RESUMO
Receptor-interacting protein kinase (RIPK)-1 is involved in RIPK3-dependent and -independent signaling pathways leading to cell death and/or inflammation. Genetic ablation of ripk1 causes postnatal lethality, which was not prevented by deletion of ripk3, caspase-8, or fadd. However, animals that lack RIPK1, RIPK3, and either caspase-8 or FADD survived weaning and matured normally. RIPK1 functions in vitro to limit caspase-8-dependent, TNFR-induced apoptosis, and animals lacking RIPK1, RIPK3, and TNFR1 survive to adulthood. The role of RIPK3 in promoting lethality in ripk1(-/-) mice suggests that RIPK3 activation is inhibited by RIPK1 postbirth. Whereas TNFR-induced RIPK3-dependent necroptosis requires RIPK1, cells lacking RIPK1 were sensitized to necroptosis triggered by poly I:C or interferons. Disruption of TLR (TRIF) or type I interferon (IFNAR) signaling delayed lethality in ripk1(-/-)tnfr1(-/-) mice. These results clarify the complex roles for RIPK1 in postnatal life and provide insights into the regulation of FADD-caspase-8 and RIPK3-MLKL signaling by RIPK1.
Assuntos
Caspase 8/metabolismo , Genes Letais , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Animais Recém-Nascidos , Apoptose , Caspase 8/genética , Morte Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Proteína de Domínio de Morte Associada a Fas/metabolismo , Fibroblastos/metabolismo , Inflamação/metabolismo , Interferons/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fatores de Necrose Tumoral/metabolismoRESUMO
The specific mechanisms that mediate CD4(+) T-cell-mediated liver injury have not been fully elucidated. CD4(+) invariant natural killer T (iNKT) cells are required for liver damage in some mouse models of hepatitis, while the chemokine receptors CXCR3 and CCR5 are considered dominant Th1 chemokine receptors involved in Th1 trafficking in inflammatory conditions. BALB/c-Tgfb1(-/-) mice spontaneously develop Th1 hepatitis. Here, we directly test the hypotheses that iNKT cells or the Th1-cell chemokine receptors CXCR3 and CCR5 are required for development of liver disease in Tgfb1(-/-) mice. Tgfb1(-/-) mouse livers exhibited significant increases in iNKT cells and in ligands for CXCR3 or CCR5. Tgfb1(-/-) mice were rendered deficient in iNKT cells, CXCR3, CCR5, or both CXCR3 and CCR5, by cross-breeding with appropriate knockout mice. Tgfb1(-/-) mice developed severe liver injury, even in the absence of functional CD1d/iNKT cells, CXCR3, CCR5, or both CXCR3 and CCR5. Liver CD4(+) T cells accumulated to high numbers, and spleen CD4(+) T-cell numbers declined, regardless of the functionality of the CXCR3/CCR5 response pathways. Similarly, dendritic cells and macrophages accumulated in Tgfb1(-/-) livers even when CXCR3 and CCR5 were knocked out. Th1-associated cytokines (IFN-γ, TNF-α, IL-2) and chemokines (CXCL9, CXCL10) were strongly overexpressed in Tgfb1(-/-) mice despite knockouts in CD1d, CXCR3, or CCR5. These studies indicate that the cellular and biochemical basis for CD4(+) T-cell-mediated injury in liver can be complex, with myriad pathways potentially involved.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Hepatite/imunologia , Fígado/patologia , Células T Matadoras Naturais/metabolismo , Receptores de Quimiocinas/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Quimiocina CXCL9/imunologia , Quimiocina CXCL9/metabolismo , Quimiocinas/imunologia , Quimiocinas/metabolismo , Modelos Animais de Doenças , Citometria de Fluxo , Hepatite/metabolismo , Hepatite/patologia , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-2/imunologia , Interleucina-2/metabolismo , Fígado/imunologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Células T Matadoras Naturais/imunologia , Receptores CCR5/genética , Receptores CCR5/imunologia , Receptores CCR5/metabolismo , Receptores CXCR3/genética , Receptores CXCR3/imunologia , Receptores CXCR3/metabolismo , Receptores de Quimiocinas/imunologia , Estatísticas não Paramétricas , Células Th1/imunologia , Células Th1/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
UNLABELLED: Immune-mediated liver injury in hepatitis is due to activated T cells producing interferon-γ (IFN-γ). It is important to identify negative feedback immune mechanisms that can regulate T cell activity. In this study, we demonstrate that liver inflammation mediated by type 1 T helper (Th1) cells can induce the accumulation of myeloid-derived suppressor cells (MDSCs), pleiomorphic cells capable of modulating T cell-mediated immunity, that heretofore have been studied almost exclusively in the context of tumor-associated inflammation. Mice deficient in the gene encoding transforming growth factor-ß1 (Tgfb1(-/-) mice) acutely develop liver necroinflammation caused by IFN-γ-producing clusters of differentiation 4-positive (CD4(+)) T cells. Liver Th1 cell accumulation was accompanied by myeloid cells expressing CD11b and Gr1, phenotypic hallmarks of MDSCs. Isolated Tgfb1(-/-) liver CD11b(+)Gr1(+) cells were functional MDSCs, readily suppressing T cell proliferation in vitro. Pharmacologic inhibitors of inducible nitric oxide (NO) synthase completely eliminated suppressor function. Suppressor function and the production of NO were dependent on cell-cell contact between MDSCs and T cells, and upon IFN-γ, and were specifically associated with the "monocytic" CD11b(+)Ly6G(-) Ly6C(hi) subset of liver Tgfb1(-/-) CD11b(+) cells. The rapid accumulation of CD11b(+)Gr1(+) cells in Tgfb1(-/-) liver was abrogated when mice were either depleted of CD4(+) T cells or rendered unable to produce IFN-γ, showing that Th1 activity induces MDSC accumulation. CONCLUSION: Th1 liver inflammation mobilizes an MDSC response that, through the production of NO, can inhibit T cell proliferation. We propose that MDSCs serve an important negative feedback function in liver immune homeostasis, and that insufficient or inappropriate activity of this cell population may contribute to inflammatory liver pathology.
