low viral mRNA persistence similar to WT mice and declining antiviral cellular immunity. Nevertheless, lesions exhibited sustained expansion providing a model of dysregulated white matter injury temporally remote from the acute CNS insult. Expanding lesions in the absence of IL-10 are characterized by sustained microglial activation and partial loss of macrophage/microglia exhibiting an acquired deactivation phenotype. Furthermore, IL-10 deficiency impaired astrocyte organization into mesh like structures at the lesion borders, but did not prevent astrocyte hypertrophy. The formation of discrete foci of demyelination in IL-10 sufficient mice correlated with IL-10 receptor expression exclusively on astrocytes in areas of demyelination suggesting a critical role for IL-10 signaling to astrocytes in limiting expansion of initial areas of white matter damage. GLIA 2015;63:2106-2120.

Distinct CD4 T-cell effects on primary versus recall CD8 T-cell responses during viral encephalomyelitis.

CD4 T-cell help is not a universal requirement for effective primary CD8 T cells but is essential to generate memory CD8 T cells capable of recall responses. This study examined how CD4 T cells affect primary and secondary anti-viral CD8 T-cell responses within the central nervous system (CNS) during encephalomyelitis induced by sublethal gliatropic coronavirus. CD4 T-cell depletion before infection did not impair peripheral expansion, interferon-γ production, CNS recruitment or initial CNS effector capacity of virus-specific CD8 T cells ex vivo. Nevertheless, impaired virus control in the absence of CD4 T cells was associated with gradually diminished CNS CD8 T-cell interferon-γ production. Furthermore, within the CD8 T-cell population short-lived effector cells were increased and memory precursor effector cells were significantly decreased, consistent with higher T-cell turnover. Transfer of memory CD8 T cells to reduce viral load in CD4-depleted mice reverted the recipient CNS CD8 T-cell phenotype to that in wild-type control mice. However, memory CD8 T cells primed without CD4 T cells and transferred into infected CD4-sufficient recipients expanded less efficiently and were not sustained in the CNS, contrasting with their helped counterparts. These data suggest that CD4 T cells are dispensable for initial expansion, CNS recruitment and differentiation of primary resident memory CD8 T cells as long as the duration of antigen exposure is limited. By contrast, CD4 T cells are essential to prolong primary CD8 T-cell function in the CNS and imprint memory CD8 T cells for recall responses.

Self-reactive CD4(+) T cells activated during viral-induced demyelination do not prevent clinical recovery.

BACKGROUND: Microbial infections have been implicated in initiating and enhancing severity of autoimmune diseases including the demyelinating disease multiple sclerosis (MS). Nevertheless, the incidence of both acute and persisting viral infections without evidence of autoimmune sequelae suggests that this process is well controlled. The conditions promoting or stemming self-reactive (SR) T cells following viral-induced tissue damage thus need to be better defined. Using a non-fatal viral mouse model of encephalomyelitis associated with demyelination and disability, yet ultimate clinical improvement, this study set out to monitor uptake and presentation of endogenous myelin antigens, as well as induction and fate of SR T cells. METHODS: Activation and central nervous system (CNS) recruitment of myelin-specific CD4 T cells was analyzed by flow cytometry during encephalomyelitis induced by a glia tropic murine coronavirus. Potential antigen-presenting cells (APC) ingesting myelin were characterized by flow cytometry and their ability to activate SR T cells tested by co-culture with carboxyfluorescein succinimidyl ester (CFSE)-labeled myelin-specific CD4 T cells. Endogenous SR T cell kinetics was analyzed within both cervical lymph nodes and CNS by Enzyme-Linked ImmunoSpot (ELISPOT) following viral infection. RESULTS: The data demonstrate the presence of APC capable of activating SR T cells in both draining lymph nodes and the CNS temporally correlating with overt demyelination. While both the CNS-infiltrating myeloid population and microglia ingested myelin, only CNS-infiltrating APC were capable of presenting endogenous myelin antigen to SR T cells ex vivo. Finally, SR T cell activation from the endogenous T cell repertoire was most notable when infectious virus was controlled and paralleled myelin damage. Although SR T cell accumulation peaked in the persistently infected CNS during maximal demyelination, they were not preferentially retained. Their gradual decline, despite ongoing demyelination, suggested minimal re-stimulation and pathogenic function in vivo consistent with the lack of autoimmune symptoms. CONCLUSIONS: The results demonstrate the potential for CNS tissue destruction to induce and recruit SR T cells to the injury site and support a host suppressive mechanism limiting development of autoimmunity.

