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1.
J Exp Med ; 218(2)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33216117

RESUMO

Two cosegregating single-nucleotide polymorphisms (SNPs) in human TLR4, an A896G transition at SNP rs4986790 (D299G) and a C1196T transition at SNP rs4986791 (T399I), have been associated with LPS hyporesponsiveness and differential susceptibility to many infectious or inflammatory diseases. However, many studies failed to confirm these associations, and transfection experiments resulted in conflicting conclusions about the impact of these SNPs on TLR4 signaling. Using advanced protein modeling from crystallographic data of human and murine TLR4, we identified homologous substitutions of these SNPs in murine Tlr4, engineered a knock-in strain expressing the D298G and N397I TLR4 SNPs homozygously, and characterized in vivo and in vitro responses to TLR4 ligands and infections in which TLR4 is implicated. Our data provide new insights into cellular and molecular mechanisms by which these SNPs decrease the TLR4 signaling efficiency and offer an experimental approach to confirm or refute human data possibly confounded by variables unrelated to the direct effects of the SNPs on TLR4 functionality.


Assuntos
Lipopolissacarídeos/genética , Polimorfismo de Nucleotídeo Único/genética , Receptor 4 Toll-Like/genética , Animais , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Camundongos , Transdução de Sinais/genética
2.
Trends Immunol ; 40(6): 492-510, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31053495

RESUMO

Long noncoding RNAs (lncRNAs) are key molecules that regulate gene expression in a variety of organisms. LncRNAs can drive different transcriptional and post-transcriptional events that impact cellular functions. Recent studies have identified many lncRNAs associated with immune cell development and activation; however, an understanding of their functional role in host immunity to infection is just emerging. Here, we provide a detailed and updated review of the functional roles of lncRNAs in regulating mammalian immune responses during host-pathogen interactions, because these functions may be either beneficial or detrimental to the host. With increased mechanistic insight into the roles of lncRNAs, it may be possible to design and/or improve lncRNA-based therapies to treat a variety of infectious and inflammatory diseases.


Assuntos
Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunomodulação/genética , RNA Longo não Codificante/genética , Animais , Resistência à Doença/genética , Resistência à Doença/imunologia , Genoma , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Humanos
3.
Artigo em Inglês | MEDLINE | ID: mdl-32039056

RESUMO

Long non-coding RNAs (lncRNAs) have emerged as key regulators of gene expression in several biological systems. The long intergenic RNA-erythroid pro-survival (lincRNA-EPS) has been shown to play a critical role in restraining inflammatory gene expression. However, the function of lincRNA-EPS during bacterial infections remains unknown. Here, we demonstrate that following infection with the intracellular bacterium Listeria monocytogenes, both mouse macrophages and dendritic cells lacking lincRNA-EPS exhibit an enhanced expression of proinflammatory cytokine genes, as well as an increased expression of the inducible nitric oxide synthase (iNos) and nitric oxide (NO) production. Importantly, we found that lincRNA-EPS-/- mice intraperitoneally infected with L. monocytogenes exhibit lower bacterial burdens in spleen and liver and produce more NO than control mice. Furthermore, lincRNA-EPS-/- mice are less susceptible to a lethal dose of L. monocytogenes than wild type (WT) mice. Collectively these findings show that lincRNA-EPS suppresses host protective NO expression and impairs the host defense against L. monocytogenes infection.


Assuntos
Citocinas/antagonistas & inibidores , Células Dendríticas/imunologia , Regulação para Baixo , Listeria monocytogenes/imunologia , Macrófagos/imunologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , Animais , Carga Bacteriana , Células Cultivadas , Citocinas/genética , Modelos Animais de Doenças , Tolerância Imunológica , Imunidade Inata , Listeriose/imunologia , Listeriose/microbiologia , Listeriose/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Teóricos , Óxido Nítrico Sintase Tipo II/genética , RNA Longo não Codificante/genética , Análise de Sobrevida
4.
J Leukoc Biol ; 104(4): 811-820, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29749650

RESUMO

IL-1 receptor-associated kinase (IRAK) 4 is a central enzyme of the TLR pathways. This study tested the hypothesis that IRAK4 kinase activity is prerequisite for regulating innate immunity during infections with intracellular bacteria. To this end, we analyzed responses of macrophages obtained from mice expressing wild-type (WT) IRAK4 or its kinase-inactive K213M mutant (IRAK4KI ) upon infection with intracellular bacteria Listeria monocytogenes or Mycobacterium smegmatis. In contrast to robust induction of cytokines by macrophages expressing kinase-sufficient IRAK4, IRAK4KI macrophages expressed decreased TNF-α, IL-6, IL-1ß, and C-C motif chemokine ligand 5 upon infection with L. monocytogenes or M. smegmatis. Bacterial infection of IRAK4KI macrophages led to attenuated activation of IRAK1, MAPKs and NF-κB, impaired induction of inducible NO synthase mRNA and secretion of NO, but resulted in elevated microbial burdens. Compared with WT animals, systemic infection of IRAK4KI mice with M. smegmatis or L. monocytogenes resulted in decreased levels of serum IL-6 and CXCL-1 but increased bacterial burdens in the spleen and liver. Thus, a loss of IRAK4 kinase activity underlies deficient cytokine and microbicidal responses during infection with intracellular bacteria L. monocytogenes or M. smegmatis via impaired activation of IRAK1, MAPKs, and NF-κB but increases bacterial burdens, correlating with decreased induction of NO.


