RESUMO
BACKGROUND: Gender-biased outcomes are associated with acute kidney injury (AKI) and human and animal studies have shown that females are preferentially protected from renal ischemia. However, the reason for this is not known. One clue might lie with pattern recognition receptors (PRRs), which are triggers of ischemic injury when ligated by molecules in the ischemic milieu. Several PRR families are expressed by renal tubular epithelial cells (RTEs) and incite cell death signaling and production of pro-inflammatory molecules. Blockade of specific PRRs (e.g., TLR2, NOD1, NOD2, and NLRP3) provides highly significant protection from ischemic RTE injury. As a first step to understand gender-biased outcomes of AKI, we tested whether constitutive gender-based differences exist in expression of these PRRS in RTEs. METHODS: To determine whether PRR expression differences exist, primary RTEs isolated from male and female WT kidneys were examined by FACS, qPCR, and Western Blot for expression of TLR2, NOD1, NOD2, and NLRP3 inflammasome components. RESULTS: No RTE gender-based differences in TLR2, NOD1, NOD2, NLRP3, or ASC were found. RTEs from female kidneys had approximately half the mRNA, but the same protein concentration of pro-caspase-1 compared to RTEs isolated from male kidneys. CONCLUSIONS: Our findings indicate that intrinsic gender differences in RTE expression of TLR2, NOD1, NOD2, NLRP3, and ASC are not responsible for the gender-biased outcomes observed in ischemia/reperfusion injury. The lower caspase-1 mRNA expression in RTEs from females warrants further exploration of additional upstream signals that might differentially regulate caspase-1 in male vs. female RTEs.
Assuntos
Inflamassomos/metabolismo , Túbulos Renais/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Receptor 2 Toll-Like/metabolismo , Animais , Caspase 1/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Feminino , Expressão Gênica , Inflamassomos/imunologia , Interleucina-6/genética , Interleucina-6/metabolismo , Rim/imunologia , Rim/lesões , Rim/metabolismo , Túbulos Renais/citologia , Túbulos Renais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/metabolismo , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/metabolismo , Caracteres Sexuais , Receptor 2 Toll-Like/genéticaRESUMO
Nucleotide-binding oligomerization domain (Nod)-containing proteins Nod1 and Nod2 play important roles in the innate immune response to pathogenic microbes, but mounting data suggest these pattern recognition receptors might also play key roles in adaptive immune responses. Targeting Nod1 and Nod2 signaling pathways in T cells is likely to provide a new strategy to modify inflammation in a variety of disease states, particularly those that depend on Ag-induced T cell activation. To better understand how Nod1 and Nod2 proteins contribute to adaptive immunity, this study investigated their role in alloantigen-induced T cell activation and asked whether their absence might impact in vivo alloresponses using a severe acute graft versus host disease model. The study provided several important observations. We found that the simultaneous absence of Nod1 and Nod2 primed T cells for activation-induced cell death. T cells from Nod1 × 2-/- mice rapidly underwent cell death upon exposure to alloantigen. The Nod1 × 2-/- T cells had sustained p53 expression that was associated with downregulation of its negative regulator MDM2. In vivo, mice transplanted with an inoculum containing Nod1 × 2-/- T cells were protected from severe graft versus host disease. The results show that the simultaneous absence of Nod1 and Nod2 is associated with accelerated T cell death upon alloantigen encounter, suggesting these proteins might provide new targets to ameliorate T cell responses in a variety of inflammatory states, including those associated with bone marrow or solid organ transplantation.
