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Background: Nucleotide-binding oligomerization domain 1 (NOD1) and NOD2 are involved in carcinogenic processes by recognizing bacterial cell wall components and triggering inflammation. This study explored the association between genetic variations in NOD1 and NOD2 and susceptibility to hepatocellular carcinoma (HCC) and its progression in a Moroccan population., Methods: Genotyping of NOD1 rs2075820 (C>T) and NOD2 rs718226 (A>G) was performed using the TaqMan allelic discrimination assay in 467 Moroccan individuals. The cohort included 156 patients with hepatocellular carcinoma (HCC), 155 patients with liver cirrhosis (LC) diagnosed with HBV, HCV, or MASLD, and 156 controls., Results: The NOD1 rs2075820 variant showed no association with HCC susceptibility or progression, which is consistent with in silico predictions. However, the NOD2 rs718226 G allele and GG genotype were more common in the HCC group compared to the cirrhosis and control groups. Individuals with the homozygous G variant had a 2-fold higher risk for HCC (ORad = 2.12; CI=1.01-4.44; Pad = 0.04). Those with the GG genotype also had an increased risk of HCC (GG vs. AG+AA ORad = 2.28; CI=1.15-4.54; Pad = 0.016). Furthermore, GG genotype carriers had a significantly higher risk of HCC progression (OR ad  = 2.58; CI=1.26-5.31; P ad ​ = 0.031). Individuals with the rs718226 minor allele had a significantly elevated risk of progressing from LC to HCC (OR ad  = 1.50; CI=1.07-2.09; P ad  = 0.016). Stratification analysis indicated that men had a higher risk of HCC progression compared to women (OR ad  = 4.63; CI=1.53-14.00 vs. OR ad  = 2.73; CI=1.05-7.09)., Conclusion: The NOD1 rs2075820 polymorphism does not appear to be a genetic risk factor for susceptibility to HCC. In contrast, the non-coding NOD2 rs718226 variant significantly increases HCC susceptibility and promotes liver cancer progression in the Moroccan population. Further studies involving larger cohorts are warranted to definitively confirm or refute the effects of NOD1 and NOD2 genetic variants on liver cancer susceptibility and progression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

NOD1, plays a pivotal role in immune responses against bacterial as well as viral invasions. While the downstream signaling pathway of NOD1 is well understood in mammals, its characterization in lower vertebrates remains elusive. In present study, an effort was made to identify and characterize downstream signaling cascade of NOD1 in response to LPS, a potential ligand of NOD1 in teleosts, in spotted snakehead. In addition, the temporal effect of LPS on transcriptional modulation of NOD1 and its downstream signaling molecule RIPK2 was investigated. Docking studies revealed well conserved leucine rich domains of NOD1 that could bind with LPS. Further, NACHT-ATP interactions revealed differences in ATP binding motifs within the NACHT domain in spotted snakehead compared to those reported in other fish species and mammals pointing towards species-specific nature of ATP interactions within the NACHT domain. Further, it was revealed that the ssNOD1-CARD domain interacts with the CARD domain of downstream signaling molecule ssRIPK. Interestingly, LPS treatment modulated the expression of both, ssNOD1 and ssRIPK2 in a time-dependent manner., Competing Interests: Declaration of competing interest The authors report there are no competing interests to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bladder cancer (BCa) is a lethal malignancy of the urinary system and exhibits a poor prognosis. Chaperonin-containing tailless complex polypeptide 1 subunit 3 (CCT3) acts as an oncogene in various tumors, whereas its effect on BCa remains unknown. We identified the ferroptosis-associated differentially expressed genes through bioinformatic analysis and selected CCT3 for further verification. The levels of cell viability, apoptosis, migration, invasion, and proliferation were measured to clarify the effect of silencing CCT3 on BCa cells. Then we evaluated the role of CCT3 knockdown in vivo. Ferroptosis was assessed by the expression detection of the ferroptosis-related proteins. The underlying mechanism was predicted by RNA sequencing and verified by an agonist for nucleotide-binding and oligomerization domain 1 (NOD1). Western blotting was conducted to detect the protein expression of NOD1, nuclear factor kappa B (NF-κB) inhibitor alpha (IκBα), and phospho-IκBα (p-IκBα). In vitro, down-regulation of CCT3 suppressed the cell viability, migration, invasion, and proliferation, as well as induced apoptosis of BCa cells. In vivo, silencing CCT3 elevated the body weight of mice and suppressed the BCa progression. In addition, CCT3 knockdown could induce ferroptosis in vitro and in vivo. CCT3 knockdown suppressed the expression of NOD1 and p-IκBα/IκBα and the NOD1 agonist could reverse the effect of CCT3 suppression on BCa in vitro and in vivo. In summary, our findings demonstrate that silencing CCT3 inhibits BCa via induction of ferroptosis and suppression of the NOD1-NF-κB pathway., (© 2024. The Author(s).)

