RESUMEN
MATERIALS AND METHODS: Locomotor outcomes in perforin-deficient (Pfp-/-) mice and wild-type littermate controls were measured after severe compression injury of the lower thoracic spinal cord up to six weeks after injury. RESULTS: According to both the Basso mouse scale score and single frame motion analysis, motor recovery of Pfp-/- mice was similar to wild-type controls. Interestingly, immunohistochemical analysis of cell types at six weeks after injury showed enhanced cholinergic reinnervation of spinal motor neurons caudal to the lesion site and neurofilament-positive structures at the injury site in Pfp-/- mice, whereas numbers of microglia/macrophages and astrocytes were decreased compared with controls. CONCLUSIONS: We conclude that, although, loss of perforin does not change the locomotor outcome after injury, it beneficially affects diverse cellular features, such as number of axons, cholinergic synapses, astrocytes and microglia/macrophages at or caudal to the lesion site. Perforin's ability to contribute to Rag2's influence on locomotion was observed in mice doubly deficient in perforin and Rag2 which recovered better than Rag2-/- or Pfp-/- mice, suggesting that natural killer cells can cooperate with T- and B-cells in spinal cord injury.
Asunto(s)
Locomoción/fisiología , Regeneración Nerviosa/fisiología , Proteínas Citotóxicas Formadoras de Poros/fisiología , Traumatismos de la Médula Espinal/enzimología , Traumatismos de la Médula Espinal/inmunología , Traumatismos de la Médula Espinal/fisiopatología , Animales , Conducta Animal/fisiología , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Citotóxicas Formadoras de Poros/deficienciaRESUMEN
NOD-like receptor (NLR), family pyrin domain containing 3 (NLRP3) assembles a protein complex known as the NLRP3 inflammasome upon sensing certain pathogen products or sterile danger signals. Gain-of-function mutations such as the D301N substitution in NLRP3, which cause its constitutive activation (NLRP3CA) also results in inflammasome assembly. This inflammasome processes prointerleukin-1 ß (proIL-1ß) and proIL-18 into bioactive IL-1ß and IL-18, respectively, and cleaves gasdermin D (GSDMD). GSDMD amino-terminal fragments form plasma membrane pores that facilitate the secretion of IL-1ß and IL-18 and lead to the inflammatory cell death pyroptosis. Accordingly, GSDMD inactivation results in negligible spontaneous inflammation in various experimental models such as in Nlrp3CA/+ mice lacking GSDMD (Nlrp3CA/+;Gsdmd−/− mice). Here, we found that Nlrp3CA/+;Gsdmd−/− mice, when challenged with LPS or TNF-α, still secreted IL-1ß and IL-18, indicating inflammasome activation independent of GSDMD. Accordingly, Gsdmd−/− macrophages failed to secrete IL-1ß and undergo pyroptosis when briefly exposed to NLRP3 inflammasome activators but released these cytokines when persistently activated. Sustained NLRP3 inflammasome induced caspase-8/-3 and GSDME cleavage and IL-1ß maturation in vitro in Gsdmd−/− macrophages. Thus, a salvage inflammatory pathway involving caspase-8/-3GSDME was activated after NLRP3 activation when the canonical NLRP3-GSDMD signaling was blocked. Consistent with genetic data, the active metabolite of FDA-approved disulfiram CuET, which inhibited GSDMD and GSDME cleavage in macrophages, reduced the severe inflammation and tissue damage that occurred in the Nlrp3CA/+ mice. Thus, NLRP3 inflammasome activation overwhelms the protection afforded by GSDMD deficiency, rewiring signaling cascades through mechanisms that include GSDME to propagate inflammation.
Asunto(s)
Inflamasomas/inmunología , Inflamación/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Proteínas de Unión a Fosfato/inmunología , Proteínas Citotóxicas Formadoras de Poros/inmunología , Animales , Células Cultivadas , Inflamación/patología , Ratones , Ratones Congénicos , Ratones Noqueados , Ratones Transgénicos , Proteínas de Unión a Fosfato/deficiencia , Proteínas Citotóxicas Formadoras de Poros/deficienciaRESUMEN
Crohn's disease (CD) is a kind of refractory intestinal inflammatory diseases. Pyroptosis was recently identified as a gasdermin-mediated proinflammatory cell death. However, it is unclear whether gasdermin-mediated pyroptosis participates in the pathogenesis of CD. Here, we show that the pyroptosis-inducing fragment GSDME N-terminal is obviously detected in the inflamed colonic mucosa but not in the uninflamed mucosa of patients with CD, suggesting that GSDME-mediated pyroptosis may be correlated with intestinal mucosal inflammation in CD. To investigate the role of GSDME in colitis development, Gsdme-/- mice and wild-type (WT) littermate controls were treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS) to induce colitis. We found that Gsdme-/- mice exhibit less-severe intestinal inflammation than WT controls do. Furthermore, our results indicate that GSDME-mediated epithelial-cell pyroptosis induces intestinal inflammation through the release of proinflammatory intracellular contents. In summary, we show that GSDME participates in the pathogenesis of CD through GSDME-mediated pyroptosis to release proinflammatory cytokines.
