Nucleotide-binding Oligomerization Domain 2 Expression Research Articles

NOD2 contributes to Parvimonas micra-induced bone resorption in diabetic rats with experimental periodontitis.

Type 2 diabetes mellitus (T2DM) may affect the oral microbial community, exacerbating periodontal inflammation; however, its pathogenic mechanisms remain unclear. As nucleotide-binding oligomerization domain 2 (NOD2) plays a crucial role in the activation during periodontitis (PD), it is hypothesized that changes in the oral microbial community due to diabetes enhance periodontal inflammation through the activation of NOD2. We collected subgingival plaque from 180 subjects who were categorized into two groups based on the presence or absence of T2DM. The composition of oral microbiota was detected by 16S rRNA high-throughput sequencing. In animal models of PD with or without T2DM, we assessed alveolar bone resorption by micro-computerized tomography and used immunohistochemistry to detect NOD2 expression in alveolar bone. Primary osteoblasts were cultured in osteogenic induction medium with high or normal glucose and treated with inactivated bacteria. After 24h of inactivated bacteria intervention, the osteogenic differentiation ability was detected by alkaline phosphatase (ALP) staining, and the expressions of NOD2 and interleukin-12 (IL-6) were detected by western blot. The relative abundance of Parvimonas and Filifactor in the T2DM group was increased compared to the group without T2DM. In animal models, alveolar bone mass was decreased in PD, particularly in T2DM with PD (DMPD) group, compared to controls. Immunohistochemistry revealed NOD2 in osteoblasts from the alveolar bone in both the PD group and DMPD group, especially in the DMPD group. In vitro, intervention with inactivated Parvimonas significantly reduced ALP secretion of primary osteoblasts in high glucose medium, accompanied by increased expression of NOD2 and IL-6. The results suggest that T2DM leading to PD may be associated with the activation of NOD2 by Parvimonas.

Two variants of NOD2 gene polymorphisms and their relation with Tuberculosis in Andhra Pradesh population, India

IntroductionTuberculosis (TB) remains one of the major infectious diseases even after the emergence of new diseases in the present era. Few polymorphic gene studies on Nucleotide-binding Oligomerization Domain 2 (NOD2) gene reveal that they are associated with the risk of TB in some populations. An attempt has been made to find such association in the Andhra Population of India. MethodsTwo variants (rs2066847 and rs2066842) of NOD2 gene were analyzed genotypically using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR–RFLP) analysis, on TB patients and negative controls. Statistical tests like Chi-square, Odds ratio (OR) and 95 % Confidence Interval (CI) were considered to evaluate the susceptibility of the NOD2 gene polymorphism with TB. ResultsThe analysis of the results obtained revealed that the two variants of NOD2 gene rs2066842 (OR = 1.278; 95%CI = 0.436–3.746; P = 0.653), and rs2066847 (OR = 0.809; 95%CI = 0.015–41.227; P = 0.915) do not attribute to the risk of TB in this population. ConclusionThe final results of the study reveal that these two polymorphisms of the NOD2 gene do not show an association with the susceptibility of TB in the Andhra Population of India. Further studies in different ethnic groups with larger sample sizes are necessary to evaluate the proper associative gene expression of NOD2 with TB.

Association of the rs2111234, rs3135499, rs8057341 polymorphisms in the NOD2 gene with leprosy: A case-control study in the Norte de Santander, Colombia population.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. The development of leprosy involves several factors, including the causative agent, the individual host's immune response, environmental factors, and the genetic background of the host. Specifically, the host's innate immune response, encoded by genes, determines their susceptibility to developing leprosy post-infection. Polymorphic variants in the nucleotide-binding oligomerization domain 2 (NOD2) gene are associated with leprosy among populations in a variety of endemic areas around the world. Colombia, a country located in the tropical zone, has several leprosy-endemic regions, including Norte de Santander. The aim of this study was to analyze the rs7194886, rs2111234, rs3135499, and rs8057341 single nucleotide polymorphisms (SNPs) in the NOD2 gene using a case-control study to determine whether they confer greater or lesser susceptibility to the development of leprosy. The TaqMan qPCR amplification system was used for SNPs detection. An association between the A-rs8057341 SNP (p = 0,006286) and resistance to leprosy was found. However, the rs3135499 (p = 0,9063) and rs2111234 (p = 0.1492) were not found to be associated with leprosy susceptibility. In addition, the rs7194886 SNP was not found to be in Hardy-Weinberg equilibrium (HWE) in the study population. The GAG haplotype, consisting of SNPs rs2111234-G, rs3135499-A, and rs8057341G, acts as a susceptibility factor for the development of leprosy in women. SNPs rs3135499 and rs8057341 are functionally related to decreased NOD2 expression according to an in-silico analysis. The SNPs rs8057341-A was related with resistance to leprosy and the haplotype rs2111234-G, rs3135499-A and rs8057341-G SNPs was related with susceptibility in the Norte de Santander Colombia, studied population.

