RESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Catepsina G/metabolismo , Células Dendríticas/metabolismo , Haptenos/metabolismo , Interleucina-12/biossíntese , Neutrófilos/enzimologia , Pele/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Dermatite de Contato/imunologia , Dermatite de Contato/metabolismo , Feminino , Haptenos/imunologia , Interleucina-12/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/metabolismo , Receptores de Interleucina-8B/deficiência , Receptores de Interleucina-8B/imunologia , Receptores de Interleucina-8B/metabolismo , Pele/imunologiaRESUMO
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.
Assuntos
Doenças Autoimunes/imunologia , Encefalomielite/imunologia , Receptores de Citocinas/metabolismo , Linfócitos T Reguladores/metabolismo , Animais , Doenças Autoimunes/tratamento farmacológico , Sistema Nervoso Central/imunologia , Avaliação Pré-Clínica de Medicamentos , Encefalomielite/tratamento farmacológico , Fatores de Transcrição Forkhead/metabolismo , Interleucinas/metabolismo , Interleucinas/uso terapêutico , Camundongos Transgênicos , Receptores de Citocinas/genética , Receptores de InterleucinaRESUMO
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.
Assuntos
Linfócitos B/virologia , Sistema Nervoso Central/virologia , Infecções por Coronavirus/imunologia , Encefalomielite/virologia , Ativação Linfocitária/imunologia , Animais , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Movimento Celular/imunologia , Sistema Nervoso Central/imunologia , Infecções por Coronavirus/virologia , Encefalomielite/imunologia , Camundongos Endogâmicos C57BLRESUMO
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.
Assuntos
Sistema Nervoso Central/patologia , Encefalomielite Autoimune Experimental/patologia , Macrófagos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anticorpos/farmacologia , Antígenos CD/metabolismo , Barreira Hematoencefálica/fisiopatologia , Proteínas de Ligação ao Cálcio/metabolismo , Permeabilidade Capilar/genética , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Interferon gama/genética , Interferon gama/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Glicoproteína Mielina-Oligodendrócito/toxicidade , Neuroglia/patologia , Infiltração de Neutrófilos/efeitos dos fármacos , Infiltração de Neutrófilos/genética , Fragmentos de Peptídeos/toxicidade , Fator de Necrose Tumoral alfa/imunologiaRESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por Coronavirus/imunologia , Encefalite Viral/imunologia , Imunidade Celular , Imunidade Inata , Vírus Elberfeld do Camundongo/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Animais , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/patologia , Encefalite Viral/genética , Encefalite Viral/patologia , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon beta/genética , Interferon beta/imunologia , Vírus Elberfeld do Camundongo/genética , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genéticaRESUMO
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.
Assuntos
Linfócitos B/imunologia , Sistema Nervoso Central/imunologia , Quimiocina CXCL13/imunologia , Infecções por Coronavirus/imunologia , Encefalomielite/imunologia , Animais , Linfócitos B/patologia , Movimento Celular/imunologia , Infecções por Coronavirus/patologia , Encefalomielite/patologia , Feminino , Switching de Imunoglobulina/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
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.
Assuntos
Sistema Nervoso Central/imunologia , Infecções por Coronavirus/imunologia , Encefalomielite Aguda Disseminada/imunologia , Interleucina-10/imunologia , Interleucinas/imunologia , Vírus da Hepatite Murina/imunologia , Transdução de Sinais/imunologia , Animais , Sistema Nervoso Central/patologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/patologia , Doenças Desmielinizantes , Encefalomielite Aguda Disseminada/genética , Encefalomielite Aguda Disseminada/patologia , Interleucina-10/genética , Interleucinas/genética , Camundongos , Camundongos Knockout , Transdução de Sinais/genética , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologiaRESUMO
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.
RESUMO
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.
RESUMO
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.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Doenças Desmielinizantes/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Antígeno CD11b/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Doenças Desmielinizantes/virologia , Imunoterapia Adotiva , Linfonodos/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Bainha de Mielina/patologia , Recuperação de Função FisiológicaRESUMO
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.
Assuntos
Astrócitos/efeitos dos fármacos , Encefalomielite Autoimune Experimental/imunologia , Interferon gama/farmacologia , Interleucina-6/antagonistas & inibidores , Ativação de Macrófagos/efeitos dos fármacos , Microglia/efeitos dos fármacos , Animais , Anticorpos Neutralizantes/uso terapêutico , Doenças Desmielinizantes/patologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Proteína Glial Fibrilar Ácida/genética , Gliose/imunologia , Humanos , Interleucina-6/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Esclerose Múltipla/tratamento farmacológico , Regiões Promotoras Genéticas/genéticaRESUMO
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.
