Activation Of Nucleotide-binding Oligomerization Domain Research Articles

Intrathecal gastrodin alleviates allodynia in a rat spinal nerve ligation model through NLRP3 inflammasome inhibition

BackgroundGastrodin (GAS), a main bioactive component of the herbal plant, Gastrodia elata Blume, has shown to have beneficial effects on neuroinflammatory diseases such as Alzheimer’s disease in animal studies and migraine in clinical studies. Inflammasome is a multimeric protein complex having a core of pattern recognition receptor and has been implicated in the development of neuroinflammatory diseases. Gastrodin has shown to modulate the activation of nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome. This study investigated the effects of GAS on the intensity of mechanical allodynia and associated changes in NLRP3 inflammasome expression at the spinal level using L5/6 spinal nerve ligation model (SNL) in rats.MethodsIntrathecal (IT) catheter implantation and SNL were used for drug administration and pain model in male Sprague-Dawley rats. The effect of gastrodin or MCC950 (NLRP3 inflammasome inhibitor) on mechanical allodynia was measured by von Frey test. Changes in NLRP3 inflammasome components and interleukin-1β (IL-1β) and cellular expression were examined in the spinal cord and dorsal root ganglion.ResultsThe expression of NLRP3 inflammasome components was found mostly in the neurons in the spinal cord and dorsal root ganglion. The protein and mRNA levels of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, and IL-1β were upregulated in SNL animals compared to Sham animals. IT administration of GAS significantly attenuated the expression of NLRP3 inflammasome and the intensity of SNL-induced mechanical allodynia. NLRP3 inflammasome inhibitor, MCC950, also attenuated the intensity of allodynia, but the effect is less strong and shorter than that of GAS.ConclusionsExpression of NLRP3 inflammasome and IL-1β is greatly increased and mostly found in the neurons at the spinal level in SNL model, and IT gastrodin exerts a significant anti-allodynic effect in SNL model partly through suppressing the expression of NLRP3 inflammasome.

Dietary tryptophan alleviates intestinal inflammation caused by long photoperiod via gut microbiota derived tryptophan metabolites-NLRP3 pathway in broiler chickens

Light pollution is a potential risk factor for intestinal health. Tryptophan plays an important role in the inhibition of intestinal inflammation. However, the mechanism of tryptophan in alleviating intestinal inflammation caused by long photoperiod is still unclear. This study investigated the anti-inflammatory effect of dietary tryptophan on intestinal inflammatory damage induced by long photoperiod and its potential mechanism in broiler chickens. We found that dietary tryptophan mitigated long photoperiod-induced intestinal tissue inflammatory damage and inhibited the activation of Nucleotide-Binding Oligomerization Domain, Leucine-Rich Repeat and Pyrin Domain-Containing 3 inflammasome. Moreover, dietary tryptophan significantly increased the relative abundance of Faecalibacterium, Enterococcus, and Lachnospiraceae_NC2004_group were significantly decreased the relative abundance of Ruminococcus_torques_group and norank_f_UCG-010 under the condition of long photoperiod (P < 0.05). The results of tryptophan targeted metabolomics show that tryptophan significantly increased indole-3-acetic acid (IAA) and indole-3 lactic acid (ILA), and significantly decreased xanthurenic acid (XA) under long photoperiod (P < 0.05). In conclusion, the results indicated that dietary tryptophan alleviates intestinal inflammatory damage caused by long photoperiod via the inhibition of Nucleotide-Binding Oligomerization Domain, Leucine-Rich Repeat and Pyrin Domain-Containing 3 inflammasome activation, which was mediated by tryptophan metabolites. Therefore, tryptophan supplementation could be a promising way to protect the intestine health under the condition of long photoperiod.

Activation of nucleotide-binding oligomerization domain 2 by muramyl dipeptide negatively regulates Toll-like receptor 9-mediated colonic inflammation through the induction of deubiquitinating enzyme A expression.