Asunto(s)
Enfermedad de Crohn/patología , Inflamación/patología , Intestinos/patología , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Piroptosis , Animales , Colitis/inducido químicamente , Colitis/inmunología , Colitis/metabolismo , Colitis/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Proteína HMGB1/metabolismo , Hematopoyesis , Humanos , Mucosa Intestinal/patología , Masculino , Ratones Endogámicos C57BL , Proteínas Citotóxicas Formadoras de Poros/deficiencia , Índice de Severidad de la Enfermedad , Ácido TrinitrobencenosulfónicoRESUMEN
Cellular inflammasome activation causes caspase-1 cleavage of the pore-forming protein gasdermin D (GSDMD) with subsequent pyroptotic cell death and cytokine release. Here, we clarify the ambiguous role of the related family member gasdermin E (GSDME) in this process. Inflammasome stimulation in GSDMD-deficient cells led to apoptotic caspase cleavage of GSDME. Endogenous GSDME activation permitted sublytic, continuous interleukin-1ß (IL-1ß) release and membrane leakage, even in GSDMD-sufficient cells, whereas ectopic expression led to pyroptosis with GSDME oligomerization and complete liberation of IL-1ß akin to GSDMD pyroptosis. We find that NLRP3 and NLRP1 inflammasomes ultimately rely concurrently on both gasdermins for IL-1ß processing and release separately from their ability to induce cell lysis. Our study thus identifies GSDME as a conduit for IL-1ß release independent of its ability to cause cell death.
Asunto(s)
Inflamasomas/genética , Interleucina-1beta/genética , Macrófagos/inmunología , Proteínas de Unión a Fosfato/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Piroptosis/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Caspasa 1/genética , Caspasa 1/inmunología , Caspasa 3/genética , Caspasa 3/inmunología , Línea Celular Transformada , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Inflamasomas/inmunología , Interleucina-1beta/inmunología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Proteínas de Unión a Fosfato/deficiencia , Proteínas de Unión a Fosfato/inmunología , Proteínas Citotóxicas Formadoras de Poros/deficiencia , Proteínas Citotóxicas Formadoras de Poros/inmunología , Piroptosis/efectos de los fármacos , Piroptosis/inmunología , Salmonella typhimurium/química , Salmonella typhimurium/patogenicidad , Transducción de Señal , Células THP-1RESUMEN
Immunotherapeutic strategies are increasingly important in neuro-oncology, and the elucidation of escape mechanisms that lead to treatment resistance is crucial. We investigated the impact of immune pressure on the clonal dynamics and immune escape signature by comparing glioma growth in immunocompetent versus immunodeficient mice. Glioma-bearing WT and Pd-1-/- mice survived significantly longer than immunodeficient Pfp-/- Rag2-/- mice. While tumors in Pfp-/- Rag2-/- mice were highly polyclonal, immunoedited tumors in WT and Pd-1-/- mice displayed reduced clonality with emergence of immune escape clones. Tumor cells in WT mice were distinguished by an IFN-γ-mediated response signature with upregulation of genes involved in immunosuppression. Tumor-infiltrating stromal cells, which include macrophages/microglia, contributed even more strongly to the immunosuppressive signature than the actual tumor cells. The identified murine immune escape signature was reflected in human patients and correlated with poor survival. In conclusion, immune pressure profoundly shapes the clonal composition and gene regulation in malignant gliomas.