Assuntos
Linfócitos B/imunologia , Infecções por Coronavirus/imunologia , Coronavirus/imunologia , Encefalomielite/imunologia , Switching de Imunoglobulina/imunologia , Imunoglobulina D/imunologia , Imunoglobulina M/imunologia , Animais , Células Produtoras de Anticorpos/imunologia , Células Produtoras de Anticorpos/virologia , Células Apresentadoras de Antígenos/imunologia , Linfócitos B/virologia , Encéfalo/imunologia , Encéfalo/virologia , Diferenciação Celular/imunologia , Movimento Celular/imunologia , Infecções por Coronavirus/virologia , Encefalomielite/virologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Viral/imunologia , Medula Espinal/imunologia , Medula Espinal/virologiaRESUMO
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.
Assuntos
Sistema Nervoso Central/virologia , Encefalite/veterinária , Interferon-alfa/genética , Interferon beta/genética , Macrófagos/imunologia , Vírus da Hepatite Murina/fisiologia , Proteínas/imunologia , Doenças dos Roedores/imunologia , Animais , Proteínas Reguladoras de Apoptose , Sistema Nervoso Central/imunologia , Encefalite/genética , Encefalite/imunologia , Encefalite/virologia , Feminino , Interferon-alfa/imunologia , Interferon beta/imunologia , Macrófagos/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Vírus da Hepatite Murina/genética , Proteínas/genética , Proteínas de Ligação a RNA , Doenças dos Roedores/genética , Doenças dos Roedores/virologia , Tropismo ViralRESUMO
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.
Assuntos
Células Produtoras de Anticorpos/imunologia , Astrócitos/metabolismo , Movimento Celular , Quimiocina CXCL10/metabolismo , Infecções por Coronavirus/imunologia , Coronavirus/imunologia , Encefalomielite/imunologia , Animais , Células Produtoras de Anticorpos/efeitos dos fármacos , Células Produtoras de Anticorpos/fisiologia , Astrócitos/imunologia , Quimiocina CXCL10/deficiência , Quimiocina CXCL10/imunologia , Coronavirus/patogenicidade , Infecções por Coronavirus/virologia , Encefalomielite/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Interferon protects mice from vesicular stomatitis virus (VSV) infection and pathogenesis; however, it is not known which of the numerous interferon-stimulated genes (ISG) mediate the antiviral effect. A prominent family of ISGs is the interferon-induced with tetratricopeptide repeats (Ifit) genes comprising three members in mice, Ifit1/ISG56, Ifit2/ISG54 and Ifit3/ISG49. Intranasal infection with a low dose of VSV is not lethal to wild-type mice and all three Ifit genes are induced in the central nervous system of the infected mice. We tested their potential contributions to the observed protection of wild-type mice from VSV pathogenesis, by taking advantage of the newly generated knockout mice lacking either Ifit2 or Ifit1. We observed that in Ifit2 knockout (Ifit2(-/-)) mice, intranasal VSV infection was uniformly lethal and death was preceded by neurological signs, such as ataxia and hind limb paralysis. In contrast, wild-type and Ifit1(-/-) mice were highly protected and survived without developing such disease. However, when VSV was injected intracranially, virus replication and survival were not significantly different between wild-type and Ifit2(-/-) mice. When administered intranasally, VSV entered the central nervous system through the olfactory bulbs, where it replicated equivalently in wild-type and Ifit2(-/-) mice and induced interferon-ß. However, as the infection spread to other regions of the brain, VSV titers rose several hundred folds higher in Ifit2(-/-) mice as compared to wild-type mice. This was not caused by a broadened cell tropism in the brains of Ifit2(-/-) mice, where VSV still replicated selectively in neurons. Surprisingly, this advantage for VSV replication in the brains of Ifit2(-/-) mice was not observed in other organs, such as lung and liver. Pathogenesis by another neurotropic RNA virus, encephalomyocarditis virus, was not enhanced in the brains of Ifit2(-/-) mice. Our study provides a clear demonstration of tissue-, virus- and ISG-specific antiviral action of interferon.