Mutations in nucleotide-binding oligomerization domain 2 (NOD2) are associated with Crohn's disease (CD). Although NOD2 activation contributes to the maintenance of intestinal homeostasis through the negative regulation of pro-inflammatory cytokine responses mediated by Toll-like receptors (TLRs), the effects of NOD2 activation on interferon (IFN)-α responses induced by TLR9 have been poorly defined. To explore the cross-talk between NOD2 and TLR9, human monocytes or dendritic cells (DCs) were stimulated with NOD2 and/or TLR9 ligands to measure IFN-α production. The severity of dextran sodium sulfate (DSS)-induced colitis was compared in mice treated with NOD2 and/or TLR9 ligands. Expression of IFN-α and IFN-stimulated genes (ISGs) was examined in the colonic mucosa of patients with inflammatory bowel disease (IBD). NOD2 activation reduced TLR9-induced IFN-α production by monocytes and DCs in a deubiquitinating enzyme A (DUBA)-dependent manner. Activation of DUBA induced by the co-stimulation of TLR9 and NOD2 inhibited Lys63-linked polyubiquitination of TRAF3 and suppressed TLR9-mediated IFN-α production. NOD2 activation in hematopoietic cells protected mice from TLR9-induced exacerbation of DSS-induced colitis by down-regulating IFN-α responses and up-regulating DUBA expression. Colonic mucosa of patients with active and remitted IBD phases was characterized by the enhanced and reduced expression of ISGs, respectively. Expression levels of IFN-α and IL-6 positively correlated in the active colonic mucosa of patients with ulcerative colitis and CD, whereas DUBA expression inversely correlated with that of IFN-α in patients with CD. Collectively, these data suggest that DUBA-dependent negative effect of NOD2 on TLR9-mediated IFN-α responses contributes to the maintenance of intestinal homeostasis.

Intermedin1-53 Inhibits NLRP3 Inflammasome Activation by Targeting IRE1α in Cardiac Fibrosis.

Intermedin (IMD), a paracrine/autocrine peptide, protects against cardiac fibrosis. However, the underlying mechanism remains poorly understood. Previous study reports that activation of nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome contributes to cardiac fibrosis. In this study, we aimed to investigate whether IMD mitigated cardiac fibrosis by inhibiting NLRP3. Cardiac fibrosis was induced by angiotensin II (Ang II) infusion for 2weeks in rats. Western blot, real-time PCR, histological staining, immunofluorescence assay, RNA sequencing, echocardiography, and hemodynamics were used to detect the role and the mechanism of IMD in cardiac fibrosis. Ang II infusion resulted in rat cardiac fibrosis, shown as over-deposition of myocardial interstitial collagen and cardiac dysfunction. Importantly, NLRP3 activation and endoplasmic reticulum stress (ERS) were found in Ang II-treated rat myocardium. Ang II infusion decreased the expression of IMD and increased the expression of the receptor system of IMD in the fibrotic rat myocardium. IMD treatment attenuated the cardiac fibrosis and improved cardiac function. In addition, IMD inhibited the upregulation of NLRP3 markers and ERS markers induced by Ang II. In vitro, IMD knockdown by small interfering RNA significantly promoted the Ang II-induced cardiac fibroblast and NLRP3 activation. Moreover, silencing of inositol requiring enzyme 1 α (IRE1α) blocked the effects of IMD inhibiting fibroblast and NLRP3 activation. Pre-incubation with PKA pathway inhibitor H89 blocked the effects of IMD on the anti-ERS, anti-NLRP3, and anti-fibrotic response. In conclusion, IMD alleviated cardiac fibrosis by inhibiting NLRP3 inflammasome activation through suppressing IRE1α via the cAMP/PKA pathway.

Therapeutic potential of Nlrp1 inflammasome, Caspase-1, or Caspase-6 against Alzheimer disease cognitive impairment.