Human induced pluripotent stem cell (iPSC)-derived macrophages (IDMs) present a valuable substitute for monocyte-derived macrophages (MDMs) in order to study inflammation pathways in vitro. Through optimization of an IDM differentiation protocol, a six-fold increase in the production yield of myeloid progenitors was achieved. The derived IDMs were further characterized with respect to nucleotide-binding oligomerization domain (NOD) and receptor-interacting serine/threonine-protein kinase 2 (RIPK2) signaling, a key regulatory pathway for autoimmune diseases. The IDM cells recapitulated MDM biology with respect to the proinflammatory chemokine and inflammatory cytokine fingerprint more closely than THP-1 cells. When assessing RIPK2 modulation effect on tumor necrosis factor α (TNF-α), a cardinal mediator of inflammation, a similar pharmacological effect of RIPK2 inhibitors was observed in IDMs and MDMs. Additionally, IDMs and MDMs displayed a similar transcription and pathway profile in response to NOD1/2 stimulation and pharmacological inhibition of RIPK2. In summary, the enhanced myeloid production yield in the improved IDM differentiation protocol offers new opportunities for utilizing physiologically relevant macrophage models in the context of inflammatory diseases., Competing Interests: Declaration of competing interest Authors were either employees of Boehringer Ingelheim Pharma GmbH & Co KG or of Boehringer Ingelheim Pharmaceuticals, Inc., at the time this study was performed. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

As part of its pathogenesis, Salmonella enterica serovar Typhimurium delivers effector proteins into host cells. One effector is SspH2, a member of the so-called novel E3 ubiquitin ligase family, that interacts with and enhances, NOD1 pro-inflammatory signaling, though the underlying mechanisms are unclear. Here, we report that SspH2 interacts with multiple members of the NLRC family to enhance pro-inflammatory signaling by targeted ubiquitination. We show that SspH2 modulates host innate immunity by interacting with both NOD1 and NOD2 in mammalian epithelial cell culture via the NF-κB pathway. Moreover, purified SspH2 and NOD1 directly interact, where NOD1 potentiates SspH2 E3 ubiquitin ligase activity. Mass spectrometry and mutational analyses identified four key lysine residues in NOD1 that are required for its enhanced activation by SspH2, but not its basal activity. These critical lysine residues are positioned in the same region of NOD1 and define a surface on the receptor that appears to be targeted by SspH2. Overall, this work provides evidence for post-translational modification of NOD1 by ubiquitin and uncovers a unique mechanism of spatially selective ubiquitination to enhance the activation of an archetypal NLR.