Assuntos
Imunidade Inata , Síndromes de Imunodeficiência/imunologia , Quinases Associadas a Receptores de Interleucina-1/imunologia , Listeriose/imunologia , Macrófagos/microbiologia , Infecções por Mycobacterium não Tuberculosas/imunologia , Animais , Citocinas/metabolismo , Feminino , Síndromes de Imunodeficiência/genética , Quinases Associadas a Receptores de Interleucina-1/deficiência , Quinases Associadas a Receptores de Interleucina-1/genética , Listeria monocytogenes , Listeriose/enzimologia , Fígado/microbiologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Infecções por Mycobacterium não Tuberculosas/enzimologia , Mycobacterium smegmatis , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Doenças da Imunodeficiência Primária , Baço/microbiologia
5.
J Biol Chem ; 292(25): 10685-10695, 2017 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-28442574

RESUMO

Toll-like receptor 2 (TLR2) plays a critical role in host defenses against mycobacterial infections. The R753Q TLR2 polymorphism has been associated with increased incidence of tuberculosis and infections with non-tuberculous mycobacteria in human populations, but the mechanisms by which this polymorphism affects TLR2 signaling are unclear. In this study, we determined the impact of the R753Q TLR2 polymorphism on macrophage sensing of Mycobacterium smegmatis Upon infection with M. smegmatis, macrophages from knock-in mice harboring R753Q TLR2 expressed lower levels of TNF-α, IL-1ß, IL-6, and IL-10 compared with cells from WT mice, but both R753Q TLR2- and WT-derived macrophages exhibited comparable bacterial burdens. The decreased cytokine responses in R753Q TLR2-expressing macrophages were accompanied by impaired phosphorylation of IL-1R-associated kinase 1 (IRAK-1), p38, ERK1/2 MAPKs, and p65 NF-κB, suggesting that the R753Q TLR2 polymorphism alters the functions of the myeloid differentiation primary response protein 88 (MyD88)-IRAK-dependent signaling axis. Supporting this notion, HEK293 cells stably transfected with YFP-tagged R753Q TLR2 displayed reduced recruitment of MyD88 to TLR2, decreased NF-κB activation, and impaired IL-8 expression upon exposure to M. smegmatis Collectively, our results indicate that the R753Q polymorphism alters TLR2 signaling competence, leading to impaired MyD88-TLR2 assembly, reduced phosphorylation of IRAK-1, diminished activation of MAPKs and NF-κB, and deficient induction of cytokines in macrophages infected with M. smegmatis.


Assuntos
Imunidade Inata , Macrófagos/imunologia , Mutação de Sentido Incorreto , Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium smegmatis/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Polimorfismo Genético , Receptor 2 Toll-Like/imunologia , Substituição de Aminoácidos , Animais , Citocinas/genética , Citocinas/imunologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Camundongos Mutantes , Infecções por Mycobacterium não Tuberculosas/genética , Fator 88 de Diferenciação Mieloide/genética , Receptor 2 Toll-Like/genética
6.
Eur J Immunol ; 47(5): 880-891, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28295231

RESUMO

Interleukin-1 receptor-associated kinase (IRAK) 4 mediates host defense against infections. As an active kinase, IRAK4 elicits full spectra of myeloid differentiation primary response protein (MyD) 88-dependent responses, while kinase-inactive IRAK4 induces a subset of cytokines and negative regulators whose expression is not regulated by mRNA stability. IRAK4 kinase activity is critical for resistance against Streptococcus pneumoniae, but its involvement in autoimmunity is incompletely understood. In this study, we determined the role of IRAK4 kinase activity in murine lupus. Lupus development in BXSB mice expressing the Y chromosome autoimmunity accelerator (Yaa) increased basal and Toll-like receptor (TLR) 4/7-induced phosphorylation of mitogen-activated protein kinases, p65 nuclear factor-κB (NF-κB), enhanced tumor necrosis factor (TNF)-α and C-C motif chemokine ligand (CCL) 5 gene expression in splenic macrophages, but decreased levels of Toll-interacting protein and IRAK-M, without affecting IRAK4 or IRAK1 expression. Mice harboring kinase-inactive IRAK4 on the lupus-prone Yaa background manifested blunted TLR signaling in macrophages and reduced glomerulonephritis, splenomegaly, serum anti-nuclear antibodies, numbers of splenic macrophages, total and TNF-α+ dendritic cells, activated T- and B-lymphocytes, and lower TNF-α expression in macrophages compared with lupus-prone mice with functional IRAK4. Thus, IRAK4 kinase activity contributes to murine lupus and could represent a new therapeutic target.


