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1.
Nat Immunol ; 25(7): 1183-1192, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38872000

RESUMO

Natural killer (NK) cells function by eliminating virus-infected or tumor cells. Here we identified an NK-lineage-biased progenitor population, referred to as early NK progenitors (ENKPs), which developed into NK cells independently of common precursors for innate lymphoid cells (ILCPs). ENKP-derived NK cells (ENKP_NK cells) and ILCP-derived NK cells (ILCP_NK cells) were transcriptionally different. We devised combinations of surface markers that identified highly enriched ENKP_NK and ILCP_NK cell populations in wild-type mice. Furthermore, Ly49H+ NK cells that responded to mouse cytomegalovirus infection primarily developed from ENKPs, whereas ILCP_NK cells were better IFNγ producers after infection with Salmonella and herpes simplex virus. Human CD56dim and CD56bright NK cells were transcriptionally similar to ENKP_NK cells and ILCP_NK cells, respectively. Our findings establish the existence of two pathways of NK cell development that generate functionally distinct NK cell subsets in mice and further suggest these pathways may be conserved in humans.


Assuntos
Diferenciação Celular , Células Matadoras Naturais , Células Matadoras Naturais/imunologia , Animais , Camundongos , Humanos , Diferenciação Celular/imunologia , Camundongos Endogâmicos C57BL , Imunidade Inata , Antígeno CD56/metabolismo , Muromegalovirus/imunologia , Linhagem da Célula/imunologia , Interferon gama/metabolismo , Interferon gama/imunologia , Células Progenitoras Linfoides/metabolismo , Células Progenitoras Linfoides/citologia , Células Progenitoras Linfoides/imunologia , Camundongos Knockout , Células Cultivadas
2.
Immunity ; 57(5): 1019-1036.e9, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38677292

RESUMO

Group 3 innate lymphoid cells (ILC3) are the major subset of gut-resident ILC with essential roles in infections and tissue repair, but how they adapt to the gut environment to maintain tissue residency is unclear. We report that Tox2 is critical for gut ILC3 maintenance and function. Gut ILC3 highly expressed Tox2, and depletion of Tox2 markedly decreased ILC3 in gut but not at central sites, resulting in defective control of Citrobacter rodentium infection. Single-cell transcriptional profiling revealed decreased expression of Hexokinase-2 in Tox2-deficient gut ILC3. Consistent with the requirement for hexokinases in glycolysis, Tox2-/- ILC3 displayed decreased ability to utilize glycolysis for protein translation. Ectopic expression of Hexokinase-2 rescued Tox2-/- gut ILC3 defects. Hypoxia and interleukin (IL)-17A each induced Tox2 expression in ILC3, suggesting a mechanism by which ILC3 adjusts to fluctuating environments by programming glycolytic metabolism. Our results reveal the requirement for Tox2 to support the metabolic adaptation of ILC3 within the gastrointestinal tract.


Assuntos
Citrobacter rodentium , Infecções por Enterobacteriaceae , Glicólise , Proteínas HMGB , Imunidade Inata , Linfócitos , Camundongos Knockout , Animais , Camundongos , Adaptação Fisiológica/imunologia , Citrobacter rodentium/imunologia , Infecções por Enterobacteriaceae/imunologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/metabolismo , Hexoquinase/metabolismo , Hexoquinase/genética , Interleucina-17/metabolismo , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos Endogâmicos C57BL , Transativadores/metabolismo , Transativadores/genética , Proteínas HMGB/genética , Proteínas HMGB/imunologia , Proteínas HMGB/metabolismo
4.
Nature ; 626(8001): 1102-1107, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38355795