Asunto(s)
Neoplasias Encefálicas/inmunología , Glioma/inmunología , Escape del Tumor/inmunología , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Evolución Clonal/genética , Evolución Clonal/inmunología , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Femenino , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Glioma/genética , Glioma/patología , Humanos , Inmunocompetencia , Huésped Inmunocomprometido , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Citotóxicas Formadoras de Poros/deficiencia , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/inmunología , Escape del Tumor/genética , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
Increasing evidence points to a key role for NK cells in controlling adaptive immune responses. In studies examining the role of CD1d on CD4+ T cell responses, we found that a line of CD1d-deficient mice on the C57BL/6J background had a homozygous 129 locus on chromosome 6 containing the entire NK cell gene cluster. Mice possessing this locus (C57BL/6.NKC129) displayed a >10-fold reduction in antigen-specific CD4+ T cell responses after intracranial infection with lymphocytic choriomeningitis virus (LCMV). Neither parental strain displayed defects in viral-specific CD4+ T cell responses. Interestingly, following infection, increased numbers of NK cells accumulated in the lymph nodes of C57BL/6.NKC129 mice and displayed enhanced in vivo functionality. Moreover, depletion of NK cells with anti-asialo-GM-1 antibody in C57BL/6.NKC129 mice resulted in a >20-fold increase in viral-specific CD4+ T cell responses. Mechanistically, we found that dendritic cell antigen presentation and early type I IFN production were significantly decreased in C57BL/6.NKC129 mice, but were restored in perforin-deficient C57BL/6.NKC129 mice or following NK depletion. Together, these data reveal that the variable genomic regions containing the activating/inhibitory NK cell receptors are key determinants of antigen-specific CD4+ T cell responses, controlling type I IFN production and the antigen-presenting capacity of dendritic cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Sitios Genéticos , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Receptores de Células Asesinas Naturales/genética , Animales , Presentación de Antígeno/genética , Antígenos CD1d/genética , Células Dendríticas/inmunología , Interferón Tipo I/biosíntesis , Células Asesinas Naturales/inmunología , Ganglios Linfáticos/inmunología , Activación de Linfocitos/genética , Coriomeningitis Linfocítica/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Citotóxicas Formadoras de Poros/deficiencia , Proteínas Citotóxicas Formadoras de Poros/genéticaRESUMEN
CD8(+) T cells use contact-dependent cytolysis of target cells to protect the host against intracellular pathogens. We have previously shown that CD8(+) T cells and perforin are required to protect against the extracellular pathogen Yersinia pseudotuberculosis. Here we establish an experimental system where CD8(+) T cells specific to a single model antigen are the only memory response present at time of challenge. Using mice immunized with a vaccine strain of Listeria monocytogenes that expresses secreted ovalbumin (Lm-OVA), we show that OVA-specific CD8(+) T cells are generated and provide limited protection against challenge with virulent OVA(+)Y. pseudotuberculosis. Perforin expression by OVA-specific CD8(+) T cells was required, as Lm-OVA-immunized perforin-deficient mice showed higher bacterial burden as compared to Lm-OVA-immunized perforin-sufficient mice. Surprisingly, antigen-specific T cell protection waned over time, as Lm-OVA-immune mice eventually succumbed to Yersinia infection. Kinetic analysis of infection in mice with and without OVA-specific CD8(+) T cells revealed that bacterial numbers increased sharply in OVA-naïve mice until death, while OVA-immune mice held bacterial burden to a lower level throughout the duration of illness until death. Clonal analysis of bacterial populations in OVA-naïve and OVA-immune mice at distinct time points revealed equivalent and severe bottle-neck effects for bacteria in both sets of mice immediately after intravenous challenge, demonstrating a dominant role for other aspects of the immune system regardless of CD8(+) T cell status. These studies indicate that CD8(+) T cells against a single antigen can restrict Y. pseudotuberculosis colonization in a perforin-dependent manner, but ultimately are insufficient in their ability to provide sterilizing immunity and protect against death.
Asunto(s)
Vacunas Bacterianas/administración & dosificación , Linfocitos T CD8-positivos/inmunología , Proteínas Citotóxicas Formadoras de Poros/inmunología , Infecciones por Yersinia pseudotuberculosis/inmunología , Yersinia pseudotuberculosis/patogenicidad , Animales , Antígenos/administración & dosificación , Antígenos/genética , Antígenos/inmunología , Carga Bacteriana , Linfocitos T CD8-positivos/microbiología , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Femenino , Expresión Génica , Memoria Inmunológica , Listeria monocytogenes/química , Listeria monocytogenes/inmunología , Hígado/inmunología , Hígado/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/administración & dosificación , Ovalbúmina/genética , Ovalbúmina/inmunología , Proteínas Citotóxicas Formadoras de Poros/deficiencia , Proteínas Citotóxicas Formadoras de Poros/genética , Análisis de Supervivencia , Yersinia pseudotuberculosis/efectos de los fármacos , Yersinia pseudotuberculosis/crecimiento & desarrollo , Infecciones por Yersinia pseudotuberculosis/microbiología , Infecciones por Yersinia pseudotuberculosis/mortalidad , Infecciones por Yersinia pseudotuberculosis/prevención & controlRESUMEN
Emerging evidence suggests that immunological mechanisms underlie metabolic control of adipose tissue. Here, we have shown the regulatory impact of a rare subpopulation of dendritic cells, rich in perforin-containing granules (perf-DCs). Using bone marrow transplantation to generate animals selectively lacking perf-DCs, we found that these chimeras progressively gained weight and exhibited features of metabolic syndrome. This phenotype was associated with an altered repertoire of T cells residing in adipose tissue and could be completely prevented by T cell depletion in vivo. A similar impact of perf-DCs on inflammatory T cells was also found in a well-defined model of multiple sclerosis, experimental autoimmune encephlalomyelitis (EAE). Thus, perf-DCs probably represent a regulatory cell subpopulation critical for protection from metabolic syndrome and autoimmunity.