Assuntos
Autoimunidade , Citocinas/imunologia , Síndromes de Imunodeficiência , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Nefrite Lúpica/imunologia , Animais , Quimiocina CCL5/genética , Quinases Associadas a Receptores de Interleucina-1/deficiência , Macrófagos/imunologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fosforilação , Doenças da Imunodeficiência Primária , Transdução de Sinais , Baço/citologia , Baço/imunologia , Receptores Toll-Like/imunologia , Fator de Necrose Tumoral alfa/metabolismo
7.
J Leukoc Biol ; 100(5): 927-941, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27343013

RESUMO

TLRs play a critical role in the detection of microbes and endogenous "alarmins" to initiate host defense, yet they can also contribute to the development and progression of inflammatory and autoimmune diseases. To avoid pathogenic inflammation, TLR signaling is subject to multilayer regulatory control mechanisms, including cooperation with coreceptors, post-translational modifications, cleavage, cellular trafficking, and interactions with negative regulators. Nucleic acid-sensing TLRs are particularly interesting in this regard, as they can both recognize host-derived structures and require internalization of their ligand as a result of intracellular sequestration of the nucleic acid-sensing TLRs. This review summarizes the regulatory mechanisms of TLRs, including regulation of their access to ligands, receptor folding, intracellular trafficking, and post-translational modifications, as well as how altered control mechanism could contribute to inflammatory and autoimmune disorders.


Assuntos
Transdução de Sinais/imunologia , Receptores Toll-Like/imunologia , Imunidade Adaptativa , Alarminas/imunologia , Animais , Dimerização , Endossomos/imunologia , Glicosilação , Humanos , Imunidade Inata , Infecções/imunologia , Ligantes , Ácidos Nucleicos/imunologia , Fosforilação , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Transporte Proteico , Relação Estrutura-Atividade , Receptores Toll-Like/ultraestrutura , Ubiquitinação
8.
J Leukoc Biol ; 99(6): 839-50, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26965636

RESUMO

Sensing of microbial pathogens and endogenous "alarmins" by macrophages and dendritic cells is reliant on pattern recognition receptors, including membrane-associated TLRs, cytosolic nucleotide-binding and oligomerization domain leucine-rich repeat-containing receptors, retinoic acid-inducible gene I-like receptors, and absent in melanoma 2-like receptors. Engagement of TLRs elicits signaling pathways that activate inflammatory genes whose expression is regulated by chromatin-modifying complexes and transcription factors. Long noncoding RNAs have emerged as new regulators of inflammatory mediators in the immune system. They are expressed in macrophages, dendritic cells, neutrophils, NK cells, and T- and B-lymphocytes and are involved in immune cell differentiation and activation. Long noncoding RNAs act via repression or activation of transcription factors, modulation of stability of mRNA and microRNA, regulation of ribosome entry and translation of mRNAs, and controlling components of the epigenetic machinery. In this review, we focus on recent advances in deciphering the mechanisms by which long noncoding RNAs regulate TLR-driven responses in macrophages and dendritic cells and discuss the involvement of long noncoding RNAs in endotoxin tolerance, autoimmune, and inflammatory diseases. The dissection of the role of long noncoding RNAs will improve our understanding of the mechanisms of regulation of inflammation and may provide new targets for therapeutic intervention.


Assuntos
Imunidade Inata , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Receptores Toll-Like/metabolismo , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/patologia , Humanos , Inflamação/genética , Inflamação/patologia
9.
J Innate Immun ; 8(2): 171-84, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26457672

RESUMO

Endotoxin tolerance protects the host by limiting excessive 'cytokine storm' during sepsis, but compromises the ability to counteract infections in septic shock survivors. It reprograms Toll-like receptor (TLR) 4 responses by attenuating the expression of proinflammatory cytokines without suppressing anti-inflammatory and antimicrobial mediators, but the mechanisms of reprogramming remain unclear. In this study, we demonstrate that the induction of endotoxin tolerance in human monocytes, THP-1 and MonoMac-6 cells inhibited lipopolysaccharide (LPS)-mediated phosphorylation of Lyn, c-Src and their recruitment to TLR4, but increased total protein phosphatase (PP) activity and the expression of protein tyrosine phosphatase (PTP) 1B, PP2A, PTP nonreceptor type (PTPN) 22 and mitogen-activated protein kinase phosphatase (MKP)-1. Chemical PP inhibitors, okadaic acid, dephostatin and cantharidic acid markedly decreased or completely abolished LPS tolerance, indicating the importance of phosphatases in endotoxin tolerization. Overexpression of PTPN22 decreased LPS-mediated nuclear factor (NF)-x03BA;B activation, p38 phosphorylation and CXCL8 gene expression, while PTPN22 ablation upregulated LPS-induced p65 NF-x03BA;B and p38 phosphorylation and the expression of TNF-α and pro-IL-1ß mRNA, indicating PTPN22 as an inhibitor of TLR4 signaling. Thus, LPS tolerance interferes with TLR4 signaling by inhibiting Lyn and c-Src phosphorylation and their recruitment to TLR4, while increasing the phosphatase activity and expression of PP2A, PTPN22, PTP1B and MKP1.


Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Tolerância Imunológica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Monócitos/imunologia , Fosfoproteínas Fosfatases/imunologia , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/imunologia , Quinases da Família src/imunologia , Proteína Tirosina Quinase CSK , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica/imunologia , Humanos , Tolerância Imunológica/genética , Lipopolissacarídeos/imunologia , Fosfoproteínas Fosfatases/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/genética , Quinases da Família src/genética
10.
J Leukoc Biol ; 98(6): 963-74, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26310831

RESUMO

Development of endotoxin tolerance in macrophages during sepsis reprograms Toll-like receptor 4 signaling to inhibit proinflammatory cytokines without suppressing anti-inflammatory and antimicrobial mediators and protects the host from excessive inflammation and tissue damage. However, endotoxin tolerance renders septic patients immunocompromised and unable to control secondary infections. Although previous studies have revealed the importance of several negative regulators of Toll-like receptor signaling in endotoxin tolerance, the role of Pellino proteins has not been addressed. The present report shows that the induction of endotoxin tolerance in vivo in mice and in vitro in human monocytes and THP-1 and MonoMac-6 macrophages increases the expression of Pellino-3. Overexpression of Pellino-3 in human embryonic kidney 293/Toll-like receptor 2 or 293/Toll-like receptor 4/myeloid differentiation factor-2 cells inhibited Toll-like receptor 2/4-mediated activation of nuclear factor-κB and induction of CXCL-8 mRNA, and Pellino-3 ablation increased these responses. Pellino-3-deficient THP-1 cells had elevated Toll-like receptor 2/4-driven tumor necrosis factor-α, interleukin-6 mRNA, and Toll-like receptor 4-driven CCL5 gene expression in response to Toll-like receptor agonists and heat-killed Escherichia coli and Staphylococcus aureus, cytokines controlled by the MyD88 and Toll-interleukin-1R domain-containing protein inducing interferon-ß-mediated pathways, respectively. In addition, deficiency in Pellino-3 slightly increased phagocytosis of heat-killed bacteria. Transfected Pellino-3 inhibited nuclear factor-κB activation driven by overexpression of MyD88, TIR domain-containing adapter inducing interferon-ß, interleukin-1R-associated kinase-1, and tumor necrosis factor receptor activator of nuclear factor-κB-binding kinase-1, TGF-ß-activated kinase 1, and tumor necrosis factor receptor-associated factor-6, and inhibited interleukin-1R-associated kinase 1 modifications and tumor necrosis factor receptor activator of nuclear factor-κB-binding kinase 1 phosphorylation. Finally, Pellino-3 ablation in THP-1 decreased the extent of endotoxin tolerization. Thus, Pellino-3 is involved in endotoxin tolerance and functions as a negative regulator of Toll-like receptor 2/4 signaling.


Assuntos
Tolerância Imunológica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Linhagem Celular Tumoral , Citocinas/genética , Citocinas/imunologia , Escherichia coli/imunologia , Humanos , Tolerância Imunológica/genética , Camundongos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Staphylococcus aureus/imunologia , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Ubiquitina-Proteína Ligases/genética
11.
J Biol Chem ; 290(31): 19218-32, 2015 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-26082489

RESUMO

Endotoxin tolerance reprograms Toll-like receptor (TLR) 4-mediated macrophage responses by attenuating induction of proinflammatory cytokines while retaining expression of anti-inflammatory and antimicrobial mediators. We previously demonstrated deficient TLR4-induced activation of IL-1 receptor-associated kinase (IRAK) 4, IRAK1, and TANK-binding kinase (TBK) 1 as critical hallmarks of endotoxin tolerance, but mechanisms remain unclear. In this study, we examined the role of the E3 ubiquitin ligase Pellino-1 in endotoxin tolerance and TLR signaling. LPS stimulation increased Pellino-1 mRNA and protein expression in macrophages from mice injected with saline and in medium-pretreated human monocytes, THP-1, and MonoMac-6 cells, whereas endotoxin tolerization abrogated LPS inducibility of Pellino-1. Overexpression of Pellino-1 in 293/TLR2 and 293/TLR4/MD2 cells enhanced TLR2- and TLR4-induced nuclear factor κB (NF-κB) and expression of IL-8 mRNA, whereas Pellino-1 knockdown reduced these responses. Pellino-1 ablation in THP-1 cells impaired induction of myeloid differentiation primary response protein (MyD88), and Toll-IL-1R domain-containing adapter inducing IFN-ß (TRIF)-dependent cytokine genes in response to TLR4 and TLR2 agonists and heat-killed Escherichia coli and Staphylococcus aureus, whereas only weakly affecting phagocytosis of heat-killed bacteria. Co-expressed Pellino-1 potentiated NF-κB activation driven by transfected MyD88, TRIF, IRAK1, TBK1, TGF-ß-activated kinase (TAK) 1, and TNFR-associated factor 6, whereas not affecting p65-induced responses. Mechanistically, Pellino-1 increased LPS-driven K63-linked polyubiquitination of IRAK1, TBK1, TAK1, and phosphorylation of TBK1 and IFN regulatory factor 3. These results reveal a novel mechanism by which endotoxin tolerance re-programs TLR4 signaling via suppression of Pellino-1, a positive regulator of MyD88- and TRIF-dependent signaling that promotes K63-linked polyubiquitination of IRAK1, TBK1, and TAK1.


