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1.
Nat Commun ; 11(1): 1978, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332737

RESUMO

There is the notion that infection with a virulent intestinal pathogen induces generally stronger mucosal adaptive immunity than the exposure to an avirulent strain. Whether the associated mucosal inflammation is important or redundant for effective induction of immunity is, however, still unclear. Here we use a model of auxotrophic Salmonella infection in germ-free mice to show that live bacterial virulence factor-driven immunogenicity can be uncoupled from inflammatory pathogenicity. Although live auxotrophic Salmonella no longer causes inflammation, its mucosal virulence factors remain the main drivers of protective mucosal immunity; virulence factor-deficient, like killed, bacteria show reduced efficacy. Assessing the involvement of innate pathogen sensing mechanisms, we show MYD88/TRIF, Caspase-1/Caspase-11 inflammasome, and NOD1/NOD2 nodosome signaling to be individually redundant. In colonized animals we show that microbiota metabolite cross-feeding may recover intestinal luminal colonization but not pathogenicity. Consequent immunoglobulin A immunity and microbial niche competition synergistically protect against Salmonella wild-type infection.


Assuntos
Imunidade nas Mucosas , Mucosa Intestinal/microbiologia , Infecções por Salmonella/microbiologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Antígenos de Bactérias , Caspase 1/metabolismo , Caspases Iniciadoras/metabolismo , Proliferação de Células , Microbioma Gastrointestinal , Imunidade Inata , Imunoglobulina A/imunologia , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Fator 88 de Diferenciação Mieloide/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Salmonella typhimurium/patogenicidade , Transdução de Sinais , Virulência , Fatores de Virulência
2.
Am J Physiol Endocrinol Metab ; 318(4): E579-E585, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32101030

RESUMO

Defining the host receptors and metabolic consequences of bacterial components can help explain how the microbiome influences metabolic diseases. Bacterial peptidoglycans that activate nucleotide-binding oligomerization domain-containing (NOD)1 worsen glucose control, whereas NOD2 activation improves glycemia. Receptor-interacting serine/threonine-protein kinase 2 (RIPK2) is required for innate immunity instigated by NOD1 and NOD2. The role of RIPK2 in the divergent effects of NOD1 versus NOD2 on blood glucose was unknown. We found that whole body deletion of RIPK2 negated all effects of NOD1 or NOD2 activation on blood glucose during an acute, low level endotoxin challenge in mice. It was known that NOD1 in hematopoietic cells participates in insulin resistance and metabolic inflammation in obese mice. It was unknown if RIPK2 in hematopoietic cells is required for the glucose-lowering and anti-inflammatory effects of NOD2 activation. We hypothesized that RIPK2 in nonhematopoietic cells dictated the glycemic effects of NOD2 activation. We found that whole body deletion of RIPK2 prevented the glucose-lowering effects of repeated NOD2 activation that were evident during a glucose tolerance test (GTT) in high-fat diet (HFD)-fed wild-type (WT) mice. NOD2 activation lowered glucose during a GTT and lowered adipose tissue inflammation in mice with RIPK2 deleted in hematopoietic cells. We conclude that RIPK2 in nonhematopoietic cells mediates the glucose lowering and anti-inflammatory effects of NOD2-activating postbiotics. We propose a model where lipopolysaccharides and NOD1 ligands synergize in hematopoietic cells to promote insulin resistance but NOD2 activation in nonhematopoietic cells promotes RIPK2-dependent immune tolerance and lowering of inflammation and insulin resistance.


Assuntos
Glicemia/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Microbiota , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Ativação Metabólica , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Dieta Hiperlipídica , Teste de Tolerância a Glucose , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética
3.
PLoS One ; 14(11): e0224738, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31697716