Assuntos
Ativação Linfocitária , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Linfócitos T/imunologia , Linfócitos T/fisiologia , Imunidade Adaptativa , Animais , Morte Celular , Modelos Animais de Doenças , Regulação para Baixo , Genes p53/genética , Genes p53/imunologia , Doença Enxerto-Hospedeiro/imunologia , Imunidade Inata , Isoantígenos/imunologia , Camundongos , Proteína Adaptadora de Sinalização NOD1/deficiência , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/deficiência , Proteína Adaptadora de Sinalização NOD2/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/imunologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Receptores de Reconhecimento de Padrão/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de SinaisRESUMO
Pattern recognition receptors (PRRs) play an important role in host anti-donor responses to transplanted tissue. A key trigger of the host alloresponse involves recognition of foreign antigen presented on activated antigen presenting cells by the host T cells. Emerging data suggest that PRR blockade can abrogate host anti-donor responses by interfering with activation of antigen presenting cells, particularly activation of dendritic cells. Our study asked whether blockade of a well-characterized family of intracellular PRRs, the NOD family, interfered with alloantigen recognition and allograft rejection. We found that deletion of either NOD1 or NOD2 in antigen presenting cells (APCs) had no effect on induction of T cell proliferation to alloantigen, but that simultaneous deletion of NOD1 and NOD2 significantly inhibited T cell responses. There was however no effect of the NOD deletion on skin graft rejection when NOD1×NOD2 skin was transplanted onto allogeneic hosts or when WT skin was transplanted onto NOD1×NOD2 deficient recipients. The conclusion of this study is that in vitro alloresponses are negatively impacted by the simultaneous deletion of NOD1 and NOD2, but that allograft rejection across a stringent allo barrier is not affected. Our results suggest that the NOD family members, NOD1 and NOD2, play a collaborative role in T cell activation by alloantigen and that their blockade in vitro can inhibit T cell responses.
Assuntos
Deleção de Genes , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/genética , Aloenxertos , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Isoantígenos/imunologia , Ativação Linfocitária , Camundongos , Transplante de Pele , Linfócitos T/imunologia , Linfócitos T/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Pattern recognition receptors (PRRs) are now recognized to be key triggers of injury in a variety of renal diseases. Several families of these receptors are present in the kidney, and recent data suggest that they are differentially expressed and regulated in the kidney. This study evaluated the interaction between two distinct PRRs that are expressed in the kidney, i.e. TLR2 (Toll-like receptor 2) and the NLRP3 inflammasome. The regulation and activation of these receptors in primary renal tubular epithelial (RTE) cells from murine kidneys were evaluated. RTE cells were extracted from WT and NLRP3-mutant mice and treated ex vivo with ligands specific for TLR2 or NLRP3. We found that TLR2 upregulated NLRP3 as well as its substrate IL-1ß, and that signaling through the NLRP3 inflammasome induced RTE cell necrosis. The results of this study suggest a previously unknown interaction between TLR2 and NLRP3 in primary RTE cells and highlight the importance of the cross talk that occurs in kidney-related PRRs. Understanding how PRRs are regulated is important for the design of rationale therapeutic strategies to modulate these receptors in renal disease.
Assuntos
Proteínas de Transporte/fisiologia , Células Epiteliais/metabolismo , Inflamassomos/fisiologia , Túbulos Renais/metabolismo , Receptor 2 Toll-Like/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Apoptose , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Células Cultivadas , Interleucina-1beta/fisiologia , Túbulos Renais/citologia , Ligantes , Lipoproteínas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR , Necrose , Nigericina/farmacologia , Cultura Primária de Células , Transdução de Sinais/fisiologia , Regulação para CimaRESUMO
The site 1 protease, encoded by Mbtps1, mediates the initial cleavage of site 2 protease substrates, including sterol regulatory element binding proteins and CREB/ATF transcription factors. We demonstrate that a hypomorphic mutation of Mbtps1 called woodrat (wrt) caused hypocholesterolemia, as well as progressive hypopigmentation of the coat, that appears to be mechanistically unrelated. Hypopigmentation was rescued by transgenic expression of wild-type Mbtps1, and reciprocal grafting studies showed that normal pigmentation depended upon both cell-intrinsic or paracrine factors, as well as factors that act systemically, both of which are lacking in wrt homozygotes. Mbtps1 exhibited a maternal-zygotic effect characterized by fully penetrant embryonic lethality of maternal-zygotic wrt mutant offspring and partial embryonic lethality (~40%) of zygotic wrt mutant offspring. Mbtps1 is one of two maternal-zygotic effect genes identified in mammals to date. It functions nonredundantly in pigmentation and embryogenesis.