Assuntos
Proteínas Nucleares/fisiologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Animais , Células HEK293 , Humanos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase Quinases/metabolismo , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Monócitos/metabolismo , NF-kappa B/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/metabolismo , Ativação Transcricional/imunologia , Ubiquitinação
12.
Immunol Rev ; 266(1): 109-22, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26085210

RESUMO

Pellinos are a family of E3 ubiquitin ligases discovered for their role in catalyzing K63-linked polyubiquitination of Pelle, an interleukin-1 (IL-1) receptor-associated kinase homolog in the Drosophila Toll pathway. Subsequent studies have revealed the central and non-redundant roles of mammalian Pellino-1, Pellino-2, and Pelino-3 in signaling pathways emanating from IL-1 receptors, Toll-like receptors, NOD-like receptors, T- and B-cell receptors. While Pellinos ability to interact with many signaling intermediates suggested their scaffolding roles, recent findings in mice expressing ligase-inactive Pellinos demonstrated the importance of Pellino ubiquitin ligase activity. Cell-specific functions of Pellinos have emerged, e.g. Pellino-1 being a negative regulator in T lymphocytes and a positive regulator in myeloid cells, and details of molecular regulation of receptor signaling by various members of the Pellino family have been revealed. In this review, we summarize current information about Pellino-mediated regulation of signaling by pattern recognition receptors, T-cell and B-cell receptors and tumor necrosis factor receptors, and discuss Pellinos roles in sepsis and infectious diseases, as well as in autoimmune, inflammatory, and allergic disorders. We also provide our perspective on the potential of targeting Pellinos with peptide- or small molecule-based drug compounds as a new therapeutic approach for septic shock and autoimmune pathologies.


Assuntos
Linfócitos B/imunologia , Imunoterapia , Proteínas Nucleares/metabolismo , Linfócitos T/imunologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Humanos , Terapia de Alvo Molecular , Especificidade de Órgãos , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais , Ubiquitinação
13.
J Leukoc Biol ; 94(2): 291-300, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23695305

RESUMO

Prior exposure to LPS induces "endotoxin tolerance" that reprograms TLR4 responses to subsequent LPS challenge by altering expression of inflammatory mediators. Endotoxin tolerance is thought to limit the excessive cytokine storm and prevent tissue damage during sepsis but renders the host immunocompromised and susceptible to secondary infections. Tolerance initiated via one TLR can affect cellular responses to challenge via the same TLR ("homotolerance") or through different TLRs ("heterotolerance"). IRAK4, an essential component of the MyD88-dependent pathway, functions as a kinase and an adapter, activating subsets of divergent signaling pathways. In this study, we addressed mechanistically the role of IRAK4 kinase activity in TLR4- and TLR2-induced tolerance using macrophages from WT versus IRAK4(KDKI) mice. Whereas IRAK4 kinase deficiency decreased LPS signaling, it did not prevent endotoxin tolerance, as endotoxin pretreatment of WT and IRAK4(KDKI) macrophages inhibited LPS-induced MAPK phosphorylation, degradation of IκB-α and recruitment of p65 to the TNF-α promoter, expression of proinflammatory cytokines, and increased levels of A20 and IRAK-M. Pretreatment of WT macrophages with Pam3Cys, a TLR2-TLR1 agonist, ablated p-p38 and p-JNK in response to challenge with Pam3Cys and LPS, whereas IRAK4(KDKI) macrophages exhibited attenuated TLR2-elicited homo- and heterotolerance at the level of MAPK activation. Thus, IRAK4 kinase activity is not required for the induction of endotoxin tolerance but contributes significantly to TLR2-elicited homo- and heterotolerance.


Assuntos
Endotoxinas/imunologia , Quinases Associadas a Receptores de Interleucina-1/fisiologia , Lipopolissacarídeos/toxicidade , Macrófagos Peritoneais/imunologia , Receptor 2 Toll-Like/fisiologia , Receptor 4 Toll-Like/fisiologia , Animais , Cisteína Endopeptidases , Citocinas/biossíntese , Citocinas/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Tolerância a Medicamentos , Endotoxinas/toxicidade , Ativação Enzimática , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Quinases Associadas a Receptores de Interleucina-1/biossíntese , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipoproteínas/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/fisiologia , NF-kappa B/metabolismo , Doenças da Imunodeficiência Primária , Transdução de Sinais , Receptor 2 Toll-Like/agonistas , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
14.
J Interferon Cytokine Res ; 33(9): 467-84, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23675778

