NOD2 Expression Research Articles

Chidan Tuihuang granule modulates gut microbiota to influence NOD1/RIPK2 pathway in cholestatic liver injury recovery

BackgroundCholestatic liver injury (CLI), which occurs if bile acids are imbalanced and the liver becomes inflamed, is difficult to treat effectively ObjectiveWe investigated how the Chinese patent medicine Chidan Tuihuang granule (CDTH) ameliorates cholestatic liver injury with a focus on its effects on the NOD1/RIPK2 pathway and intestinal flora MethodsWe used an ANIT-induced SD rat model of CLI to evaluate the therapeutic effects of CDTH. The experimental design included control, model, UDCA (ursodeoxycholic acid) and CDTH treatment groups. UHPLC-Q-Orbitrap-HRMS was used to analyse the blood components of CDTH. The efficacy of CDTH was assessed by liver function tests, histopathological examination (HE and TUNEL staining), transmission electron microscopy, and ELISA to measure apoptosis and inflammatory markers. Mechanistic insights were obtained using transcriptomics and RT-qPCR, while alterations in the expression of key proteins were studied using western blotting, immunohistochemistry, and immunofluorescence. Furthermore, the impact of CDTH on the gut microbiota and its associated metabolite, meso-2,6-diaminopimelic acid (DAP), which is linked to NOD1 activation, was examined and confirmed through in vitro ResultsThe experimental results demonstrated a notable elevation in serum levels of AST, ALT, ALP, TBA, TBIL, and DBIL in the rats belonging to the model group, accompanied by the infiltration of inflammatory cells, hepatocyte degeneration, and necrosis in the liver tissue. CDTH administration significantly improved liver function and cholestasis indicators. Transmission electron microscopy and TUNEL staining revealed a marked reduction in liver cell apoptosis with CDTH treatment. ELISA results showed that CDTH effectively reduced inflammatory markers. Transcriptomic analysis showed that CDTH inhibited the NOD1/RIPK2 pathway, resulting in a significant decrease in the expression of NOD1, RIPK2 and associated genes in liver tissue. Gut microbiota analysis demonstrated that CDTH regulated intestinal flora structure, reducing the abundance of DAP-producing Gram-negative bacteria such as lactobacilli. In vitro experiments confirmed that CDTH enhanced cell viability by downregulating the DAP-mediated NOD1/RIPK2 signaling pathway secreted by intestinal bacteria ConclusionCDTH ameliorated liver damage in cholestatic rats by inhibiting the NOD1/RIPK2 signaling pathway through regulation of gut flora and downregulation of DAP metabolites.

NOD2 reduces the chemoresistance of melanoma by inhibiting the TYMS/PLK1 signaling axis

AbstractNucleotide-binding oligomerization domain 2 (NOD2) is an immune sensor crucial for eliciting the innate immune responses. Nevertheless, discrepancies exist regarding the effect of NOD2 on different types of cancer. This study aimed to investigate these function of NOD2 in melanoma and its underlying mechanisms. We have validated the tumor suppressor effect of NOD2 in melanoma. NOD2 inhibited the proliferation of melanoma cells, hindering their migration and invasion while promoting the onset of apoptosis. Our study showed that NOD2 expression is closely related to pyrimidine and folate metabolism. NOD2 inhibits thymidylate synthase (TYMS) expression by promoting K48-type ubiquitination modification of TYMS, thereby decreasing the resistance of melanoma cells to 5-fluorouracil (5-FU) and capecitabine (CAP). TYMS was identified to form a complex with Polo-like Kinase 1 (PLK1) and activate the PLK1 signaling pathway. Furthermore, we revealed that the combination of the PLK1 inhibitor volasertib (BI6727) with 5-FU or CAP had a synergistic effect repressing the proliferation, migration, and autophagy of melanoma cells. Overall, our research highlights the protective role of NOD2 in melanoma and suggests that targeting NOD2 and the TYMS/PLK1 signaling axis is a high-profile therapy that could be a prospect for melanoma treatment.

Supplementation with Akkermansia muciniphila improved intestinal barrier and immunity in zebrafish (Danio rerio)