The sequential activation of Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing protein 1 (Nlrp1) inflammasome, Caspase-1 (Casp1), and Caspase-6 (Casp6) is implicated in primary human neuron cultures and Alzheimer Disease (AD) neurodegeneration. To validate the Nlrp1-Casp1-Casp6 pathway in vivo, the APPSwedish/Indiana J20 AD transgenic mouse model was generated on either a Nlrp1, Casp1 or Casp6 null genetic background and mice were studied at 4-5 months of age. Episodic memory deficits assessed with novel object recognition were normalized by genetic ablation of Nlrp1, Casp1, or Casp6 in J20 mice. Spatial learning deficits, assessed with the Barnes Maze, were normalized in genetically ablated J20, whereas memory recall was normalized in J20/Casp1-/- and J20/Casp6-/-, and improved in J20/Nlrp1-/- mice. Hippocampal CA1 dendritic spine density of the mushroom subtype was reduced in J20, and normalized in genetically ablated J20 mice. Reduced J20 hippocampal dentate gyrus and CA3 synaptophysin levels were normalized in genetically ablated J20. Increased Iba1+-microglia in the hippocampus and cortex of J20 brains were normalized by Casp1 and Casp6 ablation and reduced by Nlrp1 ablation. Increased pro-inflammatory cytokines, TNF-α and CXCL1, in the J20 hippocampus were normalized by Nlrp1 or Casp1 genetic ablation. CXCL1was also normalized by Casp6 genetic ablation. IFN-γ was increased and total amyloid β peptide was decreased in genetically ablated Nlrp1, Casp1 or Casp6 J20 hippocampi. We conclude that Nlrp1, Casp1, or Casp6 are implicated in AD-related cognitive impairment, inflammation, and amyloidogenesis. These results indicate that Nlrp1, Casp1, and Casp6 represent rational therapeutic targets against cognitive impairment and inflammation in AD.

GSK669, a NOD2 receptor antagonist, inhibits thrombosis and oxidative stress via targeting platelet GPVI

Background and PurposePreviously, we discovered that the activation of nucleotide-binding oligomerization domain 2 (NOD2) enhances platelet activation. We here investigated the antiplatelet and antithrombotic potential of GSK669, a NOD2 antagonist. Experimental ApproachEffects of GSK669 on platelet functions, reactive oxygen species (ROS) and proinflammatory cytokine generation were detected. NOD2-/- platelets were used to confirm GSK669 target. The interaction between GSK669 and glycoprotein VI (GPVI) was detected using surface plasmon resonance (SPR) spectroscopy. GPVI downstream signaling was examined by Western blot. The antithrombotic and antioxidative effects were investigated using mouse mesenteric arteriole thrombosis model and pulmonary embolism model. Key ResultsGSK669 significantly inhibits platelet proinflammatory cytokine release induced by muramyl dipeptide, platelet aggregation, ATP release, and ROS generation induced by collagen and collagen related peptide (CRP). Platelet spreading and clot retraction are also inhibited. GSK669 also decreases collagen-induced phosphorylation of Src, Syk, PLCγ2, and Akt. The antiplatelet effect of GSK669 is NOD2-independent and mediated by GPVI antagonism. Consistent with its antiplatelet activity as a GPVI antagonist, GSK669 inhibits platelet adhesion on collagen in flow condition. Notably, GSK669 inhibits mouse mesenteric arteriole thrombosis similarly to aspirin without bleeding. The antithrombotic effect of GSK669 is further confirmed in the pulmonary embolism model; decreased malonaldehyde (MDA) and increased superoxide dismutase (SOD) levels in mouse plasma reveal a significant antioxidant effect of GSK669. Conclusion and ImplicationsBeyond its anti-inflammatory effect as a NOD2 antagonist, GSK669 is also an efficient and safe antiplatelet agent combined with antioxidant effect by targeting GPVI. An antiplatelet agent bearing antioxidative and anti-inflammatory activities without bleeding risk may have therapeutic advantage over current antiplatelet drugs for atherothrombosis.

Trimethylamine N-Oxide Exacerbates Cardiac Fibrosis via Activating the NLRP3 Inflammasome.