RESUMO

Tissue-type plasminogen activator (tPA) is a major activator of fibrinolysis, which also attenuates the pro-inflammatory activity of lipopolysaccharide (LPS) in bone marrow-derived macrophages (BMDMs) and in vivo in mice. The activity of tPA as an LPS response modifier is independent of its proteinase activity and instead, dependent on the N-methyl-D-aspartate Receptor (NMDA-R), which is expressed by BMDMs. The major Toll-like receptor (TLR) for LPS is TLR4. Herein, we show that enzymatically-inactive (EI) tPA blocks the response of mouse BMDMs to selective TLR2 and TLR9 agonists, rapidly reversing IκBα phosphorylation and inhibiting expression of TNFα, CCL2, interleukin-1ß, and interleukin-6. The activity of EI-tPA was replicated by activated α2-macroglobulin, which like EI-tPA, signals through an NMDA-R-dependent pathway. EI-tPA failed to inhibit cytokine expression by BMDMs in response to agonists that target the Pattern Recognition Receptors (PRRs), NOD1 and NOD2, providing evidence for specificity in the function of EI-tPA. Macrophages isolated from the peritoneal space (PMs), without adding eliciting agents, expressed decreased levels of cell-surface NMDA-R compared with BMDMs. These cells were unresponsive to EI-tPA in the presence of LPS. However, when PMs were treated with CSF-1, the abundance of cell-surface NMDA-R increased and the ability of EI-tPA to neutralize the response to LPS was established. We conclude that the anti-inflammatory activity of EI-tPA is selective for TLRs but not all PRRs. The ability of macrophages to respond to EI-tPA depends on the availability of cell surface NMDA-R, which may be macrophage differentiation-state dependent.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/patologia , Ativador de Plasminogênio Tecidual/farmacologia , Receptores Toll-Like/antagonistas & inibidores , Animais , Citocinas/metabolismo , Humanos , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Testes de Neutralização , Proteína Adaptadora de Sinalização NOD1/agonistas , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/agonistas , Proteína Adaptadora de Sinalização NOD2/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Toll-Like/metabolismo
5.
Int J Mol Sci ; 20(17)2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31480368

RESUMO

THP-1 cells express high levels of native functional nucleotide-binding oligomerization domain 1 (NOD1), NOD2, and Toll-like receptor 4 (TLR4) receptors, and have often been used for investigating the immunomodulatory effects of small molecules. We postulated that they would represent an ideal cell-based model for our study, the aim of which was to develop a new in vitro tool for functional characterization of NOD antagonists. NOD antagonists were initially screened for their effect on NOD agonist-induced interleukin-8 (IL-8) release. Next, we examined the extent to which the selected NOD antagonists block the NOD-TLR4 synergistic crosstalk by measuring the effect of NOD antagonism on tumor necrosis factor-α (TNF-α) secretion from doubly activated THP-1 cells. Overall, the results obtained indicate that pro-inflammatory cytokine secretion from THP-1 provides a valuable, simple and reproducible in vitro tool for functional characterization of NOD antagonists.


Assuntos
Citocinas/biossíntese , Mediadores da Inflamação/metabolismo , Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD2/antagonistas & inibidores , Sobrevivência Celular , Humanos , Proteína Adaptadora de Sinalização NOD1/agonistas , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/agonistas , Proteína Adaptadora de Sinalização NOD2/metabolismo , Células THP-1 , Receptor 4 Toll-Like/metabolismo
6.
Cell Biochem Funct ; 37(7): 464-473, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31396989

RESUMO

We sought to explore the functions and modulated factors of NOD1 in normal decidual stromal cells (DSCs) derived from the first trimester pregnancy and whether existed different expression of NOD1 between normal and unexplained recurrent pregnancy loss (URPL) in DSCs. Twenty-six patients with normal pregnancies that required abortion and 12 URPL patients at first trimester were enrolled for the study. As a result, we found lower levels of NOD1 in the DSCs derived from URPL compared with those from normal early trimester pregnancy. Furthermore, increased NOD1 expression in the normal DSCs induced apoptosis and increased monocyte chemotactic protein-1 (MCP-1) and IL-1ß (interleukin 1 beta) secretion but decreased their invasion capacity. In addition, several cytokines such as IL-1ß, tumour necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin-17 (IL-17) were present at the maternal-fetal interface in RPL and were found to regulate NOD1 expression in primary DSCs. Our study indicates that RPL may be associated with NOD1 aberrant expression in DSCs, which plays a significant role in maintaining pregnancy via infection control and regulation of immune responses that might affect the pregnancy outcome. We expect that our results will bring more comprehensively understanding about the connection between NOD1 and RPL for researchers.