Akkermansia muciniphila (Akk), a second-generation probiotic known for its ability to regulate intestinal function in mammals, is not yet fully understood in the context of aquaculture. This study aims to investigate the effects of different forms of Akk on intestinal barrier function and immune response in zebrafish (Danio rerio) under high-fat diet conditions. The experimental groups included a control group, a high-fat diet group, an Akk group, and a group receiving various concentrations of pasteurized Akkermansia muciniphila (P-Akk) along with a high-fat diet. Evaluation methods included histological examination with hematoxylin and eosin staining, ultrastructural analysis using transmission electron microscopy, real-time fluorescence quantitative analysis, and transcriptome sequencing technology. The results showed that both the Akk and P-Akk groups exhibited a significant increase in villi number and length compared to the high-fat group. Furthermore the expression levels of claudin, claudin-2, occludin A, occludin B, and other genes were significantly upregulated, while the expression levels of intestinal proinflammatory factors genes and proteins were significantly downregulated. Compared to the high-fat group, the Akk group showed a more complete and well-preserved nucleus, mitochondria, and tight junction structures. Additionally, the morphology of intestinal epithelial microvilli in the medium and high concentration Akk group was complete and dense. The expressions of tlr2 and nf-κb were upregulated, while the expressions of myd88 and nod2 were downregulated in the medium- and high-concentration Akk groups. Akk may improve immune dysfunction in high-fat fed zebrafish through the TLR2/NF-κB signaling pathway, which requires further study. Transcriptome analysis revealed significant upregulation of the immune-related gene pigr, significant downregulation of stat3, and significant upregulation of the intercellular adhesion molecule f11r. In conclusion, dietary Akk supplementation alleviated intestinal barrier damage and immune dysfunction in high-fat zebrafish. This study provides important insights into the potential use of Akk in fish and lays the foundation for further studies on its role in fish immunity.

Pyroptosis-associated genes and tumor immune response in endometrial cancer

The occurrence and progression of tumors are linked to the process of pyroptosis. However, the precise involvement of pyroptosis-associated genes (PRGs) in endometrial cancer (EC) remains uncertain. 29 PRGs were identified as being either up-regulated or down-regulated in EC. PRGs subgroup analysis demonstrated distinct survival outcomes and diverse responses to chemotherapy and immune checkpoint blockade therapy. A higher expression of GPX4 and NOD2, coupled with lower levels of CASP6, PRKACA, and NLRP2, were found to be significantly associated with higher overall survival (OS) rates (p < 0.05). Conversely, lower expression of NOD2 was linked to lower progression-free survival (p = 0.021) and advanced tumor stage(p = 0.0024). NOD2, NLRP2, and TNM stages were identified as independent prognostic factors (p < 0.001). The LASSO prognostic model exhibited a notable decrease in OS among EC patients in the high-risk score group (ROC-AUC10-years: 0.799, p = 0.00644). Furthermore, NOD2 displayed a positive correlation with the infiltration of immune cells and the expression of immune checkpoints (p < 0.001). GPX4 and CASP6 are significantly associated with TMB and MSI (RTMB = 0.39; RMSI = 0.23). Additionally, a substantial upregulation of NOD2 was confirmed in both EC cells and tissue, indicating a positive relationship between advanced TNM stage (p < 0.0001) and infiltration of M1 phenotype macrophages. Nonetheless, its impact on patient OS did not reach statistical significance (p = 0.141). Our findings have contributed to the advancement of a prognostic model for EC patients. NOD2 receptor-mediated pyroptosis mechanism potentially regulates tumor immunity and promotes the transformation of macrophages from the M2 phenotype to the M1 phenotype, which significantly impacts the progression of EC.

Short-peptide-based enteral nutrition affects rats MDP translocation and protects against gut-lung injury via the PepT1-NOD2-beclin-1 pathway in vivo.

Peptide transporter 1 (PepT1) transports bacterial oligopeptide products and induces inflammation of the bowel. Nutritional peptides compete for the binding of intestinal bacterial products to PepT1. We investigated the mechanism of short-peptide-based enteral nutrition (SPEN) on the damage to the gut caused by the bacterial oligopeptide product muramyl dipeptide (MDP), which is transported by PepT1. The gut-lung axis is a shared mucosal immune system, and immune responses and disorders can affect the gut-respiratory relationship. Sprague-Dawley rats were gavaged with solutions containing MDP, MDP + SPEN, MDP + intact-protein-based enteral nutrition (IPEN), glucose as a control, or glucose with GSK669 (a NOD2 antagonist). Inflammation, mitochondrial damage, autophagy, and apoptosis were explored to determine the role of the PepT1-nucleotide-binding oligomerization domain-containing protein 2 (NOD2)-beclin-1 signaling pathway in the small intestinal mucosa. MDP and proinflammatory factors of lung tissue were explored to determine that MDP can migrate to lung tissue and cause inflammation. Induction of proinflammatory cell accumulation and intestinal damage in MDP gavage rats was associated with increased NOD2 and Beclin-1 mRNA expression. IL-6 and TNF-α expression and apoptosis were increased, and mitochondrial damage was severe, as indicated by increased mtDNA in the MDP group compared with controls. MDP levels and expression of proinflammatory factors in lung tissue increased in the MDP group compared with the control group. SPEN, but not IPEN, eliminated these impacts. Gavage of MDP to rats resulted in damage to the gut-lung axis. SPEN reverses the adverse effects of MDP. The PepT1-NOD2-beclin-1 pathway plays a role in small intestinal inflammation, mitochondrial damage, autophagy, and apoptosis.