NOD2 is involved in regulating odontogenic differentiation of DPSCs suppressed by MDP through NF-κB/p65 signaling.

Dental pulp stem cells (DPSCs) are well known for their capable of both self-renewal and multilineage differentiation. Dental tissue diseases, include caries, are often accompanied by inflammatory microenvironment, and muramyl dipeptide (MDP) is involved in the inflammatory stimuli to influence the differentiation of DPSCs. Nucleotide-binding oligomerization domain 2 (NOD2), a member of the cytosolic Nod-like receptor (NLR) family, plays a key role in inflammatory homeostasis regulation, but the role of NOD2 in DPSCs differentiation under inflammatory is still unclear. In this study, we identified that MDP suppressed odontogenic differentiation of DPSCs via NOD2/ NF-κB/p65 signaling pathway. Alizarin red staining and ALP activity showed the odontogenic differentiation was suppressed by MDP in a concentration-dependent manner, and the expression of dentin differentiation marker protein dentin matrix protein 1 (DMP-1) and dentin Sialophosphoprotein (DSPP) also indicated the same results. The expression of NOD2 increased gradually with the concentration of MDP as well as the phosphorylation and nuclear translocation of p65, which meant NF-κB signaling pathway was activated. Further, the interference of NOD2 inhibited the phosphorylation and nuclear translocation of p65 and reversed the MDP-mediated decrease of odontoblast differentiation of DPSCs. Our study showed that MDP can inhibit the odontoblast differentiation of DPSCs in a concentration-dependent manner. The NF-κB signaling pathway was activated by increasing expression of NOD2. Interference of NOD2 reversed the negative ability odontoblast differentiation of DPSCs in the inflammatory environment. Our study might provide a theoretical basis for the clinical treatment for dentinogenesis of DPSCs.

Association of rs3135500 and rs3135499 Polymorphisms in the MicroRNAbinding Site of Nucleotide-binding Oligomerization Domain 2 (NOD2) Gene with Susceptibility to Rheumatoid Arthritis

The nucleotide-binding oligomerization domain 2 (NOD2) is the key regulator of inflammatory responses and has been involved in the pathogenesis of rheumatoid arthritis (RA). Laboratory and in silico evaluations have demonstrated that some polymorphisms in 3ˊUTR of NOD2 gene could influence the secondary structure of this region and similarly thermodynamic features of hybridization site and finally deregulate the expression of NOD2. In the current study, for the first time, we evaluated the possible association between single nucleotide polymorphisms (SNPs) rs3135500 and rs3135499 in the NOD2 gene with RA risk in the Iranian population. One hundred and fifteen patients with RA and 120 healthy subjects were recruited in this case-control study. Genotyping of rs3135500 and rs3135499 polymorphisms were accomplished using the real‑time polymerase chain reaction high resolution melting (HRM) method. We found a substantial association of AA and AG genotypes in rs3135500 with the risk of RA (AA vs GG; OR=5.547; 95%CI [2.564-11.999]; p<0.001 and AG vs GG; OR=2.179; 95%CI [1.145-4.147]; p=0.017). Moreover, in the patient group, there was a significant relationship between the increased concentration of erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) with rs3135500 (A allele) (p<0.05). However, there were no important associations between rs3135499 with the risk of RA (p>0.05). However, we found a noteworthy association of the C allele in rs3135499 with an increased level of CRP in patients (p>0.05). Our findings propose a considerable association between NOD2 polymorphisms with increased risk of RA and disease activity.

Blue Light Induces Impaired Autophagy through Nucleotide-Binding Oligomerization Domain 2 Activation on the Mouse Ocular Surface.