Assuntos
Decídua/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Primeiro Trimestre da Gravidez/metabolismo , Células Estromais/metabolismo , Adulto , Apoptose/efeitos dos fármacos , Células Cultivadas , Citocinas/análise , Citocinas/metabolismo , Decídua/citologia , Decídua/efeitos dos fármacos , Ácido Diaminopimélico/análogos & derivados , Ácido Diaminopimélico/farmacologia , Feminino , Humanos , Proteína Adaptadora de Sinalização NOD1/genética , Gravidez , Primeiro Trimestre da Gravidez/efeitos dos fármacos , Células Estromais/efeitos dos fármacos
7.
Science ; 365(6448)2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31273097

RESUMO

Multiple cytosolic innate sensors form large signalosomes after activation, but this assembly needs to be tightly regulated to avoid accumulation of misfolded aggregates. We found that the eIF2α kinase heme-regulated inhibitor (HRI) controls NOD1 signalosome folding and activation through a process requiring eukaryotic initiation factor 2α (eIF2α), the transcription factor ATF4, and the heat shock protein HSPB8. The HRI/eIF2α signaling axis was also essential for signaling downstream of the innate immune mediators NOD2, MAVS, and TRIF but dispensable for pathways dependent on MyD88 or STING. Moreover, filament-forming α-synuclein activated HRI-dependent responses, which suggests that the HRI pathway may restrict toxic oligomer formation. We propose that HRI, eIF2α, and HSPB8 define a novel cytosolic unfolded protein response (cUPR) essential for optimal innate immune signaling by large molecular platforms, functionally homologous to the PERK/eIF2α/HSPA5 axis of the endoplasmic reticulum UPR.


Assuntos
Citosol/enzimologia , Citosol/imunologia , Imunidade Inata , Proteínas Serina-Treonina Quinases/fisiologia , Resposta a Proteínas não Dobradas/imunologia , Fator 4 Ativador da Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Linhagem Celular , Fator de Iniciação 2 em Eucariotos/metabolismo , Fibroblastos , Proteínas de Choque Térmico/metabolismo , Humanos , Listeria/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Chaperonas Moleculares/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Proteína Adaptadora de Sinalização NOD1/química , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteínas Serina-Treonina Quinases/genética , Salmonella/imunologia , Infecções por Salmonella , Shigella/imunologia , Transdução de Sinais
8.
Curr Top Microbiol Immunol ; 421: 159-177, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31123889

RESUMO

The human pathogen Helicobacter pylori interacts intimately with gastric epithelial cells to induce inflammatory responses that are a hallmark of the infection. This inflammation is a critical precursor to the development of peptic ulcer disease and gastric cancer. A major driver of this inflammation is a type IV secretion system (T4SS) encoded by the cag pathogenicity island (cagPAI), present in a subpopulation of more virulent H. pylori strains. The cagPAI T4SS specifically activates signalling pathways in gastric epithelial cells that converge on the transcription factor, nuclear factor-κB (NF-κB), which in turn upregulates key immune and inflammatory genes, resulting in various host responses. It is now clear that H. pylori possesses several mechanisms to activate NF-κB in gastric epithelial cells and, moreover, that multiple signalling pathways are involved in these responses. Two of the dominant signalling pathways implicated in NF-κB-dependent responses in epithelial cells are nucleotide-binding oligomerisation domain 1 (NOD1) and a newly described pathway involving alpha-kinase 1 (ALPK1) and tumour necrosis factor (TNF) receptor-associated factor (TRAF)-interacting protein with forkhead-associated domain (TIFA). Although the relative roles of these two pathways in regulating NF-κB-dependent responses still need to be clearly defined, it is likely that they work cooperatively and non-redundantly. This chapter will give an overview of the various mechanisms and pathways involved in H. pylori induction of NF-κB-dependent responses in gastric epithelial cells, including a 'state-of-the-art' review on the respective roles of NOD1 and ALPK1/TIFA pathways in these responses.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Infecções por Helicobacter/imunologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/imunologia , Imunidade Inata , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais , Humanos , NF-kappa B/metabolismo
9.
J Biol Chem ; 294(22): 9007-9015, 2019 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-30996003