Assuntos
Antígeno CD11b/fisiologia , Hepatite/imunologia , Interferon gama/fisiologia , Fator de Crescimento Transformador beta1/fisiologia , Animais , Linfócitos T CD4-Positivos/imunologia , Comunicação Celular , Proliferação de Células/efeitos dos fármacos , Fígado/imunologia , Camundongos , Camundongos Knockout , Células Mieloides/imunologia , Células Progenitoras Mieloides/fisiologia , Óxido Nítrico/fisiologia , Receptores de Quimiocinas/biossíntese , Células Th1/imunologiaRESUMO
Fulminant inflammation in the liver is often accompanied by the accumulation of IFN-gamma-producing T cells. The BALB/c-Tgfb1(-/-) mouse exhibits extensive, spontaneously developing necroinflammation in the liver, accompanied by the accumulation of IFN-gamma-producing CD4(+) and CD8(+) T cells. Liver damage depends on the presence of an intact Ifng gene. We determined the relevant cellular source(s) of IFN-gamma. In Tgfb1(-/-) liver, CD4(+) T cells were more numerous than CD8(+) T cells and NK cells, and produced more IFN-gamma. Depletion of CD4(+) T cells eliminated both the elevation in plasma IFN-gamma and aspartate aminotransferase, whereas depletion of CD8(+) T cells did not. Rag1(-/-)Tgfb1(-/-) mice exhibited neither IFN-gamma elevation nor tissue damage, indicating that NK cells are not sufficient. IFN-gamma was required for strong overexpression of class II genes but not for CD4(+) T cell activation, oligoclonal expansion, or accumulation in the liver. The T cell inhibitory molecule PD-L1 was strongly expressed in Tgfb1(-/-) livers, ruling out a lack of PD-L1 expression as an explanation for aberrant liver T cell activation. Finally, whereas Tgfb1(-/-) CD4(+) T cells overexpressed Fas ligand, hepatocellular damage was observed in Fas(lpr/lpr)Tgfb1(-/-) mice, indicating that liver pathology is Fas independent. We conclude that liver damage in this model of fulminant autoimmune hepatitis is driven by CD4(+) T cell production of IFN-gamma, is independent of both CD8(+) T cells and the Fas ligand/Fas pathway, and is not explained by a lack of PD-L1 expression.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Hepatite Autoimune/imunologia , Hepatite Autoimune/patologia , Falência Hepática Aguda/imunologia , Falência Hepática Aguda/patologia , Receptor fas , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Modelos Animais de Doenças , Proteína Ligante Fas/genética , Proteína Ligante Fas/fisiologia , Hepatite Autoimune/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Interferon gama/deficiência , Interferon gama/genética , Interferon gama/fisiologia , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Falência Hepática Aguda/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Fator de Crescimento Transformador beta1/genética , Receptor fas/genética , Receptor fas/fisiologiaRESUMO
BACKGROUND/AIMS: BALB/c mice with a homozygous deficiency in the Tgfb1 gene are a model of fulminant autoimmune hepatitis (AIH), spontaneously and rapidly developing Th1-mediated IFN-gamma-dependent necroinflammatory liver disease. We sought to understand the molecular basis for fulminant Th1 liver disease and the specific role of the Ifng gene. METHODS: Global gene expression in livers from BALB/c Tgfb1(-/-) mice with and without an intact Ifng gene was assessed by microarray analysis. Expression patterns were confirmed by quantitative reverse transcriptase-polymerase chain reaction. Gene ontology clustering analysis was performed to identify altered pathways. The contributions of Ifng to altered expression pathways were quantified. RESULTS: Over 100 genes were strongly (>10-fold) upregulated, most encoding proteins involved in immune function/response. Chemokines were the most prominently upregulated group, with eight chemokine genes upregulated >10-fold. Ifng was necessary for the upregulation of CXC chemokines gene, but not of CC chemokine genes. By quantitative analysis, Ifng's role in liver gene upregulation varied greatly among overexpressed genes. CONCLUSIONS: Gene expression changes indicate a particularly important and heretofore unappreciated role for chemokines in fulminant AIH. Ifng has an important role in expression of some but not all genes. Ifng is dichotomous in the regulation of distinct chemokine subfamilies: specifically, Ifng is critical for overexpression of specific CXCL genes but dispensable for overexpression of specific CCL genes. These results provide a clearer understanding of the role of Ifng in the molecular basis of necroinflammatory liver disease.
