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1.
Biomolecules ; 12(12)2022 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-36551159

RESUMO

Secretory leucoprotease inhibitor (SLPI) has multifaceted functions, including inhibition of protease activity, antimicrobial functions, and anti-inflammatory properties. In this study, we show that SLPI plays a role in controlling pulmonary Pseudomonas aeruginosa infection. Mice lacking SLPI were highly susceptible to P. aeruginosa infection, however there was no difference in bacterial burden. Utilising a model of P. aeruginosa LPS-induced lung inflammation, human recombinant SLPI (hrSLPI) administered intraperitoneally suppressed the recruitment of inflammatory cells in the bronchoalveolar lavage fluid (BALF) and resulted in reduced BALF and serum levels of inflammatory cytokines and chemokines. This anti-inflammatory effect of hrSLPI was similarly demonstrated in a systemic inflammation model induced by intraperitoneal injection of LPS from various bacteria or lipoteichoic acid, highlighting the broad anti-inflammatory properties of hrSLPI. Moreover, in bone-marrow-derived macrophages, hrSLPI reduced LPS-induced phosphorylation of p-IkB-α, p-IKK-α/ß, p-P38, demonstrating that the anti-inflammatory effect of hrSLPI was due to the inhibition of the NFκB and MAPK pathways. In conclusion, administration of hrSLPI attenuates excessive inflammatory responses and is therefore, a promising strategy to target inflammatory diseases such as acute respiratory distress syndrome or sepsis and could potentially be used to augment antibiotic treatment.


Assuntos
Inflamação , Infecções por Pseudomonas , Inibidor Secretado de Peptidases Leucocitárias , Animais , Humanos , Camundongos , Inflamação/metabolismo , Inflamação/microbiologia , Lipopolissacarídeos , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/terapia , Inibidor Secretado de Peptidases Leucocitárias/administração & dosagem , Inibidor Secretado de Peptidases Leucocitárias/metabolismo , Proteínas Recombinantes/administração & dosagem
2.
JCI Insight ; 6(12)2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34032637

RESUMO

Evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) is a protein with roles in early development, activation of the transcription factor NF-κB, and production of mitochondrial reactive oxygen species (mROS) that facilitates clearance of intracellular bacteria like Salmonella. ECSIT is also an important assembly factor for mitochondrial complex I. Unlike the murine form of Ecsit (mEcsit), we demonstrate here that human ECSIT (hECSIT) is highly labile. To explore whether the instability of hECSIT affects functions previously ascribed to its murine counterpart, we created a potentially novel transgenic mouse in which the murine Ecsit gene is replaced by the human ECSIT gene. The humanized mouse has low levels of hECSIT protein, in keeping with its intrinsic instability. Whereas low-level expression of hECSIT was capable of fully compensating for mEcsit in its roles in early development and activation of the NF-κB pathway, macrophages from humanized mice showed impaired clearance of Salmonella that was associated with reduced production of mROS. Notably, severe cardiac hypertrophy was manifested in aging humanized mice, leading to premature death. The cellular and molecular basis of this phenotype was delineated by showing that low levels of human ECSIT protein led to a marked reduction in assembly and activity of mitochondrial complex I with impaired oxidative phosphorylation and reduced production of ATP. Cardiac tissue from humanized hECSIT mice also showed reduced mitochondrial fusion and more fission but impaired clearance of fragmented mitochondria. A cardiomyocyte-intrinsic role for Ecsit in mitochondrial function and cardioprotection is also demonstrated. We also show that cardiac fibrosis and damage in humans correlated with low expression of human ECSIT. In summary, our findings identify a role for ECSIT in cardioprotection, while generating a valuable experimental model to study mitochondrial dysfunction and cardiac pathophysiology.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Cardiomegalia , Miocárdio , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Células Cultivadas , Humanos , Macrófagos/metabolismo , Camundongos , Mitocôndrias/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , NF-kappa B/genética , NF-kappa B/metabolismo
3.
Nat Commun ; 9(1): 1560, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29674674

RESUMO

The NLRP3 inflammasome has an important function in inflammation by promoting the processing of pro-IL-1ß and pro-IL-18 to their mature bioactive forms, and by inducing cell death via pyroptosis. Here we show a critical function of the E3 ubiquitin ligase Pellino2 in facilitating activation of the NLRP3 inflammasome. Pellino2-deficient mice and myeloid cells have impaired activation of NLRP3 in response to toll-like receptor priming, NLRP3 stimuli and bacterial challenge. These functions of Pellino2 in the NLRP3 pathway are dependent on Pellino2 FHA and RING-like domains, with Pellino2 promoting the ubiquitination of NLRP3 during the priming phase of activation. We also identify a negative function of IRAK1 in the NLRP3 inflammasome, and describe a counter-regulatory relationship between IRAK1 and Pellino2. Our findings reveal a Pellino2-mediated regulatory signaling system that controls activation of the NLRP3 inflammasome.


Assuntos
Inflamassomos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteínas Nucleares/imunologia , Animais , Humanos , Inflamassomos/genética , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Domínios Proteicos , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/fisiologia , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , Ubiquitinação
4.
PLoS Pathog ; 10(2): e1003960, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24586164

RESUMO

Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.


Assuntos
Infecções por Vírus Epstein-Barr/imunologia , Evasão da Resposta Imune/imunologia , Transdução de Sinais/imunologia , Receptores Toll-Like/imunologia , Proteínas Virais Reguladoras e Acessórias/imunologia , Ensaio de Imunoadsorção Enzimática , Infecções por Vírus Epstein-Barr/metabolismo , Citometria de Fluxo , Imunofluorescência , Herpesvirus Humano 4 , Humanos , Imunidade Inata , Immunoblotting , Receptores Toll-Like/metabolismo , Transfecção , Proteínas Virais Reguladoras e Acessórias/metabolismo
5.
Nat Immunol ; 13(11): 1055-62, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23042151

RESUMO

Toll-like receptors (TLRs) sense pathogen-associated molecules and respond by inducing cytokines and type I interferon. Here we show that genetic ablation of the E3 ubiquitin ligase Pellino3 augmented the expression of type I interferon but not of proinflammatory cytokines in response to TLR3 activation. Pellino3-deficient mice had greater resistance against the pathogenic and lethal effects of encephalomyocarditis virus (EMCV). TLR3 signaling induced Pellino3, which in turn interacted with and ubiquitinated TRAF6. This modification suppressed the ability of TRAF6 to interact with and activate IRF7, resulting in downregulation of type I interferon expression. Our findings highlight a new physiological role for Pellino3 and define a new autoregulatory network for controlling type I interferon expression.


Assuntos
Infecções por Cardiovirus/imunologia , Regulação da Expressão Gênica , Interferon Tipo I/imunologia , Receptor 3 Toll-Like/imunologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Infecções por Cardiovirus/genética , Infecções por Cardiovirus/mortalidade , Infecções por Cardiovirus/virologia , Vírus da Encefalomiocardite/imunologia , Homeostase , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/imunologia , Interferon Tipo I/genética , Camundongos , Camundongos Knockout , Transdução de Sinais , Taxa de Sobrevida , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/imunologia , Receptor 3 Toll-Like/genética , Ubiquitina/genética , Ubiquitina/imunologia , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
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