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1.
PLoS Biol ; 13(10): e1002269, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26440998

RESUMO

Sepsis, an exaggerated systemic inflammatory response, remains a major medical challenge. Both hyperinflammation and immunosuppression are implicated as causes of morbidity and mortality. Dendritic cell (DC) loss has been observed in septic patients and in experimental sepsis models, but the role of DCs in sepsis, and the mechanisms and significance of DC loss, are poorly understood. Here, we report that mice with selective deletion of the glucocorticoid receptor (GR) in DCs (GR(CD11c-cre)) were highly susceptible to LPS-induced septic shock, evidenced by elevated inflammatory cytokine production, hypothermia, and mortality. Neutralizing anti-IL-12 antibodies prevented hypothermia and death, demonstrating that endogenous GC-mediated suppression of IL-12 is protective. In LPS-challenged GR(CD11c-cre) mice, CD8(+) DCs were identified as the major source of prolonged IL-12 production, which correlated with elevations of NK cell-derived IFN-γ. In addition, the loss of GR in CD11c(+) cells rescued LPS-induced loss of CD8(+) DCs but not other DC subsets. Unlike wild-type animals, exposure of GR(CD11c-cre) mice to low-dose LPS did not induce CD8(+) DC loss or tolerance to subsequent challenge with high dose, but neutralization of IL-12 restored the ability of low-dose LPS to tolerize. Therefore, endogenous glucocorticoids blunt LPS-induced inflammation and promote tolerance by suppressing DC IL-12 production.


Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Células Dendríticas/efeitos dos fármacos , Glucocorticoides/agonistas , Interleucina-12/antagonistas & inibidores , Receptores de Glucocorticoides/agonistas , Choque Séptico/prevenção & controle , Animais , Anti-Inflamatórios não Esteroides/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Antígeno CD11c/genética , Antígeno CD11c/metabolismo , Células Cultivadas , Cruzamentos Genéticos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Relação Dose-Resposta a Droga , Feminino , Glucocorticoides/antagonistas & inibidores , Glucocorticoides/sangue , Glucocorticoides/metabolismo , Imunidade Inata/efeitos dos fármacos , Interleucina-12/sangue , Interleucina-12/metabolismo , Lipopolissacarídeos/toxicidade , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Glucocorticoides/antagonistas & inibidores , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Choque Séptico/imunologia , Choque Séptico/metabolismo , Choque Séptico/patologia , Transdução de Sinais/efeitos dos fármacos , Organismos Livres de Patógenos Específicos , Baço/efeitos dos fármacos , Baço/imunologia , Baço/metabolismo , Baço/patologia
2.
J Immunol ; 191(12): 6231-40, 2013 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-24244017

RESUMO

Optineurin is a widely expressed polyubiquitin-binding protein that has been implicated in regulating cell signaling via its NF-κB essential modulator-homologous C-terminal ubiquitin (Ub)-binding region. Its functions are controversial, with in vitro studies finding that optineurin suppressed TNF-mediated NF-κB activation and virus-induced activation of IFN regulatory factor 3 (IRF3), whereas bone marrow-derived macrophages (BMDMs) from mice carrying an optineurin Ub-binding point mutation had normal TLR-mediated NF-κB activation and diminished IRF3 activation. We have generated a mouse model in which the entire Ub-binding C-terminal region is deleted (Optn(470T)). Akin to C-terminal optineurin mutations found in patients with certain neurodegenerative diseases, Optn(470T) was expressed at substantially lower levels than the native protein, allowing assessment not only of the lack of Ub binding, but also of protein insufficiency. Embryonic lethality with incomplete penetrance was observed for 129 × C57BL/6 Optn(470T/470T) mice, but after further backcrossing to C57BL/6, offspring viability was restored. Moreover, the mice that survived were indistinguishable from wild type littermates and had normal immune cell distributions. Activation of NF-κB in Optn(470T) BMDM and BM-derived dendritic cells with TNF or via TLR4, T cells via the TCR, and B cells with LPS or anti-CD40 was normal. In contrast, optineurin and/or its Ub-binding function was necessary for optimal TANK binding kinase 1 and IRF3 activation, and both Optn(470T) BMDMs and bone marrow-derived dendritic cells had diminished IFN-ß production upon LPS stimulation. Importantly, Optn(470T) mice produced less IFN-ß upon LPS challenge. Therefore, endogenous optineurin is dispensable for NF-κB activation but necessary for optimal IRF3 activation in immune cells.


Assuntos
Proteínas do Olho/fisiologia , Fator Regulador 3 de Interferon/metabolismo , Subpopulações de Linfócitos/metabolismo , Macrófagos/metabolismo , NF-kappa B/metabolismo , Animais , Proteínas de Ciclo Celular , Quimera , Cruzamentos Genéticos , Células Dendríticas/imunologia , Proteínas do Olho/genética , Regulação da Expressão Gênica/imunologia , Genes Letais , Células HEK293 , Humanos , Imunidade Inata , Interferon beta/biossíntese , Interferon beta/genética , Lipopolissacarídeos/farmacologia , Subpopulações de Linfócitos/imunologia , Macrófagos/imunologia , Masculino , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos B/imunologia , Deleção de Sequência , Transdução de Sinais , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitinação
4.
Genes Dev ; 20(3): 282-96, 2006 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-16452502

