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1.
Cell ; 185(2): 283-298.e17, 2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-35021065

RESUMO

Gasdermins are a family of structurally related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while its association with genetic susceptibility to chronic mucosal inflammatory disorders is well established, little is known about its functional relevance during active disease states. Herein, we report increased GSDMB in inflammatory bowel disease, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in activated epithelial cells and organoids but instead point to increased proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display hyper-adhesiveness and enhanced formation of vinculin-based focal adhesions dependent on PDGF-A-mediated FAK phosphorylation. Importantly, carriage of disease-associated GSDMB SNPs confers functional defects, disrupting epithelial restitution/repair, which, altogether, establishes GSDMB as a critical factor for restoration of epithelial barrier function and the resolution of inflammation.


Assuntos
Células Epiteliais/metabolismo , Células Epiteliais/patologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Piroptose , Sequência de Bases , Estudos de Casos e Controles , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Células Epiteliais/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Células HEK293 , Células HT29 , Humanos , Doenças Inflamatórias Intestinais/genética , Metotrexato/farmacologia , Mutação/genética , Fosforilação/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único/genética , Piroptose/efeitos dos fármacos , Piroptose/genética , Reprodutibilidade dos Testes , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Regulação para Cima/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Cicatrização/genética
3.
Cell ; 168(5): 789-800.e10, 2017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-28235196

RESUMO

The molecular basis of the incomplete penetrance of monogenic disorders is unclear. We describe here eight related individuals with autosomal recessive TIRAP deficiency. Life-threatening staphylococcal disease occurred during childhood in the proband, but not in the other seven homozygotes. Responses to all Toll-like receptor 1/2 (TLR1/2), TLR2/6, and TLR4 agonists were impaired in the fibroblasts and leukocytes of all TIRAP-deficient individuals. However, the whole-blood response to the TLR2/6 agonist staphylococcal lipoteichoic acid (LTA) was abolished only in the index case individual, the only family member lacking LTA-specific antibodies (Abs). This defective response was reversed in the patient, but not in interleukin-1 receptor-associated kinase 4 (IRAK-4)-deficient individuals, by anti-LTA monoclonal antibody (mAb). Anti-LTA mAb also rescued the macrophage response in mice lacking TIRAP, but not TLR2 or MyD88. Thus, acquired anti-LTA Abs rescue TLR2-dependent immunity to staphylococcal LTA in individuals with inherited TIRAP deficiency, accounting for incomplete penetrance. Combined TIRAP and anti-LTA Ab deficiencies underlie staphylococcal disease in this patient.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana/deficiência , Receptores de Interleucina-1/deficiência , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/imunologia , Ácidos Teicoicos/metabolismo , Imunidade Adaptativa , Criança , Feminino , Fibroblastos/metabolismo , Humanos , Imunidade Inata , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Masculino , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/genética , Monócitos/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Linhagem , Fagócitos/metabolismo , Mutação Puntual , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Receptores de Interleucina-1/análise , Receptores de Interleucina-1/genética , Infecções Estafilocócicas/tratamento farmacológico , Ácidos Teicoicos/imunologia , Receptor 2 Toll-Like/metabolismo , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo
4.
Nat Immunol ; 19(4): 354-365, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29563620

RESUMO

Mechanisms that degrade inflammatory mRNAs are well known; however, stabilizing mechanisms are poorly understood. Here, we show that Act1, an interleukin-17 (IL-17)-receptor-complex adaptor, binds and stabilizes mRNAs encoding key inflammatory proteins. The Act1 SEFIR domain binds a stem-loop structure, the SEFIR-binding element (SBE), in the 3' untranslated region (UTR) of Cxcl1 mRNA, encoding an inflammatory chemokine. mRNA-bound Act1 directs formation of three compartmentally distinct RNA-protein complexes (RNPs) that regulate three disparate events in inflammatory-mRNA metabolism: preventing mRNA decay in the nucleus, inhibiting mRNA decapping in P bodies and promoting translation. SBE RNA aptamers decreased IL-17-mediated mRNA stabilization in vitro, IL-17-induced skin inflammation and airway inflammation in a mouse asthma model, thus providing a therapeutic strategy for autoimmune diseases. These results reveal a network in which Act1 assembles RNPs on the 3' UTRs of select mRNAs and consequently controls receptor-mediated mRNA stabilization and translation during inflammation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Inflamação/imunologia , Interleucina-17/metabolismo , Estabilidade de RNA/fisiologia , Transdução de Sinais/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Regulação da Expressão Gênica/imunologia , Inflamação/metabolismo , Interleucina-17/imunologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Receptores de Interleucina-17/metabolismo
5.
EMBO J ; 42(1): e110780, 2023 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-36373462