RESUMO

Toll-like receptors (TLRs) are germ-line-encoded innate immune sensors that recognize conserved microbial structures and host alarmins and signal expression of MHC proteins, costimulatory molecules, and inflammatory mediators by macrophages, neutrophils, dendritic cells, and other cell types. These processes activate immediate and early mechanisms of innate host defense, as well as initiate and orchestrate adaptive immune responses. Several single-nucleotide polymorphisms (SNPs) within the TLR genes have been associated with altered susceptibility to infectious, inflammatory, and allergic diseases, and have been found to play a role in tumorigenesis. Critical advances in our understanding of innate immune functions and genome-wide association studies (GWAS) have uncovered complex interactions of genetic polymorphisms within TLRs and environmental factors. However, conclusions obtained in the course of such analyses are restricted by limited power of many studies that is likely to explain controversial findings. Further, linkages to certain ethnic backgrounds, gender, and the presence of multigenic effects further complicate the interpretations of how the TLR SNPs affect immune responses. For many TLRs, the molecular mechanisms by which SNPs impact receptor functions remain unknown. In this review, I have summarized current knowledge about the TLR polymorphisms, their impact on TLR signaling, and associations with various inflammatory, infectious, allergic diseases and cancers, and discussed the directions of future scientific research.


Assuntos
Doenças Transmissíveis/genética , Hipersensibilidade/genética , Inflamação/genética , Neoplasias/genética , Polimorfismo Genético , Receptores Toll-Like/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Mutação , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais , Receptores Toll-Like/metabolismo
15.
J Biol Chem ; 287(45): 38327-37, 2012 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-22992740

RESUMO

The R753Q polymorphism in the Toll-IL-1 receptor domain of Toll-like receptor 2 (TLR2) has been linked to increased incidence of tuberculosis and other infectious diseases, but the mechanisms by which it affects TLR2 functions are unclear. Here, we studied the impact of the R753Q polymorphism on TLR2 expression, hetero-dimerization with TLR6, tyrosine phosphorylation, and recruitment of myeloid differentiation primary response protein (MyD) 88 and MyD88 adapter-like (Mal). Complementation of HEK293 cells with transfected WT or R753Q TLR2 revealed their comparable total levels and only minimal changes in cell surface expression of the mutant species. Notably, even a 100-fold increase in amounts of transfected R753Q TLR2 versus WT variant did not overcome the compromised ability of the mutant TLR2 to activate nuclear factor κB (NF-κB), indicating that a minimal decrease in cell surface levels of the R753Q TLR2 cannot account for the signaling deficiency. Molecular modeling studies suggested that the R753Q mutation changes the electrostatic potential of the DD loop and results in a discrete movement of the residues critical for protein-protein interactions. Confirming these predictions, biochemical assays demonstrated that R753Q TLR2 exhibits deficient agonist-induced tyrosine phosphorylation, hetero-dimerization with TLR6, and recruitment of Mal and MyD88. These proximal signaling deficiencies correlated with impaired capacities of the R753Q TLR2 to mediate p38 phosphorylation, NF-κB activation, and induction of IL-8 mRNA in transfected HEK293 cells challenged with inactivated Mycobacterium tuberculosis or mycobacterial components. Thus, the R753Q polymorphism renders TLR2 signaling-incompetent by impairing its tyrosine phosphorylation, dimerization with TLR6, and recruitment of Mal and MyD88.


Assuntos
Fator 88 de Diferenciação Mieloide/metabolismo , Polimorfismo Genético , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 6 Toll-Like/metabolismo , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/imunologia , Animais , Células Cultivadas , Expressão Gênica , Células HEK293 , Humanos , Immunoblotting , Interleucina-8/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Knockout , Microscopia Confocal , Modelos Moleculares , Mycobacterium tuberculosis/imunologia , Fator 88 de Diferenciação Mieloide/genética , NF-kappa B/metabolismo , Fosforilação , Multimerização Proteica , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/química , Receptor 6 Toll-Like/química , Receptor 6 Toll-Like/genética , Tirosina/metabolismo
16.
J Immunol ; 188(9): 4506-15, 2012 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-22474023