RESUMO

Studies of mammalian genes activated in response to an acute stimulus have suggested diverse mechanisms through which chromatin structure and nucleosome remodeling events contribute to inducible gene transcription. However, because of this diversity, the logical organization of the genome with respect to nucleosome remodeling and gene induction has remained obscure. Numerous proinflammatory genes are rapidly induced in macrophages in response to microbial infection. Here, we show that in lipopolysaccharide-stimulated macrophages, the catalytic BRG1/BRM subunits of the SWI/SNF class of ATP-dependent nucleosome remodeling complexes are consistently required for the activation of secondary response genes and primary response genes induced with delayed kinetics, but not for rapidly induced primary response genes. Surprisingly, a Mi-2beta complex was selectively recruited along with the SWI/SNF complexes to the control regions of secondary response and delayed primary response genes, with the Mi-2beta complex acting antagonistically to limit the induction of these gene classes. SWI/SNF and Mi-2beta complexes influenced cell size in a similarly antagonistic manner. These results provide insight into the differential contributions of nucleosome remodeling complexes to the rapid induction of defined classes of mammalian genes and reveal a robust anti-inflammatory function of Mi-2beta.


Assuntos
Adenosina Trifosfatases/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Regulação da Expressão Gênica , Inflamação/metabolismo , Macrófagos/metabolismo , Nucleossomos/metabolismo , Fatores de Transcrição/fisiologia , Animais , Células Cultivadas , Proteínas Cromossômicas não Histona/antagonistas & inibidores , DNA Helicases , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Cinética , Lipopolissacarídeos , Camundongos , Microscopia de Fluorescência , Modelos Biológicos , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Ativação Transcricional
5.
Infect Immun ; 70(6): 3085-93, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12011002

RESUMO

The locus of enterocyte effacement (LEE) is a chromosomal pathogenicity island that encodes the proteins involved in the formation of the attaching and effacing lesions by enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). The LEE comprises 41 open reading frames organized in five major operons, LEE1, LEE2, LEE3, tir (LEE5), and LEE4, which encode a type III secretion system, the intimin adhesin, the translocated intimin receptor (Tir), and other effector proteins. The first gene of LEE1 encodes the Ler regulator, which activates all the other genes within the LEE. We previously reported that the LEE genes were activated by quorum sensing through Ler (V. Sperandio, J. L. Mellies, W. Nguyen, S. Shin, and J. B. Kaper, Proc. Natl. Acad. Sci. USA 96:15196-15201, 1999). In this study we report that a putative regulator in the E. coli genome is itself activated by quorum sensing. This regulator is encoded by open reading frame b3243; belongs to the LysR family of regulators; is present in EHEC, EPEC, and E. coli K-12; and shares homology with the AphB and PtxR regulators of Vibrio cholerae and Pseudomonas aeruginosa, respectively. We confirmed the activation of b3243 by quorum sensing by using transcriptional fusions and renamed this regulator quorum-sensing E. coli regulator A (QseA). We observed that QseA activated transcription of ler and therefore of the other LEE genes. An EHEC qseA mutant had a striking reduction of type III secretion activity, which was complemented when qseA was provided in trans. Similar results were also observed with a qseA mutant of EPEC. The QseA regulator is part of the regulatory cascade that regulates EHEC and EPEC virulence genes by quorum sensing.


Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli O157/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fosfoproteínas , Transativadores/metabolismo , Ativação Transcricional , Sequência de Aminoácidos , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Escherichia coli/genética , Dados de Sequência Molecular , Óperon , Regiões Promotoras Genéticas , Homologia de Sequência de Aminoácidos , Transativadores/classificação , Transativadores/genética , Fatores de Transcrição
6.
Mol Microbiol ; 43(6): 1577-89, 2002 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11952906

RESUMO

In pathogenic Vibrio cholerae, the transmembrane DNA-binding protein ToxR co-ordinates the expression of over 20 genes, including those encoding important virulence factors such as cholera toxin and the toxin-co-regulated pilus. The outer membrane protein OmpT is the only member of the ToxR regulon known to be repressed by ToxR. In this study, we examined the environmental conditions that regulate OmpT expression and demonstrated that ompT transcription is upregulated 14-fold when the bacteria enter late log phase from early log phase. Deletion of the crp gene completely abolishes OmpT expression. Comparison of ompT transcription levels in the isogenic crp-, toxR- and crp-toxR- mutants revealed that (i) in the absence of ToxR, constitutive high-level ompT transcription is dependent on cAMP receptor protein (CRP); (ii) ToxR not only interferes with CRP-dependent ompT activation, but also abolishes the CRP-independent, basal level ompT transcription; thus, the mechanism by which ToxR represses ompT transcription involves both antiactivation and direct repression; (iii) both CRP and ToxR are required for the regulation of OmpT expression by growth phase. To provide further insights into the molecular mecha-nism of CRP-dependent activation of ompT transcription, we demonstrated that CRP-dependent activation requires a CRP binding site centred at -310 of the ompT promoter, without which the interaction of CRP with other CRP binding site(s) more proximal to the promoter results in repression. Mutations in two regions on CRP (AR1 and AR2) that directly contact RNA polymerase (RNAP) abolish activation, suggesting direct interaction of CRP with RNAP from -310 of the ompT promoter via DNA looping.


Assuntos
Proteínas de Bactérias , Proteína Receptora de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Porinas/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Vibrio cholerae/crescimento & desenvolvimento , Meios de Cultura/química , Proteína Receptora de AMP Cíclico/genética , Proteínas de Ligação a DNA/genética , Concentração Osmolar , Porinas/genética , Fatores de Transcrição/genética , Transcrição Gênica , Vibrio cholerae/genética , Vibrio cholerae/metabolismo
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