RESUMO

IL-1ß can exit the cytosol as an exosomal cargo following inflammasome activation in intestinal epithelial cells (IECs) in a Gasdermin D (GSDMD)-dependent manner. The mechanistic connection linking inflammasome activation and the biogenesis of exosomes has so far remained largely elusive. Here, we report the Ras GTPase-activating-like protein IQGAP1 functions as an adaptor, bridging GSDMD to the endosomal sorting complexes required for transport (ESCRT) machinery to promote the biogenesis of pro-IL-1ß-containing exosomes in response to NLPR3 inflammasome activation. We identified IQGAP1 as a GSDMD-interacting protein through a non-biased proteomic analysis. Functional investigation indicated the IQGAP1-GSDMD interaction is required for LPS and ATP-induced exosome release. Further analysis revealed that IQGAP1 serves as an adaptor which bridges GSDMD and associated IL-1ß complex to Tsg101, a component of the ESCRT complex, and enables the packaging of GSDMD and IL-1ß into exosomes. Importantly, this process is dependent on an LPS-induced increase in GTP-bound CDC42, a small GTPase known to activate IQGAP1. Taken together, this study reveals IQGAP1 as a link between inflammasome activation and GSDMD-dependent, ESCRT-mediated exosomal release of IL-1ß.


Assuntos
Exossomos , Inflamassomos , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Gasderminas , Exossomos/metabolismo , Proteínas ras/metabolismo , Lipopolissacarídeos/farmacologia , Proteômica , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Interleucina-1beta/metabolismo , Piroptose
6.
Nat Immunol ; 14(1): 72-81, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23202271

RESUMO

Act1 is an essential adaptor in interleukin 17 (IL-17)-mediated signaling and is recruited to the receptor for IL-17 after stimulation with IL-17. Here we found that Act1 was a 'client' protein of the molecular chaperone hsp90. The D10N variant of Act1 (Act1(D10N)) that is linked to susceptibility to psoriasis was defective in its interaction with hsp90, which resulted in a global loss of Act1 function. Act1-deficient mice modeled the mechanistic link between loss of Act1 function and susceptibility to psoriasis. Although Act1 was necessary for IL-17-mediated inflammation, Act1-deficient mice had a hyperactive response of the T(H)17 subset of helper T cells and developed spontaneous IL-22-dependent skin inflammation. In the absence of IL-17 signaling, IL-22 was the main contributor to skin inflammation, which provides a molecular mechanism for the association of Act1(D10N) with psoriasis susceptibility.


Assuntos
Conexina 43/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Fragmentos de Peptídeos/metabolismo , Psoríase/imunologia , Células Th17/imunologia , Animais , Linhagem Celular , Conexina 43/genética , Conexina 43/imunologia , Modelos Animais de Doenças , Predisposição Genética para Doença , Humanos , Interleucina-17/metabolismo , Camundongos , Camundongos Knockout , Chaperonas Moleculares/genética , Mutação/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Polimorfismo Genético , Ligação Proteica/genética , Ligação Proteica/imunologia , Psoríase/genética , Transdução de Sinais
7.
J Immunol ; 209(10): 1860-1869, 2022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36426949