Asunto(s)
Autoinmunidad/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Inflamación/inmunología , Síndrome Metabólico/inmunología , Proteínas Citotóxicas Formadoras de Poros/análisis , Tejido Adiposo/inmunología , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Traslado Adoptivo , Animales , Antígenos de Diferenciación/análisis , Antígeno CD11c/análisis , Linfocitos T CD8-positivos/patología , Linfocitos T CD8-positivos/trasplante , Células Clonales/inmunología , Gránulos Citoplasmáticos/química , Células Dendríticas/clasificación , Células Dendríticas/ultraestructura , Dieta Alta en Grasa/efectos adversos , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Femenino , Inflamación/patología , Depleción Linfocítica , Masculino , Síndrome Metabólico/etiología , Síndrome Metabólico/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Obesidad/inmunología , Obesidad/patología , Fenotipo , Proteínas Citotóxicas Formadoras de Poros/deficiencia , Proteínas Citotóxicas Formadoras de Poros/genética , Quimera por Radiación , Autotolerancia/inmunologíaRESUMEN
Exogenously administered glucocorticoids enhance eosinophil and neutrophil granulocyte production from murine bone-marrow. A hematological response dependent on endogenous glucocorticoids underlies bone-marrow eosinophilia induced by trauma or allergic sensitization/challenge. We detected a defect in granulopoiesis in nonsensitized, perforin-deficient mice. In steady-state conditions, perforin- (Pfp-) deficient mice showed significantly decreased bone-marrow and blood eosinophil and neutrophil counts, and colony formation in response to GM-CSF, relative to wild-type controls of comparable age and/or weight. By contrast, peripheral blood or spleen total cell and lymphocyte numbers were not affected by perforin deficiency. Dexamethasone enhanced colony formation by GM-CSF-stimulated progenitors from wild-type controls, but not Pfp mice. Dexamethasone injection increased bone-marrow eosinophil and neutrophil counts in wild-type controls, but not Pfp mice. Because perforin is expressed in effector lymphocytes, we examined whether this defect would be corrected by transferring wild-type lymphocytes into perforin-deficient recipients. Short-term reconstitution of the response to dexamethasone was separately achieved for eosinophils and neutrophils by transfer of distinct populations of splenic lymphocytes from nonsensitized wild-type donors. Transfer of the same amount of splenic lymphocytes from perforin-deficient donors was ineffective. This demonstrates that the perforin-dependent, granulopoietic response to dexamethasone can be restored by transfer of innate lymphocyte subpopulations.
Asunto(s)
Dexametasona/farmacología , Granulocitos/efectos de los fármacos , Granulocitos/inmunología , Linfocitos/inmunología , Proteínas Citotóxicas Formadoras de Poros/deficiencia , Animales , Dexametasona/administración & dosificación , Eosinófilos/citología , Eosinófilos/inmunología , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Granulocitos/citología , Linfocitos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/citología , Neutrófilos/inmunología , Proteínas Citotóxicas Formadoras de Poros/genéticaRESUMEN
Multiple myeloma (MM) is an age-dependent hematological malignancy. Evaluation of immune interactions that drive MM relies on in vitro experiments that do not reflect the complex cellular stroma involved in MM pathogenesis. Here we used Vk*MYC transgenic mice, which spontaneously develop MM, and demonstrated that the immune system plays a critical role in the control of MM progression and the response to treatment. We monitored Vk*MYC mice that had been crossed with Cd226 mutant mice over a period of 3 years and found that CD226 limits spontaneous MM development. The CD226-dependent anti-myeloma immune response against transplanted Vk*MYC MM cells was mediated both by NK and CD8+ T cells through perforin and IFN-γ pathways. Moreover, CD226 expression was required for optimal antimyeloma efficacy of cyclophosphamide (CTX) and bortezomib (Btz), which are both standardly used to manage MM in patients. Activation of costimulatory receptor CD137 with mAb (4-1BB) exerted strong antimyeloma activity, while inhibition of coinhibitory receptors PD-1 and CTLA-4 had no effect. Taken together, the results of this study provide in vivo evidence that CD226 is important for MM immunosurveillance and indicate that specific immune components should be targeted for optimal MM treatment efficacy. As progressive immunosuppression associates with MM development, strategies aimed to increase immune functions may have important therapeutic implications in MM.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/fisiología , Vigilancia Inmunológica/inmunología , Mieloma Múltiple/inmunología , Proteínas de Neoplasias/fisiología , Animales , Anticuerpos Monoclonales/uso terapéutico , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/inmunología , Antineoplásicos/uso terapéutico , Ácidos Borónicos/uso terapéutico , Bortezomib , Linfocitos T CD8-positivos/inmunología , Antígeno CTLA-4/antagonistas & inhibidores , Cruzamientos Genéticos , Ciclofosfamida/uso terapéutico , Progresión de la Enfermedad , Genes myc , Predisposición Genética a la Enfermedad , Inmunoterapia , Interferón gamma/deficiencia , Interferón gamma/genética , Interferón gamma/fisiología , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Trasplante de Neoplasias , Proteínas Citotóxicas Formadoras de Poros/deficiencia , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/fisiología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Pirazinas/uso terapéutico , Receptores Virales/deficiencia , Receptores Virales/genética , Receptores Virales/fisiología , Carga Tumoral , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/antagonistas & inhibidores , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunologíaRESUMEN
Immunosuppressive pretransplantation conditioning is essential for donor cell engraftment in allogeneic bone marrow transplantation (BMT). The role of residual postconditioning recipient immunity in determining engraftment is poorly understood. We examined the role of recipient perforin in the kinetics of donor cell engraftment. MHC-mismatched BMT mouse models demonstrated that both the rate and proportion of donor lymphoid cell engraftment and expansion of effector memory donor T cells in both spleen and BM were significantly increased within 5 to 7 days post-BMT in perforin-deficient (pfn(-/-)) recipients, compared with wild-type. In wild-type recipients, depletion of natural killer (NK) cells before BMT enhanced donor lymphoid cell engraftment to that seen in pfn(-/-) recipients. This demonstrated that a perforin-dependent, NK-mediated, host-versus-graft (HVG) effect limits the rate of donor engraftment and T cell activation. Radiation-resistant natural killer T (NKT) cells survived in the BM of lethally irradiated mice and may drive NK cell activation, resulting in the HVG effect. Furthermore, reduced pretransplant irradiation doses in pfn(-/-) recipients permitted long-term donor lymphoid cell engraftment. These findings suggest that suppression of perforin activity or selective depletion of recipient NK cells before BMT could be used to improve donor stem cell engraftment, in turn allowing for the reduction of pretransplant conditioning.