Exendin-4 blockade of T1R2/T1R3 activation improves Pseudomonas aeruginosa-related pneumonia in an animal model of chemically induced diabetes

ObjectivePoorly controlled diabetes frequently exacerbates lung infection, thereby complicating treatment strategies. Recent studies have shown that exendin-4 exhibits not only hypoglycemic but also anti-inflammatory properties. This study aimed to explore the role of exendin-4 in lung infection with diabetes, as well as its association with NOD1/NF-κB and the T1R2/T1R3 sweet taste receptor.Methods16HBE human bronchial epithelial cells cultured with 20 mM glucose were stimulated with lipopolysaccharide (LPS) isolated from Pseudomonas aeruginosa (PA). Furthermore, Sprague‒Dawley rats were fed a high-fat diet, followed by intraperitoneal injection of streptozotocin and intratracheal instillation of PA. The levels of TNF-α, IL-1β and IL-6 were evaluated using ELISAs and RT‒qPCR. The expression of T1R2, T1R3, NOD1 and NF-κB p65 was assayed using western blotting and immunofluorescence staining. Pathological changes in the lungs of the rats were observed using hematoxylin and eosin (H&E) staining.ResultsAt the same dose of LPS, the 20 mM glucose group produced more proinflammatory cytokines (TNF-α, IL-1β and IL-6) and had higher levels of T1R2, T1R3, NOD1 and NF-κB p65 than the normal control group (with 5.6 mM glucose). However, preintervention with exendin-4 significantly reduced the levels of the aforementioned proinflammatory cytokines and signaling molecules. Similarly, diabetic rats infected with PA exhibited increased levels of proinflammatory cytokines in their lungs and increased expression of T1R2, T1R3, NOD1 and NF-κB p65, and these effects were reversed by exendin-4.ConclusionsDiabetic hyperglycemia can exacerbate inflammation during lung infection, promote the increase in NOD1/NF-κB, and promote T1R2/T1R3. Exendin-4 can ameliorate PA-related pneumonia with diabetes and overexpression of NOD1/NF-κB. Additionally, exendin-4 suppresses T1R2/T1R3, potentially through its hypoglycemic effect or through a direct mechanism. The correlation between heightened expression of T1R2/T1R3 and an intensified inflammatory response in lung infection with diabetes requires further investigation.

Enterococcus faecalis Extracellular Vesicles Promote Apical Periodontitis

Enterococcus faecalis is an important contributor to the persistence of chronic apical periodontitis. However, the mechanism by which E. faecalis infection in the root canals and dentinal tubules affects periapical tissue remains unclear. Bacterial extracellular vesicles (EVs) act as natural carriers of microbe-associated molecular patterns (MAMPs) and have recently attracted considerable attention. In this study, we investigated the role of EVs derived from E. faecalis in the pathogenesis of apical periodontitis. We observed that E. faecalis EVs can induce inflammatory bone destruction in the periapical areas of mice. Double-labeling immunofluorescence indicated that M1 macrophage infiltration was increased by E. faecalis EVs in apical lesions. Moreover, in vitro experiments demonstrated the internalization of E. faecalis EVs into macrophages. Macrophages tended to polarize toward the M1 profile after treatment with E. faecalis EVs. Pattern recognition receptors (PRRs) can recognize MAMPs of bacterial EVs and, in turn, trigger inflammatory responses. Thus, we performed further mechanistic exploration, which showed that E. faecalis EVs considerably increased the expression of NOD2, a cytoplasmic PRR, and that inhibition of NOD2 markedly reduced macrophage M1 polarization induced by E. faecalis EVs. RIPK2 ubiquitination is a major downstream of NOD2. We also observed increased RIPK2 ubiquitination in macrophages treated with E. faecalis EVs, and E. faecalis EV-induced macrophage M1 polarization was notably alleviated by the RIPK2 ubiquitination inhibitor. Our study revealed the potential for EVs to be considered a virulence factor of E. faecalis and found that E. faecalis EVs can promote macrophage M1 polarization via NOD2/RIPK2 signaling. To our knowledge, this is the first report to investigate apical periodontitis development from the perspective of bacterial vesicles and demonstrate the role and mechanism of E. faecalis EVs in macrophage polarization. This study expands our understanding of the pathogenic mechanism of E. faecalis and provides novel insights into the pathogenesis of apical periodontitis.

A modified BCG with depletion of enzymes associated with peptidoglycan amidation induces enhanced protection against tuberculosis in mice.