RESUMO

IL-17A plays an important role in the pathogenesis of asthma, particularly the neutrophilic corticosteroid (CS)-resistant subtype of asthma. Clinical studies suggest that a subset of asthma patients, i.e., Th17/IL-17A-mediated (type 17) CS-resistant neutrophilic asthma, may improve with Th17/IL-17A pathway blockade. However, little is known about the mechanisms underlying type 17 asthma and CS response. In this article, we show that blood levels of lipocalin-2 (LCN2) and serum amyloid A (SAA) levels are positively correlated with IL-17A levels and are not inhibited by high-dose CS usage in asthma patients. In airway cell culture systems, IL-17A induces these two secreted proteins, and their induction is enhanced by CS. Furthermore, plasma LCN2 and SAA levels are increased in mice on a preclinical type 17 asthma model, correlated to IL-17A levels, and are not reduced by glucocorticoid (GC). In the mechanistic studies, we identify CEBPB as the critical transcription factor responsible for the synergistic induction of LCN2 and SAA by IL-17A and GC. IL-17A and GC collaboratively regulate CEBPB at both transcriptional and posttranscriptional levels. The posttranscriptional regulation of CEBPB is mediated in part by Act1, the adaptor and RNA binding protein in IL-17A signaling, which directly binds CEBPB mRNA and inhibits its degradation. Overall, our findings suggest that blood LCN2 and SAA levels may be associated with a type 17 asthma subtype and provide insight into the molecular mechanism of the IL-17A-Act1/CEBPB axis on these CS-resistant genes.


Assuntos
Asma , Interleucina-17 , Camundongos , Animais , Interleucina-17/genética , Asma/tratamento farmacológico , Asma/patologia , Células Th17/patologia , Transdução de Sinais , Glucocorticoides
8.
Nat Immunol ; 12(9): 853-60, 2011 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-21822258

RESUMO

Interleukin 17 (IL-17) promotes the expression of chemokines and cytokines via the induction of gene transcription and post-transcriptional stabilization of mRNA. We show here that IL-17 enhanced the stability of chemokine CXCL1 mRNA and other mRNAs through a pathway that involved the adaptor Act1, the adaptors TRAF2 or TRAF5 and the splicing factor SF2 (also known as alternative splicing factor (ASF)). TRAF2 and TRAF5 were necessary for IL-17 to signal the stabilization of CXCL1 mRNA. Furthermore, IL-17 promoted the formation of complexes of TRAF5-TRAF2, Act1 and SF2 (ASF). Overexpression of SF2 (ASF) shortened the half-life of CXCL1 mRNA, whereas depletion of SF2 (ASF) prolonged it. SF2 (ASF) bound chemokine mRNA in unstimulated cells, whereas the SF2 (ASF)-mRNA interaction was much lower after stimulation with IL-17. Our findings define an IL-17-induced signaling pathway that links to the stabilization of selected mRNA species through Act1, TRAF2-TRAF5 and the RNA-binding protein SF2 (ASF).


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quimiocina CXCL1/metabolismo , Inflamação/imunologia , Interleucina-17 , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/imunologia , Fator 5 Associado a Receptor de TNF/metabolismo , Células Th17/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Processamento Alternativo , Animais , Quimiocina CXCL1/genética , Quimiocina CXCL1/imunologia , Feminino , Meia-Vida , Células HeLa , Humanos , Inflamação/genética , Inflamação/metabolismo , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-17/farmacologia , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Processamento Pós-Transcricional do RNA , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Fatores de Processamento de Serina-Arginina , Fator 5 Associado a Receptor de TNF/genética , Fator 5 Associado a Receptor de TNF/imunologia , Células Th17/metabolismo , Transcrição Gênica
9.
Nat Immunol ; 12(9): 844-52, 2011 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-21822257

RESUMO

Interleukin 17 (IL-17) is critical in the pathogenesis of inflammatory and autoimmune diseases. Here we report that Act1, the key adaptor for the IL-17 receptor (IL-7R), formed a complex with the inducible kinase IKKi after stimulation with IL-17. Through the use of IKKi-deficient mice, we found that IKKi was required for IL-17-induced expression of genes encoding inflammatory molecules in primary airway epithelial cells, neutrophilia and pulmonary inflammation. IKKi deficiency abolished IL-17-induced formation of the complex of Act1 and the adaptors TRAF2 and TRAF5, activation of mitogen-activated protein kinases (MAPKs) and mRNA stability, whereas the Act1-TRAF6-transcription factor NF-κB axis was retained. IKKi was required for IL-17-induced phosphorylation of Act1 on Ser311, adjacent to a putative TRAF-binding motif. Substitution of the serine at position 311 with alanine impaired the IL-17-mediated Act1-TRAF2-TRAF5 interaction and gene expression. Thus, IKKi is a kinase newly identified as modulating IL-17 signaling through its effect on Act1 phosphorylation and consequent function.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Quimiocina CXCL1/imunologia , Quinase I-kappa B , Neutrófilos/imunologia , Pneumonia/imunologia , Transdução de Sinais/imunologia , Células Th17/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Quinase I-kappa B/deficiência , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-17/farmacologia , Pulmão , Camundongos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neutrófilos/metabolismo , Fosforilação , Pneumonia/genética , Pneumonia/metabolismo , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro , Receptores de Interleucina-17/imunologia , Fator 5 Associado a Receptor de TNF/imunologia , Fator 5 Associado a Receptor de TNF/metabolismo , Células Th17/metabolismo
10.
Immunity ; 36(5): 821-33, 2012 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-22608496