RESUMO

NOD1 and NOD2 are intracellular sensors of bacterial peptidoglycan that belong to the Nod-like receptor family of innate immune proteins. In addition to their role as direct bacterial sensors, it was proposed that the nucleotide-binding oligomerization domain (NOD) proteins could detect endoplasmic reticulum (ER) stress induced by thapsigargin, an inhibitor of the sarcoplasmic or endoplasmic reticulum calcium ATPase family that pumps Ca2+ into the ER, resulting in pro-inflammatory signaling. Here, we confirm that thapsigargin induces NOD-dependent pro-inflammatory signaling in epithelial cells. However, the effect was specific to thapsigargin, as tunicamycin and the subtilase cytotoxin SubAB from Shiga toxigenic Escherichia coli, which induce ER stress by other mechanisms, did not induce cytokine expression. The calcium ionophore A23187 also induced NOD-dependent signaling, and calcium chelators demonstrated a role for both intracellular and extracellular calcium in mediating thapsigargin-induced and NOD-dependent pro-inflammatory signaling, in part through the activation of plasma membrane-associated calcium release-activated channels. Moreover, our results demonstrate that both endocytosis and the addition of serum to the cell culture medium were required for thapsigargin-mediated NOD activation. Finally, we analyzed cell culture grade fetal calf serum as well as serum from laboratory mice using HPLC and MS identified the presence of various peptidoglycan fragments. We propose that cellular perturbations that affect intracellular Ca2+ can trigger internalization of peptidoglycan trace contaminants found in culture serum, thereby stimulating pro-inflammatory signaling. The presence of peptidoglycan in animal serum suggests that a homeostatic function of NOD signaling may have been previously overlooked.


Assuntos
Citocinas/metabolismo , Estresse do Retículo Endoplasmático , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Peptidoglicano/sangue , Calcimicina/química , Calcimicina/farmacologia , Cálcio/química , Cálcio/metabolismo , Quimiocina CXCL1/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Técnicas de Inativação de Genes , Células HCT116 , Humanos , Interleucina-8/metabolismo , Proteína Adaptadora de Sinalização NOD1/deficiência , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/deficiência , Proteína Adaptadora de Sinalização NOD2/genética , Transdução de Sinais/efeitos dos fármacos , Tapsigargina/farmacologia
10.
Microb Pathog ; 131: 53-64, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30940608

RESUMO

Polymorphonuclear neutrophils (PMNs) are indispensable in fighting infectious microbes by adopting various antimicrobial strategies including phagocytosis and neutrophil extracellular traps (NETs). Although the role and importance of PMNs in periodontal disease are well established, the specific molecular mechanisms involved in NET formation are yet to be characterized. In the present study, we sought to determine the role of periodontal pathogen on NET formation by utilizing Fusobacterium nucleatum. Our data demonstrates that F. nucleatum activates neutrophils and induces robust NETosis in a time-dependent manner via the upregulation of the Nucleotide oligomerization domain 1 (NOD1) and NOD2 receptors. Furthermore, CRISPR/Cas9 knockout of HL-60 cells and the use of ligands/inhibitors confirmed the involvement of NOD1 and NOD2 receptors in F. nucleatum-mediated NET formation. When treated with NOD1 and NOD2 inhibitors, we observed a significant downregulation of peptidylarginine deiminase 4 (PAD4) activity. In addition, neutrophils showed a significant increase and decrease of myeloperoxidase (MPO) and neutrophil elastase (NE) when treated with NOD1/NOD2 ligands and inhibitors, respectively. Taken together, CRISPR/Cas9 knockout of NOD1/NOD2 HL-60 cells and inhibitors of NOD signaling confirmed the role of NLRs in F. nucleatum-mediated NETosis. Our data demonstrates an important pathway linking NOD1 and NOD2 to NETosis by F. nucleatum, a prominent microbe in periodontal biofilms. This is the first study to elucidate the role of NOD-like receptors in NETosis and their downstream signaling network.


Assuntos
Fusobacterium nucleatum/patogenicidade , Neutrófilos/imunologia , Neutrófilos/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Periodontite/metabolismo , Biofilmes , Sistemas CRISPR-Cas/genética , Regulação para Baixo , Células HL-60 , Histonas/metabolismo , Humanos , Elastase de Leucócito/metabolismo , Periodontite/microbiologia , Peroxidase/metabolismo , Fagocitose , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas/metabolismo , Transdução de Sinais
11.
Res Vet Sci ; 124: 310-316, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31030118

RESUMO

NOD-like receptors (NLRs) play a key role in the innate immune system, acting as a second line of surveillance against pathogens. NLRs detect particular bacteria that have gained access to the cytoplasm, evading recognition by other pattern recognition receptors, such as Toll-like receptors. It has been demonstrated that coding sequence-single nucleotide polymorphisms may alter the ligand recognition ability of NLRs, affecting their pathogen-sensing function. However, there have been no data relating to the identification and functional analysis of SNPs in porcine NLR promoters. We examined the promoter sequences of the porcine NOD1 and NOD2 genes with the aim to identify and to evaluate the effect of genetic variations on promoter activity. Six SNPs in NOD1 and three SNPs in NOD2 were identified. Luciferase reporter gene assays showed significant differences in promoter activity between allele variants of NOD1 -920G>A (NC_010460.4:g.42431413G>A) and NOD2 -1670G>A (NC_010448.4:g.34169122T>C) SNPs. The results suggest that promoter polymorphisms could modify the expression levels of porcine NOD1 and NOD2 genes.


