Neutrophil Cathepsin G Regulates Dendritic Cell Production of IL-12 during Development of CD4 T Cell Responses to Antigens in the Skin.

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten skin sensitization and challenge. Sensitization of wild-type (WT) mice induces hapten-reactive effector CD8 T cells producing IFN-γ and IL-17- and IL-4-producing CD4 T cells that cannot mediate CHS. Although CXCR2-dependent Ly6G+ (neutrophil) cell recruitment into hapten-challenged skin is required to direct effector CD8 T cell infiltration into the challenge site to elicit CHS, 2,4-dinitrofluorobenezene (DNFB) sensitization of CXCR2-/- mice and neutrophil-depleted WT mice induced both hapten-reactive CD4 and CD8 T cells producing IFN-γ and IL-17. CD4 T cell-mediated CHS responses were not generated during DNFB sensitization of neutrophil-depleted WT mice treated with anti-IL-12 mAb or neutrophil-depleted IL-12-/- mice. Neutrophil depletion during DNFB sensitization of WT mice markedly increased IL-12-producing hapten-primed dendritic cell numbers in the skin-draining lymph nodes. Sensitization of mice lacking the neutrophil serine protease cathepsin G (CG)-induced hapten-reactive CD4 and CD8 T cells producing IFN-γ and IL-17 with elevated and elongated CHS responses to DNFB challenge. Induction of CHS effector CD4 T cells producing IFN-γ in neutrophil-depleted WT mice was eliminated by s.c. injection of active, but not inactivated, CG during sensitization. Thus, hapten skin sensitization induces neutrophil release of CG that systemically inhibits hapten-presenting dendritic cell production of IL-12 and the development of hapten-reactive CD4 T cells to IFN-γ-producing CHS effector cells.

Treg-specific IL-27Rα deletion uncovers a key role for IL-27 in Treg function to control autoimmunity.

Dysregulated Foxp3+ Treg functions result in uncontrolled immune activation and autoimmunity. Therefore, identifying cellular factors modulating Treg functions is an area of great importance. Here, using Treg-specific Il27ra-/- mice, we report that IL-27 signaling in Foxp3+ Tregs is essential for Tregs to control autoimmune inflammation in the central nervous system (CNS). Following experimental autoimmune encephalomyelitis (EAE) induction, Treg-specific Il27ra-/- mice develop more severe EAE. Consistent with the severe disease, the numbers of IFNγ- and IL-17-producing CD4 T cells infiltrating the CNS tissues are greater in these mice. Treg accumulation in the inflamed CNS tissues is not affected by the lack of IL-27 signaling in Tregs, suggesting a functional defect of Il27ra-/- Tregs. IL-10 production by conventional CD4 T cells and their CNS accumulation are rather elevated in Treg-specific Il27ra-/- mice. Analysis with Treg fate-mapping reporter mice further demonstrates that IL-27 signaling in Tregs may control stability of Foxp3 expression. Finally, systemic administration of recombinant IL-27 in Treg-specific Il27ra-/- mice fails to ameliorate the disease even in the presence of IL-27-responsive conventional CD4 T cells. These findings uncover a previously unknown role of IL-27 in regulating Treg function to control autoimmune inflammation.

An optimized method for enumerating CNS derived memory B cells during viral-induced inflammation.