RESUMO

Plasma cells produce large quantities of antibodies and so play essential roles in immune protection1. Plasma cells, including a long-lived subset, reside in the bone marrow where they depend on poorly defined microenvironment-linked survival signals1. We show that bone marrow plasma cells use the ligand-gated purinergic ion channel P2RX4 to sense extracellular ATP released by bone marrow osteoblasts through the gap-junction protein pannexin 3 (PANX3). Mutation of Panx3 or P2rx4 each caused decreased serum antibodies and selective loss of bone marrow plasma cells. Compared to their wild-type counterparts, PANX3-null osteoblasts secreted less extracellular ATP and failed to support plasma cells in vitro. The P2RX4-specific inhibitor 5-BDBD abrogated the impact of extracellular ATP on bone marrow plasma cells in vitro, depleted bone marrow plasma cells in vivo and reduced pre-induced antigen-specific serum antibody titre with little posttreatment rebound. P2RX4 blockade also reduced autoantibody titre and kidney disease in two mouse models of humoral autoimmunity. P2RX4 promotes plasma cell survival by regulating endoplasmic reticulum homeostasis, as short-term P2RX4 blockade caused accumulation of endoplasmic reticulum stress-associated regulatory proteins including ATF4 and B-lineage mutation of the pro-apoptotic ATF4 target Chop prevented bone marrow plasma cell demise on P2RX4 inhibition. Thus, generating mature protective and pathogenic plasma cells requires P2RX4 signalling controlled by PANX3-regulated extracellular ATP release from bone marrow niche cells.


Assuntos
Trifosfato de Adenosina , Células da Medula Óssea , Plasmócitos , Animais , Camundongos , Trifosfato de Adenosina/metabolismo , Autoanticorpos/imunologia , Autoimunidade/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Linhagem da Célula , Conexinas/genética , Conexinas/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Mutação , Osteoblastos/metabolismo , Plasmócitos/citologia , Plasmócitos/imunologia , Plasmócitos/metabolismo , Receptores Purinérgicos P2X4/metabolismo , Transdução de Sinais
5.
J Immunol ; 203(3): 686-695, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31243087

RESUMO

The thymus is critical for the establishment of the adaptive immune system and the development of a diverse T cell repertoire. T cell development depends upon cell-cell interactions with epithelial cells in the thymus. The thymus is composed of two different types of epithelial cells: cortical and medullary epithelial cells. Both of these express and critically depend on the transcription factor Foxn1 Foxn1 is also expressed in the hair follicle, and disruption of Foxn1 function in mice results in severe thymic developmental defects and the hairless (nude) phenotype. Despite its importance, little is known about the direct regulation of Foxn1 expression. In this study, we identify a cis-regulatory element (RE) critical for expression of Foxn1 in mouse thymic epithelial cells but dispensable for expression in hair follicles. Analysis of chromatin accessibility, histone modifications, and sequence conservation identified regions within the first intron of Foxn1 that possessed the characteristics of REs. Systematic knockout of candidate regions lead us to identify a 1.6 kb region that, when deleted, results in a near total disruption of thymus development. Interestingly, Foxn1 expression and function in the hair follicle were unaffected. RNA fluorescent in situ hybridization showed a near complete loss of Foxn1 mRNA expression in the embryonic thymic bud. Our studies have identified a genomic RE with thymic-specific control of Foxn1 gene expression.


Assuntos
Células Epiteliais/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Elementos Reguladores de Transcrição/genética , Linfócitos T/imunologia , Timo/metabolismo , Animais , Fatores de Transcrição Forkhead/biossíntese , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Folículo Piloso/metabolismo , Camundongos , Camundongos Knockout , Camundongos Nus , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Linfócitos T/citologia , Timo/citologia
6.
PLoS Biol ; 14(6): e1002502, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27337557

RESUMO

[This corrects the article DOI: 10.1371/journal.pbio.1000518.].