RESUMO

Interleukin-25 (IL-25 or IL-17E), a member of the structurally related IL-17 family, functions as an important mediator of T helper 2 cell-type (type 2) responses. We examined the cell type-specific role of IL-25-induced Act1-mediated signaling in protective immunity against helminth infection. Targeted Act1 deficiency in epithelial cells resulted in a marked delay in worm expulsion and abolished the expansion of the Lin(-)c-Kit(+) innate cell population in the mesenteric lymph node, lung, and liver. Th2 cell-inducing cytokine (IL-25 and IL-33) expression were reduced in the intestinal epithelial cells from the infected and IL-25-injected epithelial-specific Act1-deficient mice. Adoptive transfer of Lin(-)c-Kit(+) cells or combined injection of IL-25 and IL-33 restored the type 2 responses in these mice. Taken together, these results suggest that epithelial-specific Act1 mediates the expansion of the Lin(-)c-Kit(+) innate cell population through the positive-feedback loop of IL-25, initiating the type 2 immunity against helminth infection.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Células Epiteliais/imunologia , Helmintíase/imunologia , Helmintos/imunologia , Interleucinas/imunologia , Células Th2/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem da Célula , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Helmintíase/metabolismo , Helmintos/metabolismo , Imunidade Inata/imunologia , Interleucinas/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Fígado/citologia , Fígado/imunologia , Fígado/metabolismo , Pulmão/citologia , Pulmão/imunologia , Pulmão/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Th2/metabolismo
11.
Immunity ; 37(5): 800-12, 2012 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-23142783

RESUMO

Interleukin-1 (IL-1)-induced activation of the mTOR kinase pathway has major influences on Th17 cell survival, proliferation, and effector function. Via biochemical and genetic approaches, the kinases IKKi and GSK3α were identified as the critical intermediate signaling components for IL-1-induced AKT activation, which in turn activated mTOR. Although insulin-induced AKT activation is known to phosphorylate and inactivate GSK3α and GSK3ß, we found that GSK3α but not GSK3ß formed a constitutive complex to phosphorylate and suppress AKT activation, showing that a reverse action from GSK to AKT can take place. Upon IL-1 stimulation, IKKi was activated to mediate GSK3α phosphorylation at S21, thereby inactivating GSK3α to promote IL-1-induced AKT-mTOR activation. Thus, IKKi has a critical role in Th17 cell maintenance and/or proliferation through the GSK-AKT-mTOR pathway, implicating the potential of IKKi as a therapeutic target.


Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Quinase I-kappa B/metabolismo , Interleucina-1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Células Th17/metabolismo , Animais , Processos de Crescimento Celular/fisiologia , Ativação Enzimática , Quinase 3 da Glicogênio Sintase/imunologia , Glicogênio Sintase Quinase 3 beta , Insulina/imunologia , Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Transdução de Sinais , Serina-Treonina Quinases TOR/imunologia , Células Th17/citologia , Células Th17/enzimologia , Células Th17/imunologia
12.
J Immunol ; 202(5): 1540-1548, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30683702