RESUMO

Asp(299)Gly (D299G) and, to a lesser extent, Thr(399)Ile (T399I) TLR4 polymorphisms have been associated with gram-negative sepsis and other infectious diseases, but the mechanisms by which they affect TLR4 signaling are unclear. In this study, we determined the impact of the D299G and T399I polymorphisms on TLR4 expression, interactions with myeloid differentiation factor 2 (MD2), LPS binding, and LPS-mediated activation of the MyD88- and Toll/IL-1R resistance domain-containing adapter inducing IFN-ß (TRIF) signaling pathways. Complementation of human embryonic kidney 293/CD14/MD2 transfectants with wild-type (WT) or mutant yellow fluorescent protein-tagged TLR4 variants revealed comparable total TLR4 expression, TLR4-MD2 interactions, and LPS binding. FACS analyses with anti-TLR4 Ab showed only minimal changes in the cell-surface levels of the D299G TLR4. Cells transfected with D299G TLR4 exhibited impaired LPS-induced phosphorylation of p38 and TANK-binding kinase 1, activation of NF-κB and IFN regulatory factor 3, and induction of IL-8 and IFN-ß mRNA, whereas T399I TLR4 did not cause statistically significant inhibition. In contrast to WT TLR4, expression of the D299G mutants in TLR4(-/-) mouse macrophages failed to elicit LPS-mediated induction of TNF-α and IFN-ß mRNA. Coimmunoprecipitation revealed diminished LPS-driven interaction of MyD88 and TRIF with the D299G TLR4 species, in contrast to robust adapter recruitment exhibited by WT TLR4. Thus, the D299G polymorphism compromises recruitment of MyD88 and TRIF to TLR4 without affecting TLR4 expression, TLR4-MD2 interaction, or LPS binding, suggesting that it interferes with TLR4 dimerization and assembly of intracellular docking platforms for adapter recruitment.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/imunologia , Macrófagos/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Polimorfismo Genético/imunologia , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Células Cultivadas , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Lipopolissacarídeos/farmacologia , Antígeno 96 de Linfócito/genética , Antígeno 96 de Linfócito/imunologia , Antígeno 96 de Linfócito/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/genética , NF-kappa B/imunologia , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Fosforilação/imunologia , Proteínas Quinases/genética , Proteínas Quinases/imunologia , Proteínas Quinases/metabolismo , Multimerização Proteica/genética , Multimerização Proteica/imunologia , Transdução de Sinais/genética , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
17.
J Virol ; 86(13): 7216-26, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22532679

RESUMO

Lymphocytic choriomeningitis virus (LCMV), the prototype arenavirus, and Lassa virus (LASV), the causative agent of Lassa fever (LF), have extensive strain diversity and significant variations in pathogenicity for humans and experimental animals. The WE strain of LCMV (LCMV-WE), but not the Armstrong (Arm) strain, induces a fatal LF-like disease in rhesus macaques. We also demonstrated that LASV infection of human macrophages and endothelial cells resulted in reduced levels of proinflammatory cytokines. Here we have shown that cells infected with LASV or with LCMV-WE suppressed Toll-like receptor 2 (TLR2)-dependent proinflammatory cytokine responses. The persisting isolate LCMV clone 13 (CL13) also failed to stimulate interleukin-6 (IL-6) in macrophages. In contrast, nonpathogenic Mopeia virus, which is a genetic relative of LASV and LCMV-Arm induced robust responses that were TLR2/Mal dependent, required virus replication, and were enhanced by CD14. Superinfection experiments demonstrated that the WE strain of LCMV inhibited the Arm-mediated IL-8 response during the early stage of infection. In cells transfected with the NF-κB-luciferase reporter, infection with LCMV-Arm resulted in the induction of NF-κB, but cells infected with LCMV-WE and CL13 did not. These results suggest that pathogenic arenaviruses suppress NF-κB-mediated proinflammatory cytokine responses in infected cells.


Assuntos
Citocinas/antagonistas & inibidores , Evasão da Resposta Imune , Vírus Lassa/patogenicidade , Vírus da Coriomeningite Linfocítica/patogenicidade , Proteínas da Mielina/antagonistas & inibidores , Proteolipídeos/antagonistas & inibidores , Receptor 2 Toll-Like/antagonistas & inibidores , Animais , Linhagem Celular , Humanos , Vírus Lassa/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Proteínas de Membrana Transportadoras/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas da Mielina/imunologia , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina , NF-kappa B/antagonistas & inibidores , NF-kappa B/imunologia , Proteolipídeos/imunologia , Receptor 2 Toll-Like/imunologia
18.
J Leukoc Biol ; 90(6): 1141-8, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21934070

RESUMO

TLRs mediate host defense against microbial pathogens by eliciting production of inflammatory mediators and activating expression of MHC, adhesion, and costimulatory molecules. Endotoxin tolerance limits excessive TLR-driven inflammation during sepsis and reprograms macrophage responses to LPS, decreasing expression of proinflammatory cytokines without inhibiting anti-inflammatory and antimicrobial mediators. Molecular mechanisms of reprogramming of TLR4 signaling upon in vivo induction of endotoxin tolerance are incompletely understood. We used an in vivo model of endotoxin tolerance, whereby C57BL/6 mice were i.p.-inoculated with LPS or PBS, followed by in vitro challenge of peritoneal or splenic macrophages with LPS to examine activation of IRAK4 and expression of negative regulatory molecules. Administration of LPS in vivo-induced endotoxin tolerance in peritoneal and splenic macrophages, as evidenced by decreased degradation of IκBα, suppressed phosphorylation of p38 and reduced expression of TNF-α, IL-6, and KC mRNA upon in vitro LPS challenge. Macrophages from control and endotoxin-tolerant mice exhibited comparable TLR4 mRNA levels and similar expression of IL-1RA and IL-10 genes. Endotoxin tolerization in vivo blocked TLR4-driven IRAK4 phosphorylation and activation in macrophages, while increasing expression of IRAK-M, SHIP-1, A20 mRNA, and A20 protein. Thus, induction of endotoxin tolerance in vivo inhibits expression of proinflammatory mediators via impaired activation of IRAK4, p38, and NF-κB and increases expression of negative regulators of TLR4 pathways.