Background/Aims: Gut microbiota has been reported to correlate with a higher mortality and worse prognosis of cardiovascular diseases. Trimethylamine N-oxide (TMAO) is a gut microbiota-dependent metabolite of specific dietary nutrients, which is linked to cardiac fibrosis. Recent reports have suggested that the activation of Nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome contributed to cardiac fibrosis. However, whether TMAO mediates cardiac fibrosis via activating NLRP3 inflammasome remains unclear.Methods and Results: To determine the role of TMAO–mediated cardiac fibrosis, we established mouse models of doxorubicin (DOX)-induced cardiac fibrosis with or without TMAO in drinking water. TMAO exacerbated DOX-induced cardiac dysfunction, heart weight and cardiac fibrosis manifested by enhanced collagen accumulation, higher profibrotic levels and elevated inflammatory factors as well as NLRP3 inflammasome activation. Using primary cultured mouse cardiac fibroblast, our results indicated that TMAO promoted proliferation, migration and collagen secretion in a dose-dependent manner by TGF-β/Smad3 signaling. Furthermore, TMAO treatment induced NLRP3 inflammasome activation including oxidative stress in cultured cardiac fibroblast. Importantly, the silencing of NLRP3 presented a protection effect against cardiac fibrosis including cellular proliferation, migration and collagen deposition in vitro.Conclusion: Our data suggested that TMAO aggravated DOX-induced mouse cardiac fibrosis, at least in part, through activation of the NLRP3 inflammasome, providing a new potential target for preventing the progression of cardiac fibrosis.

IL-1β Plays an Important Role in Pressure Overload-Induced Atrial Fibrillation in Mice.

Hypertension is one risk for atrial fibrillation (AF) and induces cardiac inflammation. Recent evidence indicates that pressure overload-induced ventricular structural remodeling is associated with the activation of nucleotide binding-oligomerization domain (NOD)-like receptor P3 (NLRP3) inflammasomes, including an apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We hypothesized that NLRP3 inflammasomes are an initial sensor for danger signals in pressure overload-induced atrial remodeling, leading to AF. Transverse aortic constriction (TAC) or a sham procedure was performed in mice deficient for ASC-/- and interleukin-1β (IL-1β-/-). One week after the procedure, electrical left atrial burst pacing from the esophagus was performed for 30 s to induce AF. IL-1β, monocyte chemotactic protein 1 (MCP-1), connective tissue growth factor (CTGF), and collagen 1 gene expression were also examined. The electrical burst pacing induced AF in TAC-operated wild-type (WT) (p < 0.001) and ASC-/- (p < 0.05) mice, compared to no AF in the sham-operated WT and ASC-/- mice, respectively. In contrast, the number of mice in which sustained AF was induced was similar between TAC-operated IL-1β-/- and sham-operated IL-1β-/- mice (p > 0.05). The expression of all genes tested was increased in TAC-operated WT and ASC-/- mice compared with sham-operated WT and ASC-/- mouse atria, respectively. CTGF and collagen 1, but not MCP-1, gene expressions were increased in TAC-operated IL-1β-/- mouse atria compared with sham-operated WT and IL-1β-/- mouse atria. In contrast, the IL-1β gene was not detected in either TAC-operated or sham-operated IL-1β-/- mouse atria. These results suggest that an IL-1β activation pathway, different from NLRP3 inflammasomes, plays an important role in pressure overload-induced sustained AF.

Targeting Oxidative Stress and Mitochondrial Dysfunction in the Treatment of Impaired Wound Healing: A Systematic Review.

Wound healing is a well-tuned biological process, which is achieved via consecutive and overlapping phases including hemostasis, inflammatory-related events, cell proliferation and tissue remodeling. Several factors can impair wound healing such as oxygenation defects, aging, and stress as well as deleterious health conditions such as infection, diabetes, alcohol overuse, smoking and impaired nutritional status. Growing evidence suggests that reactive oxygen species (ROS) are crucial regulators of several phases of healing processes. ROS are centrally involved in all wound healing processes as low concentrations of ROS generation are required for the fight against invading microorganisms and cell survival signaling. Excessive production of ROS or impaired ROS detoxification causes oxidative damage, which is the main cause of non-healing chronic wounds. In this context, experimental and clinical studies have revealed that antioxidant and anti-inflammatory strategies have proven beneficial in the non-healing state. Among available antioxidant strategies, treatments using mitochondrial-targeted antioxidants are of particular interest. Specifically, mitochondrial-targeted peptides such as elamipretide have the potential to mitigate mitochondrial dysfunction and aberrant inflammatory response through activation of nucleotide-binding oligomerization domain (NOD)-like family receptors, such as the pyrin domain containing 3 (NLRP3) inflammasome, nuclear factor-kappa B (NF-κB) signaling pathway inhibition, and nuclear factor (erythroid-derived 2)-like 2 (Nrf2).