In this study, we investigated the effects of blue light exposure on nucleotide-binding oligomerization domain 2 (NOD2) expression on the mouse ocular surface and evaluated the role of NOD2 activation in light-induced cell death. Mice were divided into wild-type (WT), NOD2-knock out (KO), WT + blue light (WT + BL), and NOD2-KO + blue light (NOD2-KO + BL) groups, and the mice in the WT+BL and NOD2-KO + BL groups were exposed to blue light for 10 days. After 10 days of blue light exposure, increased reactive oxygen species and malondialdehyde were observed in the WT + BL and NOD2-KO + BL groups, and the WT + BL group showed a higher expression of NOD2 and autophagy related 16 like 1. Although both WT+BL and NOD2-KO + BL groups showed an increase in the expression of light chain 3-II, NOD2-KO + BL mice had a significantly lower p62 expression than WT + BL mice. In addition, NOD2-KO+BL mice had significantly lower corneal epithelial damage and apoptosis than WT + BL mice. In conclusion, blue light exposure can induce impaired autophagy by activation of NOD2 on the ocular surface. In addition, the reactive oxygen species (ROS)–NOD2–autophagy related 16 like 1 (ATG16L) signaling pathway may be involved in the blue-light-induced autophagy responses, resulting in corneal epithelial apoptosis.

NOD2 inhibits tumorigenesis and increases chemosensitivity of hepatocellular carcinoma by targeting AMPK pathway

Nucleotide binding oligomerization domain 2 (NOD2) is a recognized innate immune sensor which can initiate potent immune response against pathogens. Many innate immune sensors have been reported to be of great importance in carcinogenesis. However, the role of NOD2 in cancer is not well understood. Here we investigated the role of NOD2 in the development of hepatocellular carcinoma (HCC). We demonstrated that NOD2 deficiency promoted hepatocarcinogenesis in N-nitrosodiethylamine (DEN)/carbon tetrachloride (CCl4) induced HCC mice model and xenograft tumor model. In vitro investigation showed that NOD2 acted as a tumor suppressor and inhibited proliferation, colony formation and invasion of HCC cells. Clinical investigation showed that NOD2 expression was completely lost or significantly downregulated in clinical HCC tissues, and loss of NOD2 expression was significantly correlated with advanced disease stages. Further investigation showed that NOD2 exerted its anti-tumor effect through activating adenosine 5′-monophosphate (AMP) -activated protein kinase (AMPK) signaling pathway, and NOD2 significantly enhanced the sensitivity of HCC cells to sorafenib, lenvatinib and 5-FU treatment through activating AMPK pathway induced apoptosis. Moreover, we demonstrated that NOD2 activated AMPK pathway by directly binding with AMPKα-LKB1 complex, which led to autophagy-mediated apoptosis of HCC cells. Altogether, this study showed that NOD2 acted as a tumor suppressor as well as a chemotherapeutic regulator in HCC cells by directly activating AMPK pathway, which indicated a potential therapeutic strategy for HCC treatment by upregulating NOD2-AMPK signaling axis.

NOD2 regulates microglial inflammation through the TAK1-NF-κB pathway and autophagy activation in murine pneumococcal meningitis

Streptococcus pneumoniae is responsible for pneumococcal meningitis, with significant mortality and morbidity worldwide. Microglial inflammation plays a vital role in meningitis. The peptidoglycan sensor NOD2 (nucleotide-binding oligomerization domain 2) has been identified to promote microglia activation, but the role in autophagy following pneumococcal meningitis remains unclear. In the present study, we investigated the role of NOD2 in microglial inflammation and autophagy, as well as related signaling pathways, during S. pneumonia infection. NOD2 expression was knocked down by the injection of lentivirus-mediated short-hairpin RNA (shRNA). Our results revealed that NOD2 promotes microglial inflammation by increasing inflammatory mediators. We also showed that the TAK1-NF-κB pathway is involved in this process. In addition, NOD2 increased the expression of autophagy-related proteins and induced autophagosome formation. Rapamycin and 3-MA were utilized to assess the role of autophagy in microglial inflammation induced by S. pneumonia. We demonstrated that autophagy serves as a cellular defense mechanism to reduce inflammatory mediators. Similar to the in vitro results, NOD2 induced inflammation and autophagy in the brain in a mouse meningitis model. Moreover, NOD2 silencing significantly reduced brain edema and improved the neurological function of pneumococcal meningitis mice. Taken together, these data demonstrate that NOD2 promotes microglial inflammation and autophagy in murine pneumococcal meningitis, and the TAK1-NF-κB pathway is involved in microglial activation.