RESUMO
Cytoplasmic innate immune receptors are important therapeutic targets for diseases associated with overproduction of proinflammatory cytokines. One cytoplasmic receptor complex, the Nlrp3 inflammasome, responds to an extensive array of molecules associated with cellular stress. Under normal conditions, Nlrp3 is autorepressed, but in the presence of its ligands, it oligomerizes, recruits apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc), and triggers caspase 1 activation and the maturation of proinflammatory cytokines such as IL-1ß and IL-18. Because ischemic tissue injury provides a potential source for Nlrp3 ligands, our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in components of the Nlrp3 inflammasome (Nlrp3(-/-) and Asc(-/-) mice). To examine the role of the inflammasome in renal ischemia-reperfusion injury (IRI) we also tested its downstream targets caspase 1, IL-1ß, and IL-18. Both Nlrp3 and Asc were highly expressed in renal tubular epithelium of humans and mice, and the absence of Nlrp3, but not Asc or the downstream inflammasome targets, dramatically protected from kidney IRI. We conclude that Nlrp3 contributes to renal IRI by a direct effect on renal tubular epithelium and that this effect is independent of inflammasome-induced proinflammatory cytokine production.
Assuntos
Injúria Renal Aguda/metabolismo , Proteínas de Transporte/metabolismo , Células Epiteliais/metabolismo , Inflamassomos/metabolismo , Traumatismo por Reperfusão/metabolismo , Injúria Renal Aguda/imunologia , Animais , Apoptose/imunologia , Proteínas Reguladoras de Apoptose , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/imunologia , Caspase 1/imunologia , Caspase 1/metabolismo , Células Cultivadas , Citocinas/metabolismo , Proteínas do Citoesqueleto/imunologia , Proteínas do Citoesqueleto/metabolismo , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/imunologia , Humanos , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Inflamassomos/imunologia , Túbulos Renais , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Traumatismo por Reperfusão/imunologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Nucleotide-binding oligomerization domain (Nod) 1 and Nod2 are members of a family of intracellular innate sensors that participate in innate immune responses to pathogens and molecules released during the course of tissue injury, including injury induced by ischemia. Ischemic injury to the kidney is characterized by renal tubular epithelial apoptosis and inflammation. Among the best studied intracellular innate immune receptors known to contribute to apoptosis and inflammation are Nod1 and Nod2. Our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in Nod1, Nod2, Nod(1 x 2), and in their downstream signaling molecule receptor-interacting protein 2. We found that Nod1 and Nod2 were present in renal tubular epithelial cells in both mouse and human kidneys and that the absence of these receptors in mice resulted in protection from kidney ischemia reperfusion injury. Significant protection from kidney injury was seen with a deficiency of Nod2 and receptor-interacting protein 2, and the simultaneous deficiency of Nod1 and Nod2 provided even greater protection. We conclude that the intracellular sensors Nod1 and Nod2 play an important role in the pathogenesis of acute ischemic injury of the kidney, although possibly through different mechanisms.
Assuntos
Células Epiteliais/metabolismo , Perfilação da Expressão Gênica , Túbulos Renais/metabolismo , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/genética , Animais , Apoptose/genética , Apoptose/fisiologia , Transplante de Medula Óssea , Quimiocinas/metabolismo , Citocinas/metabolismo , Feminino , Humanos , Marcação In Situ das Extremidades Cortadas , Rim/irrigação sanguínea , Rim/metabolismo , Túbulos Renais/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/fisiopatologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Quimeras de Transplante/sangue , Quimeras de Transplante/genéticaRESUMO
TLRs are an evolutionarily conserved family of cell membrane proteins believed to play a significant role in innate immunity and the response to tissue injury, including that induced by ischemia. TLR signaling pathways activate transcription factors that regulate expression of prosurvival proteins, as well as proinflammatory cytokines and chemokines through one of two proximal adapter proteins, MyD88 or Toll/IL-1R domain-containing adaptor-inducing IFN-beta (Trif). Our study defines the constitutive protein expression of TLR2 in kidneys of humans and mice, and provides insight into the signaling mechanisms by which a deficiency of TLR2 protects from ischemic organ injury. Our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in TLR2, MyD88, Trif, and MyD88xTrif. TLR2 protein was evident in many cell types in the kidney, including renal tubules of the outer stripe of the medulla, glomeruli, and in the renal vasculature. The pattern of protein expression was similar in humans and mice. The absence of TLR2, MyD88, and MyD88xTrif conferred both physiologic and histologic protection against sublethal ischemia at 24 h. Interestingly, TLR2-deficient mice were better protected from ischemic renal injury than those deficient for the adapter protein MyD88, raising the intriguing possibility that TLR-2-dependent/MyD88-independent pathways also contribute to kidney injury. We conclude that TLR2 protein is constitutively expressed in the kidney and plays an important role in the pathogenesis of acute ischemic injury by signaling both MyD88-dependent and MyD88-independent pathways.