WITHDRAWN: Tetramethylpyrazine ameliorates renal inflammation via autophagy induction in acute kidney injury

// Rui Xin 1,* , Long Jin 2,* , Luying Liu 1,* , Lin Wang 3 , Yun Hou 1 , Xianghui Meng 4 , Yanxiao Xiang 5 , Xuhong Sun 1 , Weidong Jiang 3 , Xiaojie Wu 1 , Lixia Zhang 6 , Zhenzhen Du 6 , Xiying Luan 1 and Pengchao Du 1,* 1 College of Basic Medical, Binzhou Medical University, Yantai, Shandong, China 2 The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning, China 3 The Second Hospital of Shandong University, Jinan, Shandong, China 4 Central hospital of ZiBo (GaoQing branch courts), Zibo, Shandong, China 5 Qilu Hospital, Shandong University, Jinan, Shandong, China 6 Binzhou Medical University, Yantai, Shandong, China * These authors have contributed equally to this work Correspondence to: Pengchao Du, email: pengchaodu@163.com Keywords : NOD2; autophagy; TMP; acute kidney injury Received: July 27, 2017 Accepted: December 01, 2017 Epub: January 06, 2018 Abstract Tetramethylpyrazine was used to alleviate clinical renal injury in acute kidney injury, while the underlying molecular mechanism was poorly known. The activation of nucleotide-binding oligomerization domain containing 2 (NOD2) was focused on. In the present study, we showed that [ 1 ] tetramethylpyrazine protected against acute kidney injury through inhibition of the upregulation of NOD2 expression after ischemia/reperfusion injury in rat models, in vivo ; [ 2 ] tetramethylpyrazine could significantly inhibit the upregulation of NOD2 expression induced by CoCl2 treatment and induce autophagy strongly in vitro ; [ 3 ] furtherly, the effect of tetramethylpyrazine on reducing NOD2 expression was blocked by autophagy inhibition in part. For the first time, these results provided evidence that tetramethylpyrazine could inhibit inflammation mediated by NOD2 to protect against renal injury in acute kidney injury via autophagy induction partly, which helped design a new approach to develop therapeutic strategies for prevention of deterioration of renal function and for treatment of acute kidney injury.

The Role of the p38-MNK-eIF4E Signaling Axis in TNF Production Downstream of the NOD1 Receptor.

Activation of nucleotide-binding oligomerization domain (NOD) 1 and NOD2 by muropeptides triggers a complex transcriptional program in innate immune cells. However, little is known about posttranscriptional regulation of NOD1- and NOD2-dependent responses. When stimulated with a prototypic NOD1 agonist, N-acetylglucosaminyl-N-acetylmuramyl-l-alanyl-d-isoglutamyl-meso-diaminopimelic acid (GM-triDAP), human monocyte-derived macrophages (MDM) produced an order of magnitude more TNF, IL-6, and pro-IL-1β than did monocyte-derived dendritic cells (MDDC), despite similar NOD1 expression, similar cytokine mRNA kinetics, and comparable responses to LPS. TNF production by GM-triDAP-activated MDM was independent of autocrine IL-1. However, GM-triDAP-activated MDM translated TNF mRNA more efficiently than did MDDC. As an underlying mechanism, NOD1 triggering in MDM caused a more potent and long-lasting activation of the signaling axis involving p38 MAPK, MAPK-interacting kinase (MNK), and eukaryotic translation initiation factor 4E, which is a critical regulator of translation. Furthermore, MNK controlled TNF mRNA abundance in MDDC and MDM upon NOD1 triggering. NOD1-dependent responses were more sensitive to MNK inhibition than were TLR4-dependent responses. These results demonstrate the importance of the p38-MNK-eukaryotic translation initiation factor 4E axis in TNF production downstream of NOD1.