While KRAS mutation is the leading cause of low survival rates in lung cancer bone metastasis patients, effective treatments are still lacking. Here, we identified homeobox C10 (HOXC10) as a lynchpin in pan-KRAS-mutant lung cancer bone metastasis. Through RNA-seq approach and patient tissue studies, we demonstrated that HOXC10 expression was dramatically increased. Genetic depletion of HOXC10 preferentially impeded cell proliferation and migration in vitro. The bioluminescence imaging and micro-CT results demonstrated that inhibition of HOXC10 significantly reduced bone metastasis of KRAS-mutant lung cancer in vivo. Mechanistically, the transcription factor HOXC10 activated NOD1/ERK signaling pathway to reprogram epithelial-mesenchymal transition (EMT) and bone microenvironment by activating the NOD1 promoter. Strikingly, inhibition of HOXC10 in combination with STAT3 inhibitor was effective against KRAS-mutant lung cancer bone metastasis by triggering ferroptosis. Taken together, these findings reveal that HOXC10 effectively alleviates pan-KRAS-mutant lung cancer with bone metastasis in the NOD1/ERK axis-dependent manner, and support further development of an effective combinatorial strategy for this kind of disease., (© 2024. The Author(s).)

Objective: Poorly 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., Methods: 16HBE 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., Results: At 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., Conclusions: Diabetic 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., (© 2024. The Author(s).)

Background: Patients with diabetes are particularly susceptible to Legionella pneumophila (LP) infection, but the exact pathogenesis of LP infection in diabetic patients is still not fully understood. Herein, we investigated the effect of diabetes on immune function during LP infection in vitro and in vivo., Methods: The time course of LP infection in macrophages under normal and high-glucose (HG) conditions was examined in vitro. Western blot was used to determine nucleotide-binding oligomerization domain 1 (NOD1), kinase 1/2 (ERK1/2), mitogen-activated protein kinase p38 (MAPK p38), and c-Jun N-terminal kinases (JNK). Enzyme-linked immunosorbent assay (ELISA) was used to assess the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Cell Counting Kit-8 (CCK8) assay assessed U937 cell viability after treating cells with different concentrations of high sugar medium and ML130 (NOD1 inhibitor). For the in vivo study, normal and streptozocin-induced diabetic guinea pigs were infected with LP for 6, 24, and 72 h, after which NOD1, MAPK-related signals, TNF-α, and IL-6 expression in lung tissues were assessed using immunohistochemistry, western blot, and RT-PCR., Results: HG attenuated the upregulation of NOD1 expression and reduced TNF-α and IL-6 secretion caused by LP compared with LP-infected cells exposed to normal glucose levels (all p < 0.05). In diabetic guinea pigs, HG inhibited the upregulation of NOD1 expression in lung tissues and the activation of p38, ERK1/2, and cJNK caused by LP infection compared to control pigs (all p < 0.05)., Conclusion: HG attenuates the response of macrophages to LP infection by inhibiting NOD1 upregulation and the activation of MAPK signaling., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Tumor-resident microbiota in breast cancer promotes cancer initiation and malignant progression. However, targeting microbiota to improve the effects of breast cancer therapy has not been investigated in detail. Here, we evaluated the microbiota composition of breast tumors and found that enterotoxigenic Bacteroides fragilis (ETBF) was highly enriched in the tumors of patients who did not respond to taxane-based neoadjuvant chemotherapy. ETBF, albeit at low biomass, secreted the toxic protein BFT-1 to promote breast cancer cell stemness and chemoresistance. Mechanistic studies showed that BFT-1 directly bound to NOD1 and stabilized NOD1 protein. NOD1 was highly expressed on ALDH+ breast cancer stem cells (BCSCs) and cooperated with GAK to phosphorylate NUMB and promote its lysosomal degradation, thereby activating the NOTCH1-HEY1 signaling pathway to increase BCSCs. NOD1 inhibition and ETBF clearance increase the chemosensitivity of breast cancer by impairing BCSCs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Higher Education Press.)