7.
J Autoimmun ; 90: 39-48, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29439835

RESUMO

Dendritic cell (DC)-mediated T cell tolerance deficiencies contribute to the pathogenesis of autoimmune diseases such as type 1 diabetes. Delivering self-antigen to dendritic-cell inhibitory receptor-2 (DCIR2)+ DCs can delay but not completely block diabetes development in NOD mice. These DCIR2-targeting antibodies induce tolerance via deletion and anergy, but do not increase islet-specific Tregs. Because low-dose IL-2 (LD-IL-2) administration can preferentially expand Tregs, we tested whether delivering islet-antigen to tolerogenic DCIR2+ DCs along with LD-IL-2 would boost islet-specific Tregs and further block autoimmunity. But, surprisingly, adding LD-IL-2 did not increase efficacy of DC-targeted antigen to inhibit diabetes. Here we show the effects of LD-IL-2, with or without antigen delivery to DCIR2+ DCs, on both polyclonal and autoreactive Treg and conventional T cells (Tconv). As expected, LD-IL-2 increased total Tregs, but autoreactive Tregs required both antigen and IL-2 stimulation for optimal expansion. Also, islet-specific Tregs had lower CD25 expression and IL-2 sensitivity, while islet-specific Tconv had higher CD25 expression, compared to polyclonal populations. LD-IL-2 increased activation and expansion of Tconv, and was more pronounced for autoreactive cells after treatment with IL-2 + islet-antigen. Therefore, LD-IL-2 therapy, especially when combined with antigen stimulation, may not optimally activate and expand antigen-specific Tregs in chronic autoimmune settings.


Assuntos
Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Interleucina-2/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Apresentação de Antígeno , Autoantígenos/imunologia , Autoimunidade , Células Cultivadas , Feminino , Humanos , Tolerância Imunológica , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Receptores de Superfície Celular/metabolismo
8.
J Immunol ; 196(5): 2031-40, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26826238

RESUMO

Innate immune signals help break self-tolerance to initiate autoimmune diseases such as type 1 diabetes, but innate contributions to subsequent regulation of disease progression are less clear. Most studies have measured in vitro innate responses of GM-CSF dendritic cells (DCs) that are functionally distinct from conventional DCs (cDCs) and do not reflect in vivo DC subsets. To determine whether autoimmune NOD mice have alterations in type 1 IFN innate responsiveness, we compared cDCs from prediabetic NOD and control C57BL/6 (B6) mice stimulated in vivo with the TLR9 ligand CpG, a strong type 1 IFN inducer. In response to CpG, NOD mice produce more type 1 IFN and express higher levels of CD40, and NOD monocyte DCs make more TNF. However, the overall CpG-induced transcriptional response is muted in NOD cDCs. Of relevance the costimulatory proteins CD80/CD86, signals needed for regulatory T cell homeostasis, are upregulated less on NOD cDCs. Interestingly, NOD Rag1(-/-) mice also display a defect in CpG-induced CD86 upregulation compared with B6 Rag1(-/-), indicating this particular innate alteration precedes adaptive autoimmunity. The impaired response in NOD DCs is likely downstream of the IFN-α/ß receptor because DCs from NOD and B6 mice show similar CpG-induced CD86 levels when anti-IFN-α/ß receptor Ab is added. IFN-α-induced nuclear localization of activated STAT1 is markedly reduced in NOD CD11c(+) cells, consistent with lower type 1 IFN responsiveness. In conclusion, NOD DCs display altered innate responses characterized by enhanced type 1 IFN and activation of monocyte-derived DCs but diminished cDC type 1 IFN response.


Assuntos
Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/imunologia , Imunidade Inata/imunologia , Interferon Tipo I/imunologia , Fator de Transcrição STAT1/imunologia , Tolerância a Antígenos Próprios/imunologia , Transporte Ativo do Núcleo Celular , Animais , Western Blotting , Linhagem da Célula , Núcleo Celular/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Microscopia Confocal , Monócitos/citologia , Monócitos/imunologia , Oligodesoxirribonucleotídeos/imunologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Fator de Transcrição STAT1/metabolismo , Receptor Toll-Like 9/agonistas
9.
Proc Natl Acad Sci U S A ; 111(38): 13930-5, 2014 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-25201955