Mechanisms by which Mycobacterium tuberculosis (Mtb) evades pathogen recognition receptor activation during infection may offer insights for the development of improved tuberculosis (TB) vaccines. Whilst Mtb elicits NOD-2 activation through host recognition of its peptidoglycan-derived muramyl dipeptide (MDP), it masks the endogenous NOD-1 ligand through amidation of glutamate at the second position in peptidoglycan side-chains. As the current BCG vaccine is derived from pathogenic mycobacteria, a similar situation prevails. To alleviate this masking ability and to potentially improve efficacy of the BCG vaccine, we used CRISPRi to inhibit expression of the essential enzyme pair, MurT-GatD, implicated in amidation of peptidoglycan side-chains. We demonstrate that depletion of these enzymes results in reduced growth, cell wall defects, increased susceptibility to antibiotics, altered spatial localization of new peptidoglycan and increased NOD-1 expression in macrophages. In cell culture experiments, training of a human monocyte cell line with this recombinant BCG yielded improved control of Mtb growth. In the murine model of TB infection, we demonstrate that depletion of MurT-GatD in BCG, which is expected to unmask the D-glutamate diaminopimelate (iE-DAP) NOD-1 ligand, yields superior prevention of TB disease compared to the standard BCG vaccine. In vitro and in vivo experiments in this study demonstrate the feasibility of gene regulation platforms such as CRISPRi to alter antigen presentation in BCG in a bespoke manner that tunes immunity towards more effective protection against TB disease.

Functional characterization of NOD1 from golden pompano Trachinotus ovatus

Fish rely on innate immune system for immunity, and nucleotide-binding oligomerization domain-like receptors (NLRs) are a vital group of receptor for recognition. In the present study, NOD1 gene was cloned and characterized from golden pompano Trachinotus ovatus, a commercially important aquaculture fish species. The ORF of T. ovatus NOD1 was 2820 bp long, encoding 939 amino acid residues with a highly conserved domains containing CARD-NACHT-LRRs. Phylogenetic analysis revealed that the T. ovatus NOD1 clustered with those of fish and separated from those of birds and mammals. T. ovatus NOD1 has wide tissue distribution with the highest expression in gills. Bacterial challenges (Streptococcus agalactiae and Vibrio alginolyticus) significantly up-regulated the expression of NOD1 with different response time. The results of T. ovatus NOD1 ligand recognition and signaling pathway analysis revealed that T. ovatus NOD1 could recognize iE-DAP at the concentration of ≧ 100 ng/mL and able to activate NF-κB signaling pathway. This study confirmed that NOD1 play a crucial role in the innate immunity of T. ovatus. The findings of this study improve our understanding on the immune function of NOD1 in teleost, especially T. ovatus.

C-type lectin 4 of Toxocara canis activates NF-ĸB and MAPK pathways by modulating NOD1/2 and RIP2 in murine macrophages in vitro.

Toxocara canis is a parasitic zoonose that is distributed worldwide and is one of the two pathogens causing toxocariasis. After infection, it causes serious public health and safety problems, which pose significant veterinary and medical challenges. To better understand the regulatory effects of T. canis infection on the host immune cells, murine macrophages (RAW264.7) were incubated with recombinant T. canis C-type lectin 4 (rTc-CTL-4) protein in vitro. The quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to analyze the nucleotide-binding oligomerization domain-containing protein 1/2 (NOD1/2), receptor-interacting protein 2 (RIP2), nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB), and mitogen-activated protein kinase (MAPK) on mRNA level and protein expression level in macrophages. Our results indicated that 10μg/mL rTc-CTL-4 protein could modulate the expression of NOD1, NOD2, and RIP2 at both the transcriptional and translational levels. The protein translation levels of NF-κB, P-p65, p38, and P-p38 in macrophages were also modulated by rTc-CTL-4 protein. Macrophages were co-incubated with rTc-CTL-4 protein after siRNA silencing of NOD1, NOD2, and RIP2. The expression levels of NF-κB, P-p65, p38, and P-p38 were significantly changed compared with the negative control groups (Neg. Ctrl.). Taken together, rTc-CTL-4 protein seemed to act on NOD1/2-RIP2-NF-κB and MAPK signaling pathways in macrophages and might activate MAPK and NF-κB signaling pathways by regulating NOD1, NOD2, and RIP2. The insights from the above studies could contribute to our understanding of immune recognition and regulatory mechanisms of T. canis infection in the host animals.

MURAMYL DIPEPTIDE CAUSES MITOCHONDRIAL DYSFUNCTION AND INTESTINAL INFLAMMATORY CYTOKINE RESPONSES IN RATS.