Inflammatory bowel disease (IBD) is a chronic inflammatory condition involving dysregulated immune responses and imbalances in the gut microbiota in genetically susceptible individuals. Current therapies for IBD often have significant side-effects and limited success, prompting the search for novel therapeutic strategies. Microbiome-based approaches aim to restore the gut microbiota balance towards anti-inflammatory and mucosa-healing profiles. Extracellular vesicles (EVs) from beneficial gut microbes are emerging as potential postbiotics. Serotonin plays a crucial role in intestinal homeostasis, and its dysregulation is associated with IBD severity. Our study investigated the impact of EVs from the probiotic Nissle 1917 (EcN) and commensal E. coli on intestinal serotonin metabolism under inflammatory conditions using an IL-1β-induced inflammation model in Caco-2 cells. We found strain-specific effects. Specifically, EcN EVs reduced free serotonin levels by upregulating SERT expression through the downregulation of miR-24, miR-200a, TLR4, and NOD1. Additionally, EcN EVs mitigated IL-1β-induced changes in tight junction proteins and oxidative stress markers. These findings underscore the potential of postbiotic interventions as a therapeutic approach for IBD and related pathologies, with EcN EVs exhibiting promise in modulating serotonin metabolism and preserving intestinal barrier integrity. This study is the first to demonstrate the regulation of miR-24 and miR-200a by probiotic-derived EVs.

Neisseria gonorrhoeae , a human restricted pathogen, releases inflammatory peptidoglycan (PG) fragments that contribute to the pathophysiology of pelvic inflammatory disease. The genus Neisseria is also home to multiple species of human- or animal-associated Neisseria that form part of the normal microbiota. Here we characterized PG release from the human-associated nonpathogenic species Neisseria lactamica and Neisseria mucosa and animal-associated Neisseria from macaques and wild mice. An N. mucosa strain and an N. lactamica strain were found to release limited amounts of the proinflammatory monomeric PG fragments. However, a single amino acid difference in the PG fragment permease AmpG resulted in increased PG fragment release in a second N. lactamica strain examined. Neisseria isolated from macaques also showed substantial release of PG monomers. The mouse colonizer Neisseria musculi exhibited PG fragment release similar to that seen in N. gonorrhoeae with PG monomers being the predominant fragments released. All the human-associated species were able to stimulate NOD1 and NOD2 responses. N. musculi was a poor inducer of mouse NOD1, but ldcA mutation increased this response. The ability to genetically manipulate N. musculi and examine effects of different PG fragments or differing amounts of PG fragments during mouse colonization will lead to a better understanding of the roles of PG in Neisseria infections. Overall, we found that only some nonpathogenic Neisseria have diminished release of proinflammatory PG fragments, and there are differences even within a species as to types and amounts of PG fragments released., Competing Interests: The authors declare no conflict of interest.

NOD1 and NOD2 as two representative members of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family play important roles in antimicrobial immunity. However, transcription mechanism of nod1 and nod2 and their signal circle are less understood in teleost fish. In this study, with the cloning of card9 and ripk2 in Chinese perch, the interaction between NOD1, NOD2, and CARD9 and RIPK2 were revealed through coimmunoprecipitation and immunofluorescence assays. The overexpression of NOD1, NOD2, RIPK2 and CARD9 induced significantly the promoter activity of NF-κB, IFNh and IFNc. Furthermore, it was found that nod1 and nod2 were induced by poly(I:C), type I IFNs, RLR and even NOD1/NOD2 themselves through the ISRE site of their proximal promoters. It is thus indicated that nod1 and nod2 can be classified also as ISGs due to the presence of ISRE in their proximal promoter, and their expression can be mechanistically controlled through PRR pathway as well as through IFN signaling in antiviral immune response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Peng, Wang, Li, Li, Wu, Zhang, Li, Liu, Nie and Chen.)

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., Competing Interests: MS, PU, KO, AA, MP, WB No competing interests declared, BK Senior editor, eLife, (© 2024, Shaku et al.)