Assuntos
Encéfalo/virologia , Proteínas/metabolismo , Estomatite Vesicular/imunologia , Vírus da Estomatite Vesicular Indiana/patogenicidade , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Vírus da Encefalomiocardite/imunologia , Vírus da Encefalomiocardite/patogenicidade , Feminino , Interferon beta/metabolismo , Fígado/virologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas/genética , Proteínas de Ligação a RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Estomatite Vesicular/patologia , Estomatite Vesicular/virologia , Vírus da Estomatite Vesicular Indiana/imunologia , Replicação ViralRESUMO
Naive T cells undergo robust proliferation in lymphopenic conditions, whereas they remain quiescent in steady-state conditions. However, a mechanism by which naive T cells are kept from proliferating under steady-state conditions remains unclear. In this study, we report that memory CD4 T cells are able to limit naive T cell proliferation within lymphopenic hosts by modulating stimulatory functions of dendritic cells (DC). The inhibition was mediated by IL-27, which was primarily expressed in CD8(+) DC subsets as the result of memory CD4 T cell-DC interaction. IL-27 appeared to be the major mediator of inhibition, as naive T cells deficient in IL-27R were resistant to memory CD4 T cell-mediated inhibition. Finally, IL-27-mediated regulation of T cell proliferation was also observed in steady-state conditions as well as during Ag-mediated immune responses. We propose a new model for maintaining peripheral T cell homeostasis via memory CD4 T cells and CD8(+) DC-derived IL-27 in vivo.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Antígenos CD8 , Proliferação de Células , Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Memória Imunológica , Interleucinas/imunologia , Modelos Imunológicos , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Comunicação Celular/genética , Comunicação Celular/imunologia , Células Dendríticas/metabolismo , Regulação da Expressão Gênica/genética , Homeostase/genética , Homeostase/imunologia , Interleucinas/biossíntese , Interleucinas/genética , Camundongos , Camundongos KnockoutRESUMO
Intraperitoneal injection of the Gram-negative bacterial endotoxin lipopolysaccharide (LPS) elicits a rapid innate immune response. While this systemic inflammatory response can be destructive, tolerable low doses of LPS render the brain transiently resistant to subsequent injuries. However, the mechanism by which microglia respond to LPS stimulation and participate in subsequent neuroprotection has not been documented. In this study, we first established a novel LPS treatment paradigm where mice were injected intraperitoneally with 1.0 mg/kg LPS for four consecutive days to globally activate CNS microglia. By using a reciprocal bone marrow transplantation procedure between wild-type and Toll-like receptor 4 (TLR4) mutant mice, we demonstrated that the presence of LPS receptor (TLR4) is not required on hematogenous immune cells but is required on cells that are not replaced by bone marrow transplantation, such as vascular endothelia and microglia, to transduce microglial activation and neuroprotection. Furthermore, we showed that activated microglia physically ensheathe cortical projection neurons, which have reduced axosomatic inhibitory synapses from the neuronal perikarya. In line with previous reports that inhibitory synapse reduction protects neurons from degeneration and injury, we show here that neuronal cell death and lesion volumes are significantly reduced in LPS-treated animals following experimental brain injury. Together, our results suggest that activated microglia participate in neuroprotection and that this neuroprotection is likely achieved through reduction of inhibitory axosomatic synapses. The therapeutic significance of these findings rests not only in identifying neuroprotective functions of microglia, but also in establishing the CNS location of TLR4 activation.
Assuntos
Lesões Encefálicas/tratamento farmacológico , Lipopolissacarídeos/administração & dosagem , Microglia/efeitos dos fármacos , Fármacos Neuroprotetores/administração & dosagem , Receptor 4 Toll-Like/metabolismo , Animais , Antígenos CD/metabolismo , Apoptose/efeitos dos fármacos , Transplante de Medula Óssea , Lesões Encefálicas/patologia , Lesões Encefálicas/cirurgia , Bromodesoxiuridina/metabolismo , Proliferação de Células/efeitos dos fármacos , Sistema Nervoso Central/citologia , Sistema Nervoso Central/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Esquema de Medicação , Citometria de Fluxo , Imunidade Inata/efeitos dos fármacos , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise em Microsséries , Microglia/metabolismo , Microglia/ultraestrutura , Microscopia Imunoeletrônica , Córtex Motor/patologia , Córtex Motor/ultraestrutura , Sinapses/genética , Sinapses/metabolismo , Sinapses/ultraestrutura , Receptor 4 Toll-Like/deficiência , Quimeras de TransplanteRESUMO