P5443NOD2 knock down worsens diastolic dysfunction in murine angiotensin II-induced heart failure

Abstract Background Heart failure with preserved ejection fraction (HFpEF) is associated with cardiac inflammatory responses, indicating a potential role of the immune system in the pathology of diastolic dysfunction. The cytoplasmatic pattern recognition receptor, nucleotide binding oligomerization domain 2 (NOD2) belongs to the innate immune system and induces among others the NLRP3 inflammasome, known to be involved in myocarditis and coronary heart disease. Purpose The aim of this study was to explore the role of NOD2 in Angiotensin II (AngII)-induced diastolic heart failure. Methods In NOD2−/− knock down and C57Bl6/j-wild type (WT) mice, diastolic dysfunction was induced by subcutaneous administration of 1.4mg/kg*day–1 AngII. Twenty-one days after first AngII administration, left ventricular (LV) function was evaluated by pressure tip catheter. Cardiac fibrosis, inflammation, and the expression of NOD2 and the NLRP3 component Apoptosis-associated speck like protein containing a caspase recruitment domain (ASC) were determined via immunohistochemistry, real-time PCR or Western Blot. Results LV NOD2 mRNA expression was 2.3-fold (p<0.0005) and 1.9-fold (p<0.0005) lower in NOD2−/− control and NOD2−/− AngII mice compared to their respective WT littermates. In parallel, LV protein expression of the downstream NLRP3 component Apoptosis-associated speck like protein containing a caspase recruitment domain (ASC) was 1.5-fold (p<0.05) lower in NOD2−/− AngII mice versus WT AngII mice, whereas LV protein IL-1β levels were unchanged. LV diastolic dysfunction was more pronounced in NOD2−/− AngII mice versus WT AngII mice, as displayed by a 19% (p<0.05) increased LV relaxation time and 24% (p<0.057) impaired dP/dtmin, with no changes in the ejection fraction (EF: NOD2−/− AngII 72.5%±5.4 versus WT AngII 65.6±3.5). In parallel, LV presence of CD68-positive cells was 1.8-fold (p<0.05) higher in NOD2−/− AngII compared to WT AngII mice. Concomitantly, NOD2−/− AngII mice displayed 1.3-fold (p<0.05) and 1.7-fold (p<0.05) higher LV mRNA expression of the chemokine macrophage inflammatory protein (MIP)-2 and monocyte chemotactant protein (MCP)-1 compared to WT AngII mice, respectively. Furthermore, cardiac interstitial fibrosis in NOD2−/− mice with AngII-induced diastolic dysperformance was more pronounced versus the WT AngII group, as indicated by a 2.0-fold (p<0.0005), 2.0-fold, and 1.6-fold (p<0.05) higher LV ColI/ColIII ratio, and TGF-β and TIMP-1 mRNA expression, respectively. Conclusion NOD2−/− deteriorates LV diastolic dysfunction and worsens pathophysiological key mechanisms in mice with AngII-induced diastolic heart failure.

RICK/RIP2 is a NOD2-independent nodal point of gut inflammation.

Previous studies have shown that inhibition of receptor-interacting serine/threonine kinase (RICK) (also known as RIP2) results in amelioration of experimental colitis. This role has largely been attributed to nucleotide-binding oligomerization domain 2 (NOD2) signaling since the latter is considered a major inducer of RICK activation. In this study, we explored the molecular mechanisms accounting for RICK-mediated inhibition of inflammatory bowel disease (IBD). In an initial series of studies focused on trinitrobenzene sulfonic acid (TNBS)-colitis and dextran sodium sulfate (DSS)-colitis we showed that down-regulation of intestinal RICK expression in NOD2-intact mice by intra-rectal administration of a plasmid expressing RICK-specific siRNA was accompanied by down-regulation of pro-inflammatory cytokine responses in the colon and protection of the mice from experimental colitis. Somewhat surprisingly, intra-rectal administration of RICK-siRNA also inhibited TNBS-colitis and DSS-colitis in NOD2-deficient and in NOD1/NOD2-double deficient mice. In complementary studies of humans with IBD we found that expression of RICK, cellular inhibitor of apoptosis protein 2 (cIAP2) and downstream signaling partners were markedly increased in inflamed tissue of IBD compared to controls without marked elevations of NOD1 or NOD2 expression. In addition, the increase in RICK expression correlated with disease activity and pro-inflammatory cytokine responses. These studies thus suggest that NOD1- or NOD2-independenent activation of RICK plays a major role in both murine experimental colitis and human IBD.