RESUMO

Novel inhibitor of histone acetyltransferase repressor (NIR) is a transcriptional corepressor with inhibitor of histone acetyltransferase activity and is a potent suppressor of p53. Although NIR deficiency in mice leads to early embryonic lethality, lymphoid-restricted deletion resulted in the absence of double-positive CD4(+)CD8(+) thymocytes, whereas bone-marrow-derived B cells were arrested at the B220(+)CD19(-) pro-B-cell stage. V(D)J recombination was preserved in NIR-deficient DN3 double-negative thymocytes, suggesting that NIR does not affect p53 function in response to physiologic DNA breaks. Nevertheless, the combined deficiency of NIR and p53 provided rescue of DN3L double-negative thymocytes and their further differentiation to double- and single-positive thymocytes, whereas B cells in the marrow further developed to the B220(+)CD19(+) pro-B-cell stage. Our results show that NIR cooperate with p53 to impose checkpoint for the generation of mature B and T lymphocytes.


Assuntos
Diferenciação Celular/imunologia , Proteínas Repressoras/imunologia , Timócitos/imunologia , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Diferenciação Celular/genética , Quebras de DNA , Camundongos , Células Precursoras de Linfócitos B/citologia , Proteínas Repressoras/genética , Timócitos/citologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia
10.
J Biol Chem ; 290(36): 22076-84, 2015 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-26224629

RESUMO

NF-κB essential modulator (NEMO) and cylindromatosis protein (CYLD) are intracellular proteins that regulate the NF-κB signaling pathway. Although mice with either CYLD deficiency or an alteration in the zinc finger domain of NEMO (K392R) are born healthy, we found that the combination of these two gene defects in double mutant (DM) mice is early embryonic lethal but can be rescued by the absence of TNF receptor 1 (TNFR1). Notably, NEMO was not recruited into the TNFR1 complex of DM cells, and consequently NF-κB induction by TNF was severely impaired and DM cells were sensitized to TNF-induced cell death. Interestingly, the TNF signaling defects can be fully rescued by reconstitution of DM cells with CYLD lacking ubiquitin hydrolase activity but not with CYLD mutated in TNF receptor-associated factor 2 (TRAF2) or NEMO binding sites. Therefore, our data demonstrate an unexpected non-catalytic function for CYLD as an adapter protein between TRAF2 and the NEMO zinc finger that is important for TNF-induced NF-κB signaling during embryogenesis.


Assuntos
Desenvolvimento Embrionário/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Proteínas Supressoras de Tumor/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/metabolismo , Ligação Proteica , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Dedos de Zinco/genética
11.
Proc Natl Acad Sci U S A ; 110(13): 5127-32, 2013 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-23493554

RESUMO

Mutations in the TNF family of proteins have been associated with inherited forms of immune deficiency. Using an array-based sequencing assay, we identified an autosomal-dominant deficiency in TNF-like weak inducer of apoptosis (TWEAK; TNFSF12) in a kindred with recurrent infection and impaired antibody responses to protein and polysaccharide vaccines. This mutation occurs in the sixth exon of TWEAK and results in the amino acid substitution R145C within the conserved TNF-homology domain of the full-length protein. TWEAK mutant protein formed high molecular weight aggregates under nonreducing conditions, suggesting an increased propensity for intermolecular interactions. As a result, mutant TWEAK associated with B-cell-activating factor (BAFF) protein and down-regulated the BAFF-mediated activation of the noncanonical NF-κB pathway through inhibition of p100 processing to p52, resulting in inhibition of BAFF-dependent B-cell survival and proliferation. As BAFF mediates T-cell-independent isotype switching and B-cell survival, our data implicate TWEAK as a disease-susceptibility gene for a humoral immunodeficiency.


