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1.
J Clin Invest ; 131(17)2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34623328

RESUMO

Properly balancing microbial responses by the innate immune system through pattern recognition receptors (PRRs) is critical for intestinal immune homeostasis. Ring finger protein 186 (RNF186) genetic variants are associated with inflammatory bowel disease (IBD). However, functions for the E3 ubiquitin ligase RNF186 are incompletely defined. We found that upon stimulation of the PRR nucleotide-binding oligomerization domain containing 2 (NOD2) in human macrophages, RNF186 localized to the ER, formed a complex with ER stress sensors, ubiquitinated the ER stress sensor activating transcription factor 6 (ATF6), and promoted the unfolded protein response (UPR). These events, in turn, led to downstream signaling, cytokine secretion, and antimicrobial pathway induction. Importantly, RNF186-mediated ubiquitination of K152 on ATF6 was required for these outcomes, highlighting a key role for ATF6 ubiquitination in PRR-initiated functions. Human macrophages transfected with the rare RNF186-A64T IBD risk variant and macrophages from common rs6426833 RNF186 IBD risk carriers demonstrated reduced NOD2-induced outcomes, which were restored by rescuing UPR signaling. Mice deficient in RNF186 or ATF6 demonstrated a reduced UPR in colonic tissues, increased weight loss, and less effective clearance of bacteria with dextran sodium sulfate-induced injury and upon oral challenge with Salmonella Typhimurium. Therefore, we identified that RNF186 was required for PRR-induced, UPR-associated signaling leading to key macrophage functions; defined that RNF186-mediated ubiquitination of ATF6 was essential for these functions; and elucidated how RNF186 IBD risk variants modulated these outcomes.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Fator 6 Ativador da Transcrição/química , Fator 6 Ativador da Transcrição/deficiência , Fator 6 Ativador da Transcrição/genética , Animais , Estresse do Retículo Endoplasmático , Variação Genética , Interações entre Hospedeiro e Microrganismos , Humanos , Imunidade Inata , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Adaptadora de Sinalização NOD2/metabolismo , Receptores de Reconhecimento de Padrão/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Fatores de Risco , Transdução de Sinais , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
2.
Proc Natl Acad Sci U S A ; 118(32)2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34353900

RESUMO

Balancing microbial-induced cytokines and microbial clearance is critical at mucosal sites such as the intestine. How the inflammatory bowel disease (IBD)-associated gene RNF186 regulates this balance is unclear. We found that macrophages from IBD-risk rs6426833 carriers in the RNF186 region showed reduced cytokines to stimulation through multiple pattern recognition receptors (PRRs). Upon stimulation of PRRs, the E3-ubiquitin ligase RNF186 promoted ubiquitination of signaling complex molecules shared across PRRs and those unique to select PRRs. Furthermore, RNF186 was required for PRR-initiated signaling complex assembly and downstream signaling. RNF186, along with its intact E3-ubiquitin ligase activity, was required for optimal PRR-induced antimicrobial reactive oxygen species, reactive nitrogen species, and autophagy pathways and intracellular bacterial clearance in human macrophages and for bacterial clearance in intestinal myeloid cells. Cells transfected with the rare RNF186-A64T IBD-risk variant and macrophages from common rs6426833 RNF186 IBD-risk carriers demonstrated a reduction in these RNF186-dependent outcomes. These studies identify mechanisms through which RNF186 regulates innate immunity and show that RNF186 IBD-risk variants demonstrate a loss of function in PRR-initiated outcomes.


Assuntos
Doenças Inflamatórias Intestinais/patologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Receptores de Reconhecimento de Padrão/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Citocinas/metabolismo , Humanos , Imunidade Inata , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/microbiologia , Intestinos/citologia , Macrófagos/patologia , Células Mieloides/metabolismo , Células Mieloides/patologia , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Polimorfismo de Nucleotídeo Único , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
3.
Cell Mol Gastroenterol Hepatol ; 11(1): 249-272, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32827707