As an ancient species with both conservation and commercial value, Sturgeon's inflammatory regulation mechanism is a research point. Nucleotide-binding and oligomerization domain-containing proteins 1 and 2 (NOD1/2) are classical intracellular pattern recognition receptors (PRRs) in immunity of anti-bacterial infection. However, the characterization and function of NOD1/2 in Sturgeon are still unclear. In this study, we analyzed the synteny relationship of NOD1/2 genes between Acipenser ruthenus and representative fishes at the genome-level. Results showed that the ArNOD2 collinear genes pair was present in all representative fishes. The duplicated ArNOD1/2 genes were under purifying selection during evolution as indicated by their Ka/Ks values. To explore the function of NOD1/2, we further investigated their expression patterns and the effects of pathogenic infection, PAMPs treatment, and siRNA interference in Acipenser baerii, the sibling species of A. ruthenus. Results showed that both AbNOD1/2 were expressed at early developmental stages and in different tissues. Pathogenic infection in vivo and PAMPs treatment in vitro demonstrated that AbNOD1/2 could respond to pathogen stimulation. siRNA interference with AbNOD1/2 inhibited expression levels of RIPK2 and inflammatory cytokines compared to the control group after iE-DAP or MDP treatment. This study hinted that the AbNOD1/2 could stimulate the inflammatory cytokines response during evolutionary processes., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Background: Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) plays a pivotal role in inducing metabolic inflammation in diabetes. Additionally, the NOD1 ligand disrupts the equilibrium of bone marrow-derived hematopoietic stem/progenitor cells, a process that has immense significance in the development of diabetic retinopathy (DR). We hypothesized that NOD1 depletion impedes the advancement of DR by resolving bone marrow dysfunction., Methods: We generated NOD1 -/- -Akita double-mutant mice and chimeric mice with hematopoietic-specific NOD1 depletion to study the role of NOD1 in the bone marrow-retina axis., Results: Elevated circulating NOD1 activators were observed in Akita mice after 6 months of diabetes. NOD1 depletion partially restored diabetes-induced structural changes and retinal electrical responses in NOD1 -/- -Akita mice. Loss of NOD1 significantly ameliorated the progression of diabetic retinal vascular degeneration, as determined by acellular capillary quantification. The preventive effect of NOD1 depletion on DR is linked to bone marrow phenotype alterations, including a restored HSC pool and a shift in hematopoiesis toward myelopoiesis. We also generated chimeric mice with hematopoietic-specific NOD1 ablation, and the results further indicated that NOD1 had a protective effect against DR. Mechanistically, loss of hematopoietic NOD1 resulted in reduced bone marrow-derived macrophage infiltration and decreased CXCL1 and CXCL2 secretion within the retina, subsequently leading to diminished neutrophil chemoattraction and NETosis., Conclusions: The results of our study unveil, for the first time, the critical role of NOD1 as a trigger for a hematopoietic imbalance toward myelopoiesis and local retinal inflammation, culminating in DR progression. Targeting NOD1 in bone marrow may be a potential strategy for the prevention and treatment of DR., (© 2024. The Author(s).)

The human Nod-like receptor protein NOD1 is a well-described pattern-recognition receptor (PRR) with diverse functions. NOD1 associates with F-actin and its protein levels are upregulated in metastatic cancer cells. A hallmark of cancer cells is their ability to migrate, which involves actin remodelling. Using chemotaxis and wound healing assays, we show that NOD1 expression correlated with the migration rate and chemotactic index in the cervical carcinoma cell line HeLa. The effect of NOD1 in cell migration was independent of the downstream kinase RIPK2 and NF-ĸB activity. Additionally, NOD1 negatively regulated the phosphorylation status of cofilin, which inhibits actin turnover. Co-immunoprecipitation assays identified HCLS1-associated protein X-1 (HAX-1) as a previously unknown interaction partner of NOD1. Silencing of HAX-1 expression reduced the migration behaviour to similar levels as NOD1 knockdown, and simultaneous knockdown of NOD1 and HAX-1 showed no additive effect, suggesting that both proteins act in the same pathway. In conclusion, our data revealed an important role of the PRR NOD1 in regulating cell migration as well as chemotaxis in human cervical cancer cells and identified HAX-1 as a protein that interacts with NOD1 and is involved in this signalling pathway., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