BACKGROUND: CNS inflammation resulting from infection, injury, or neurodegeneration leads to accumulation of diverse B cell subsets. Although antibody secreting cells (ASC) within the inflamed CNS have been extensively examined, memory B cell (Bmem) characterization has been limited as they do not secrete antibody without stimulation. Moreover, unlike human Bmem, reliable surface markers for murine Bmem remain elusive. NEW METHOD: Using a viral encephalomyelitis model we developed a modified limiting dilution in vitro stimulation assay to convert CNS-derived virus specific Bmem into ASC. COMPARISON WITH EXISTING METHODS: Stimulation methods established for lymphoid tissue cells using prolonged stimulation with viral lysate resulted in substantial ASC loss and minimal Bmem to ASC conversion of CNS-derived cells. By varying stimulation duration, TLR activators, and culture supplements, we achieved optimal conversion by culturing cells with TLR7/8 agonist R848 in the presence of feeder cells for 2days. RESULTS: Flow cytometry markers CD38 and CD73 characterizing murine Bmem from lymphoid tissue showed more diverse expression patterns on corresponding CNS-derived B cell subsets. Using the optimized TLR7/8 stimulation protocol, we compared virus-specific IgG Bmem versus pre-existing ASC within the brain and spinal cord. Increasing Bmem frequencies during chronic infection mirrored kinetics of ASC. However, despite initially similar Bmem and ASC accumulation, Bmem prevailed in the brain, but were lower than ASC in the spinal cord during persistence. CONCLUSION: Simultaneous enumeration of antigen-specific Bmem and ASC using the Bmem assay optimized for CNS-derived cells enables characterization of temporal changes during microbial or auto-antigen induced neuroinflammation.

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.

Differential Regulation of Self-reactive CD4+ T Cells in Cervical Lymph Nodes and Central Nervous System during Viral Encephalomyelitis.

Viral infections have long been implicated as triggers of autoimmune diseases, including multiple sclerosis (MS), a central nervous system (CNS) inflammatory demyelinating disorder. Epitope spreading, molecular mimicry, cryptic antigen, and bystander activation have been implicated as mechanisms responsible for activating self-reactive (SR) immune cells, ultimately leading to organ-specific autoimmune disease. Taking advantage of coronavirus JHM strain of mouse hepatitis virus (JHMV)-induced demyelination, this study demonstrates that the host also mounts counteractive measures to specifically limit expansion of endogenous SR T cells. In this model, immune-mediated demyelination is associated with induction of SR T cells after viral control. However, their decline during persisting infection, despite ongoing demyelination, suggests an active control mechanism. Antigen-specific IL-10-secreting CD4+ T cells (Tr1) and Foxp3+ regulatory T cells (Tregs), both known to control autoimmunity and induced following JHMV infection, were assessed for their relative in vivo suppressive function of SR T cells. Ablation of Foxp3+ Tregs in chronically infected DEREG mice significantly increased SR CD4+ T cells within cervical lymph nodes (CLN), albeit without affecting their numbers or activation within the CNS compared to controls. In contrast, infected IL-27 receptor deficient (IL-27R-/-) mice, characterized by a drastic reduction of Tr1 cells, revealed that SR CD4+ T cells in CLN remained unchanged but were specifically increased within the CNS. These results suggest that distinct Treg subsets limit SR T cells in the draining lymph nodes and CNS to maximize suppression of SR T-cell-mediated autoimmune pathology. The JHMV model is thus valuable to decipher tissue-specific mechanisms preventing autoimmunity.

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.

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.

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.

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.

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.

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.

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.

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.

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.

PKR mediated regulation of inflammation and IL-10 during viral encephalomyelitis.

Double-stranded RNA-dependent protein kinase (PKR) regulates antiviral activity, immune responses, apoptosis and neurotoxicity. Gliatropic coronavirus infection induced PKR activation in infected as well uninfected cells within the central nervous system (CNS). However, PKR deficiency only modestly increased viral replication and did not affect IFN-α/ß or IL-1ß expression. Despite reduced Il-6, Ccl5, and Cxcl10 mRNA, protein levels remained unaltered. Furthermore, PKR deficiency selectively reduced IL-10 production in CD4, but not CD8 T cells, without affecting CNS pathology. The results demonstrate the ability of PKR to balance neuroinflammation by selectively modulating key cytokines and chemokines in CNS resident and CD4 T cells.

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.

Role of CD25(+) CD4(+) T cells in acute and persistent coronavirus infection of the central nervous system.