RESUMO

IL-17A is a critical proinflammatory cytokine for the pathogenesis of asthma including neutrophilic pulmonary inflammation and airway hyperresponsiveness. In this study, by cell type-specific deletion of IL-17R and adaptor Act1, we demonstrated that IL-17R/Act1 exerts a direct impact on the contraction of airway smooth muscle cells (ASMCs). Mechanistically, IL-17A induced the recruitment of Rab35 (a small monomeric GTPase) and DennD1C (guanine nucleotide exchange factor [GEF]) to the IL-17R/Act1 complex in ASMCs, resulting in activation of Rab35. Rab35 knockdown showed that IL-17A-induced Rab35 activation was essential for protein kinase Cα (PKCα) activation and phosphorylation of fascin at Ser39 in ASMCs, allowing F-actin to interact with myosin to form stress fibers and enhance the contraction induced by methacholine. PKCα inhibitor or Rab35 knockdown indeed substantially reduced IL-17A-induced stress fiber formation in ASMCs and attenuated IL-17A-enhanced, methacholine-induced contraction of airway smooth muscle. Taken together, these data indicate that IL-17A promotes airway smooth muscle contraction via direct recruitment of Rab35 to IL-17R, followed by PKCα activation and stress fiber formation.


Assuntos
Interleucina-17/metabolismo , Músculo Liso/metabolismo , Proteína Quinase C-alfa/antagonistas & inibidores , Receptores de Interleucina-17/metabolismo , Fibras de Estresse/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Interleucina-17/antagonistas & inibidores , Interleucina-17/deficiência , Camundongos , Camundongos Knockout , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Proteína Quinase C-alfa/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptores de Interleucina-17/antagonistas & inibidores , Fibras de Estresse/efeitos dos fármacos , Proteínas rab de Ligação ao GTP/antagonistas & inibidores
13.
Immunity ; 32(1): 54-66, 2010 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-20060329

RESUMO

Interleukin-1 (IL-1)-mediated signaling in T cells is essential for T helper 17 (Th17) cell differentiation. We showed here that SIGIRR, a negative regulator of IL-1 receptor and Toll-like receptor signaling, was induced during Th17 cell lineage commitment and governed Th17 cell differentiation and expansion through its inhibitory effects on IL-1 signaling. The absence of SIGIRR in T cells resulted in increased Th17 cell polarization in vivo upon myelin oligodendrocyte glycoprotein (MOG(35-55)) peptide immunization. Recombinant IL-1 promoted a marked increase in the proliferation of SIGIRR-deficient T cells under an in vitro Th17 cell-polarization condition. Importantly, we detected increased IL-1-induced phosphorylation of JNK and mTOR kinase in SIGIRR-deficient Th17 cells compared to wild-type Th17 cells. IL-1-induced proliferation was abolished in mTOR-deficient Th17 cells, indicating the essential role of mTOR activation. Our results demonstrate an important mechanism by which SIGIRR controls Th17 cell expansion and effector function through the IL-1-induced mTOR signaling pathway.


Assuntos
Diferenciação Celular/imunologia , Interleucina-17/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Receptores de Interleucina-1/imunologia , Linfócitos T Auxiliares-Indutores/citologia , Animais , Linhagem da Célula/imunologia , Proliferação de Células , Separação Celular , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Ativação Enzimática/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Immunoblotting , Imunoprecipitação , Interleucina-17/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Interleucina-1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Serina-Treonina Quinases TOR , Transfecção
14.
EMBO J ; 32(4): 583-96, 2013 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-23376919

RESUMO

Toll-like receptors transduce their signals through the adaptor molecule MyD88 and members of the IL-1R-associated kinase family (IRAK-1, 2, M and 4). IRAK-1 and IRAK-2, known to form Myddosomes with MyD88-IRAK-4, mediate TLR7-induced TAK1-dependent NFκB activation. IRAK-M was previously known to function as a negative regulator that prevents the dissociation of IRAKs from MyD88, thereby inhibiting downstream signalling. However, we now found that IRAK-M was also able to interact with MyD88-IRAK-4 to form IRAK-M Myddosome to mediate TLR7-induced MEKK3-dependent second wave NFκB activation, which is uncoupled from post-transcriptional regulation. As a result, the IRAK-M-dependent pathway only induced expression of genes that are not regulated at the post-transcriptional levels (including inhibitory molecules SOCS1, SHIP1, A20 and IκBα), exerting an overall inhibitory effect on inflammatory response. On the other hand, through interaction with IRAK-2, IRAK-M inhibited TLR7-mediated production of cytokines and chemokines at translational levels. Taken together, IRAK-M mediates TLR7-induced MEKK3-dependent second wave NFκB activation to produce inhibitory molecules as a negative feedback for the pathway, while exerting inhibitory effect on translational control of cytokines and chemokines.