Assuntos
Linfócitos B/imunologia , Doenças Genéticas Inatas/imunologia , Predisposição Genética para Doença , Síndromes de Imunodeficiência/imunologia , Mutação de Sentido Incorreto , Fatores de Necrose Tumoral/imunologia , Adulto , Substituição de Aminoácidos , Fator Ativador de Células B/genética , Fator Ativador de Células B/imunologia , Linfócitos B/patologia , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Criança , Pré-Escolar , Citocina TWEAK , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Feminino , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/patologia , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/patologia , Masculino , Subunidade p52 de NF-kappa B/genética , Subunidade p52 de NF-kappa B/imunologia , Fatores de Necrose Tumoral/genética
12.
Immunology ; 143(4): 640-50, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24954893

RESUMO

We have previously shown that interleukin-2 (IL-2) inhibits dendritic cell (DC) development from mouse bone marrow (BM) precursors stimulated with the ligand for FMS-like tyrosine kinase 3 receptor (Flt3L), and have provided evidence that this inhibition occurs at the monocyte DC precursor stage of DC development. Here, we explored the mechanism of IL-2-mediated inhibition of DC development. First, we showed that these in vitro cultures accurately model DCs that develop in vivo by comparing gene and protein expression of the three main Flt3L-induced DC subsets from the BM, CD11b(+) and CD24(+) conventional DCs (cDCs) and plasmacytoid DCs (pDCs) with their respective ex vivo spleen DC subsets (CD11b(+), CD8(+) and pDCs). Next, gene expression changes were quantified in Flt3L DC subsets that developed in the presence of IL-2. These changes included increased expression of Bcl2l11, which encodes the apoptosis-inducing protein Bim, and decreased expression of Flt3 (CD135), the receptor that initiates DC development. Interleukin-2 also significantly reduced Flt3 protein expression on all three Flt3L DC subsets, and attenuated Flt3L-induced STAT3 phosphorylation in DCs. Based on these data, we hypothesized that decreased Flt3 signalling may divert BM precursors down monocyte and macrophage lineages. Indeed, addition of IL-2 led to increases in Flt3(-) cells, including cKit(+) Ly6C(+) CD11b(-) populations consistent with the recently identified committed monocyte/macrophage progenitor. Therefore, IL-2 can inhibit DC development via decreased signalling through Flt3 and increased monocyte/macrophage development.


Assuntos
Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Interleucina-2/farmacologia , Macrófagos/metabolismo , Monócitos/metabolismo , Células Progenitoras Mieloides/metabolismo , Tirosina Quinase 3 Semelhante a fms/genética , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Citocinas/genética , Células Dendríticas/imunologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição STAT3/metabolismo , Tirosina Quinase 3 Semelhante a fms/metabolismo
13.
PLoS Biol ; 8(10): e1000518, 2010 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-21048983

RESUMO

Chromosomal translocations between loci encoding MALT1 and c-IAP2 are common in MALT lymphomas. The resulting fusion proteins lack the c-IAP2 RING domain, the region responsible for its ubiquitin protein ligase (E3) activity. Ectopic expression of the fusion protein activates the canonical NF-κB signaling cascade, but how it does so is controversial and how it promotes MALT lymphoma is unknown. Considering recent reports implicating c-IAP1 and c-IAP2 E3 activity in repression of non-canonical NF-κB signaling, we asked if the c-IAP2/MALT fusion protein can initiate non-canonical NF-κB activation. Here we show that in addition to canonical activation, the fusion protein stabilizes NIK and activates non-canonical NF-κB. Canonical but not non-canonical activation depended on MALT1 paracaspase activity, and expression of E3-inactive c-IAP2 activated non-canonical NF-κB. Mice in which endogenous c-IAP2 was replaced with an E3-inactive mutant accumulated abnormal B cells with elevated non-canonical NF-κB and had increased numbers of B cells with a marginal zone phenotype, gut-associated lymphoid hyperplasia, and other features of MALT lymphoma. Thus, the c-IAP2/MALT1 fusion protein activates NF-κB by two distinct mechanisms, and loss of c-IAP2 E3 activity in vivo is sufficient to induce abnormalities common to MALT lymphoma.