The influence of CD25(+)CD4(+) regulatory T cells (Treg) on acute and chronic viral infection of the central nervous system (CNS) was examined using a glial tropic murine coronavirus. Treg in the CNS were highest during initial T cell mediated virus control, decreased and then remained relatively stable during persistence. Anti-CD25 treatment did not affect CNS recruitment of inflammatory cells. Viral control was initially delayed; however, neither the kinetics of viral control nor viral persistence were affected. By contrast, the absence of Treg during the acute phase resulted in increased demyelination during viral persistence. These data suggest that CNS inflammation, progression of viral control and viral persistence are relatively independent of CD25(+)CD4(+) Treg. However, their absence during acute infection alters the ability of the host to limit tissue damage.

MMP-independent role of TIMP-1 at the blood brain barrier during viral encephalomyelitis.

Infection of the CNS (central nervous system) with a sublethal neurotropic coronavirus (JHMV) induces a vigorous inflammatory response. CD4⁺ and CD8⁺ T cells are essential to control infectious virus but at the cost of tissue damage. An enigma in understanding the contribution of T cell subsets in pathogenesis resides in their distinct migration pattern across the BBB (blood brain barrier). CD4⁺ T cells transiently accumulate within the perivascular space, whereas CD8⁺ T cells migrate directly into the CNS parenchyma. As MMPs (matrix metalloproteinases) facilitate migration across the glia limitans, specific expression of the TIMP (tissue inhibitor of MMPs)-1 by CD4⁺ T cells present in the perivascular cuffs suggested that TIMP-1 is responsible for stalling CD4⁺ T cell migration into the CNS parenchyma. Using TIMP-1 deficient mice, the present data demonstrate an increase rather than a decrease in CD4⁺ T cell accumulation within the perivascular space during JHMV infection. Whereas virus control was not affected by perivascular retention of CD4⁺ T cells, disease severity was decreased and associated with reduced IFNγ (interferon γ) production. Moreover, decreased CD4⁺ T cell recruitment into the CNS parenchyma of TIMP-1 deficient mice was not associated with impaired T cell recruiting chemokines or MMP expression, and no compensation by other TIMP molecules was identified. These data suggest an MMP-independent role of TIMP-1 in regulating CD4⁺ T cell access into the CNS parenchyma during acute JHMV encephalitis.

IL-21 optimizes T cell and humoral responses in the central nervous system during viral encephalitis.

Acute coronavirus encephalomyelitis is controlled by T cells while humoral responses suppress virus persistence. This study defines the contribution of interleukin (IL)-21, a regulator of T and B cell function, to central nervous system (CNS) immunity. IL-21 receptor deficiency did not affect peripheral T cell activation or trafficking, but dampened granzyme B, gamma interferon and IL-10 expression by CNS T cells and reduced serum and intrathecal humoral responses. Viral control was already lost prior to humoral CNS responses, but demyelination remained comparable. These data demonstrate a critical role of IL-21 in regulating CNS immunity, sustaining viral persistence and preventing mortality.

Intrathecal humoral immunity to encephalitic RNA viruses.

The nervous system is the target for acute encephalitic viral infections, as well as a reservoir for persisting viruses. Intrathecal antibody (Ab) synthesis is well documented in humans afflicted by infections associated with neurological complications, as well as the demyelinating disease, multiple sclerosis. This review focuses on the origin, recruitment, maintenance, and biological relevance of Ab-secreting cells (ASC) found in the central nervous system (CNS) following experimental neurotropic RNA virus infections. We will summarize evidence for a highly dynamic, evolving humoral response characterized by temporal alterations in B cell subsets, proliferation, and differentiation. Overall local Ab plays a beneficial role via complement-independent control of virus replication, although cross or self-reactive Ab to CNS antigens may contribute to immune-mediated pathogenesis during some infections. Importantly, protective Ab exert anti-viral activity not only by direct neutralization, but also by binding to cell surface-expressed viral glycoproteins. Ab engagement of viral glycoproteins blocks budding and mediates intracellular signaling leading to restored homeostatic and innate functions. The sustained Ab production by local ASC, as well as chemokines and cytokines associated with ASC recruitment and retention, are highlighted as critical components of immune control.