RESUMO

BACKGROUND & AIMS: TNFSF15 genetic variants leading to increased TNF superfamily member 15 (TNFSF15) expression confer risk for inflammatory bowel disease (IBD), and TNFSF15 is being explored as a therapeutic target in IBD patients. Although the focus for TNFSF15-mediated inflammatory outcomes has been predominantly on its action on T cells, TNFSF15 also promotes inflammatory outcomes in human macrophages. Given the critical role for macrophages in bacterial clearance, we hypothesized that TNFSF15 promotes antimicrobial pathways in human macrophages and that macrophages from TNFSF15 IBD risk carriers with higher TNFSF15 expression have an advantage in these antimicrobial outcomes. METHODS: We analyzed protein expression, signaling, bacterial uptake, and intracellular bacterial clearance in human monocyte-derived macrophages through flow cytometry, enzyme-linked immunosorbent assay, and gentamicin protection. RESULTS: Autocrine/paracrine TNFSF15 interactions with death receptor 3 (DR3) were required for optimal levels of pattern-recognition-receptor (PRR)-induced bacterial clearance in human macrophages. TNFSF15 induced pyruvate dehydrogenase kinase 1-dependent bacterial uptake and promoted intracellular bacterial clearance through reactive oxygen species, nitric oxide synthase 2, and autophagy up-regulation. The TNFSF15-initiated TNF receptor-associated factor 2/receptor-interacting protein kinase 1/RIP3 pathway was required for mitogen-activated protein kinase and nuclear factor-κB activation, and, in turn, induction of each of the antimicrobial pathways; the TNFSF15-initiated Fas-associated protein with death domain/mucosa-associated lymphoid tissue lymphoma translocation protein 1/caspase-8 pathway played a less prominent role in antimicrobial functions, despite its key role in TNFSF15-induced cytokine secretion. Complementation of signaling pathways or antimicrobial pathways restored bacterial uptake and clearance in PRR-stimulated macrophages where TNFSF15:DR3 interactions were inhibited. Monocyte-derived macrophages from high TNFSF15-expressing rs6478108 TT IBD risk carriers in the TNFSF15 region showed increased levels of the identified antimicrobial pathways. CONCLUSIONS: We identify that autocrine/paracrine TNFSF15 is required for optimal PRR-enhanced antimicrobial pathways in macrophages, define mechanisms regulating TNFSF15-dependent bacterial clearance, and determine how the TNFSF15 IBD risk genotype modulates these outcomes.


Assuntos
Doenças Inflamatórias Intestinais/imunologia , Macrófagos/imunologia , Membro 25 de Receptores de Fatores de Necrose Tumoral/metabolismo , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Animais , Comunicação Autócrina/imunologia , Células Cultivadas , Enterococcus faecalis/imunologia , Enterococcus faecalis/isolamento & purificação , Escherichia coli/imunologia , Escherichia coli/isolamento & purificação , Predisposição Genética para Doença , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/microbiologia , Macrófagos/metabolismo , Camundongos , Comunicação Parácrina/imunologia , Cultura Primária de Células , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética
4.
J Immunol ; 205(5): 1406-1418, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32661180

RESUMO

STAT proteins can regulate both pro- and anti-inflammatory cytokine signaling. Therefore, identifying consequences of modulating expression of a given STAT is ultimately critical for determining its potential as a therapeutic target and for defining the mechanisms through which immune-mediated disease variants in STAT genes contribute to disease pathogenesis. Genetic variants in the STAT1/STAT4 region are associated with multiple immune-mediated diseases, including inflammatory bowel disease (IBD). These diseases are characterized by dysregulated cytokine secretion in response to pattern-recognition receptor (PRR) stimulation. We found that the common IBD-associated rs1517352 C risk allele increased both STAT1 and STAT4 expression in human monocyte-derived macrophages (MDMs). We therefore hypothesized that the STAT1/STAT4 variant might regulate PRR-initiated responses in a complementary and cooperative manner because of the important role of autocrine/paracrine cytokines in modulating PRR-initiated signaling. STAT1 and STAT4 were required for PRR- and live bacterial-induced secretion of multiple cytokines. These outcomes were particularly dependent on PRR-initiated autocrine/paracrine IL-12-induced STAT4 activation to generate IFN-γ, with autocrine IFN-γ then signaling through STAT1. STAT1 and STAT4 also promoted bacterial-induced cytokines in intestinal myeloid cells and PRR-enhanced antimicrobial pathways in MDMs. Importantly, MDMs from rs1517352 C IBD risk allele carriers demonstrated increased TLR4-, IFN-γ- and IL-12-induced STAT1 and STAT4 phosphorylation and cytokine secretion and increased TLR4-enhanced antimicrobial pathways. Taken together, STAT1 and STAT4 expression is coregulated by a shared genetic region, and STAT1 /STAT4-immune disease-associated variants modulate IFN-γ- and IL-12-associated outcomes, and in turn, PRR-induced outcomes, highlighting that these genes cooperate to regulate pathways relevant to disease pathogenesis.


Assuntos
Predisposição Genética para Doença/genética , Variação Genética/imunologia , Macrófagos/metabolismo , Receptores de Reconhecimento de Padrão/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT4/genética , Alelos , Linhagem Celular , Citocinas/genética , Expressão Gênica/genética , Humanos , Doenças Inflamatórias Intestinais/genética , Interferon gama/genética , Interleucina-12/genética , Células Mieloides/metabolismo , Fosforilação/genética , Transdução de Sinais/genética
5.
J Immunol ; 205(4): 1024-1038, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32690658