Assuntos
Cisteína Endopeptidases/biossíntese , Regulação para Baixo/imunologia , Endotoxinas/antagonistas & inibidores , Tolerância Imunológica , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Quinases Associadas a Receptores de Interleucina-1/biossíntese , Monoéster Fosfórico Hidrolases/biossíntese , Regulação para Cima/imunologia , Animais , Células Cultivadas , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Endotoxinas/administração & dosagem , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Inositol Polifosfato 5-Fosfatases , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/imunologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa
19.
J Biol Chem ; 286(10): 7905-7916, 2011 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-21220427

RESUMO

Endotoxin tolerance reprograms Toll-like receptor 4 responses by impairing LPS-elicited production of pro-inflammatory cytokines without inhibiting expression of anti-inflammatory or anti-microbial mediators. In septic patients, Toll-like receptor tolerance is thought to underlie decreased pro-inflammatory cytokine expression in response to LPS and increased incidence of microbial infections. The impact of endotoxin tolerance on recruitment, post-translational modifications and signalosome assembly of IL-1 receptor-associated kinase (IRAK) 4, IRAK1, TNF receptor-associated factor (TRAF) 6, TGF-ß-activated kinase (TAK) 1, and IκB kinase (IKK) γ is largely unknown. We report that endotoxin tolerization of THP1 cells and human monocytes impairs LPS-mediated receptor recruitment and activation of IRAK4, ablates K63-linked polyubiquitination of IRAK1 and TRAF6, compromises assembly of IRAK1-TRAF6 and IRAK1-IKKγ platforms, and inhibits TAK1 activation. Deficiencies in these signaling events in LPS-tolerant cells coincided with increased expression of A20, an essential deubiquitination enzyme, and sustained A20-IRAK1 associations. Overexpression of A20 inhibited LPS-induced activation of NF-κB and ablated NF-κB reporter activation driven by ectopic expression of MyD88, IRAK1, IRAK2, TRAF6, and TAK1/TAB1, while not affecting the responses induced by IKKß and p65. A20 shRNA knockdown abolished LPS tolerization of THP1 cells, mechanistically linking A20 and endotoxin tolerance. Thus, deficient LPS-induced activation of IRAK4 and TAK1, K63-linked polyubiquitination of IRAK1 and TRAF6, and disrupted IRAK1-TRAF6 and IRAK1-IKKγ assembly associated with increased A20 expression and A20-IRAK1 interactions are new determinants of endotoxin tolerance.


Assuntos
Resistência a Medicamentos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Quinase I-kappa B/metabolismo , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Lipopolissacarídeos/farmacologia , MAP Quinase Quinase Quinases/metabolismo , Monócitos/metabolismo , Proteínas Nucleares/biossíntese , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitinação/efeitos dos fármacos , Linhagem Celular , Proteínas de Ligação a DNA , Humanos , Quinase I-kappa B/genética , Quinases Associadas a Receptores de Interleucina-1/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , MAP Quinase Quinase Quinases/genética , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Proteínas Nucleares/genética , Fator 6 Associado a Receptor de TNF/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa
20.
Innate Immun ; 17(4): 375-87, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20682587

RESUMO

Macrophages and natural killer (NK) cells are important antitumor effectors by virtue of their ability to produce cytokines, chemokines and interferons (IFNs) and to mediate tumor cytotoxicity. Little is known about the impact of Toll-like receptor (TLR) and nucleotide binding and oligomerization domain (NOD)-like receptor (NLR) pathways on NK cell functions, and the role of TLRs and NLRs in macrophage activation is incompletely understood. In this study, we examined the capacities of expressed TLRs and NLRs to elicit cytokine production in human NK cells and THP1 macrophages, and to activate NK cytotoxicity against the squamous cell carcinoma of head and neck cell line Tu167 and erythroleukemia K562 cells. We found that NK cells express high levels of NOD2, NLRP3, TLR3, TLR7, and TLR9, while NOD1 was expressed at low levels. All tested NLR and TLR agonists potentiated NK cytotoxicity against Tu167 cells, whereas only poly (I:C) increased NK cytotoxicity against K562 cells. Poly (I:C) and Escherichia coli RNA markedly up-regulated TNF-α and IFN-γ expression in the NK92 cell line and human CD56(+)CD3(-) primary NK cells. High levels of NOD2, TLR7 and TLR9 proteins were observed in human THP1 cells, followed by TLR3, NOD1, and NLRP3. Stimulation of NLRP3 with E. coli RNA led to the highest induction of TNF-α, IL-6, IL-12p40, RANTES and IFN-ß, whereas TLR7, TLR3, TLR9, NOD1 and NOD2 agonists had lower effects. Our data reveal involvement of TLRs and NLRs in potentiation of antitumor cytotoxicity and cytokine-producing activities of human NK cells and macrophages.


Assuntos
Carcinoma de Células Escamosas/imunologia , Escherichia coli/imunologia , Células Matadoras Naturais/metabolismo , Macrófagos/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Escherichia coli/genética , Humanos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Células K562 , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Poli I-C/farmacologia , RNA Bacteriano/farmacologia , Receptores Toll-Like/agonistas
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