Assuntos
Citocinas/biossíntese , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Glicoproteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Receptores de Interleucina-1/metabolismo , Transdução de Sinais/fisiologia , Receptor 7 Toll-Like/metabolismo , Animais , Linhagem Celular , Citocinas/genética , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , MAP Quinase Quinase Quinase 3/genética , MAP Quinase Quinase Quinase 3/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , NF-kappa B/genética , Receptores de Interleucina-1/genética , Receptor 7 Toll-Like/genética
15.
Gastroenterology ; 149(7): 1860-1871.e8, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26344057

RESUMO

BACKGROUND & AIMS: Single immunoglobulin and toll-interleukin 1 receptor (SIGIRR), a negative regulator of the Toll-like and interleukin-1 receptor (IL-1R) signaling pathways, controls intestinal inflammation and suppresses colon tumorigenesis in mice. However, the importance of SIGIRR in human colorectal cancer development has not been determined. We investigated the role of SIGIRR in development of human colorectal cancer. METHODS: We performed RNA sequence analyses of pairs of colon tumor and nontumor tissues, each collected from 68 patients. Immunoblot and immunofluorescence analyses were used to determine levels of SIGIRR protein in primary human colonic epithelial cells, tumor tissues, and colon cancer cell lines. We expressed SIGIRR and mutant forms of the protein in Vaco cell lines. We created and analyzed mice that expressed full-length (control) or a mutant form of Sigirr (encoding SIGIRR(N86/102S), which is not glycosylated) specifically in the intestinal epithelium. Some mice were given azoxymethane (AOM) and dextran sulfate sodium to induce colitis-associated cancer. Intestinal tissues were collected and analyzed by immunohistochemical and gene expression profile analyses. RESULTS: RNA sequence analyses revealed increased expression of a SIGIRR mRNA isoform, SIGIRR(ΔE8), in colorectal cancer tissues compared to paired nontumor tissues. SIGIRR(ΔE8) is not modified by complex glycans and is therefore retained in the cytoplasm-it cannot localize to the cell membrane or reduce IL1R signaling. SIGIRR(ΔE8) interacts with and has a dominant-negative effect on SIGIRR, reducing its glycosylation, localization to the cell surface, and function. Most SIGIRR detected in human colon cancer tissues was cytoplasmic, whereas in nontumor tissues it was found at the cell membrane. Mice that expressed SIGIRR(N86/102S) developed more inflammation and formed larger tumors after administration of azoxymethane and dextran sulfate sodium than control mice; colon tissues from these mutant mice expressed higher levels of the inflammatory cytokines IL-17A and IL-6 had activation of the transcription factors STAT3 and NFκB. SIGIRR(N86/102S) expressed in colons of mice did not localize to the epithelial cell surface. CONCLUSION: Levels of SIGIRR are lower in human colorectal tumors, compared with nontumor tissues; tumors contain the dominant-negative isoform SIGIRR(ΔE8). This mutant protein blocks localization of full-length SIGIRR to the surface of colon epithelial cells and its ability to downregulate IL1R signaling. Expression of SIGIRR(N86/102S) in the colonic epithelium of mice increases expression of inflammatory cytokines and formation and size of colitis-associated tumors.


Assuntos
Colite/metabolismo , Colo/metabolismo , Neoplasias do Colo/metabolismo , Genes Dominantes , Mucosa Intestinal/metabolismo , Receptores de Interleucina-1/metabolismo , Animais , Azoximetano , Membrana Celular/metabolismo , Colite/induzido quimicamente , Colite/genética , Colite/patologia , Colo/patologia , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Citosol/metabolismo , Sulfato de Dextrana , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Glicosilação , Células HeLa , Humanos , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/patologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Receptores de Interleucina-1/genética , Transdução de Sinais , Técnicas de Cultura de Tecidos , Transfecção
16.
J Immunol ; 191(2): 640-9, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23772036