RESUMO

Common IRF5 genetic risk variants associated with multiple immune-mediated diseases are a major determinant of interindividual variability in pattern-recognition receptor (PRR)-induced cytokines in myeloid cells. However, how myeloid cell-intrinsic IRF5 regulates the multiple distinct checkpoints mediating T cell outcomes in vivo and IRF5-dependent mechanisms contributing to these distinct checkpoints are not well defined. Using an in vivo Ag-specific adoptive T cell transfer approach into Irf5-/- mice, we found that T cell-extrinsic IRF5 regulated T cell outcomes at multiple critical checkpoints, including chemokine-mediated T cell trafficking into lymph nodes and PDK1-dependent soluble Ag uptake, costimulatory molecule upregulation, and secretion of Th1 (IL-12)- and Th17 (IL-23, IL-1ß, and IL-6)-conditioning cytokines by myeloid cells, which then cross-regulated Th2 and regulatory T cells. IRF5 was required for PRR-induced MAPK and NF-κB activation, which, in turn, regulated these key outcomes in myeloid cells. Importantly, mice with IRF5 deleted from myeloid cells demonstrated T cell outcomes similar to those observed in Irf5-/- mice. Complementation of IL-12 and IL-23 was able to restore T cell differentiation both in vitro and in vivo in the context of myeloid cell-deficient IRF5. Finally, human monocyte-derived dendritic cells from IRF5 disease-associated genetic risk carriers leading to increased IRF5 expression demonstrated increased Ag uptake and increased PRR-induced costimulatory molecule expression and chemokine and cytokine secretion compared with monocyte-derived dendritic cells from low-expressing IRF5 genetic variant carriers. These data establish that myeloid cell-intrinsic IRF5 regulates multiple distinct checkpoints in T cell activation and differentiation and that these are modulated by IRF5 disease risk variants.


Assuntos
Fatores Reguladores de Interferon/metabolismo , Células Mieloides/metabolismo , Linfócitos T/metabolismo , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Regulação da Expressão Gênica/imunologia , Humanos , Fatores Reguladores de Interferon/imunologia , Linfonodos/imunologia , Linfonodos/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Monócitos/metabolismo , Células Mieloides/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Regulação para Cima/imunologia
6.
Gut ; 69(2): 264-273, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31097538

RESUMO

OBJECTIVE: The interleukin (IL)23 pathway contributes to IBD pathogenesis and is being actively studied as a therapeutic target in patients with IBD. Unexpected outcomes in these therapeutic trials have highlighted the importance of understanding the cell types and mechanisms through which IL23 regulates immune outcomes. How IL23 regulates macrophage outcomes and the consequences of the IL23R R381Q IBD-protective variant on macrophages are not well defined; macrophages are key players in IBD pathogenesis and inflammation. DESIGN: We analysed protein and RNA expression, signalling and localisation in human monocyte-derived macrophages (MDMs) through western blot, ELISA, real-time PCR, flow cytometry, immunoprecipitation and microscopy. RESULTS: IL23R was critical for optimal levels of pattern-recognition receptor (PRR)-induced signalling and cytokines in human MDMs. In contrast to the coreceptor IL12Rß1, IL23 induced dynamic IL23R cell surface regulation and this required clathrin and dynamin-mediated endocytosis and endocytic recycling-dependent pathways; these pathways were essential for IL23R-mediated outcomes. The IBD-protective IL23R R381Q variant showed distinct outcomes. Relative to IL23R R381, HeLa cells expressing IL23R Q381 showed decreased IL23R recycling and reduced assembly of IL23R Q381 with Janus kinase/signal transducer and activator of transcription pathway members. In MDMs from IL23R Q381 carriers, IL23R accumulated in late endosomes and lysosomes on IL23 treatment and cells demonstrated decreased IL23R- and PRR-induced signalling and cytokines relative to IL23R R381 MDMs. CONCLUSION: Macrophage-mediated inflammatory pathways are key contributors to IBD pathogenesis, and we identify an autocrine/paracrine IL23 requirement in PRR-initiated human macrophage outcomes and in human intestinal myeloid cells, establish that IL23R undergoes ligand-induced recycling, define mechanisms regulating IL23R-induced signalling and determine how the IBD-protective IL23R R381Q variant modulates these processes.


Assuntos
Citocinas/imunologia , Doenças Inflamatórias Intestinais/imunologia , Macrófagos/imunologia , Receptores de Interleucina/imunologia , Comunicação Autócrina/imunologia , Endocitose/imunologia , Endossomos/imunologia , Variação Genética , Humanos , Imunidade Inata/genética , Imunidade Inata/imunologia , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/prevenção & controle , Interleucina-23/imunologia , Janus Quinase 2/metabolismo , Comunicação Parácrina/imunologia , Receptores de Interleucina/genética , Receptores de Reconhecimento de Padrão/imunologia , Transdução de Sinais/imunologia
7.
Cell Rep ; 29(13): 4525-4539.e4, 2019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31875558

RESUMO

LACC1 genetic variants are associated with multiple immune-mediated diseases. However, laccase domain containing-1 (LACC1) functions are incompletely defined. We find that upon stimulation of the pattern-recognition receptor (PRR) NOD2, LACC1 localizes to the endoplasmic reticulum (ER) and forms a complex with ER-stress sensors. All three ER-stress branches, PERK, IRE1α, and ATF6, are required for NOD2-induced signaling, cytokines, and antimicrobial pathways in human macrophages. LACC1, and its localization to the ER, is required for these outcomes. Relative to wild-type (WT) LACC1, transfection of the common Val254 and rare Arg284 immune-mediated disease-risk LACC1 variants into HeLa cells and macrophages, as well as macrophages from LACC1 Val254 carriers, shows reduced NOD2-induced ER stress-associated outcomes; these downstream outcomes are restored by rescuing ER stress. Therefore, we identify ER stress to be essential in PRR-induced outcomes in macrophages, define a critical role for LACC1 in these ER stress-dependent events, and elucidate how LACC1 disease-risk variants mediate these outcomes.