CD4 T cell activation during peripheral infections not only is essential in inducing protective CD8 T cell memory but also promotes CD8 T cell function and survival. However, the contributions of CD4 T cell help to antiviral CD8 T cell immunity during central nervous system (CNS) infection are not well established. Encephalitis induced by the sublethal coronavirus JHMV was used to identify when CD4 T cells regulate CD8 T cell responses following CNS infection. Peripheral expansion of virus-specific CD8 T cells was impaired when CD4 T cells were ablated prior to infection but not at 4 days postinfection. Delayed CD4 T cell depletion abrogated CD4 T cell recruitment to the CNS but only slightly diminished CD8 T cell recruitment. Nevertheless, the absence of CNS CD4 T cells was associated with reduced gamma interferon (IFN-γ) and granzyme B expression by infiltrating CD8 T cells, increased CD8 T cell apoptosis, and impaired control of infectious virus. CD4 T cell depletion subsequent to CD4 T cell CNS migration restored CD8 T cell activity and virus control. Analysis of γc-dependent cytokine expression indicated interleukin-21 (IL-21) as a primary candidate optimizing CD8 T cell activity within the CNS. These results demonstrate that CD4 T cells play critical roles in both enhancing peripheral activation of CD8 T cells and prolonging their antiviral function within the CNS. The data highlight the necessity for temporally and spatially distinct CD4 T cell helper functions in sustaining CD8 T cell activity during CNS infection.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Coronavirus/imunologia , Encefalite Viral/imunologia , Imunidade Celular , Vírus da Hepatite Murina/fisiologia , Animais , Infecções do Sistema Nervoso Central/imunologia , Infecções do Sistema Nervoso Central/virologia , Infecções por Coronavirus/virologia , Encefalite Viral/virologia , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Vírus da Hepatite Murina/imunologiaRESUMO
Type I interferons (IFNα/ß) provide a primary defense against infection. Nevertheless, the dynamics of IFNα/ß induction and responsiveness by central nervous system (CNS) resident cells in vivo in response to viral infections are poorly understood. Mice were infected with a neurotropic coronavirus with tropism for oligodendroglia and microglia to probe innate antiviral responses during acute encephalomyelitis. Expression of genes associated with the IFNα/ß pathways was monitored in microglia and oligodendroglia purified from naïve and infected mice by fluorescent activated cell sorting. Compared with microglia, oligodendroglia were characterized by low basal expression of mRNA encoding viral RNA sensing pattern recognition receptors (PRRs), IFNα/ß receptor chains, interferon sensitive genes (ISG), as well as kinases and transcription factors critical in IFNα/ß signaling. Although PRRs and ISGs were upregulated by infection in both cell types, the repertoire and absolute mRNA levels were more limited in oligodendroglia. Furthermore, although oligodendroglia harbored higher levels of viral RNA compared with microglia, Ifnα/ß was only induced in microglia. Stimulation with the double stranded RNA analogue poly I:C also failed to induce Ifnα/ß in oligodendroglia, and resulted in reduced and delayed induction of ISGs compared with microglia. The limited antiviral response by oligodendroglia was associated with a high threshold for upregulation of Ikkε and Irf7 transcripts, both central to amplifying IFNα/ß responses. Overall, these data reveal that oligodendroglia from the adult CNS are poor sensors of viral infection and suggest they require exogenous IFNα/ß to establish an antiviral state.
Assuntos
Interferon Tipo I/metabolismo , Oligodendroglia/metabolismo , Animais , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Encefalomielite/genética , Encefalomielite/metabolismo , Encefalomielite/patologia , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Indutores de Interferon/farmacologia , Helicase IFIH1 Induzida por Interferon , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/metabolismo , Proteína Proteolipídica de Mielina/genética , Proteína Proteolipídica de Mielina/metabolismo , Oligodendroglia/efeitos dos fármacos , Poli I-C/farmacologia , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
BACKGROUND: Anti-viral CD8 T-cell activity is enhanced and prolonged by CD4 T-cell-mediated help, but negatively regulated by inhibitory B7-H1 interactions. During viral encephalomyelitis, the absence of CD4 T cells decreases CD8 T cell activity and impedes viral control in the central nervous system (CNS). By contrast, the absence of B7-H1 enhances CD8 T-cell function and accelerates viral control, but increases morbidity. However, the relative contribution of CD4 T cells to CD8 function in the CNS, in the absence of B7-H1, remains unclear. METHODS: Wild-type (WT) and B7-H1-/- mice were infected with a gliatropic coronavirus and CD4 T cells depleted to specifically block T helper function in the CNS. Flow cytometry and gene expression analysis of purified T-cell populations from lymph nodes and the CNS was used to directly monitor ex vivo T-cell effector function. The biological affects of altered T-cell responses were evaluated by analysis of viral control and spinal-cord pathology. RESULTS: Increased anti-viral activity by CD8 T cells in the CNS of B7-H1-/- mice was lost upon depletion of CD4 T cells; however, despite concomitant loss of viral control, the clinical disease was less severe. CD4 depletion in B7-H1-/- mice also decreased inducible nitric oxide synthase expression by microglia and macrophages, consistent with decreased microglia/macrophage activation and reduced interferon (IFN)-γ. Enhanced production of IFN-γ, interleukin (IL)-10 and IL-21 mRNA was seen in CD4 T cells from infected B7-H1-/- compared with WT mice, suggesting that over-activated CD4 T cells primarily contribute to the increased pathology. CONCLUSIONS: The local requirement of CD4 T-cell help for CD8 T-cell function is not overcome if B7-H1 inhibitory signals are lost. Moreover, the increased effector activity by CD8 T cells in the CNS of B7-H1-/- mice is attributable not only to the absence of B7-H1 upregulation on major histocompatibility complex class I-presenting resident target cells, but also to enhanced local CD4 T-cell function. B7-H1-mediated restraint of CD4 T-cell activity is thus crucial to dampen both CD8 T-cell function and microglia/macrophage activation, thereby providing protection from T-cell-mediated bystander damage.