Asunto(s)
Trasplante de Médula Ósea , Supervivencia de Injerto , Enfermedad Injerto contra Huésped/prevención & control , Células Asesinas Naturales/efectos de la radiación , Proteínas Citotóxicas Formadoras de Poros/inmunología , Linfocitos T/inmunología , Animales , Femenino , Expresión Génica , Reacción Huésped-Injerto/efectos de la radiación , Memoria Inmunológica , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Depleción Linfocítica , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Citotóxicas Formadoras de Poros/deficiencia , Proteínas Citotóxicas Formadoras de Poros/genética , Tolerancia a Radiación , Linfocitos T/citología , Trasplante Homólogo , Irradiación Corporal TotalRESUMEN
RATIONALE: CD4(+) natural killer T (NKT) cells augment atherosclerosis in apolipoprotein E-deficient (ApoE)(-/-) mice but their mechanisms of action are unknown. OBJECTIVES: We investigated the roles of bystander T, B, and NK cells; NKT cell-derived interferon-γ, interleukin (IL)-4, and IL-21 cytokines; and NKT cell-derived perforin and granzyme B cytotoxins in promoting CD4(+) NKT cell atherogenicity. METHODS AND RESULTS: Transfer of CD4(+) NKT cells into T- and B-cell-deficient ApoE(-/-)Rag2(-/-) mice augmented aortic root atherosclerosis by ≈75% that was ≈30% of lesions in ApoE(-/-) mice; macrophage accumulation similarly increased. Transferred NKT cells were identified in the liver and atherosclerotic lesions of recipient mice. Transfer of CD4(+) NKT cells into T-, B-cell-deficient, and NK cell-deficient ApoE(-/-)Rag2(-/-)γC(-/-) mice also augmented atherosclerosis. These data indicate that CD4(+) NKT cells can exert proatherogenic effects independent of other lymphocytes. To investigate the role of NKT cell-derived interferon-γ, IL-4, and IL-21 cytokines and perforin and granzyme B cytotoxins, CD4(+) NKT cells from mice deficient in these molecules were transferred into NKT cell-deficient ApoE(-/-)Jα18(-/-) mice. CD4(+) NKT cells deficient in IL-4, interferon-γ, or IL-21 augmented atherosclerosis in ApoE(-/-)Jα18(-/-) mice by ≈95%, ≈80%, and ≈70%, respectively. Transfer of CD4(+) NKT cells deficient in perforin or granzyme B failed to augment atherosclerosis. Apoptotic cells, necrotic cores, and proinflammatory VCAM-1 (vascular cell adhesion molecule) and MCP-1 (monocyte chemotactic protein) were reduced in mice receiving perforin-deficient NKT cells. CD4(+) NKT cells are twice as potent as CD4(+) T cells in promoting atherosclerosis. CONCLUSIONS: CD4(+) NKT cells potently promote atherosclerosis by perforin and granzyme B-dependent apoptosis that increases postapoptotic necrosis and inflammation.