NOD1 is a cytosolic immune receptor well known for recognizing intracellular bacteria and inducing innate immune responses. Upon ligand binding, it usually forms a complex with the serine/threonine kinase RIPK2 to activate the transcription factor NF-κB. Next to its role in pathogen recognition, NOD1 has been associated with cancer progression. In this regard, Hezinger et al. investigated a non-canonical role of NOD1 in cell migration. They discovered that NOD1 is crucial for the migration and chemotaxis of HeLa cells and identified HAX-1 as a novel interaction partner., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Functional dyspepsia (FD) is a multifactorial disorder. Helicobacter pylori (H. pylori)-related dyspepsia (HpD) may be considered a separate entity. Duodenal eosinophilia is a potential pathogenic mechanism in FD. However, the impact of duodenal eosinophilia and host genetic polymorphism of innate and pro-inflammatory cascade, nucleotide-binding oligomerization domain 1 (NOD-1), and interleukin-1 beta (IL-1β) in HpD was not explored., Aim: To evaluate the association of NOD1-796G>A and IL-1B-511C>T gene variants and low-grade duodenal eosinophilia in HpD., Methods: A multicenter cross-sectional study was conducted. A total of 253 patients who met Rome-IV criteria were selected before upper endoscopy and 98 patients were included after unremarkable upper endoscopy and positive H. pylori in gastric biopsies were assessed. Clinical parameters, H. pylori cagA and duodenal histology, were evaluated., Results: Sixty-four (65%) patients had epigastric pain syndrome (EPS), 24 (25%) postprandial distress syndrome (PDS), and 10 (10%) EPS/PDS overlap. FD subtypes were not associated with NOD1-796G>A and IL-1B-511C>T gene variants. Low-grade duodenal eosinophilia was significantly increased in NOD1-796 GG versus single A-allele, but not in IL-1B-511 single T-allele or CC-allele. This association is dependent of cagA infection, since harboring cagA strain was significantly associated with low-grade duodenal eosinophilia with isolated variants NOD1-796 GG and IL-1B-511 single T-allele, but not without cagA. When we performed combined polymorphism analysis with NOD1-796 GG/IL-1B-511 single T-allele, a synergistic effect on low-grade duodenal eosinophilia was found between these two loci irrespective of cagA strain status in HpD., Conclusion: Our findings suggest that low-grade duodenal eosinophilia is significantly associated with NOD1-796 GG allele specially in cagA strain and with allelic combination NOD1-796 GG/IL-1B-511 single T-allele independent of cagA strain infection in HpD patients., (© 2023 John Wiley & Sons Ltd.)