Introduction: Intestinal flora and the translocation of its products, such as muramyl dipeptide (MDP), are common causes of sepsis. MDP is a common activator of the intracellular pattern recognition receptor NOD2, and MDP translocation can cause inflammatory damage to the small intestine and systemic inflammatory responses in rats. Therefore, this study investigated the effects of MDP on the intestinal mucosa and distant organs during sepsis and the role of the NOD2/AMPK/LC3 pathway in MDP-induced mitochondrial dysfunction in the intestinal epithelium. Methods: Fifty male Sprague Dawley rats were randomly divided into five treatment groups: lipopolysaccharide (LPS) only, 1.5 and 15 mg/kg MDP+LPS, and 1.5 and 15 mg/kg MDP+short-peptide enteral nutrition (SPEN)+LPS. The total caloric intake was the same per group. The rats were euthanized 24 h after establishing the model, and peripheral blood and small intestinal mucosal and lung tissues were collected. Results: Compared to the LPS group, both MDP+LPS groups had aggravated inflammatory damage to the intestinal mucosal and lung tissues, increased IL-6 and MDP production, increased NOD2 expression, decreased AMPK and LC3 expression, increased mitochondrial reactive oxygen species production, and decreased mitochondrial membrane potential. Compared to the MDP+LPS groups, the MDP+SPEN+LPS groups had decreased IL-6 and MDP production, increased AMPK and LC3 protein expression, and protected mitochondrial and organ functions. Conclusions: MDP translocation reduced mitochondrial autophagy by regulating the NOD2/AMPK/LC3 pathway, causing mitochondrial dysfunction. SPEN protected against MDP-induced impairment of intestinal epithelial mitochondrial function during sepsis.

A30 MICROBIOTA AND NOD2 REGULATE SMALL INTESTINAL RESTITUTION THROUGH FETAL-LIKE INTESTINAL STEM CELLS

Abstract Background Inflammatory bowel disease is characterized by chronic inflammation of the gastrointestinal tract, resulting in recurrent injury to the intestinal epithelium. Restitution of the small intestinal epithelium is a coordinated response that involves the dedifferentiation of epithelial cell lineages, proliferation of Lgr5+ intestinal stem cells, and fetal-like stem cell reversion. While gut microbiota are critical mediators of intestinal inflammation, their impact on epithelial restitution remains unclear. Aims We aim to identify the how microbes regulate small intestinal epithelial restitution following damage. Our hypothesis is that gut microbiota accelerate restitution through pattern recognition receptor-driven signals following fetal-like stem cell reversion. Methods Irradiation (IR, 12Gγ) was used to induce a synchronized small intestinal epithelial restitution response in mice. Intestinal restitution kinetics were assessed transcriptionally (scRNA-Seq, qPCR) and histologically. Small intestinal organoids were used to assess epithelial restitution kinetics in vitro. Results ScRNA-Seq of small intestinal epithelial cells following IR from germ-free mice (GF), and specific pathogen-free mice (SPF) mice revealed that microbiota induced greater expression of fetal-like stem cell reversion markers, Ly6a and Clu, and greater expression of the proliferation marker, Pcna. These results were supported histologically as irradiated SPF mice observed an increase in fetal-like stem cells marked by Ly6a and Clu and an increase BrdU+ proliferating cells. ScRNA-seq and in situ hybridization highlighted that fetal-like intestinal stem cells upregulate expression of Nod2, a bacterial pattern recognition receptor. Using intestinal organoids to assess the function of Nod2, we observed that muramyl dipeptide driven Nod2-signlaing potentiates an interferon gene signature following IFNγ and TNFα co-stimulation. Further supporting the role for Nod2 in intestinal restitution, intestinal epithelium specific Nod2KO mice decreased BrdU+ proliferating cells post-IR compared to littermate controls. Conclusions Microbiota promote small intestinal restitution following IR through Nod2-signaling in fetal-like intestinal stem cells. Funding Agencies CIHR

Membrane inflammasome activation by choriodecidual Ureaplasma parvum infection without intra-amniotic infection in a Non-Human Primate model†.

Intrauterine infection is a significant cause of neonatal morbidity and mortality. Ureaplasma parvum is a microorganism commonly isolated from cases of preterm birth and preterm premature rupture of membranes (pPROM). However, the mechanisms of early stage ascending reproductive tract infection remain poorly understood. To examine inflammation in fetal (chorioamnionic) membranes we utilized a non-human primate (NHP) model of choriodecidual U. parvum infection. Eight chronically catheterized pregnant rhesus macaques underwent maternal-fetal catheterization surgery at ~105-112 days gestation and choriodecidual inoculation with U. parvum (105 CFU/mL, n =4) or sterile media (controls; n = 4) starting at 115-119 days, repeated at 5-day intervals until C-section at 136-140 days (term=167 days). The average inoculation to delivery interval was 21 days, and Ureaplasma infection of the amniotic fluid (AF) was undetectable in all animals. Choriodecidual Ureaplasma infection resulted in increased fetal membrane expression of MMP-9 and PTGS2, but did not result in preterm labor or increased concentrations of AF pro-inflammatory cytokines. However, membrane expression of inflammasome sensors, NLRP3, NLRC4, AIM2, and NOD2, and adaptor ASC (PYCARD) gene expression were significantly increased. Gene expression of IL-1β, IL-18, IL-18R1 , CASPASE-1, and pro-CASPASE-1 protein increased with Ureaplasma infection. Downstream inflammatory genes MYD88 and NFκB (Nuclear factor kappa-light-chain-enhancer of activated B cells) were also significantly upregulated. These results demonstrate that choriodecidualUreaplasma infection, can cause activation of inflammasome complexes and pathways associated with pPROM and preterm labor prior to microbes being detectable in the AF.