Assuntos
Estresse do Retículo Endoplasmático/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Macrófagos/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Fator 6 Ativador da Transcrição/genética , Fator 6 Ativador da Transcrição/imunologia , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/microbiologia , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Endorribonucleases/imunologia , Enterococcus faecalis/crescimento & desenvolvimento , Enterococcus faecalis/imunologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/imunologia , Regulação da Expressão Gênica , Células HeLa , Interações Hospedeiro-Patógeno/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Macrófagos/microbiologia , Proteína Adaptadora de Sinalização NOD2/genética , Fagocitose , Cultura Primária de Células , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Risco , Transdução de Sinais , eIF-2 Quinase/genética , eIF-2 Quinase/imunologia
8.
J Immunol ; 203(12): 3325-3338, 2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31732533

RESUMO

Genetic variants in the STAT3/STAT5A/STAT5B region are associated with immune-mediated diseases, including inflammatory bowel disease (IBD). However, how STAT3 and STAT5 regulate the critical balance between pro- and anti-inflammatory cytokines and how common disease-associated genetic variants (e.g., rs12942547) in the region modulate this balance are incompletely understood. We found that upon pattern-recognition receptor (PRR) stimulation of human monocyte-derived macrophages (MDMs), decreasing STAT3, STAT5a, and STAT5b expression led to a progressive decrease in anti-inflammatory cytokines, whereas proinflammatory cytokines initially decreased but then increased when STAT3 or STAT5 expression fell below a critical threshold. Mechanisms regulating STAT3- and STAT5-dependent inflammatory cytokine outcomes included negative feedback from autocrine/paracrine IL-10, TGF-ß, IL-4, IL-13, IL-22, and TSLP secretion and SOCS1/SOCS2/SOCS3 induction. MDMs from rs12942547 AA disease-risk carriers demonstrated increased STAT3, STAT5a, and STAT5b expression and increased PRR-induced STAT3 and STAT5 phosphorylation relative to GG MDMs. Both pro- and anti-inflammatory cytokine secretion was decreased in MDMs from GG carriers, as STAT3, STAT5a, and STAT5b expression was above the threshold for reciprocal regulation of these cytokines. Taken together, we identify that the threshold of STAT3, STAT5a, and STAT5b expression determines if PRR-induced proinflammatory cytokines are increased or decreased, define mechanisms for this reciprocal regulation, and elucidate consequences for disease variants in the STAT3/STAT5A/STAT5B region, indicating that considering signaling thresholds and targeting specific cell types might be beneficial when evaluating therapeutic interventions in this pathway.


Assuntos
Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Suscetibilidade a Doenças , Expressão Gênica , Humanos , Doenças Inflamatórias Intestinais/etiologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Fosforilação , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT5/genética
9.
J Immunol ; 202(11): 3297-3308, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31028123

RESUMO

Intestinal tissues are continuously exposed to microbial products that stimulate pattern-recognition receptors (PRRs). Ongoing PRR stimulation can confer epigenetic changes in macrophages, which can then regulate subsequent immune outcomes and adaptation to the local environment. Mechanisms leading to these changes are incompletely understood. We found that short-term stimulation of the PRR NOD2 in primary human monocyte-derived macrophages resulted in increased H3 and H4 acetylation of cytokine promoters, consistent with the increased cytokine secretion observed. However, with prolonged NOD2 stimulation, both the acetylation and cytokine secretion were dramatically decreased. Chronic NOD2 stimulation upregulated the transcription factors Twist1 and Twist2, which bound to the promoters of the histone deacetylases HDAC1 and HDAC3 and induced HDAC1 and HDAC3 expression. HDAC1 and HDAC3 then mediated histone deacetylation at cytokine promoters and, in turn, cytokine downregulation under these conditions. Similar regulation was observed upon chronic stimulation of multiple PRRs. Consistent with the chronic microbial exposure in the intestinal environment, TWIST1, TWIST2, HDAC1, and HDAC3 were upregulated in human intestinal relative to peripheral macrophages. Importantly, complementing HDAC1 and HDAC3 in Twist1/Twist2-deficient monocyte-derived macrophages restored the reduced histone acetylation on cytokine promoters and the decreased cytokine secretion with chronic NOD2 stimulation. Taken together, we identify mechanisms wherein Twist1 and Twist2 promote chromatin modifications, resulting in macrophage instruction and adaptation to conditions in the intestinal microenvironment.