Aims: Activation of specific innate immune receptors has been characterized to modulate nutrient metabolism in individual metabolic tissue directly or indirectly via secretory molecules. Activation of the nucleotide-binding oligomerization domain-containing protein 1 (NOD1) in adipocytes has been reported to induce lipolysis linked with insulin resistance and inflammatory response. These cues are positioned to modulate metabolic action in distal organs through paracrine/endocrine signaling. Here, we assessed the role of NOD1-mediated lipolysis and inflammatory response in adipocytes to affect lipid metabolism in hepatocytes., Main Methods: Human hepatoma cells (HepG2) were exposed to conditioned medium obtained from 3 T3-L1 adipocytes pretreated with NOD1 ligand (iE-DAP) and the effects on lipid accumulation, inflammation and insulin response were assessed. Activation of mechanisms leading to hepatic lipid accumulation was investigated by gene expression analysis., Key Findings: The conditioned medium from NOD1-activated 3 T3-L1 adipocytes (CM-DAP) induced lipid accumulation in HepG2 cells, driven by both lipolysis and inflammatory responses. The CM-DAP-induced lipid accumulation was independent to de novo lipogenesis and resulted from the enhanced transport of fatty acids inside and consequent increase in rate of triglycerides synthesis in hepatocytes. Moreover, CM-DAP-induced lipid accumulation instigated the expression of the markers of fatty acid oxidation and VLDL assembly for the export of triglycerides from hepatocyte. Furthermore, CM-DAP-induced lipid accumulation was associated with induction of inflammatory response and impairment of insulin signaling in HepG2 cells., Significance: Beyond showing liver-specific mechanisms to adipocytes-derived factors, our findings support the involvement of adipose tissue as a mediator in NOD1-mediated biological responses to modulate hepatic metabolism., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Subacute sclerosing panencephalitis is a progressive neurodegenerative disease that is a late complication of measles infection. However, to date, the pathogenesis of subacute sclerosing panencephalitis is still not explained; both viral and host factors seem to be associated. The present study aimed to investigate the relationship between NOD1 and NOD2 gene variants and subacute sclerosing panencephalitis. Methods: The gene variants of NOD1 (rs2075820 and rs2075818) and NOD2 (R334Q and R334W) were explored in 64 subacute sclerosing panencephalitis patients and 70 controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results: The frequencies of the AA genotype and A allele of rs2075820 ( NOD1 ; c.796G>A) polymorphism were lower in patients compared with controls ( P  = .022 and .014, respectively). The presence of the A allele of rs2075820 may be considered as a protective factor for subacute sclerosing panencephalitis. There was a significant difference between the groups in rs2075818 ( NOD1 G/C) polymorphism, and the CC genotype increased the risk of subacute sclerosing panencephalitis by 3.471-fold. The carriers of the C allele of rs2075818 (G/C) had a 1.855-fold susceptibility to subacute sclerosing panencephalitis ( P  = .018). The GC genotype might be associated with subacute sclerosing panencephalitis susceptibility in the patients compared with patients without having that haplotype ( P  = .03). Conclusions: Thus, we identified an association between subacute sclerosing panencephalitis and the rs2075820 ( NOD1 G/A) and rs2075818 ( NOD1 G/C) polymorphisms. These findings implicate a possible effect of this genetic polymorphism in susceptibility to subacute sclerosing panencephalitis, which needs to be confirmed in bigger populations.

Impaired intestinal barrier function and gut microbiota dysbiosis are believed to be related to exacerbation of acute pancreatitis (AP). As a bacterial cell wall peptidoglycan component, diaminopimelic acid (DAP) is a specific ligand of NOD1 that regulates the NOD1/RIP2/NF-kB signaling pathway. Here, we investigated the role of DAP in the crosstalk between the gut microbiota and pancreas during the occurrence of AP. Upregulation of NOD1/RIP2/NF-kB and elevated serum DAP levels were found in severe AP (SAP) model rats. The accumulation of DAP in SAP patients corroborated its ability to serve as an indicator of disease severity. Subsequently, SAP rats were treated with oral administration of the traditional Chinese medicine Qingyi Keli (QYKL) as well as neomycin, which can widely eliminate DAP-containing bacteria. Both QYKL and neomycin intervention ameliorated intestinal and pancreatic damage and systemic inflammation in SAP rats. Through 16S rDNA sequencing, we found that QYKL could rehabilitate the gut microbiota structure and selectively inhibit the overgrowth of enteric bacteria, such as Helicobacter and Lactobacillus, in SAP rats without affecting some protective strains, including Romboutsia and Allobaculum. Interestingly, we demonstrated that the decrease in serum DAP was accompanied by suppression of the NOD1/RIP2/NF-kB signaling pathway in both the intestine and pancreas of the two intervention groups. Taken together, these results suggested that the gut microbiota-DAP-NOD1/RIP2 signaling pathway might play a critical role in the progression of AP and that SAP could be alleviated via intervention in the signaling pathway. Our work provides new potential early warning indicators of SAP and targets for intervention., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Jiao, Liu, Li, Zhang, Pan, Luo, Zhang, Qi, Zhao, Yin and Shang.)