RESUMO

IL-17, a major inflammatory cytokine plays a critical role in the pathogenesis of many autoimmune inflammatory diseases. In this study, we report a new function of RNA-binding protein HuR in IL-17-induced Act1-mediated chemokine mRNA stabilization. HuR deficiency markedly reduced IL-17-induced chemokine expression due to increased mRNA decay. Act1-mediated HuR polyubiquitination was required for the binding of HuR to CXCL1 mRNA, leading to mRNA stabilization. Although IL-17 induced the coshift of Act1 and HuR to the polysomal fractions in a sucrose gradient, HuR deficiency reduced the ratio of translation-active/translation-inactive IL-17-induced chemokine mRNAs. Furthermore, HuR deletion in distal lung epithelium attenuated IL-17-induced neutrophilia. In summary, HuR functions to couple receptor-proximal signaling to posttranscriptional machinery, contributing to IL-17-induced inflammation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quimiocina CXCL1/genética , Quimiocina CXCL5/genética , Proteínas ELAV/metabolismo , Interleucina-17/metabolismo , Estabilidade de RNA , Animais , Linhagem Celular , Proteínas ELAV/genética , Células HeLa , Humanos , Inflamação/imunologia , Pulmão/metabolismo , Camundongos , Camundongos Knockout , Ligação Proteica , RNA Mensageiro/metabolismo , Mucosa Respiratória/metabolismo , Transdução de Sinais , Ubiquitinação
17.
J Exp Med ; 221(7)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38861022

RESUMO

The IL-17 receptor adaptor molecule Act1, an RNA-binding protein, plays a critical role in IL-17-mediated cancer progression. Here, we report a novel mechanism of how IL-17/Act1 induces chemoresistance by modulating redox homeostasis through epitranscriptomic regulation of antioxidant RNA metabolism. Transcriptome-wide mapping of direct Act1-RNA interactions revealed that Act1 binds to the 5'UTR of antioxidant mRNAs and Wilms' tumor 1-associating protein (WTAP), a key regulator in m6A methyltransferase complex. Strikingly, Act1's binding sites are located in proximity to m6A modification sites, which allows Act1 to promote the recruitment of elF3G for cap-independent translation. Loss of Act1's RNA binding activity or Wtap knockdown abolished IL-17-induced m6A modification and translation of Wtap and antioxidant mRNAs, indicating a feedforward mechanism of the Act1-WTAP loop. We then developed antisense oligonucleotides (Wtap ASO) that specifically disrupt Act1's binding to Wtap mRNA, abolishing IL-17/Act1-WTAP-mediated antioxidant protein production during chemotherapy. Wtap ASO substantially increased the antitumor efficacy of cisplatin, demonstrating a potential therapeutic strategy for chemoresistance.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Antioxidantes , Resistencia a Medicamentos Antineoplásicos , Homeostase , Oxirredução , RNA Mensageiro , Animais , Humanos , Camundongos , Regiões 5' não Traduzidas , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interleucina-17/metabolismo , Fatores de Processamento de RNA , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética
18.
Sci Immunol ; 9(95): eabq1558, 2024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38701190

RESUMO

Steroid resistance poses a major challenge for the management of autoimmune neuroinflammation. T helper 17 (TH17) cells are widely implicated in the pathology of steroid resistance; however, the underlying mechanisms are unknown. In this study, we identified that interleukin-1 receptor (IL-1R) blockade rendered experimental autoimmune encephalomyelitis (EAE) mice sensitive to dexamethasone (Dex) treatment. Interleukin-1ß (IL-1ß) induced a signal transducer and activator of transcription 5 (STAT5)-mediated steroid-resistant transcriptional program in TH17 cells, which promoted inflammatory cytokine production and suppressed Dex-induced anti-inflammatory genes. TH17-specific deletion of STAT5 ablated the IL-1ß-induced steroid-resistant transcriptional program and rendered EAE mice sensitive to Dex treatment. IL-1ß synergized with Dex to promote the STAT5-dependent expression of CD69 and the development of central nervous system (CNS)-resident CD69+ TH17 cells. Combined IL-1R blockade and Dex treatment ablated CNS-resident TH17 cells, reduced EAE severity, and prevented relapse. CD69+ tissue-resident TH17 cells were also detected in brain lesions of patients with multiple sclerosis. These findings (i) demonstrate that IL-1ß-STAT5 signaling in TH17 cells mediates steroid resistance and (ii) identify a therapeutic strategy for reversing steroid resistance in TH17-mediated CNS autoimmunity.