In vitro evaluation of the immunomodulatory and wakame assimilation properties of Lactiplantibacillus plantarum strains from swine milk.

The emergence and spread of antibiotic resistance threat forced to explore alternative strategies for improving the resistance to pathogens in livestock production. Probiotic lactic acid bacteria represent an alternative for this objective. In this study, seven Lactiplantibacillus plantarum strains from porcine colostrum and milk were isolated, identified and characterized in terms of their abilities to modulate immunity in porcine intestinal epithelial (PIE) cells. Then, two potential immunoregulatory strains were studied in terms of their ability to utilize and grow in wakame (Undaria pinnafida). Isolates were identified by 16S rRNA gene and evaluated by studying their interaction with PIE cells. The expressions of peptidoglycan recognition proteins (PGRPs), nucleotide-binding oligomerization domain (NODs), host defense peptides (pBD), and type I interferons (IFNs) were evaluated by RT-qPCR. The strain 4M4417 showed a remarkable capacity to differentially regulate the expression of PGRP1, PGRP3, NOD1, NOD2, and pBD1 in PIE cells. On the other hand, the strain 4M4326 was the most efficient to improve the expression of IFN-α and IFN-β in PIE cells challenged with poly (I:C). Both L. plantarum 4M4326 and 4M4417 were characterized in terms of their ability to utilize wakame. Results demonstrated that both strains efficiently grew in wakame-based broth. Our results suggest that L. planatrum 4M4326 and 4M4417 are interesting candidates to develop immunomodulatory feeds based on wakame utilization. These new immunosynbiotic feeds could help to reduce severity of intestinal infections and improve immune health status in pigs.

P1193 NOD2 and Beyond: Unmasking Tobacco-Induced Epigenetic Changes in Crohn's Disease

Abstract Background Smoking has been shown to have a very negative impact on Crohn's disease (CD). Indeed, this harmful habit induces the early onset of the disease and doubles the risk of postoperative recurrence. This research project involves a thorough analysis of omics data, with a primary focus on DNA methylation, one of the most prevalent epigenetic modifications. Our hypothesis is that tobacco-induced alterations in gene methylation pattern of subcutaneous adipose tissue (SAT), particularly within key pathways, contribute to a more adverse disease profile in smokers. Methods A comprehensive epigenome-wide analysis was conducted using the Illumina EPIC/450k array to investigate subcutaneous adipose tissue in patients with CD, encompassing both smokers and non-smokers. Utilizing tools such as dmpfinder and Bumphunter (minfi), differentially methylated positions (DMPs) and regions (DMRs) were identified. Additionally, gene expression analysis for key genes was performed to establish correlations between methylation and subsequent gene expression, not only in SAT but also in other fat depots such as visceral adipose tissue (VAT) or peripheral blood mononuclear cells (PBMCs). Results A distinctive DNA methylation pattern in the SAT of smokers compared to non-smoker CD patients was identified (Fig. 1A). Over 27,270 DMPs associated with significant biological processes related to DNA damage, inflammation, or immunity were uncovered (Fig. 1B-C). Additionally, more than 13,000 DMRs located in the promoter region were identified (Fig. 1C). The relationship between methylation levels in the promoter regions and subsequent gene expression on SAT was validated for selected genes. Specifically, NOD2 exhibited higher DNA methylation in the promoter region among smokers compared to non-smokers CD patients (p value=0.04), resulting in reduced NOD2 gene expression on SAT (p value=0.04) (Fig.1D). Consistent results were observed in other tissues such as VAT and PBMCs (Fig. 1E). Finally, we observe the implication of NOD2 in pathways related to immune response, inflammation or apoptosis and a trend indicating that lower NOD2 expression is associated with higher levels of C-reactive protein (CRP)(Fig. 1F). Conclusion Our study reveals that smoking produces DNA methylation changes on SAT of CD patients and affects the expression of several key genes involved in the pathogeny of CD. Particularly, our results unveil a new link between tobacco-smoking and lower NOD2 expression and associate this to an elevated levels of PCR, paving the way for a fresh perspective in exploring disease prognosis.