Assuntos
Macrófagos/imunologia , Proteína 1 Relacionada a Twist/metabolismo , Proteína 2 Relacionada a Twist/metabolismo , Acetilação , Animais , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Regiões Promotoras Genéticas/genética , Ligação Proteica , Receptores de Reconhecimento de Padrão/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 2 Relacionada a Twist/genética
10.
J Immunol ; 202(3): 920-930, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30593537

RESUMO

Common IFN regulatory factor 5 (IRF5) variants associated with multiple immune-mediated diseases are a major determinant of interindividual variability in pattern recognition receptor (PRR)-induced cytokines in macrophages. PRR-initiated pathways also contribute to bacterial clearance, and dysregulation of bacterial clearance can contribute to immune-mediated diseases. However, the role of IRF5 in macrophage-mediated bacterial clearance is not well defined. Furthermore, it is unclear if macrophages from individuals who are carriers of low IRF5-expressing genetic variants associated with protection for immune-mediated diseases might be at a disadvantage in bacterial clearance. We found that IRF5 was required for optimal bacterial clearance in PRR-stimulated, M1-differentiated human macrophages. Mechanisms regulated by IRF5 included inducing reactive oxygen species through p40phox, p47phox and p67phox, NOS2, and autophagy through ATG5. Complementing these pathways in IRF5-deficient M1 macrophages restored bacterial clearance. Further, these antimicrobial pathways required the activation of IRF5-dependent MAPK, NF-κB, and Akt2 pathways. Importantly, relative to high IRF5-expressing rs2004640/rs2280714 TT/TT immune-mediated disease risk-carrier human macrophages, M1-differentiated GG/CC carrier macrophages demonstrated less reactive oxygen species, NOS2, and autophagy pathway induction and, consequently, reduced bacterial clearance. Increasing IRF5 expression to the rs2004640/rs2280714 TT/TT levels restored these antimicrobial pathways. We define mechanisms wherein common IRF5 genetic variants modulate bacterial clearance, thereby highlighting that immune-mediated disease risk IRF5 carriers might be relatively protected from microbial-associated diseases.


Assuntos
Bactérias/imunologia , Fatores Reguladores de Interferon/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Autofagia , Proteína 5 Relacionada à Autofagia/genética , Diferenciação Celular , Células Cultivadas , Enterococcus faecalis/imunologia , Predisposição Genética para Doença , Variação Genética , Humanos , Fatores Reguladores de Interferon/genética , Interferon gama/farmacologia , Óxido Nítrico Sintase Tipo II/metabolismo , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Reconhecimento de Padrão/imunologia , Salmonella enterica/imunologia , Transdução de Sinais
11.
Elife ; 72018 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-30355448

RESUMO

Homeostasis at mucosal surfaces requires cross-talk between the environment and barrier epithelial cells. Disruption of barrier function typifies mucosal disease. Here we elucidate a bifunctional role in coordinating this cross-talk for the inflammatory bowel disease risk-gene INAVA. Both activities require INAVA's DUF3338 domain (renamed CUPID). CUPID stably binds the cytohesin ARF-GEF ARNO to effect lateral membrane F-actin assembly underlying cell-cell junctions and barrier function. Unexpectedly, when bound to CUPID, ARNO affects F-actin dynamics in the absence of its canonical activity as a guanine nucleotide-exchange factor. Upon exposure to IL-1ß, INAVA relocates to form cytosolic puncta, where CUPID amplifies TRAF6-dependent polyubiquitination and inflammatory signaling. In this case, ARNO binding to CUPID negatively-regulates polyubiquitination and the inflammatory response. INAVA and ARNO act similarly in primary human macrophages responding to IL-1ß and to NOD2 agonists. Thus, INAVA-CUPID exhibits dual functions, coordinated directly by ARNO, that bridge epithelial barrier function with extracellular signals and inflammation.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Mucosa/metabolismo , Mucosa/patologia , Transdução de Sinais , Actinas/metabolismo , Proteínas de Transporte/química , Membrana Celular/metabolismo , Epitélio/metabolismo , Epitélio/patologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Junções Intercelulares/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Ligação Proteica , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitinação
13.
Nat Commun ; 8: 15865, 2017 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-28656966

RESUMO

Optimal regulation of the innate immune receptor nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is essential for controlling bacterial infections and inflammatory disorders. Chronic NOD2 stimulation induces non-responsiveness to restimulation, termed NOD2-induced tolerance. Although the levels of the NOD2 adaptor, RIP2, are reported to regulate both acute and chronic NOD2 signalling, how RIP2 levels are modulated is unclear. Here we show that ZNRF4 induces K48-linked ubiquitination of RIP2 and promotes RIP2 degradation. A fraction of RIP2 localizes to the endoplasmic reticulum (ER), where it interacts with ZNRF4 under either 55 unstimulated and muramyl dipeptide-stimulated conditions. Znrf4 knockdown monocytes have sustained nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, and Znrf4 knockdown mice have reduced NOD2-induced tolerance and more effective control of Listeria monocytogenes infection. Our results thus demonstrate E3-ubiquitin ligase ZNRF4-mediated RIP2 degradation as a negative regulatory mechanism of NOD2-induced NF-κB, cytokine and anti-bacterial responses in vitro and in vivo, and identify a ZNRF4-RIP2 axis of fine-tuning NOD2 signalling to promote protective host immunity.