Evidence that NF-κB and MAPK Signaling Promotes NLRP Inflammasome Activation in Neurons Following Ischemic Stroke.

Multi-protein complexes, termed "inflammasomes," are known to contribute to neuronal cell death and brain injury following ischemic stroke. Ischemic stroke increases the expression and activation of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) Pyrin domain containing 1 and 3 (NLRP1 and NLRP3) inflammasome proteins and both interleukin (IL)-1β and IL-18 in neurons. In this study, we provide evidence that activation of either the NF-κB and MAPK signaling pathways was partly responsible for inducing the expression and activation of NLRP1 and NLRP3 inflammasome proteins and that these effects can be attenuated using pharmacological inhibitors of these two pathways in neurons and brain tissue under in vitro and in vivo ischemic conditions, respectively. Moreover, these findings provided supporting evidence that treatment with intravenous immunoglobulin (IVIg) preparation can reduce activation of the NF-κB and MAPK signaling pathways resulting in decreased expression and activation of NLRP1 and NLRP3 inflammasomes, as well as increasing expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL, in primary cortical neurons and/or cerebral tissue under in vitro and in vivo ischemic conditions. In summary, these results provide compelling evidence that both the NF-κB and MAPK signaling pathways play a pivotal role in regulating the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons and brain tissue under ischemic conditions. In addition, treatment with IVIg preparation decreased the activation of the NF-κB and MAPK signaling pathways, and thus attenuated the expression and activation of NLRP1 and NLRP3 inflammasomes in primary cortical neurons under ischemic conditions. Hence, these findings suggest that therapeutic interventions that target inflammasome activation in neurons may provide new opportunities in the future treatment of ischemic stroke.

Activation of Nucleotide-Binding Oligomerization Domain 1 (NOD1) Receptor Signaling in Labeo rohita by iE-DAP and Identification of Ligand-Binding Key Motifs in NOD1 by Molecular Modeling and Docking

The nucleotide-binding oligomerization domain 1 (NOD1) receptor recognizes various pattern-associated structures of microbes through its leucine-rich repeat (LRR) domain and activates signaling cascades to induce innate immunity. This report describes the activation of NOD1 receptor signaling by gamma-D-glutamyl-meso-diaminopimelic acid (or γ-D-Glu-mDAP [iE-DAP]) in a commercially important fish species, rohu (Labeo rohita). It also described critical motifs in the NOD1-LRR domain that could be involved in binding iE-DAP, lipopolysaccharide (LPS), and polyinosinic:polycytidylic acid (poly I:C). The activation of NOD1 receptor signaling was studied by injecting iE-DAP, and analysis of tissue samples for NOD1 and receptor-interacting serine/threonine kinase (RICK) expression was done by quantitative real-time polymerase chain reaction (qRT-PCR) assay. To identify ligand-binding motifs in NOD1, the 3D model of NOD1-LRR was generated, followed by a 6-ns molecular dynamics simulation. Molecular docking of LPS with NOD1-LRR was executed at the Hex and PatchDock servers, and iE-DAP and poly I:C in the AutoDock 4.2, FlexX 2.1, Glide 5.5, and GOLD 4.1 programs. The results of qRT-PCR revealed significant (p < 0.05) upregulation of NOD1 and RICK expression. Molecular docking revealed that the amino acid residues at LRR1-2, LRR3-7, and LRR8-9 could be involved in poly I:C, LPS, and iE-DAP binding, respectively. In fish, this is the first report describing the 3D structure of NOD1-LRR and its critical ligand-binding motifs.