Astrocyte response to IFN-γ limits IL-6-mediated microglia activation and progressive autoimmune encephalomyelitis.

BACKGROUND: Therapeutic modalities effective in patients with progressive forms of multiple sclerosis (MS) are limited. In a murine model of progressive MS, the sustained disability during the chronic phase of experimental autoimmune encephalomyelitis (EAE) correlated with elevated expression of interleukin (IL)-6, a cytokine with pleiotropic functions and therapeutic target for non-central nervous system (CNS) autoimmune disease. Sustained IL-6 expression in astrocytes restricted to areas of demyelination suggested that IL-6 plays a major role in disease progression during chronic EAE. METHODS: A progressive form of EAE was induced using transgenic mice expressing a dominant negative interferon-γ (IFN-γ) receptor alpha chain under control of human glial fibrillary acidic protein (GFAP) promoter (GFAPγR1Δ mice). The role of IL-6 in regulating progressive CNS autoimmunity was assessed by treating GFAPγR1Δ mice with anti-IL-6 neutralizing antibody during chronic EAE. RESULTS: IL-6 neutralization restricted disease progression and decreased disability, myelin loss, and axonal damage without affecting astrogliosis. IL-6 blockade reduced CNS inflammation by limiting inflammatory cell proliferation; however, the relative frequencies of CNS leukocyte infiltrates, including the Th1, Th17, and Treg CD4 T cell subsets, were not altered. IL-6 blockade rather limited the activation and proliferation of microglia, which correlated with higher expression of Galectin-1, a regulator of microglia activation expressed by astrocytes. CONCLUSIONS: These data demonstrate that astrocyte-derived IL-6 is a key mediator of progressive disease and support IL-6 blockade as a viable intervention strategy to combat progressive MS.

Progression from IgD+ IgM+ to isotype-switched B cells is site specific during coronavirus-induced encephalomyelitis.

UNLABELLED: Various infections in the central nervous system (CNS) trigger B cell accumulation; however, the relative dynamics between viral replication and alterations in distinct B cell subsets are largely unknown. Using a glia-tropic coronavirus infection, which is initiated in the brain but rapidly spreads to and predominantly persists in the spinal cord, this study characterizes longitudinal changes in B cell subsets at both infected anatomical sites. The phase of T cell-dependent, antibody-independent control of infectious virus was associated with a similar recruitment of naive/early-activated IgD(+) IgM(+) B cells into both the brain and spinal cord. This population was progressively replaced by CD138(-) IgD(-) IgM(+) B cells, isotype-switched CD138(-) IgD(-) IgM(-) memory B cells (B(mem)), and CD138(+) antibody-secreting cells (ASC). A more rapid transition to B(mem) and ASC in spinal cord than in brain was associated with higher levels of persisting viral RNA and transcripts encoding factors promoting B cell migration, differentiation, and survival. The results demonstrate that naive/early-activated B cells are recruited early during coronavirus CNS infection but are subsequently replaced by more differentiated B cells. Furthermore, viral persistence, even at low levels, is a driving force for accumulation of isotype-switched B(mem) and ASC. IMPORTANCE: Acute and chronic human CNS infections are associated with an accumulation of heterogeneous B cell subsets; however, their influence on viral load and disease is unclear. Using a glia-tropic coronavirus model, we demonstrate that the accumulation of B cells ranging from early-activated to isotype-switched differentiation stages is both temporally and spatially orchestrated. Acutely infected brains and spinal cords indiscriminately recruit a homogeneous population of early-activated B cells, which is progressively replaced by diverse, more differentiated subsets. The latter process is accelerated by elevated proinflammatory responses associated with viral persistence. The results imply that early-recruited B cells do not have antiviral function but may contribute to the inflammatory environment or act as antigen-presenting cells. Moreover, CNS viral persistence is a driving force promoting differentiated B cells with protective potential.