Assuntos
Linfócitos B/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , NF-kappa B/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linfócitos B/citologia , Proliferação de Células , Sobrevivência Celular , Técnicas de Introdução de Genes , Humanos , Proteínas Inibidoras de Apoptose/genética , Linfoma de Zona Marginal Tipo Células B/genética , Linfoma de Zona Marginal Tipo Células B/metabolismo , Camundongos , Camundongos Transgênicos , NF-kappa B/genética , Proteínas Recombinantes de Fusão/genética , Translocação Genética , Ubiquitina-Proteína Ligases/genética
14.
Proc Natl Acad Sci U S A ; 107(18): 8340-5, 2010 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-20404153

RESUMO

IL-12 and IL-23 are produced by activated antigen-presenting cells but the two induce distinct immune responses by promoting Th1 and Th17 cell differentiation, respectively. IL-23 is a heterodimeric cytokine consisting of two subunits: p40 that is shared with IL-12 and p19 unique to IL-23. In this study, we showed that the production of IL-23 but not IL-12 was negatively regulated by protein phosphatase 2A (PP2A) in dendritic cells (DC). PP2A inhibits IL-23 production by suppressing the expression of the IL-23p19 gene. Treating DC with okadaic acid that inhibits the PP2A activity or knocking down the catalytic subunit of PP2A with siRNA enhanced IL-23 but not IL-12 production. Unlike PP2A, MAP kinase phosphatase-1 or CYLD did not show an effect on IL-23 production supporting the specificity of PP2A. PP2A-mediated inhibition requires a newly made protein that is likely responsible for bringing PP2A and IKKbeta together upon LPS stimulation, which then results in the termination of IKK phosphorylation. Thus, our results uncovered an important role of the protein phosphatase in the regulation of IL-23 production and identified PP2A as a previously uncharacterized inhibitor of IL-23p19 expression in DC.


Assuntos
Células Dendríticas/imunologia , Regulação para Baixo , Subunidade p19 da Interleucina-23/imunologia , Proteína Fosfatase 2/metabolismo , Animais , Células Cultivadas , Células Dendríticas/metabolismo , Fosfatase 1 de Especificidade Dupla/deficiência , Fosfatase 1 de Especificidade Dupla/metabolismo , Quinase I-kappa B/metabolismo , Interleucina-12/biossíntese , Interleucina-12/imunologia , Subunidade p19 da Interleucina-23/biossíntese , Subunidade p19 da Interleucina-23/genética , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fosforilação , Ligação Proteica , Proteína Fosfatase 2/genética , RNA Interferente Pequeno/genética
15.
Sci Adv ; 9(46): eadg8126, 2023 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-37967174

RESUMO

Thymic epithelial cells (TEC) control T cell development and play essential roles in establishing self-tolerance. By using Foxn1-Cre-driven ablation of Klf6 gene in TEC, we identified Klf6 as a critical factor in TEC development. Klf6 deficiency resulted in a hypoplastic thymus-evident from fetal stages into adulthood-in which a dramatic increase in the frequency of apoptotic TEC was observed. Among cortical TEC (cTEC), a previously unreported cTEC population expressing the transcription factor Sox10 was relatively expanded. Within medullary TEC (mTEC), mTEC I and Tuft-like mTEC IV were disproportionately decreased. Klf6 deficiency altered chromatin accessibility and affected TEC chromatin configuration. Consistent with these defects, naïve conventional T cells and invariant natural killer T cells were reduced in the spleen. Late stages of T cell receptor-dependent selection of thymocytes were affected, and mice exhibited autoimmunity. Thus, Klf6 has a prosurvival role and affects the development of specific TEC subsets contributing to thymic function.