Correlation between toll-like receptor 4 and nucleotide-binding oligomerization domain 2 (NOD2) and pathological severity in dogs with chronic gastrointestinal diseases

Toll-like receptor 4 (TLR4), nucleotide-binding oligomerization domain 2 (NOD2), and TNF-α play important roles in human inflammatory bowel diseases. The aim of this study was to elucidate the relationship between Toll-like receptor 4, NOD2, and TNF-α and the severity of chronic gastrointestinal diseases in dogs. We examined the expression levels of TLR4, NOD2, and TNF-α in the stomach, duodenum, ileum, colon, and rectum obtained from 21 dogs with chronic gastrointestinal disease, including inflammatory bowel disease, high-grade lymphoma, food responsive enteropathy, chronic pancreatitis, low-grade lymphoma, inflammatory colorectal polyp, and chronic colitis. Next, we demonstrated whether there is good correlation between the expression levels of TLR4, NOD2, and TNF-α and the histopathological analysis of each sample. We found that the level of TLR4 expression in the ileum of dogs with chronic gastrointestinal disease was positively associated with the histopathological severity. We also found that the level of NOD2 expression in the duodenum, stomach, and rectum was positively associated with the histopathological severity. However, there was no correlation between TNF-α expression in the 5 regions tested in this study and the histopathological severity. These findings indicate that TLR4 and NOD2 are remarkably associated with the severity of chronic gastrointestinal disease in dogs.

NOD2 Expression in Intestinal Epithelial Cells Protects Toward the Development of Inflammation and Associated Carcinogenesis.

Nucleotide-binding oligomerization domain 2 (NOD2) is an intracellular pattern recognition receptor that senses bacterial peptidoglycan-conserved motifs in cytosol and stimulates host immune response including epithelial and immune cells. The association of NOD2 mutations with a number of inflammatory pathologies including Crohn’s disease (CD), graft-versus-host diseases, or Blau syndrome, highlights its pivotal role in inflammatory response and the associated-carcinogenesis development. Since its identification in 2001 and its association with CD, the role of NOD2 in epithelial cells and immune cells has been investigated extensively but the precise mechanism by which NOD2 mutations lead to CD and the associated carcinogenesis development is largely unknown. In this review, we present and discuss recent developments about the role of NOD2 inside epithelial cells on the control of the inflammatory process and its linked carcinogenesis development.

NOD2 Expression in Streptococcus pneumoniae Meningitis and Its Influence on the Blood-Brain Barrier.

Streptococcus pneumoniae meningitis is one of the most common disorders seen in clinical practice. It is believed that the brain tissue immune injury is caused by the expression of pattern-recognition receptors (PRR) which can further induce the release of other cytokines and inflammatory cascades. The aim of this study is to investigate the expression of nucleotide-binding oligomerization domain 2 (NOD2) and inflammatory factors in rat brain tissues infected with Streptococcus pneumoniae and its influence on the blood-brain barrier (BBB) permeability. Rats were given an intracranial injection of Streptococcus pneumoniae to construct the Streptococcus pneumoniae meningitis rat models. The expression change curves of NOD2 and inflammatory factors at different time points (0 h, 12 h, 24 h, 48 h, and 7 d) after Streptococcus pneumoniae were evaluated by enzyme-linked immunosorbent assay (ELISA). Western blotting analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were engaged to examine the expression of NOD2. Furthermore, the changing processes of pathological characteristics, nervous system score, cerebral oedema, and BBB permeability were observed. Our results showed that NOD2 expression began to increase in the 12 h after Streptococcus pneumoniae infection group, while the remaining inflammatory factors were not obviously increased. Meanwhile, the levels of NOD2, as well as inflammatory factors IL-1β, TNF-α, and IL-6 were markedly elevated in 24 h and 48 h infection groups, which were consistent with the increases in BBB permeability and BWC, and the positive expression of NOD2 in the infected rat brain tissues was observed using immunohistochemistry (IHC). This study suggests that NOD2 might be related to the activation of inflammation pathways and the damage to the blood-brain barrier. NOD2 and inflammatory factors have played vital roles in the pathogenesis of Streptococcus pneumoniae meningitis.

Alterations in nucleotide-binding oligomerization domain-2 expression, pathway activation, and cytokine production in Yao syndrome