Assuntos
Acetilmuramil-Alanil-Isoglutamina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Tolerância Imunológica , Proteína Adaptadora de Sinalização NOD2/metabolismo , Acetilmuramil-Alanil-Isoglutamina/imunologia , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Células HEK293 , Humanos , Tolerância Imunológica/efeitos dos fármacos , Listeria monocytogenes/patogenicidade , Listeriose/imunologia , Listeriose/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Monócitos/metabolismo , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Transdução de Sinais/fisiologia , Ubiquitinação/efeitos dos fármacos
14.
Nat Commun ; 8: 15614, 2017 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-28593945

RESUMO

Functional consequences for most inflammatory disease-associated loci are incompletely defined, including in the LACC1 (C13orf31) region. Here we show that human peripheral and intestinal myeloid-derived cells express laccase domain-containing 1 (LACC1); LACC1 is expressed in both the cytoplasm and mitochondria. Upon NOD2 stimulation of human macrophages, LACC1 associates with the NOD2-signalling complex, and is critical for optimal NOD2-induced signalling, mitochondrial ROS (mtROS) production, cytokine secretion and bacterial clearance. LACC1 constitutively associates with succinate dehydrogenase (SDH) subunit A, and amplifies pattern recognition receptor (PRR)-induced SDH activity, an important contributor to mtROS production. Relative to LACC1 Ile254, cells transfected with Crohn's disease-risk LACC1 Val254 or LACC1 with mutations of the nearby histidines (249,250) have reduced PRR-induced outcomes. Relative to LACC1 Ile254 carriers, Val254 disease-risk carrier macrophages demonstrate decreased PRR-induced mtROS, signalling, cytokine secretion and bacterial clearance. Therefore, LACC1 is critical for amplifying PRR-induced outcomes, an effect that is attenuated by the LACC1 disease-risk variant.


Assuntos
Bactérias/imunologia , Imunidade Inata/imunologia , Proteína Adaptadora de Sinalização NOD2/genética , Proteínas/imunologia , Receptores de Reconhecimento de Padrão/imunologia , Células Cultivadas , Doença de Crohn/genética , Citocinas/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos/imunologia , NF-kappa B/metabolismo , Proteínas/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Succinato Desidrogenase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
J Clin Invest ; 127(6): 2192-2205, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28436939

RESUMO

Inflammatory bowel disease (IBD) is characterized by dysregulation in both cytokines and responses to intestinal microbes, and proper regulation of pattern recognition receptor (PRR) signaling is critical for intestinal immune homeostasis. Altered functions for the IBD risk locus containing rs7554511, which encompasses the C1orf106 gene (recently named INAVA), and roles for the protein encoded by the INAVA gene are unknown. Here, we investigated the role of INAVA and INAVA genotype in regulating PRR-initiated outcomes in primary human cells. Both peripheral and intestinal myeloid cells expressed INAVA. Upon PRR stimulation, INAVA was required for optimal MAPK and NF-κB activation, cytokine secretion, and intracellular bacterial clearance. INAVA recruited 14-3-3τ, thereby contributing to recruitment of a signaling complex that amplified downstream signals and cytokines. Further, INAVA enhanced bacterial clearance by regulating reactive oxygen, reactive nitrogen, and autophagy pathways. Macrophages from rs7554511 C risk carriers expressed lower levels of INAVA RNA and protein. Lower expression was attributed in part to decreased transcription mediated directly by the intronic region containing the rs7554511 C variant. In rs7554511 C risk carrier macrophages, lower INAVA expression led to decreased PRR-induced activation of MAPK and NF-κB pathways, cytokines, and bacterial clearance pathways. Thus, IBD-associated polymorphisms in INAVA modulate PRR-initiated signaling, cytokines, and intracellular bacterial clearance, likely contributing to intestinal immune homeostasis.


Assuntos
Proteínas de Transporte/genética , Doenças Inflamatórias Intestinais/genética , Receptores de Reconhecimento de Padrão/metabolismo , Transporte Ativo do Núcleo Celular , Estudos de Casos e Controles , Citocinas/metabolismo , Enterococcus faecalis/fisiologia , Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Células HEK293 , Heterozigoto , Humanos , Sistema de Sinalização das MAP Quinases , Macrófagos/metabolismo , Macrófagos/microbiologia , Células Mieloides/metabolismo , Polimorfismo de Nucleotídeo Único , Cultura Primária de Células , Fatores de Risco , Staphylococcus aureus/fisiologia
16.
Cell Death Dis ; 8(2): e2622, 2017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-28230865

RESUMO

Efficient clearance of pro-inflammatory macrophages from tissues after resolution of a challenge is critical to prevent prolonged inflammation. Defects in clearance can contribute to conditions such as inflammatory bowel disease, and thus may be therapeutically targetable. However, the signaling pathways that induce termination of pro-inflammatory macrophages are incompletely defined. We tested whether the ErbB4 receptor tyrosine kinase, previously not known to have role in macrophage biology, is involved in this process. In vitro, pro-inflammatory activation of cultured murine and human macrophages induced ErbB4 expression; in contrast, other ErbB family members were not induced in pro-inflammatory cells, and other innate immune lineages (dendritic cells, neutrophils) did not express detectable ErbB4 levels. Treatment of activated pro-inflammatory macrophages with the ErbB4 ligand neuregulin-4 (NRG4) induced apoptosis. ErbB4 localized to the mitochondria in these cells. Apoptosis was accompanied by loss of mitochondrial membrane potential, and was dependent upon the proteases that generate the cleaved ErbB4 intracellular domain fragment, suggesting a requirement for this fragment and mitochondrial pathway apoptosis. In vivo, ErbB4 was highly expressed on pro-inflammatory macrophages but not neutrophils during experimental DSS colitis in C57Bl/6 mice. Active inflammation in this model suppressed NRG4 expression, which may allow for macrophage persistence and ongoing inflammation. Consistent with this notion, NRG4 levels rebounded during the recovery phase, and administration of exogenous NRG4 during colitis reduced colonic macrophage numbers and ameliorated inflammation. These data define a novel role for ErbB4 in macrophage apoptosis, and outline a mechanism of feedback inhibition that may promote resolution of colitis.