Transcriptome analysis identifies genetic risk markers and explores the pathogenesis for inflammatory bowel disease

Inflammatory bowel disease (IBD) is an incurable and disabling bowel disease driven by multiple risk factors that severely limit patients' quality of life. We integrated the RNA-sequencing data of 1238 IBD patients, and investigated the pathogenesis of IBD by combining transcriptional element prediction analysis and immune-related analysis. Here, we first determined that KIAA1109 is inhibited in IBD patients. The expression of KIAA1109 and NOD2, the key receptor of NOD-like receptors, showed a negative correlation. The NOD-like receptor signaling pathway is activated and exerts transcriptional regulation on the chemokines CXCL1 and CXCL2 through the activation of the transcription factors NFκB and AP1. Analysis of immune infiltration revealed that the expression of chemokines CXCL1 and CXCL2 may regulate the inflammatory response induced by immune cells. These findings suggest that the KIAA1109-NOD2-NFκB/AP1-CXCL1/CXCL2 regulatory axis is the molecular mechanism of IBD pathogenesis, which will provide a new perspective for the diagnosis, treatment and management of IBD patients.

NOD2 attenuates osteoarthritis via reprogramming the activation of synovial macrophages

ObjectiveSynovial inflammation, which precedes other pathological changes in osteoarthritis (OA), is primarily initiated by activation and M1 polarization of macrophages. While macrophages play a pivotal role in the inflammatory process of OA, the mechanisms underlying their activation and polarization remain incompletely elucidated. This study aims to investigate the role of NOD2 as a reciprocal modulator of HMGB1/TLR4 signaling in macrophage activation and polarization during OA pathogenesis.DesignWe examined NOD2 expression in the synovium and determined the impact of NOD2 on macrophage activation and polarization by knockdown and overexpression models in vitro. Paracrine effect of macrophages on fibroblast-like synoviocytes (FLS) and chondrocytes was evaluated under conditions of NOD2 overexpression. Additionally, the in vivo effect of NOD2 was assessed using collagenase VII induced OA model in mice.ResultsExpression of NOD2 was elevated in osteoarthritic synovium. In vitro experiments demonstrated that NOD2 serves as a negative regulator of HMGB1/TLR4 signaling pathway. Furthermore, NOD2 overexpression hampered the inflammatory paracrine effect of macrophages on FLS and chondrocytes. In vivo experiments revealed that NOD2 overexpression mitigated OA in mice.ConclusionsSupported by convincing evidence on the inhibitory role of NOD2 in modulating the activation and M1 polarization of synovial macrophages, this study provided novel insights into the involvement of innate immunity in OA pathogenesis and highlighted NOD2 as a potential target for the prevention and treatment of OA.

Inflammatory response modulation by epinephrine and norepinephrine

Relevance. Inflammation is a defense response of an organism to a pathogen. It appears in order to maintain homeostasis and is regulated by the immune, nervous, and endocrine systems. The hormones epinephrine and norepinephrine are produced in the adrenal medulla and in the brain, and are universal messengers that trigger the transmission of nerve impulses at synapses, and also have a receptor-mediated effect on immunocompetent cells. The aim of this study was to investigate adrenergic pathway regulation of inflammation on the neutrophil granulocytes in the presence of activators of innate immunity receptors. Materials and Methods. Neutrophil granulocytes were obtained from peripheral blood of healthy volunteers in a density gradient of Histopaque 1077 and Histopaque 1119 (Sigma Aldrich, Steinheim, Germany), and cultured in the presence of LPS, GMDP, epinephrine and norepinephrine. The amount of human neutrophil peptides 1-3 (HNP1-3) was examined using an enzyme-linked immunosorbent assay; the gene expression of TLR4, NOD2, ATF3 and A20 was determined using RT-PCR. Results and Discussion. Norepinephrine (noradrenaline) was found to decrease the synthesis of human neutrophils peptides 1-3 (HNP 1-3 defensins, alone and in the combination with agonists of TLR4 and NOD2 receptors - LPS and GMDP respectively. It was found out that there was no a statistically significant effect of epinephrine (adrenaline) on the production of HNP 1-3, including when combined with LPS and GMDP. As a result of the study, an increase in the levels of expression of the genes TLR4, NOD2 and regulator of inflammatory reactions A20 both in LPS- and GMDP- induced neutrophil culture were uncovered, while ATF3 was increased only in LPS-induced neutrophil culture. Epinephrine demonstrated the absence of a statistically significant effect on the expression of the studied genes. While norepinephrine significantly increased the expression of A20 genes. Conclusion. The data obtained shows that norepinephrine can reduce the synthesis of HNP 1-3, including the one induced by LPS and GMDP. Moreover, the ability of norepinephrine to induce the expression of A20 may play a significant role in modulation of inflammation.