Asunto(s)
Aterosclerosis/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Granzimas/deficiencia , Células T Asesinas Naturales/metabolismo , Proteínas Citotóxicas Formadoras de Poros/deficiencia , Seno Aórtico/metabolismo , Traslado Adoptivo/métodos , Animales , Aterosclerosis/inmunología , Aterosclerosis/patología , Linfocitos T CD4-Positivos/inmunología , Masculino , Ratones , Ratones Noqueados , Células T Asesinas Naturales/inmunología , Seno Aórtico/inmunología , Seno Aórtico/patologíaRESUMEN
BACKGROUND & AIMS: Biliary atresia represents obstructive cholangiopathy in infants progressing rapidly to cirrhosis and end-stage liver disease. Activated NK cells expressing Nkg2d have been linked to bile duct injury and obstruction by establishing contact with cholangiocytes. To define the mechanisms used by cytotoxic cells, we investigated the role of perforin and granzymes in a neonatal mouse model of rotavirus (RRV)-induced biliary atresia. METHODS: We used complementary cell lysis assays, flow cytometric analyses, quantitative PCRs and in vivo systems to determine the mechanisms of bile duct epithelial injury and the control of the tissue phenotype in experimental biliary atresia. RESULTS: RRV-infected hepatic NK and CD8 T cells increased the expression of perforin and injured cholangiocytes in short-term culture in a perforin-dependent fashion. However, the loss of perforin in vivo delayed but did not prevent the obstruction of bile ducts. Based on the increased expression of granzymes by perforin-deficient cytotoxic cells in long-term cytolytic assays, we found that the inhibition of granzymes by nafamostat mesilate (FUT-175) blocked cholangiocyte lysis. Administration of FUT-175 to perforin-deficient mice after RRV infection decreased the development of jaundice, minimized epithelial injury, and improved long-term survival. However, the inhibition of granzymes alone in wild-type mice was not sufficient to prevent the atresia phenotype in newborn mice. In infants with biliary atresia, hepatic Granzymes A and B mRNA, but not Perforin, increased at the time of portoenterostomy. CONCLUSIONS: Perforin and granzymes have complementary roles mediating epithelial injury by NK and CD8 T cells. The prevention of experimental biliary atresia can only be achieved by inhibiting both granules.
Asunto(s)
Atresia Biliar/etiología , Atresia Biliar/metabolismo , Granzimas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Animales , Animales Recién Nacidos , Benzamidinas , Conductos Biliares/inmunología , Conductos Biliares/patología , Atresia Biliar/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Colestasis/etiología , Colestasis/patología , Colestasis/prevención & control , Modelos Animales de Enfermedad , Granzimas/antagonistas & inhibidores , Granzimas/genética , Guanidinas/farmacología , Humanos , Lactante , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Hígado/enzimología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Proteínas Citotóxicas Formadoras de Poros/deficiencia , Proteínas Citotóxicas Formadoras de Poros/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Infecciones por Rotavirus/complicacionesRESUMEN
Defects in perforin and related genes lead to a loss of normal immune regulation and underlie hemophagocytic lymphohistiocytosis (HLH), which requires hematopoietic cell transplantation for long-term cure. However, transplantation may be complicated by the development of mixed chimerism and uncertainty regarding the risk of HLH recurrence. To help clarify this risk and investigate how perforin influences immune activation, we studied perforin-mediated immune regulation in the context of mixed chimerism using a murine model of HLH. We found that there is a distinct threshold of â¼10% to 20% perforin expression with either mixed hematopoietic or CD8(+) T cell chimerism, above which immune regulation was reestablished. These findings demonstrate that perforin-mediated immunoregulation functions in trans and are consistent with a feedback model in which cytotoxic T cells control immune activation by killing dendritic cells. These findings also suggest rational targets for maintenance of minimal posttransplant chimerism and for therapeutic strategies involving gene correction.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Células Madre Hematopoyéticas/inmunología , Linfohistiocitosis Hemofagocítica/inmunología , Linfohistiocitosis Hemofagocítica/terapia , Proteínas Citotóxicas Formadoras de Poros/inmunología , Animales , Linfocitos T CD8-positivos/citología , Células Madre Hematopoyéticas/citología , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Linfohistiocitosis Hemofagocítica/genética , Ratones , Ratones Transgénicos , Perforina , Proteínas Citotóxicas Formadoras de Poros/deficiencia , Proteínas Citotóxicas Formadoras de Poros/genética , Linfocitos T Citotóxicos/citología , Linfocitos T Citotóxicos/inmunología , Quimera por TrasplanteRESUMEN
Immune suppression by Treg has been demonstrated in a number of models, but the mechanisms of this suppression are only partly understood. Recent work has suggested that Tregs may suppress by directly killing immune cell populations in vivo in a perforin- and granzyme B-dependent manner. To establish whether perforin is necessary for the regulation of immune responses in vivo, we examined OVA-specific CD8(+) T cell responses in WT and PKO mice immunized with OVA and α-GalCer and the expansion of WT OT-I CD8(+) T cells adoptively transferred into WT or PKO mice immunized with DC-OVA. We observed similar expansion, phenotype, and effector function of CD8(+) T cells in WT and PKO mice, suggesting that CD8(+) T cells were subjected to a similar amount of regulation in the two mouse strains. In addition, when WT and PKO mice were depleted of Tregs by anti-CD25 mAb treatment before DC-OVA immunization, CD8(+) T cell proliferation, cytotoxicity, and cytokine production were increased similarly, suggesting a comparable involvement of CD25(+) Tregs in controlling T cell proliferation and effector function in these two mouse strains. These data suggest that perforin expression is not required for normal immune regulation in these models of in vivo CD8(+) T cell responses induced by immunization with OVA and α-GalCer or DC-OVA.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Depleción Linfocítica , Proteínas Citotóxicas Formadoras de Poros/deficiencia , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Células Dendríticas/metabolismo , Galactosilceramidas/metabolismo , Inmunización , Lectinas Tipo C , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/inmunología , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Receptores Inmunológicos/metabolismo , Linfocitos T ReguladoresRESUMEN