Assuntos
Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/genética , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , Sus scrofa/genética , Animais , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Análise de Sequência de DNA , Sus scrofa/metabolismo
12.
Lung ; 197(3): 377-385, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30874883

RESUMO

INTRODUCTION: Asthma is a common respiratory childhood disease that results from an interaction between genetic, environmental and immunologic factors. The implication of nucleotide-binding and oligomerization domain 1 and 2 (NOD1/CARD4, NOD2/CARD15) was highlighted in many inflammatory diseases. METHODS: In this case-control study, we analyzed the association of three NOD2 polymorphisms and one NOD1 variant, in 338 Tunisian asthmatic children and 425 healthy Controls, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. We also assessed NOD1 and NOD2 mRNA and protein levels by qRT-PCR and ELISA techniques. RESULTS: The homozygous AA genotype of rs2075820 was a risk factor for asthma (OR 2.39). The influence of the E266K variant in the presence of the heterozygous AG genotype was higher in male than female groups. The homozygous AA genotype was a risk factor associated with asthma, for patients aged between 6 and 18 years OR 2.39, IC95% (1.04-5.49) p < 0.01. The mRNA expression of NOD1, but not NOD2, was enhanced in asthma patients compared to Controls. We noted a significant difference between asthmatics and healthy controls in NOD1 protein expression (asthma patients : 31.18 ± 10.9 pg/ml, Controls: 20.10 ± 2.58 pg/ml; p < 0.001). CONCLUSIONS: The NOD1 rs2075820 variant was associated with a higher childhood asthma risk and the NOD1 expression at mRNA and protein levels was significantly increased in asthma patients.


Assuntos
Asma/genética , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/genética , RNA Mensageiro/metabolismo , Adolescente , Asma/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Tunísia
13.
Gene ; 698: 150-156, 2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-30851423

RESUMO

OBJECTIVE: Investigating the expression of the lnc RNAs screened above between normal and insulin resistant 3T3-L1 adipocytes. Addressing the mechanism underlying the regulation of inflammation response by lnc TINCR. METHODS: 3T3-L1 preadipocytes were induced to differentiate into mature adipocytes. Oil red O staining was used to find the fat droplets in mature adipocytes. Mature adipocytes were randomized to normal control group and Tri-DAP (NOD1 ligand) group. After the establishment of insulin resistance model, we used deep RNA sequencing(RNA-Seq) to identify lncRNAs that are regulated during NODI activation in mouse adipocytes. Real-time PCR was used to analyze the expression of lnc TINCR, proinflammatory IL-6, TNF-α, Cxcl1 and RIPK2 in the presence or absence of Tri-DAP(10 µg/ml). We employed siRNA against lnc TINCR to confirm its effects in inflammatory response. RESULTS: Deep RNA sequencing identified 81 lncRNAs and 167 coding genes that were significantly up-related while 78 lncRNAs and 82 coding genes that were significantly down-related greater than twofold during NOD1 activation in adipocytes. We discovered that lnc TINCR, termed lnc TINCR(Tri-DAP-inducible non-protein coding RNA) is greatly upregulated in Tri-DAP activated adipocytes. Moreover knockdown of lnc TINCR dampens the proinflammatory response (P < 0.05; in adipocytes). CONCLUSIONS: lnc TINCR is a positive regulator of inflammation-induced insulin resistance presumably via activation of NOD1 signaling pathways.


Assuntos
Inflamação/genética , Proteína Adaptadora de Sinalização NOD1/genética , RNA Longo não Codificante/genética , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Diferenciação Celular , Insulina/metabolismo , Resistência à Insulina/genética , Camundongos , Proteína Adaptadora de Sinalização NOD1/metabolismo , Fosforilação , RNA Longo não Codificante/metabolismo , RNA Mensageiro , Análise de Sequência de RNA/métodos , Transdução de Sinais
14.
Bioorg Med Chem Lett ; 29(10): 1153-1161, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30890292

RESUMO

The innate immune system is the body's first defense against invading microorganisms, relying on the recognition of bacterial-derived small molecules by host protein receptors. This recognition event and downstream immune response rely heavily on the specific chemical features of both the innate immune receptors and their bacterial derived ligands. This review presents a chemist's perspective on some of the most crucial and complex components of two receptors (NOD1 and NOD2): starting from the structural and chemical characteristics of bacterial-derived small molecules, to the specific proposed models of molecular recognition of these molecules by immune receptors, to the subsequent post-translational modifications that ultimately dictate downstream immune signaling. Recent advances in the field are discussed, as well as the potential for the development of targeted therapeutics.