Ifit2 deficiency results in uncontrolled neurotropic coronavirus replication and enhanced encephalitis via impaired alpha/beta interferon induction in macrophages.

Type I interferons (IFN-α/ß) limit viral dissemination prior to the emergence of adaptive immune responses through the concerted action of interferon-stimulated genes (ISGs). Although IFN-α/ß induction by coronaviruses is modest, it effectively limits viral spread within the central nervous system (CNS) and protects against mortality. The protective roles of specific ISGs against the mouse hepatitis virus (MHV) members of the coronaviruses are largely unknown. This study demonstrates a protective role of the ISG Ifit2 in encephalitis induced by the dual hepato- and neurotropic MHV-A59. Contrasting the mild encephalitis and 100% survival of MHV-A59-infected wild-type (wt) mice, nearly 60% of infected Ifit2(-/-) mice exhibited severe encephalitis and succumbed between 6 and 8 days postinfection. Increased clinical disease in Ifit2(-/-) mice coincided with higher viral loads and enhanced viral spread throughout the CNS parenchyma. Ifit2(-/-) mice also expressed significantly reduced IFN-α/ß and downstream ISG mRNAs Ifit1, Isg15, and Pkr, while expression of proinflammatory cytokines and chemokines was only modestly affected in the CNS. Impaired IFN-α/ß induction in the absence of Ifit2 was confirmed by ex vivo mRNA analysis of microglia and macrophages, the prominent cell types producing IFN-α/ß following MHV CNS infection. Furthermore, both IFN-α/ß mRNA and protein production were significantly reduced in MHV-infected Ifit2(-/-) relative to wt bone marrow-derived macrophages. Collectively, the data implicate Ifit2 as a positive regulator of IFN-α/ß expression, rather than direct antiviral mediator, during MHV-induced encephalitis.

Astrocyte-derived CXCL10 drives accumulation of antibody-secreting cells in the central nervous system during viral encephalomyelitis.

Microbial infections of the central nervous system (CNS) are often associated with local accumulation of antibody (Ab)-secreting cells (ASC). By providing a source of Ab at the site of infection, CNS-localized ASC play a critical role in acute viral control and in preventing viral recrudescence. Following coronavirus-induced encephalomyelitis, the CNS accumulation of ASC is chemokine (C-X-C motif) receptor 3 (CXCR3) dependent. This study demonstrates that CNS-expressed CXCR3 ligand CXCL10 is the critical chemokine regulating ASC accumulation. Impaired ASC recruitment in CXCL10(-/-) but not CXCL9(-/-) mice was consistent with reduced CNS IgG and κ-light chain mRNA and virus-specific Ab. Moreover, the few ASC recruited to the CNS in CXCL10(-/-) mice were confined to the vasculature, distinct from the parenchymal localization in wild-type and CXCL9(-/-) mice. However, neither CXCL9 nor CXCL10 deficiency diminished neutralizing serum Ab, supporting a direct role for CXCL10 in ASC migration. T cell accumulation, localization, and effector functions were also not affected in either CXCL9(-/-) or CXCL10(-/-) mice, consistent with similar control of infectious virus. There was also no evidence for dysregulation of chemokines or cytokines involved in ASC regulation. The distinct roles of CXCL9 and CXCL10 in ASC accumulation rather coincided with their differential localization. While CXCL10 was predominantly expressed by astrocytes, CXCL9 expression was confined to the vasculature/perivascular spaces. These results suggest that CXCL10 is critical for two phases: recruitment of ASC to the CNS vasculature and ASC entry into the CNS parenchyma.