We performed gene-expression profiling of PBMCs obtained from patients with familial hemophagocytic lymphohistiocytosis (FHL) to screen for biologic correlates with the genetic and/or clinical forms of this disease. Unsupervised hierarchical clustering of 167 differentially expressed probe sets, representing 143 genes, identified 3 groups of patients corresponding to the genetic forms and clinical presentations of the disease. Two clusters of up- and down-regulated genes separated patients with perforin-deficient FHL from those with unidentified genetic cause(s) of the disease. The clusterscomprised genes involved in defense/immune responses, apoptosis, zinc homeostasis, and systemic inflammation. Unsupervised hierarchical clustering partitioned patients with unknown genetic cause(s) of FHL into 2 well-distinguished subgroups. Patterns of up- and down-regulated genes separated patients with "late-onset" and "relapsing" forms of FHL from patients with an "early onset and rapidly evolving" form of the disease. A cluster was identified in patients with "late onset and relapsing" form of FHL related to B- and T-cell differentiation/survival, T-cell activation, and vesicular transport. The resulting data suggest that unique gene-expression signatures can distinguish between genetic and clinical subtypes of FHL. These differentially expressed genes may represent biomarkers that can be used as predictors of disease progression.
Asunto(s)
Linfohistiocitosis Hemofagocítica/genética , Adolescente , Edad de Inicio , Estudios de Casos y Controles , Niño , Preescolar , Estudios de Cohortes , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Humanos , Lactante , Linfohistiocitosis Hemofagocítica/clasificación , Linfohistiocitosis Hemofagocítica/inmunología , Familia de Multigenes , Mutación , Perforina , Proteínas Citotóxicas Formadoras de Poros/deficiencia , Proteínas Citotóxicas Formadoras de Poros/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Liver parenchymal cell allografts initiate both CD4-dependent and CD4-independent, CD8(+) T cell-mediated acute rejection pathways. The magnitude of allospecific CD8(+) T cell in vivo cytotoxic effector function is maximal when primed in the presence of CD4(+) T cells. The current studies were conducted to determine if and how CD4(+) T cells might influence cytotoxic effector mechanisms. METHODS: Mice were transplanted with allogeneic hepatocytes. In vivo cytotoxicity assays and various gene-deficient recipient mice and target cells were used to determine the development of Fas-, TNF-α-, and perforin-dependent cytotoxic effector mechanisms after transplantation. RESULTS: CD8(+) T cells maturing in CD4-sufficient hepatocyte recipients develop multiple (Fas-, TNF-α-, and perforin-mediated) cytotoxic mechanisms. However, CD8(+) T cells, maturing in the absence of CD4(+) T cells, mediate cytotoxicity and transplant rejection that is exclusively TNF-α/TNFR-dependent. To determine the kinetics of CD4-mediated help, CD4(+) T cells were adoptively transferred into CD4-deficient mice at various times posttransplant. The maximal influence of CD4(+) T cells on the magnitude of CD8-mediated in vivo allocytotoxicityf occurs within 48 hours. CONCLUSION: The implication of these studies is that interference of CD4(+) T cell function by disease or immunotherapy will have downstream consequences on both the magnitude of allocytotoxicity as well as the cytotoxic effector mechanisms used by allospecific CD8(+) cytolytic T cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica , Rechazo de Injerto/inmunología , Hepatocitos/trasplante , Trasplante de Hígado/inmunología , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Traslado Adoptivo , Animales , Antígenos CD4/genética , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/trasplante , Linfocitos T CD8-positivos/metabolismo , Supervivencia de Injerto , Hepatocitos/inmunología , Hepatocitos/metabolismo , Hepatocitos/patología , Trasplante de Hígado/efectos adversos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Proteínas Citotóxicas Formadoras de Poros/deficiencia , Proteínas Citotóxicas Formadoras de Poros/genética , Receptores del Factor de Necrosis Tumoral/deficiencia , Receptores del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo II del Factor de Necrosis Tumoral/deficiencia , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Transducción de Señal , Factores de Tiempo , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
The inhibitory programmed death 1 (PD-1)-programmed death ligand 1 (PD-L1) pathway contributes to the functional down-regulation of T cell responses during persistent systemic and local virus infections. The blockade of PD-1-PD-L1-mediated inhibition is considered as a therapeutic approach to reinvigorate antiviral T cell responses. Yet previous studies reported that PD-L1-deficient mice develop fatal pathology during early systemic lymphocytic choriomeningitis virus (LCMV) infection, suggesting a host protective role of T cell down-regulation. As the exact mechanisms of pathology development remained unclear, we set out to delineate in detail the underlying pathogenesis. Mice deficient in PD-1-PD-L1 signaling or lacking PD-1 signaling in CD8 T cells succumbed to fatal CD8 T cell-mediated immunopathology early after systemic LCMV infection. In the absence of regulation via PD-1, CD8 T cells killed infected vascular endothelial cells via perforin-mediated cytolysis, thereby severely compromising vascular integrity. This resulted in systemic vascular leakage and a consequential collapse of the circulatory system. Our results indicate that the PD-1-PD-L1 pathway protects the vascular system from severe CD8 T cell-mediated damage during early systemic LCMV infection, highlighting a pivotal physiological role of T cell down-regulation and suggesting the potential development of immunopathological side effects when interfering with the PD-1-PD-L1 pathway during systemic virus infections.