Assuntos
Proteína Adaptadora de Sinalização NOD1/química , Proteína Adaptadora de Sinalização NOD2/química , Bactérias/metabolismo , Humanos , Imunidade Inata , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Peptidoglicano/química , Peptidoglicano/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais
15.
Endocrinology ; 160(5): 1021-1030, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30807635

RESUMO

Insulin resistance is driven, in part, by activation of the innate immune system. We have discussed the evidence linking nucleotide-binding oligomerization domain (NOD)1, an intracellular pattern recognition receptor, to the onset and progression of obesity-induced insulin resistance. On a molecular level, crosstalk between downstream NOD1 effectors and the insulin receptor pathway inhibits insulin signaling, potentially through reduced insulin receptor substrate action. In vivo studies have demonstrated that NOD1 activation induces peripheral, hepatic, and whole-body insulin resistance. Also, NOD1-deficient models are protected from high-fat diet (HFD)-induced insulin resistance. Moreover, hematopoietic NOD1 deficiency prevented HFD-induced changes in proinflammatory macrophage polarization status, thus protecting against the development of metabolic inflammation and insulin resistance. Serum from HFD-fed mice activated NOD1 signaling ex vivo; however, the molecular identity of the activating factors remains unclear. Many have proposed that an HFD changes the gut permeability, resulting in increased translocation of bacterial fragments and increased circulating NOD1 ligands. In contrast, others have suggested that NOD1 ligands are endogenous and potentially lipid-derived metabolites produced during states of nutrient overload. Nevertheless, that NOD1 contributes to the development of insulin resistance, and that NOD1-based therapy might provide benefit, is an exciting advancement in metabolic research.


Assuntos
Imunidade Inata/imunologia , Resistência à Insulina/imunologia , Proteína Adaptadora de Sinalização NOD1/imunologia , Obesidade/imunologia , Animais , Dieta Hiperlipídica , Imunidade Inata/genética , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Camundongos , Modelos Imunológicos , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD1/metabolismo , Obesidade/genética , Obesidade/metabolismo , Receptor de Insulina/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia
16.
BMC Genomics ; 20(1): 152, 2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30791886

RESUMO

BACKGROUND: Triple negative breast cancer (TNBC) is a malignancy with very poor prognosis, due to its aggressive clinical characteristics and lack of response to receptor-targeted drug therapy. In TNBC, immune-related pathways are typically upregulated and may be associated with a better prognosis of the disease, encouraging the pursuit for immunotherapeutic options. A number of immune-related molecules have already been associated to the onset and progression of breast cancer, including NOD1 and NOD2, innate immune receptors of bacterial-derived components which activate pro-inflammatory and survival pathways. In the context of TNBC, overexpression of either NOD1or NOD2 is shown to reduce cell proliferation and increase clonogenic potential in vitro. To further investigate the pathways linking NOD1 and NOD2 signaling to tumorigenesis in TNBC, we undertook a global proteome profiling of TNBC-derived cells ectopically expressing each one of these NOD receptors. RESULTS: We have identified a total of 95 and 58 differentially regulated proteins in NOD1- and NOD2-overexpressing cells, respectively. We used bioinformatics analyses to identify enriched molecular signatures aiming to integrate the differentially regulated proteins into functional networks. These analyses suggest that overexpression of both NOD1 and NOD2 may disrupt immune-related pathways, particularly NF-κB and MAPK signaling cascades. Moreover, overexpression of either of these receptors may affect several stress response and protein degradation systems, such as autophagy and the ubiquitin-proteasome complex. Interestingly, the levels of several proteins associated to cellular adhesion and migration were also affected in these NOD-overexpressing cells. CONCLUSIONS: Our proteomic analyses shed new light on the molecular pathways that may be modulating tumorigenesis via NOD1 and NOD2 signaling in TNBC. Up- and downregulation of several proteins associated to inflammation and stress response pathways may promote activation of protein degradation systems, as well as modulate cell-cycle and cellular adhesion proteins. Altogether, these signals seem to be modulating cellular proliferation and migration via NF-κB, PI3K/Akt/mTOR and MAPK signaling pathways. Further investigation of altered proteins in these pathways may provide more insights on relevant targets, possibly enabling the immunomodulation of tumorigenesis in the aggressive TNBC phenotype.