Yao syndrome (YAOS) is a systemic autoinflammatory disease (SAID), formerly termed nucleotide-binding oligomerization domain-2 (NOD2)-associated autoinflammatory disease. Due to the recent identification of YAOS, the molecular mechanisms underlying its disease pathogenesis are unclear. With specific NOD2 variants as characteristic genotypic features of YAOS, our study examined NOD2 expression, transcript splicing, signaling pathway activation, and cytokine profiles in peripheral blood mononuclear cells (PBMCs) from 10 YAOS patients and six healthy individuals. All participants were genotyped for NOD2 variants; all YAOS patients were heterozygous for the NOD2 IVS8+158 variant (IVS8+158) and four patients also carried a concurrent NOD2 R702W variant (IVS8+158/R702W haplotype). Resembling other SAIDs, plasma levels of TNFα, IL-1β, IL-6, IFNγ, and S100A12 were unaltered in YAOS patients. Intron-8 splicing of NOD2 transcripts was unaffected by carriage of NOD2 IVS8+158. However, NOD2 transcript level and basal p38 mitogen-activated protein kinase (MAPK) activity were significantly elevated in PBMCs from IVS8+158 YAOS patients. Moreover, these patients’ cells had elevated basal IL-6 secretion that was enhanced by muramyl dipeptide (MDP) stimulation. Tocilizumab treatment of a YAOS IVS8+158 patient resulted in marked clinical improvement. In contrast, MDP-stimulated NF-κB activity was uniquely suppressed in haplotype IVS8+158/R702W patients, as was TNFα secretion. Our study demonstrates for the first time that NOD2 expression and pathway activation are aberrant in YAOS, and specific NOD2 genotypes result in distinct NOD2 expression and cytokine profiles. These findings may also help select therapeutic strategies in the future.

NOD2 promotes cell proliferation and inflammatory response by mediating expression of TSLP in human airway smooth muscle cells

The newly discovered intracytosolic pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) has been studied as an important indicator of T helper 2 (Th2) inflammation, and its effect on regulatory T (Treg) cells is likely to modulate the immune response. In this study, we attempted to study the expression of NOD2 and its impact in human airway smooth muscle cells (HASMC). Quantitative real-time PCR (qRT-PCR) was used to measure the expression level of NOD2 in HASMC and comparisons were made between those from asthmatic and non-asthmatic donors; we found that NOD2 was significantly upregulated in asthma patient tissues and cell lines. In addition, overexpression of NOD2 apparently promotes cell proliferation and migration in HASMC. Gain-of-function in vitro experiments further showed that NOD2 overexpression significantly promotes pro-inflammatory cytokine release in HASMC. Subsequent experimental analysis indicated that thymic stromal lymphopoietin (TSLP) is involved in NOD2-mediated cellular effects in HASMC. Therefore, our results indicate that NOD2 is an asthma-related factor that can promote cell proliferation and inflammatory response by mediated expression of TSLP in HASMC. Taken together, our results indicate that NOD2 could serve as a potential diagnostic biomarker and therapeutic option for human asthma in the near future.

Inflammatory responses in the Japanese pufferfish (Takifugu rubripes) head kidney cells stimulated with an inflammasome-inducing agent, nigericin

A cytosolic receptor complex called inflammasome is responsible for mounting inflammatory response by releasing pro-inflammatory cytokines, interleukin (IL)-1β and IL-18. However, inflammatory cascades mediated by the inflammasome are unknown in a lower vertebrate like fish. Therefore, in an in vitro experiment, in order to obtain a preliminary information, we conducted transcriptomic analysis of 18 cytokines including pro-inflammatory cytokines in the Japanese pufferfish (Takifugu rubripes) head kidney (HK) cells stimulated with an inflammasome-inducing agent, nigericin, and a combination of nigericin and LPS by a multiplex RT-PCR assay (GenomeLab Genetic Analysis System, GeXPS; Beckman Coulter Inc.). Furthermore, expression of IL-1β, IL-6, IL-18, nuclear factor (NF)-κB, nucleotide-binding oligomerization domain 2 (NOD2) and NOD-like receptor X1 (NLRX1) genes was examined in HK cells by a quantitative real-time PCR. Additionally, to confirm functionality of activated inflammatory immunity, we also assessed phagocytic activity, superoxide anion production (NBT assay) and lysozyme activity in the nigericin-stimulated HK cells. An increased gene expression of pro-inflammatory cytokines (IL-1β and IL-18), NF-κB and NOD2 was recorded in nigericin and combined nigericin+LPS- stimulated HK cells. Enhanced cellular (phagocytic activity and NBT assay) and humoral (lysozyme activity) immune parameters in the stimulated cells confirmed induction of inflammatory response. Results suggested probable activation of inflammasome components for processing of the inflammatory cytokines in the Japanese pufferfish.