Assuntos
Regulação da Expressão Gênica , Timócitos , Animais , Camundongos , Diferenciação Celular/genética , Cromatina/metabolismo , Células Epiteliais/metabolismo , Camundongos Endogâmicos C57BL , Timócitos/metabolismo , Timo/metabolismo
16.
J Biol Chem ; 286(3): 2236-44, 2011 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-20959462

RESUMO

NIR (novel INHAT repressor) is a transcriptional co-repressor with inhibitor of histone acetyltransferase (INHAT) activity and has previously been shown to physically interact with and suppress p53 transcriptional activity and function. However, the mechanism by which NIR suppresses p53 is not completely understood. Using a proteomic approach, we have identified the Aurora kinase B as a novel binding partner of NIR. We show that Aurora B, NIR and p53 exist in a protein complex in which Aurora B binds to NIR, thus also indirectly associates with p53. Functionally, overexpression of Aurora B or NIR suppresses p53 transcriptional activity, and depletion of Aurora B or NIR causes p53-dependent apoptosis and cell growth arrest, due to the up-regulation of p21 and Bax. We then demonstrate that Aurora B phosphorylates multiple sites in the p53 DNA-binding domain in vitro, and this phosphorylation probably also occurs in cells. Importantly, the Aurora B-mediated phosphorylation on Ser(269) or Thr(284) significantly compromises p53 transcriptional activity. Taken together, these results provide novel insight into NIR-mediated p53 suppression and also suggest an additional way for p53 regulation.


Assuntos
Apoptose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Transcrição Gênica/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Aurora Quinase B , Aurora Quinases , Deleção de Genes , Células HEK293 , Humanos , Fosforilação/fisiologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Proteína Supressora de Tumor p53/genética , Regulação para Cima/fisiologia , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
17.
J Biol Chem ; 286(47): 40520-30, 2011 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-21931165

RESUMO

CYLD is a lysine 63-deubiquitinating enzyme that inhibits NF-κB and JNK signaling. Here, we show that CYLD knock-out mice have markedly increased numbers of regulatory T cells (Tregs) in peripheral lymphoid organs but not in the thymus. In vitro stimulation of CYLD-deficient naive T cells with anti-CD3/28 in the presence of TGF-ß led to a marked increase in the number of Foxp3-expressing T cells when compared with stimulated naive control CD4(+) cells. Under endogenous conditions, CYLD formed a complex with Smad7 that facilitated CYLD deubiquitination of Smad7 at lysine 360 and 374 residues. Moreover, this site-specific ubiquitination of Smad7 was required for activation of TAK1 and p38 kinases. Finally, knockdown of Smad7 or inhibition of p38 activity in primary T cells impaired Treg differentiation. Together, our results show that CYLD regulates TGF-ß signaling function in T cells and the development of Tregs through deubiquitination of Smad7.


Assuntos
Cisteína Endopeptidases/metabolismo , Transdução de Sinais , Proteína Smad7/metabolismo , Linfócitos T Reguladores/citologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Cisteína Endopeptidases/deficiência , Cisteína Endopeptidases/genética , Enzima Desubiquitinante CYLD , Fatores de Transcrição Forkhead/genética , Técnicas de Inativação de Genes , Células HeLa , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Linfonodos/imunologia , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Ubiquitinação/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
18.
Hum Mutat ; 32(3): 318-24, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21309033

RESUMO

The covalent attachment of lysine 63-linked polyubiquitin to the zinc-finger domain of IKBKG/NEMO (also known as IKKγ) is necessary for full activation of NF-κB. Impairments of this biochemical mechanism explain the deleterious effects of hypomorphic NEMO mutations on NF-κB signaling function in humans suffering from X-linked ectodermal dysplasia and immunodeficiency. Nevertheless, the biological function of the NEMO zinc-finger domain in the regulation of mitogen-activated protein kinase (MAPK) activity is poorly understood. Here we show that dendritic cells from patients with EDI caused by a C-terminal E391X deletion of the zinc finger of NEMO exhibit impaired MAPK activation in response to lipopolysaccharide (LPS) stimulation. Interestingly, DCs from patients with a C417R missense mutation within the zinc finger domain of NEMO in which ubiquitination of NEMO is preserved are also defective in JNK and ERK activity following LPS stimulation. Our findings indicate that the structural integrity of the NEMO ZF domain is more important than its polyubiquitination for full activation of the MAPK. Furthermore, phosphorylation and polyubiquitination of upstream TAK1 were significantly reduced in the E391X zinc-finger deleted patients, indicating that the NEMO zinc finger may play an important role in assembling the proximal signaling complex for MAPK activation.