Expression of TRAF6 and pro-inflammatory cytokines through activation of TLR2, TLR4, NOD1, and NOD2 in human periodontal ligament fibroblasts

ObjectiveHuman periodontal ligament fibroblasts (HPDLFs) play a crucial role in protecting against oral bacteria in periapical tissue. Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) are two major forms of innate immune sensors that recognize microbial pathogens and initiate pro-inflammatory signalling. Tumour necrosis factor receptor-associated factor 6 (TRAF6) is an adapter protein for TLR-mediated nuclear factor-κB (NF-κB) signalling pathway activation that induces the production of pro-inflammatory cytokines. The aim of this study was to investigate the expression of TLR2, TLR4, NOD1, and NOD2 in HPDLFs. We also investigated the expression of TRAF6 and pro-inflammatory cytokines induced by the activation of TLRs and NODs. MethodsThe expression of TLR2, TLR4, NOD1, and NOD2 was measured by reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry, and immunostaining. HPDLFs were stimulated with TLR and NOD agonists. Then, the expression of TRAF6 was measured by real-time PCR and western blot. Concentrations of IL-1β, IL-6, and IL-8 in the culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Finally, by using small interfering RNA (siRNA) for TRAF6, we analysed the production of IL-1β, IL-6, and IL-8 in HPDLFs upon stimulation with TLRs and NODs agonists. ResultsWe found clear mRNA and protein expression of TLR2, TLR4, NOD1, and NOD2 in HPDLFs. The expression levels of TRAF6 and pro-inflammatory cytokines (IL-1β, IL-6, and IL-8) were markedly up-regulated upon the activation of TLRs and NODs. Furthermore, the co-activation of TLRs and NODs had synergistic effect on the production of TRAF6 and pro-inflammatory cytokines. We also found TRAF6 suppression resulted in reduced IL-1β, IL-6, and IL-8 expression upon TLR and NOD agonists challenge. ConclusionThese findings indicated that TLR2, TLR4, NOD1, and NOD2 are functional receptors in HPDLFs during innate immune responses to invading bacteria, and a combination of signalling through TLRs and NODs leads to the synergistic enhancement of inflammatory reactions in HPDLFs. In addition, TLR and NOD signalling involving TRAF6 contribute to inflammatory responses in HPDLFs.

NOD1-Mediated Mucosal Host Defense againstHelicobacter pylori

Infection of the stomach with Helicobacter pylori is an important risk factor for gastritis, peptic ulcer, and gastric carcinoma. Although it has been well established that persistent colonization by H. pylori is associated with adaptive Th1 responses, the innate immune responses leading to these Th1 responses are poorly defined. Recent studies have shown that the activation of nucleotide-binding oligomerization domain 1 (NOD1) in gastric epithelial cells plays an important role in innate immune responses against H. pylori. The detection of H. pylori-derived ligands by cytosolic NOD1 induces several host defense factors, including antimicrobial peptides, cytokines, and chemokines. In this paper, we review the molecular mechanisms by which NOD1 contributes to mucosal host defense against H. pylori infection of the stomach.

Muramyl dipeptide activation of nucleotide-binding oligomerization domain 2 protects mice from experimental colitis

The mechanisms underlying the susceptibility of individuals with caspase recruitment domain 15 (CARD15) mutations and corresponding abnormalities of nucleotide-binding oligomerization domain 2 (NOD2) protein to Crohn disease are still poorly understood. One possibility is based on previous studies showing that muramyl dipeptide (MDP) activation of NOD2 negatively regulates TLR2 responses and that absence of such regulation leads to heightened Th1 responses. We now report that administration of MDP protects mice from the development of experimental colitis by downregulating multiple TLR responses, not just TLR2. The basis of these in vivo findings was suggested by in vitro studies of DCs, in which we showed that prestimulation of cells with MDP reduces cytokine responses to multiple TLR ligands and this reduction is dependent on enhanced IFN regulatory factor 4 (IRF4) activity. Further studies of mouse models of colitis showed that this inhibitory role of IRF4 does in fact apply to MDP-mediated protection from colitis, since neither IRF4-deficient mice nor mice treated with siRNA specific for IRF4 were protected. These findings indicate that MDP activation of NOD2 regulates innate responses to intestinal microflora by downregulating multiple TLR responses and suggest that the absence of such regulation leads to increased susceptibility to Crohn disease.