O-020 Selective Enhancement of NOD2 Anti-bacterial Defenses in the Context of Crohn’s Disease

The first and most consistently replicated risk gene linked to Crohn’s Disease (CD) is nucleotide binding oligomerization domain 2 (NOD2). NOD2 plays multifaceted roles in maintaining gut homeostasis, and impairment of NOD2 function contributes to CD pathogenesis by altering gut immunity. However, therapies specifically enhancing NOD2 function do not currently exist. NOD2 is negatively regulated by carbamoyl phosphate synthetase/ aspartate transcarbamylase/ dihydroorotase (CAD), an enzyme involved in nucleotide synthesis. In vitro studies suggest that pharmacologic inhibition of CAD activity enhances NOD2-dependent signaling and anti-bacterial activity. This study examined the specific effects of CAD inhibition on NOD2-dependent immune responses and investigated whether enhancement of NOD2 function in vivo using a CAD enzyme inhibitor could be a novel therapeutic target for CD. NOD2-dependent signal transduction, target gene expression, cytokine secretion and Salmonella killing was assessed in vitro using HCT116 human epithelial cells, mouse bone marrow-derived macrophages (BMDM) and human peripheral blood monocyte-derived macrophages from healthy individuals (n = 6) and CD patients (n = 7) by immunoblot, qPCR, ELISA and gentamicin protection assay. The effect of enhanced NOD2 function by CAD inhibition was assessed in vivo using the 2.5% dextran sulfate sodium (DSS)-induced colitis model in C56BL/6 and Nod2−/− mice. Disease activity index, CAD enzyme activity, serum amyloid A levels (systemic inflammation), colonic cytokine production (local inflammation), antimicrobial peptide expression and a 12 point histologic colitis score were evaluated. Inhibition of CAD activity specifically enhanced intracellular Salmonella killing by macrophages from both healthy individuals (37.4% ± 7.0% enhancement, P = 0.006) and CD patients (29.4% ± 6.1% enhancement, P = 0.008). Enhanced bactericidal activity in response to CAD inhibition was dependent on NOD2 expression, as it was not observed in HCT116 cells treated with NOD2 RNAi or in Nod2−/− BMDM treated with CAD inhibitors. CAD inhibition stimulated autophagy induction and antimicrobial peptide expression without altering TNFα, IL-6, or IL-1β secretion in vitro. In experimental colitis models using a pharmacologic CAD inhibitor, enzyme activity was significantly inhibited (29.8 ± 5.4 versus 52.7 ± 2.9 nM, P < 0.001) and resulted in improved colitis scores (5.7 ± 0.5 versus 9.5 ± 0.9, P = 0.003) and reduced serum amyloid A levels, (489.6 ± 237 versus 1315.1 ± 326.5 ng/mL, P < 0.01). These effects were not observed in Nod2−/− mice. Systemic CAD inhibition also enhanced colonic antimicrobial peptide expression and reduced colonic cytokine secretion. Inhibition of CAD enzyme activity preferentially activates NOD2-dependent antimicrobial responses without inducing pro-inflammatory cytokine production. Stimulation of NOD2-dependent antimicrobial defenses protects against experimental colitis and reduces systemic inflammation. These findings suggest that CAD may be a future therapeutic target for CD.

Su1732 Estrogen Upregulates Expression of Cystic Fibrosis Transmembrane Conductance Regulator in Duodenal Mucosa

Duodenal brush border dual oxidase 2 (Duox2), a NADPH oxidase, generates luminal H2O2 by luminal ATP, P2Y receptor activation and intracellular Ca2+ signal. Rapid production of antimicrobial H2O2 from the duodenal surface via the ATP-P2Y-Duox2 pathway suggests the presence of luminal bacterial sensing in the duodenum. We hypothesized that the pattern recognition receptors (PRRs) Toll-like receptors (TLRs) or nucleotide-binding oligomerization domain 2 (NOD2) are involved in the regulation of duodenal antimicrobial defenses. We measured duodenal HCO3 secretion (DBS) with pH and CO2 electrodes in the perfused duodenum of anesthetized rats. The TLR2 ligand Pam3CSK4 (Pam3, 1 μg/ml), the TLR4 ligand lipopolysaccharide (LPS, 1 μg/ml), the NOD2 ligand muramyl dipeptide (MDP, 10 μg/ml) or its control peptide murabutide (10 μg/ml) were perfused. H2O2 in the perfusate was measured with the fluorogenic substrate Amplex Red. TLR2, TLR4 and NOD2 expression on the villous brush borders, with NOD2 also present in Brunner's glands was detected by immunofluorescence. Luminal perfusion of Pam3, LPS or MDP alone had little effect on DBS or H2O2 output. Co-perfusion of Pam3 or LPS with MDP, but not with murabutide, increased DBS with a parallel increase of luminal H2O2 output. In Vivo x-z confocal images demonstrated that luminal LPS +MDP rapidly increased Amplex Red fluorescence, equivalent to H2O2 generation, from the villous surface. Furthermore, the IkB kinase inhibitor TCPA1 (1 μM) or the proteasome/NFkB inhibitor MG132 (10 μM) reduced the augmented DBS and H2O2 output, suggesting the involvement of NFkB signals. These results suggest that bacteria in the duodenal lumen are sensed by PRRs, which acutely signal the release of antimicrobial H2O2, partially via NFkB signaling. Rapid protective responses to luminal bacterial attachment, rather than genomic mechanisms, may be a highly novel component of the foregut antimicrobial defense system.