Assuntos
Apoptose/fisiologia , Colo/metabolismo , Inflamação/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Macrófagos/metabolismo , Receptor ErbB-4/metabolismo , Animais , Células Cultivadas , Colite/metabolismo , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neurregulinas/metabolismo , Neutrófilos/metabolismo , Transdução de Sinais/fisiologia
17.
J Immunol ; 197(9): 3695-3704, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27664279

RESUMO

JAK2 genetic variants are associated with inflammatory bowel disease (IBD) and JAK inhibitors are being evaluated for therapy targeting immune-mediated diseases, including IBD. As JAK pathway-mediated cytokine regulation varies across cell types and stimulation conditions, we examined how JAK signaling and IBD-associated JAK2 variants regulate distinct acute and chronic microbial product exposure outcomes in human myeloid cells, consistent with the conditions of initial entry and ongoing intestinal tissue residence, respectively. Macrophages from controls and ulcerative colitis patients carrying the IBD-risk rs10758669 CC genotype showed increased JAK2 expression and nucleotide-binding oligomerization domain 2-induced JAK2 phosphorylation relative to AA carriers. Interestingly, the threshold of JAK2 expression and signaling determined pattern-recognition receptor (PRR)-induced outcomes; whereas anti-inflammatory cytokines progressively decreased with lower JAK2 expression, proinflammatory cytokines switched from decreased to increased secretion below a certain JAK2 expression threshold. Low JAK2-expressing rs10758669 AA macrophages were above this threshold; consequently, both PRR-induced pro- and anti-inflammatory cytokines were decreased. However, relative to rs10758669 CC risk carriers, AA carrier macrophages switched to increased nucleotide-binding oligomerization domain 2-induced proinflammatory cytokines at lower therapeutically used JAK inhibitor doses. Importantly, JAK inhibitors increased proinflammatory cytokines secreted by peripheral macrophages following chronic PRR stimulation and by human intestinal myeloid cells following exposure to intestinal pathogens. Mechanistically, the decreased response to and secretion of autocrine/paracrine IL-10, IL-4, IL-22 and thymic stromal lymphopoietin regulated these JAK-dependent outcomes in myeloid cells. Taken together, the JAK signaling threshold determines whether PRR-induced pro- and anti-inflammatory cytokines are reciprocally regulated in myeloid cells; consideration of JAK2 genotype and targeting of specific cell types might improve JAK-targeted therapy in immune-mediated diseases.


Assuntos
Doenças Inflamatórias Intestinais/imunologia , Janus Quinase 2/metabolismo , Macrófagos/fisiologia , Adulto , Alelos , Células Cultivadas , Citocinas/metabolismo , Feminino , Genótipo , Humanos , Mediadores da Inflamação/metabolismo , Doenças Inflamatórias Intestinais/genética , Janus Quinase 2/genética , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais , Resultado do Tratamento
18.
Cell Rep ; 16(9): 2442-55, 2016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27545875

RESUMO

Interferon regulatory factor 5 (IRF5) regulates inflammatory M1 macrophage polarization, and disease-associated IRF5 genetic variants regulate pattern-recognition-receptor (PRR)-induced cytokines. PRR-stimulated macrophages and M1 macrophages exhibit enhanced glycolysis, a central mediator of inflammation. We find that IRF5 is needed for PRR-enhanced glycolysis in human macrophages and in mice in vivo. Upon stimulation of the PRR nucleotide binding oligomerization domain containing 2 (NOD2) in human macrophages, IRF5 binds RIP2, IRAK1, and TRAF6. IRF5, in turn, is required for optimal Akt2 activation, which increases expression of glycolytic pathway genes and HIF1A as well as pro-inflammatory cytokines and M1 polarization. Furthermore, pro-inflammatory cytokines and glycolytic pathways co-regulate each other. Rs2004640/rs2280714 TT/TT IRF5 disease-risk-carrier cells demonstrate increased IRF5 expression and increased PRR-induced Akt2 activation, glycolysis, pro-inflammatory cytokines, and M1 polarization relative to GG/CC carrier macrophages. Our findings identify that IRF5 disease-associated polymorphisms regulate diverse immunological and metabolic outcomes and provide further insight into mechanisms contributing to the increasingly recognized important role for glycolysis in inflammation.