Assuntos
Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/genética , Proteoma , Proteômica , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Proliferação de Células , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Humanos , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteômica/métodos , Transcriptoma , Neoplasias de Mama Triplo Negativas/patologia
17.
Microb Pathog ; 128: 390-395, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30703473

RESUMO

Long term high-concentrate (HC) diet feeding induces subacute ruminal acidosis (SARA), which is reported to trigger a pro-inflammatory response. This study aimed to investigate the role of nucleotide-binding oligomerization domain protein 1 (NOD1) in initiating the pro-inflammatory response triggered by grain-induced SARA in the mammary gland of mid-lactating dairy cows. Twelve multiparous mid-lactating Holstein cows (455 ±â€¯28 kg) were randomly assigned into two groups to conduct the experiment for 18 weeks as follows: one group was fed a low-concentrate (LC) diet as a control (40% grain), and the other was fed an HC diet as a treatment (60% grain). Overall, the results showed that a decreased rumen pH and elevated γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) concentrations in the HC group compared with LC group. The concentration of pro-inflammatory cytokines, including interleukin (IL)-1ß, IL-6 and tumour necrosis factor-alpha (TNF-α), significantly increased in the lacteal vein of the HC group than LC group. The mRNA expression levels of NOD1, receptor-interacting protein2 (RIP2), NF-κBp65 (p65), IL-1ß, IL-6, IL-8 and TNF-α, which involved in inflammatory response, were up-regulated in the HC-induced mammary gland. The changes of the target proteins, including NOD1, p65 and pp65 presented the same tendency as those of the target genes. Collectively, long-term high concentrate feeding-induced SARA increased the rumen iE-DAP concentration which activated NOD1-NF-κB signalling pathway-dependent inflammation in the mammary gland of mid-lactating cows.


Assuntos
Acidose/veterinária , Ração Animal/efeitos adversos , Dieta/efeitos adversos , Lactação/efeitos dos fármacos , Glândulas Mamárias Animais/efeitos dos fármacos , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Acidose/metabolismo , Animais , Bovinos , Doenças dos Bovinos/metabolismo , Citocinas/sangue , Citocinas/genética , Citocinas/metabolismo , Ácido Diaminopimélico/análogos & derivados , Ácido Diaminopimélico/metabolismo , Dieta/métodos , Dieta/veterinária , Feminino , Trato Gastrointestinal/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Inflamação/induzido quimicamente , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Glândulas Mamárias Animais/metabolismo , Rúmen/química , Fator de Necrose Tumoral alfa/metabolismo
18.
ACS Chem Biol ; 14(3): 405-414, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30735346

RESUMO

The peptidoglycan fragments γ-d-glutamyl- meso-diaminopimelic acid (iE-DAP) and muramyl-dipeptide (MDP) are microbial-specific metabolites that activate intracellular pattern recognition receptors and stimulate immune signaling pathways. While extensive structure-activity studies have demonstrated that these bacterial cell wall metabolites trigger NOD1- and NOD2-dependent signaling, their direct binding to these innate immune receptors or other proteins in mammalian cells has not been established. To characterize these fundamental microbial metabolite-host interactions, we synthesized a series of peptidoglycan metabolite photoaffinity reporters and evaluated their cross-linking to NOD1 and NOD2 in mammalian cells. We show that active iE-DAP and MDP photoaffinity reporters selectively cross-linked NOD1 and NOD2, respectively, and not their inactive mutants. We also discovered MDP reporter cross-linking to Arf GTPases, which interacted most prominently with GTP-bound Arf6 and coimmunoprecipitated with NOD2 upon MDP stimulation. Notably, MDP binding to NOD2 and Arf6 was abrogated with loss-of-function NOD2 mutants associated with Crohn's disease. Our studies demonstrate peptidoglycan metabolite photoaffinity reporters can capture their cognate immune receptors in cells and reveal unpredicted ligand-induced interactions with other cellular cofactors. These photoaffinity reporters should afford useful tools to discover and characterize other peptidoglycan metabolite-interacting proteins.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Ácido Diaminopimélico/análogos & derivados , Peptidoglicano/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Parede Celular/metabolismo , Citocinas/metabolismo , Ácido Diaminopimélico/metabolismo , Células HEK293 , Humanos , Ligantes , Proteínas Mutantes/metabolismo , Mutação , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Ligação Proteica , Transdução de Sinais , Relação Estrutura-Atividade
19.
BMB Rep ; 52(6): 373-378, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30760380