Assuntos
Hepatite Autoimune/metabolismo , Interferon gama/fisiologia , Fígado/metabolismo , Animais , Quimiocinas/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta1/fisiologiaRESUMO
Gangliosides, sialic acid-containing glycosphingolipids present in most cell membranes, are thought to participate in the maintenance of immune privilege and tumor-induced immunosuppression. However, the mechanisms responsible for their immunomodulatory activity remain poorly understood. The purpose of this study was to investigate whether gangliosides are able to modulate the balance of type-1/type-2 T cell responses and to characterize the cellular mechanisms involved. The effects of different gangliosides on anti-CD3-stimulated murine splenocytes and purified T cells were studied. The presence of gangliosides during T cell activation reduced the expression of interferon-gamma (IFN-gamma) and enhanced that of interleukin (IL)-4, suggesting a shift toward a type-2 response. Intracellular cytokine staining demonstrated that gangliosides inhibited IFN-gamma production in CD4+, CD8+, and natural killer (NK)1.1+ cell populations and enhanced IL-4 in CD4+ T cells. The ganglioside-mediated enhancement in IL-4 production was independent of changes in endogenous IFN-gamma, did not occur with cells from CD1d-deficient mice, and was partially inhibited by anti-CD1d antibodies. The inhibitory effects on IFN-gamma were independent of endogenous IL-4 or the presence of NKT cells and were unaffected by anti-CD1d antibodies. These results suggest that gangliosides may modify the immunological environment by promoting immune deviation in favor of type-2 T cell responses.
Assuntos
Gangliosídeos/imunologia , Tolerância Imunológica/imunologia , Imunidade Celular/imunologia , Imunossupressores/imunologia , Células Th2/imunologia , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Feminino , Gangliosídeos/farmacologia , Tolerância Imunológica/efeitos dos fármacos , Imunidade Celular/efeitos dos fármacos , Imunossupressores/farmacologia , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-4/imunologia , Interleucina-4/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Células Th2/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
Glycosaminoglycans (GAGs) located on cellular membranes and the extracellular matrix (ECM) are able to interact with chemokines and pro-inflammatory cytokines, leading to local cytokine/chemokine accumulation. The tissue-bound cytokines/chemokines function in promoting leukocyte migration and activation, contributing to local inflammation. Hence, targeting of GAG-cytokine interactions may provide an avenue for the attenuation of inflammatory responses. A cationic peptide (MC2) derived from the heparin-binding sequence of mouse IFN-gamma was previously shown by our laboratory to delay allograft rejection in an animal model. In order to further investigate potential anti-inflammatory properties of the MC2 peptide, we have studied its activity in an acute peritoneal inflammation model. Groups of C57Bl/6 mice were injected intraperitoneally with either ConA or thioglycollate and treated with saline (control), the MC2 peptide or two control cationic peptides, poly-l-lysine (PLL) and poly-l-arginine (PLA). Treatment with the MC2 peptide, but not PLA or PLL, resulted in statistically significant reductions in total cell numbers, concentration of total proteins and concentrations of pro-inflammatory cytokines (TNFalpha, IL-6 or IL-1 beta) in peritoneal lavage fluids, without alterations to the qualitative cellular composition of the exudate. These results suggest that targeting GAG-cytokine interaction is a viable approach to reduce inflammation.
Assuntos
Anti-Inflamatórios/farmacologia , Citocinas/fisiologia , Glicosaminoglicanos/fisiologia , Inflamação/tratamento farmacológico , Interferon gama/farmacologia , Fragmentos de Peptídeos/farmacologia , Proteínas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Líquido Ascítico/citologia , Líquido Ascítico/metabolismo , Células Cultivadas , Concanavalina A , Citocinas/metabolismo , Feminino , Glicosaminoglicanos/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , TioglicolatosRESUMO
Myeloid derived suppressor cells (MDSC) were first described nearly two decades ago. Until recently, however, descriptions of MDSC populations were found almost exclusively in animal models of cancer or in cancer patients. Over the last few years, an increasing number of reports have been published describing populations of myeloid cells with MDSC-like properties in murine models of autoimmune disease. In contrast to the proposed deleterious role of MDSC in cancer--where these cells likely inhibit tumor immunity--in the context of autoimmunity, MDSC have the potential to suppress the autoimmune response, thereby limiting tissue injury. A logical corollary of this hypothesis is that a failure of endogenous MDSC to appropriately control autoimmune T cell responses in vivo may actually contribute to the pathogenesis of autoimmune disease.