Assuntos
Glicólise/genética , Fatores Reguladores de Interferon/genética , Macrófagos/metabolismo , Mutação , Proteínas Proto-Oncogênicas c-akt/genética , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Adjuvantes Imunológicos/farmacologia , Animais , Diferenciação Celular , Regulação da Expressão Gênica , Glicólise/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fatores Reguladores de Interferon/deficiência , Fatores Reguladores de Interferon/metabolismo , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Cultura Primária de Células , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo
19.
Gut ; 65(11): 1799-1811, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-26215868

RESUMO

OBJECTIVE: IBD is characterised by dysregulated intestinal immune homeostasis and cytokine secretion. In the intestine, properly regulating pattern recognition receptor (PRR)-mediated signalling and cytokines is crucial given the ongoing host-microbial interactions. TPL2 (MAP3K8, COT) contributes to PRR-initiated pathways, yet the mechanisms for TPL2 signalling contributions in primary human myeloid cells are incompletely understood and its role in intestinal myeloid cells is poorly defined. Furthermore, functional consequences for the IBD-risk locus rs1042058 in TPL2 are unknown. METHODS: We analysed protein, cytokine and RNA expression, and signalling in human monocyte-derived macrophages (MDMs) through western blot, ELISA, real-time PCR and flow cytometry. RESULTS: PRR-induced cytokine secretion was increased in MDMs from rs1042058 TPL2 GG risk individuals. TPL2 activation by the Crohn's disease-associated PRR nucleotide-oligomerisation domain (NOD)2 required PKC, and IKKß, IKKα and IKKγ signalling. TPL2, in turn, significantly enhanced NOD2-induced ERK, JNK and NFκB signalling. We found that another major mechanism for the TPL2 contribution to NOD2 signalling was through ERK-dependent and JNK-dependent caspase-1 and caspase-8 activation, which in turn, led to early autocrine interleukin (IL)-1ß and IL-18 secretion and amplification of long-term cytokines. Importantly, Salmonella typhimurium-induced cytokines from human intestinal myeloid-derived cells required TPL2 as well as autocrine IL-1ß and IL-18. Finally, rs1042058 GG risk carrier MDMs from healthy individuals and patients with Crohn's disease had increased TPL2 expression and NOD2-initiated TPL2 phosphorylation, ERK, JNK and NFκB activation, and early autocrine IL-1ß and IL-18 secretion. CONCLUSIONS: Taken together, the rs1042058 GG IBD-risk polymorphism in TPL2 results in a gain-of-function by increasing TPL2 expression and signalling, thereby amplifying PRR-initiated outcomes.


Assuntos
Caspase 1/metabolismo , Caspase 8/metabolismo , Doença de Crohn , Intestinos/imunologia , MAP Quinase Quinase Quinases/genética , Macrófagos/metabolismo , Proteínas Proto-Oncogênicas/genética , Técnicas de Cultura de Células , Doença de Crohn/genética , Doença de Crohn/imunologia , Citocinas/metabolismo , Regulação da Expressão Gênica , Humanos , Interleucina-1beta/metabolismo , Polimorfismo Genético , Receptores de Reconhecimento de Padrão , Transdução de Sinais/imunologia
20.
Proc Natl Acad Sci U S A ; 112(33): 10461-6, 2015 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-26240347

RESUMO

Inflammatory bowel disease (IBD) is characterized by dysregulated host:microbial interactions and cytokine production. Host pattern recognition receptors (PRRs) are critical in regulating these interactions. Multiple genetic loci are associated with IBD, but altered functions for most, including in the rs713875 MTMR3/HORMAD2/LIF/OSM region, are unknown. We identified a previously undefined role for myotubularin-related protein 3 (MTMR3) in amplifying PRR-induced cytokine secretion in human macrophages and defined MTMR3-initiated mechanisms contributing to this amplification. MTMR3 decreased PRR-induced phosphatidylinositol 3-phosphate (PtdIns3P) and autophagy levels, thereby increasing PRR-induced caspase-1 activation, autocrine IL-1ß secretion, NFκB signaling, and, ultimately, overall cytokine secretion. This MTMR3-mediated regulation required the N-terminal pleckstrin homology-GRAM domain and Cys413 within the phosphatase domain of MTMR3. In MTMR3-deficient macrophages, reducing the enhanced autophagy or restoring NFκB signaling rescued PRR-induced cytokines. Macrophages from rs713875 CC IBD risk carriers demonstrated increased MTMR3 expression and, in turn, decreased PRR-induced PtdIns3P and autophagy and increased PRR-induced caspase-1 activation, signaling, and cytokine secretion. Thus, the rs713875 IBD risk polymorphism increases MTMR3 expression, which modulates PRR-induced outcomes, ultimately leading to enhanced PRR-induced cytokines.


Assuntos
Autofagia , Caspase 1/metabolismo , Citocinas/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Transdução de Sinais , Alelos , Ativação Enzimática , Regulação da Expressão Gênica , Predisposição Genética para Doença , Genótipo , Homeostase , Humanos , Inflamação/metabolismo , Doenças Inflamatórias Intestinais/genética , Leucócitos Mononucleares/citologia , Ligantes , Macrófagos/metabolismo , Monócitos/citologia , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Fatores de Risco , Receptores Toll-Like/metabolismo
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