RESUMO

The nucleotide-binding and oligomerization domain (NOD) is an innate pattern recognition receptor that recognizes pathogen- and damage-associated molecular patterns. The 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) is a matrix degradation product found in the synovial fluids of patients with osteoarthritis (OA). We investigated whether NOD2 was involved in 29-kDa FN-f-induced pro-catabolic gene expression in human chondrocytes. The expression of mRNA and protein was measured using quantitative real-time polymerase chain reaction (qrt-PCR) and Western blot analysis. Small interfering RNAs were used for knockdown of NOD2 and toll-like receptor 2 (TLR-2). An immunoprecipitation assay was performed to examine protein interactions. The NOD2 levels in human OA cartilage were much higher than in normal cartilage. NOD1 and NOD2 expression, as well as pro-inflammatory cytokines, including interleukin-1beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α), were upregulated by 29-kDa FN-f in human chondrocytes. NOD2 silencing showed that NOD2 was involved in the 29-kDa FN-f-induced expression of TLR-2. Expressions of IL-6, IL-8, matrix metalloproteinase (MMP)-1, -3, and -13 were also suppressed by TLR-2 knockdown. Furthermore, NOD2 and TLR-2 knockdown data demonstrated that both NOD2 and TLR-2 modulated the expressions of their adaptors, receptorinteracting protein 2 (RIP2) and myeloid differentiation 88, in 29-kDa FN-f-treated chondrocytes. 29-kDa FN-f enhanced the interaction of NOD2, RIP2 and transforming growth factor beta-activated kinase 1 (TAK1), an indispensable signaling intermediate in the TLR-2 signaling pathway, and activated nuclear factor-κB (NF-κB), subsequently leading to increased expressions of pro-inflammatory cytokines and cartilagedegrading enzymes. These results demonstrate that 29-kDa FN-f modulated pro-catabolic responses via cross-regulation of NOD2 and TLR-2 signaling pathways. [BMB Reports 2019; 52(6): 373-378].


Assuntos
Condrócitos/metabolismo , Fibronectinas/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Osteoartrite/metabolismo , Cartilagem/metabolismo , Células Cultivadas , Citocinas/metabolismo , Fibronectinas/genética , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Articulações/metabolismo , Metaloendopeptidases/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Fragmentos de Peptídeos/metabolismo , Transdução de Sinais , Receptor 2 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
20.
FASEB J ; 33(3): 3912-3921, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30496704

RESUMO

Atherosclerosis is a chronic disease characterized by vascular lipid retention and inflammation, and pattern recognition receptors (PRRs) are important contributors in early stages of the disease. Given the implication of the intracellular PRR nucleotide-binding oligomerization domain 1 (NOD1) in cardiovascular diseases, we investigated its contribution to early atherosclerosis. We evidenced NOD1 induction in atherosclerotic human and mouse tissues, predominantly in vascular endothelial cells. Accordingly, NOD1 genetic inactivation in Apoe-/- mice reduced not only atherosclerosis burden, but also monocyte and neutrophil accumulation in atheromata. Of note, in the presence of either peptidoglycan or oxidized LDLs, endothelial NOD1 triggered VCAM-1 up-regulation through the RIP2-NF-κB axis in an autocrine manner, enhancing firm adhesion of both sets of myeloid cells to the inflamed micro- and macrovasculature in vivo. Our data define a major proatherogenic role for endothelial NOD1 in early leukocyte recruitment to the athero-prone vasculature, thus introducing NOD1 as an innovative therapeutic target and potential prognostic molecule.-González-Ramos, S., Paz-García, M., Rius, C., del Monte-Monge, A., Rodríguez, C., Fernández-García, V., Andrés, V., Martínez-González, J., Lasunción, M. A., Martín-Sanz, P., Soehnlein, O., Boscá, L. Endothelial NOD1 directs myeloid cell recruitment in atherosclerosis through VCAM-1.


Assuntos
Aterosclerose/metabolismo , Movimento Celular , Endotélio Vascular/metabolismo , Células Mieloides/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/patologia , Comunicação Autócrina , Células Cultivadas , Humanos , Lipoproteínas LDL/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/fisiologia , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo
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