Su1731 Novel Nongenomic Bacterial Component Sensing in Duodenum via Pattern Recognition Receptors

Duodenal brush border dual oxidase 2 (Duox2), a NADPH oxidase, generates luminal H2O2 by luminal ATP, P2Y receptor activation and intracellular Ca2+ signal. Rapid production of antimicrobial H2O2 from the duodenal surface via the ATP-P2Y-Duox2 pathway suggests the presence of luminal bacterial sensing in the duodenum. We hypothesized that the pattern recognition receptors (PRRs) Toll-like receptors (TLRs) or nucleotide-binding oligomerization domain 2 (NOD2) are involved in the regulation of duodenal antimicrobial defenses. We measured duodenal HCO3 secretion (DBS) with pH and CO2 electrodes in the perfused duodenum of anesthetized rats. The TLR2 ligand Pam3CSK4 (Pam3, 1 μg/ml), the TLR4 ligand lipopolysaccharide (LPS, 1 μg/ml), the NOD2 ligand muramyl dipeptide (MDP, 10 μg/ml) or its control peptide murabutide (10 μg/ml) were perfused. H2O2 in the perfusate was measured with the fluorogenic substrate Amplex Red. TLR2, TLR4 and NOD2 expression on the villous brush borders, with NOD2 also present in Brunner's glands was detected by immunofluorescence. Luminal perfusion of Pam3, LPS or MDP alone had little effect on DBS or H2O2 output. Co-perfusion of Pam3 or LPS with MDP, but not with murabutide, increased DBS with a parallel increase of luminal H2O2 output. In Vivo x-z confocal images demonstrated that luminal LPS +MDP rapidly increased Amplex Red fluorescence, equivalent to H2O2 generation, from the villous surface. Furthermore, the IkB kinase inhibitor TCPA1 (1 μM) or the proteasome/NFkB inhibitor MG132 (10 μM) reduced the augmented DBS and H2O2 output, suggesting the involvement of NFkB signals. These results suggest that bacteria in the duodenal lumen are sensed by PRRs, which acutely signal the release of antimicrobial H2O2, partially via NFkB signaling. Rapid protective responses to luminal bacterial attachment, rather than genomic mechanisms, may be a highly novel component of the foregut antimicrobial defense system.

Effects of muramyl dipeptide on the differentiation of human dental pulp cells

To investigate the nucleotide-binding oligomerization domain-2 (NOD-2) gene expression in deep caries and the effects of NOD-2 agonist muramyl dipeptide (MDP) on the differentiation of human dental pulp cells (hDPC). NOD-2 gene level in deep caries and healthy pulp tissue was determined by real-time quantitative polymerase chain reaction (realtime-PCR). Realtime-PCR, Western blotting and immunofluorescence were performed to evaluate NOD-2 gene and protein expression. Dentin sialoprotein (DSP) protein level was assessed when hDPC were challenged by different concentrations of MDP for 24 hours, and sialophosphoprotein (DSPP), osteocalcin (OCN) mRNA and osteopontin (OPN) protein level were detected at different time points after incubation with 0.1 mg/L MDP. NOD-2 mRNA level was higher in pulp tissue of deep caries (0.2610 ± 0.0824) than that in healthy controls (0.0024 ± 0.0002), P < 0.05. The expression of NOD-2 gene and protein increased in a time denpendent manner upon stimulation with MDP. Immunofluorescence confirmed that NOD-2 protein was located in cytoplasm. Moreover, 0.1 mg/L MDP augmented DSP protein level. DSPP and OCN mRNA were elevated with time and reached the peak at 12 h and down-regulated. OPN protein level also increased with time. Dental pulp NOD-2 expression are up-regulated in pulp tissue of deep caries. MDP may be related to the differentiation of hDPC.