Assuntos
Células Dendríticas/metabolismo , Displasia Ectodérmica/enzimologia , Displasia Ectodérmica/genética , Quinase I-kappa B/genética , Síndromes de Imunodeficiência/enzimologia , Síndromes de Imunodeficiência/genética , Sistema de Sinalização das MAP Quinases/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células Cultivadas , Citocinas/biossíntese , Displasia Ectodérmica/imunologia , Doenças Genéticas Ligadas ao Cromossomo X , Humanos , Quinase I-kappa B/química , Quinase I-kappa B/metabolismo , Síndromes de Imunodeficiência/imunologia , Lipopolissacarídeos/imunologia , MAP Quinase Quinase Quinases/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , NF-kappa B/metabolismo , Doenças da Imunodeficiência Primária , Deleção de Sequência , Ubiquitinação , Dedos de Zinco/genética
19.
Curr Biol ; 17(16): 1438-43, 2007 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-17702576

RESUMO

NF-kappaB essential modulator (NEMO), the regulatory subunit of the IkappaB kinase (IKK) that activates NF-kappaB, is essential for NF-kappaB activation. NEMO was recently found to contain a region that preferentially binds Lys (K)63-linked but not K48-linked polyubiquitin (polyUb) chains, and the ability of NEMO to bind to K63-linked polyUb RIP (receptor-interacting protein) is necessary for efficient tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation. Optineurin is a homolog of NEMO, and mutations in the optineurin gene are found in a subset of patients with glaucoma, a neurodegenerative disease involving the loss of retinal ganglion cells. Although optineurin shares considerable homology with NEMO, in resting cells, it is not present in the high-molecular-weight complex containing IKKalpha and IKKbeta, and optineurin cannot substitute for NEMO in lipopolysaccharide (LPS)-induced NF-kappaB activation. On the other hand, the overexpression of optineurin blocks the protective effect of E3-14.7K on cell death caused by the overexpression of TNFalpha receptor 1 (TNFR1). Here we show that optineurin has a K63-linked polyUb-binding region similar to that of NEMO, and like NEMO, it bound K63- but not K48-linked polyUb. Optineurin competitively antagonized NEMO's binding to polyUb RIP, and its overexpression inhibited TNFalpha-induced NF-kappaB activation. This competition occurs at physiologic protein levels because microRNA silencing of optineurin resulted in markedly enhanced TNFalpha-induced NF-kappaB activity. These results reveal a physiologic role for optineurin in dampening TNFalpha signaling, and this role might provide an explanation for its association with glaucoma.


Assuntos
Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fator de Transcrição TFIIIA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas de Ciclo Celular , Glaucoma/metabolismo , Humanos , Proteínas de Membrana Transportadoras , Ubiquitina/metabolismo
20.
Front Immunol ; 11: 470, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32265924

RESUMO

The transcription factor TCF-1 (encoded by Tcf7) plays critical roles in several lineages of hematopoietic cells. In this study, we examined the molecular basis for Tcf7 regulation in T cells, innate lymphoid cells, and migratory conventional dendritic cells that we find express Tcf7. We identified a 1 kb regulatory element crucial for the initiation of Tcf7 expression in T cells and innate lymphoid cells, but dispensable for Tcf7 expression in Tcf7-expressing dendritic cells. Within this region, we identified a Notch binding site important for the initiation of Tcf7 expression in T cells but not in innate lymphoid cells. Our work establishes that the same regulatory element is used by distinct transcriptional controllers to initiate Tcf7 expression in T cells and ILCs.


Assuntos
Fator 1-alfa Nuclear de Hepatócito/metabolismo , Linfócitos/imunologia , Elementos Reguladores de Transcrição/genética , Linfócitos T/fisiologia , Animais , Diferenciação Celular , Células Cultivadas , Regulação da Expressão Gênica , Fator 1-alfa Nuclear de Hepatócito/genética , Imunidade Inata , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
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