Assuntos
Dexametasona , Encefalomielite Autoimune Experimental , Interleucina-1beta , Fator de Transcrição STAT5 , Células Th17 , Animais , Células Th17/imunologia , Fator de Transcrição STAT5/metabolismo , Fator de Transcrição STAT5/imunologia , Camundongos , Interleucina-1beta/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Camundongos Endogâmicos C57BL , Resistência a Medicamentos , Transdução de Sinais/imunologia , Camundongos Knockout , Doenças Neuroinflamatórias/imunologia , Doenças Neuroinflamatórias/tratamento farmacológico , Feminino , Humanos
19.
J Exp Med ; 204(5): 1025-36, 2007 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-17470642

RESUMO

IRAK4 is a member of IL-1 receptor (IL-1R)-associated kinase (IRAK) family and has been shown to play an essential role in Toll-like receptor (TLR)-mediated signaling. We recently generated IRAK4 kinase-inactive knock-in mice to examine the role of kinase activity of IRAK4 in TLR-mediated signaling pathways. The IRAK4 kinase-inactive knock-in mice were completely resistant to lipopolysaccharide (LPS)- and CpG-induced shock, due to impaired TLR-mediated induction of proinflammatory cytokines and chemokines. Although inactivation of IRAK4 kinase activity did not affect the levels of TLR/IL-1R-mediated nuclear factor kappaB activation, a reduction of LPS-, R848-, and IL-1-mediated mRNA stability contributed to the reduced cytokine and chemokine production in bone marrow-derived macrophages from IRAK4 kinase-inactive knock-in mice. Both TLR7- and TLR9-mediated type I interferon production was abolished in plasmacytoid dendritic cells isolated from IRAK4 knock-in mice. In addition, influenza virus-induced production of interferons in plasmacytoid DCs was also dependent on IRAK4 kinase activity. Collectively, our results indicate that IRAK4 kinase activity plays a critical role in TLR-dependent immune responses.


Assuntos
Imunidade Inata/imunologia , Quinases Associadas a Receptores de Interleucina-1/imunologia , Transdução de Sinais/imunologia , Receptores Toll-Like/imunologia , Animais , Northern Blotting , Western Blotting , Citocinas/metabolismo , Primers do DNA , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Vírus da Influenza A/imunologia , Quinases Associadas a Receptores de Interleucina-1/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
20.
J Immunol ; 187(6): 3155-64, 2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21856933

RESUMO

The cellular and molecular mechanisms driven by IL-25 and its cognate receptor IL-17RB necessary for the promotion of Th2-mediating pathogenic pulmonary inflammation remains to be defined. We have previously reported the critical role of the U-box-type E3 ubiquitin ligase Act1 (1) for the downstream signaling of the IL-17 cytokine family including the Th2-promoting cytokine IL-25 (IL-17E) (2). In this study, we report that IL-25-driven but not conventional IL-4-driven Th2 polarization and cytokine production is impaired in Act1-deficient T cells. Also, Act1 deficiency in the T cell compartment results in the abrogation of eosinophilic airway infiltration as well as airway hyperresponsiveness in mouse models of Ag-induced airway inflammation. The in vivo generation of Ag-specific Th2 cytokine-producing cells is defective in the absence of Act1 expression in T cells after OVA/aluminum hydroxide immunization. Notably, the production of OVA-specific IgG(1) but not IgG(2a) or IgE is also impaired. At the molecular level, we report that IL-25-mediated induction of Th2 master regulator GATA-3 and the transcription factor GFI-1 is attenuated in Act1-deficient T cells. Taken together, our findings indicate that Act1 expression in T cells is required for cellular and humoral Th2-mediated allergic responses and the development of airway hyperresponsiveness, in part, through Act1's function in IL-25-induced development of Th2 T cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Hipersensibilidade/imunologia , Interleucinas/imunologia , Pneumonia/imunologia , Células Th2/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Hipersensibilidade/metabolismo , Imuno-Histoquímica , Interleucinas/metabolismo , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Pneumonia/metabolismo , Transdução de Sinais/imunologia , Células Th2/metabolismo
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