256 Effects of Lactobacillus Fermentate Replacing Bacitracin on Biomarkers Associated with Bacterial Cell Wall Recognition and Intestinal Integrity in Pigs

Abstract Two experiments were conducted to evaluate the efficacy of Lactobacillus fermentate (LBF) as postbiotic on immune response and barrier function in jejunal mucosa of nursery pigs challenged with F18+Escherichia coli. Sixty-four pigs (32 barrows and 32 gilts) weaned at 21 d of age with 7.9 ± 0.5 kg in Exp. 1 (n = 32) and 6.6 ± 0.7 kg in Exp. 2 (n = 32) were allotted based on a randomized complete block design with sex and initial BW as blocks. The treatments were NC: no challenge/no supplement; PC: E. coli challenge/no supplement; AGP: E. coli challenge/bacitracin (30 g/t feed); and PBT: E. coli challenge/postbiotic (2 kg/t feed). Pigs were fed a basal diet for 28 d, formulated to meet the nutrient requirements of NRC (2012). At d 7, the challenged pigs were orally inoculated with F18+E. coli. At d 28, pigs were euthanized to collect jejunal tissue to analyze gene expressions involved in bacterial cell wall recognition, immune response, and intestinal barrier function. Data were analyzed using the Proc MIXED of SAS 9.4. The PC tended to reduce (P = 0.097) the relative expression of PGLYRP2A (1.12 to 0.76) and increased (P &amp;lt; 0.05) TLR4 (0.98 to 1.67) and NOD1 (0.99 to 1.56) compared with NC. The AGP tended to increase (P = 0.074) PGLYRP2A (0.76 to 1.15) and tended to reduce (P = 0.099) PGLYRP3 (1.21 to 0.82) compared with PC. The PBT increased (P &amp;lt; 0.05) PGLYRP4 (0.86 to 1.53) compared with PC. The AGP tended to reduce (P = 0.085) TLR4 (1.67 to 0.98) compared with PC. The AGP and the PBT reduced (P &amp;lt; 0.05) NOD1 (1.56 to 0.98 and 0.97, respectively) compared with PC. The PC tended to increase (P = 0.079) CD14 (1.13 to 2.13) compared with NC, however, PBT tended to reduce (P = 0.091) it (2.13 to 1.27) compared with PC. The AGP increased (P &amp;lt; 0.05) interferon-γ (1.13 to 1.21) and the PBT tended to increase (P = 0.096) it (1.13 to 1.63) compared with PC. The AGP tended to increase (P = 0.082) claudin-1 (0.85 to 1.81) compared with PC. In conclusion, F18+E. coli challenge upregulated the expression of pathogen recognition genes, including TLR4, CD14, and NOD1, which can increase the production of cytokines associated with pro-inflammatory response. The AGP showed a trend towards increasing the intestinal barrier function, possibly by increasing the expression of interferon-γ and reducing the expression of genes associated with pathogen recognition. The PBT enhanced the immunocompetence of nursery pigs by increasing the expression of interferon-γ and PGLYRP4, and reducing the expression of genes associated with pathogen recognition including NOD1 and CD14, which may indicate a reduction of the pathogen invasion.

E3 ligase SOCS3 regulates NOD2 expression by ubiquitin proteasome system in lung cancer progression.

Despite lung cancer is one of the leading causes of cancer-related deaths, it remains hard to discover effective diagnostic and therapeutic approaches. Moreover, the five-year survival rate is relatively lower than other tumors. So urgent needs for finding a new theranostic target to treat lung cancer effectively. This study aims to present SOCS3 and NOD2 proteins as novel targets for diagnosis and therapy. We first confirmed SOCS3 expression level in patients' tissues. Then, we applied knockdown and overexpression of SOCS3 on lung cancer cell lines and performed proliferation, migration, and invasion assay. After that, we found NOD2 is a target of SOCS3 and introduced overexpression of NOD2 to A549 for verifying reduced tumorigenicity of lung cancer cells. We identified protein expression level of SOCS3 was frequently higher in tumor tissues than adjacent normal tissues. Truly, overexpression of SOCS3 promoted proliferation, migration, and invasion capacity of lung cancer cells. We found that SOCS3 interacts with NOD2 and SOCS3 ubiquitinates NOD2 directly. Furthermore, lung cancer tissues with higher SOCS3 expression showed lower NOD2 expression. We confirmed overexpression of NOD2 leads to suppressed tumorigenicity of lung cancer cells, and these effects occurred through MAPK pathway. Collectively, our work reveals novel roles of SOCS3 in lung tumorigenesis and proposes SOCS3 as a promising biomarker candidate for therapeutic and diagnostic target for lung cancer.