Asunto(s)
Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/fisiopatología , Receptor de Muerte Celular Programada 1/fisiología , Choque/fisiopatología , Animales , Antígeno B7-H1/deficiencia , Antígeno B7-H1/genética , Antígeno B7-H1/fisiología , Linfocitos T CD8-positivos/inmunología , Permeabilidad Capilar/fisiología , Modelos Animales de Enfermedad , Endotelio Vascular/patología , Antígenos H-2/genética , Antígenos H-2/fisiología , Antígeno de Histocompatibilidad H-2D , Hipotensión/etiología , Hipotensión/fisiopatología , Coriomeningitis Linfocítica/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas Citotóxicas Formadoras de Poros/deficiencia , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/fisiología , Receptor de Muerte Celular Programada 1/deficiencia , Receptor de Muerte Celular Programada 1/genética , Edema Pulmonar/etiología , Edema Pulmonar/fisiopatología , Choque/inmunología , Choque/prevención & control , Transducción de SeñalRESUMEN
Contact hypersensitivity (CHS) is a T cell response to hapten skin challenge of sensitized individuals proposed to be mediated by hapten-primed CD8 cytolytic T cells. Effector CD8 T cell recruitment into hapten challenge sites to elicit CHS requires prior CXCL1- and CXCL2-mediated neutrophil infiltration into the site. We investigated whether neutrophil activities directing hapten-primed CD8 T cell skin infiltration in response to 2,4-dinitro-1-fluorobenzene (DNFB) required Fas ligand (FasL) and perforin expression. Although DNFB sensitization of gld/perforin-/- mice induced hapten-specific CD8 T cells producing IFN-γ and IL-17, these T cells did not infiltrate the DNFB challenge site to elicit CHS but did infiltrate the challenge site and elicit CHS when transferred to hapten-challenged naive wild-type recipients. Hapten-primed wild-type CD8 T cells, however, did not elicit CHS when transferred to naive gld/perforin-/- recipients. Wild-type bone marrow neutrophils expressed FasL and perforin, and when transferred to sensitized gld/perforin-/- mice, they restored hapten-primed CD8 T cell infiltration into the challenge site and CHS. The FasL/perforin-mediated activity of wild-type neutrophils induced the expression of T cell chemoattractants, CCL1, CCL2, and CCL5, within the hapten-challenged skin. These results indicate FasL/perforin-independent functions of hapten-primed CD8 T cells in CHS and identify new functions for neutrophils in regulating effector CD8 T cell recruitment and immune responses in the skin.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Movimiento Celular/inmunología , Dermatitis por Contacto/inmunología , Dinitrofluorobenceno/administración & dosificación , Proteína Ligando Fas/genética , Neutrófilos/inmunología , Proteínas Citotóxicas Formadoras de Poros/genética , Piel/inmunología , Animales , Antígenos/administración & dosificación , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Movimiento Celular/genética , Dermatitis por Contacto/patología , Dinitrofluorobenceno/inmunología , Modelos Animales de Enfermedad , Oído Externo , Proteína Ligando Fas/biosíntesis , Femenino , Regulación de la Expresión Génica/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/biosíntesis , Proteínas Citotóxicas Formadoras de Poros/deficienciaRESUMEN
Memory CD4+ T cells combat viral infection and contribute to protective immune responses through multiple mechanisms, but how these pathways interact is unclear. We found that several pathways involving memory CD4+ T cells act together to effectively clear influenza A virus (IAV) in otherwise unprimed mice. Memory CD4+ T cell protection was enhanced through synergy with naive B cells or CD8+ T cells and maximized when both were present. However, memory CD4+ T cells protected against lower viral doses independently of other lymphocytes through production of IFN-γ. Moreover, memory CD4+ T cells selected for epitope-specific viral escape mutants via a perforin-dependent pathway. By deconstructing protective immunity mediated by memory CD4+ T cells, we demonstrated that this population simultaneously acts through multiple pathways to provide a high level of protection that ensures eradication of rapidly mutating pathogens such as IAV. This redundancy indicates the need for reductionist approaches for delineating the individual mechanisms of protection mediated by memory CD4+ T cells responding to pathogens.