The human-restricted pathogen Neisseria meningitidis, which is best known for causing invasive meningococcal disease, has a nonpathogenic lifestyle as an asymptomatic colonizer of the human naso- and oropharyngeal space. N. meningitidis releases small peptidoglycan (PG) fragments during growth. It was demonstrated previously that N. meningitidis releases low levels of tripeptide PG monomer, which is an inflammatory molecule recognized by the human intracellular innate immune receptor NOD1. In the present study, we demonstrated that N. meningitidis released more PG-derived peptides than PG monomers. Using a reporter cell line overexpressing human NOD1, we showed that N. meningitidis activates NOD1 using PG-derived peptides. The generation of such peptides required the presence of the periplasmic N- acetylmuramyl-l-alanine amidase AmiC and the outer membrane lipoprotein NlpD. AmiC and NlpD were found to function in cell separation, and mutation of either amiC or nlpD resulted in large clumps of unseparated N. meningitidis cells instead of the characteristic diplococci. Using stochastic optical reconstruction microscopy, we demonstrated that FLAG epitope-tagged NlpD localized to the septum, while similarly tagged AmiC was found at the septum in some diplococci but was distributed around the cell in most cases. In a human whole-blood infection assay, an nlpD mutant was severely attenuated and showed particular sensitivity to complement. Thus, in N. meningitidis, the cell separation proteins AmiC and NlpD are necessary for NOD1 stimulation and survival during infection of human blood.

Background: Metastatic cervical squamous cell carcinoma (CSCC) has poor prognosis and is recalcitrant to the current treatment strategies, which warrants the necessity to identify novel prognostic markers and therapeutic targets. Given that CSCC is a virus-induced malignancy, we hypothesized that the pattern recognition receptors (PRRs) involved in the innate immune response likely play a critical role in tumor development., Methods: A bioinformatics analysis, qPCR, IHC, immunofluorescence, and WB were performed to determine the expression of NOD1/NOD2. The biological characteristics of overexpression NOD1 or NOD2 CSCC cells were compared to parental cells: proliferation, migration/invasion and cytokines secretion were examined in vitro through CCK8/colony formation/cell cycle profiling/cell counting, wound healing/transwell, and ELISA assays, respectively. The proliferative and metastatic capacity of overexpression NOD1 or NOD2 CSCC cells were also evaluated in vivo. FCM, mRNA and protein arrays, ELISA, and WB were used to identify the mechanisms involved, while novel pharmacological treatment were evaluated in vitro and in vivo. Quantitative variables between two groups were compared by Student's t test (normal distribution) or Mann-Whitney U test (non-normal distribution), and one-way or two-way ANOVA was used for comparing multiple groups. Pearson χ 2 test or Fisher's exact test was used to compare qualitative variables. Survival curves were plotted by the Kaplan-Meier method and compared by the log-rank test. P values of < 0.05 were considered statistically significant., Results: NOD1 was highly expressed in CSCC with lymph-vascular space invasion (LVSI, P < 0.01) and lymph node metastasis (LM, P < 0.01) and related to worse overall survival (OS, P = 0.016). In vitro and in vivo functional assays revealed that the upregulation of NOD1 or NOD2 in CSCC cells promoted proliferation, invasion, and migration. Mechanistically, NOD1 and NOD2 exerted their oncogenic effects by activating NF-κb and ERK signaling pathways and enhancing IL-8 secretion. Inhibition of the IL-8 receptor partially abrogated the effects of NOD1/2 on CSCC cells., Conclusions: NOD1/2-NF-κb/ERK and IL-8 axis may be involved in the progression of CSCC; the NOD1 significantly enhanced the progression of proliferation and metastasis, which leads to a poor prognosis. Anti-IL-8 was identified as a potential therapeutic target for patients with NOD1 high tumor., (© 2022. The Author(s).)

Background: The intracellular NOD-like receptor (NLR) family of pathogen recognition receptors (PRRa) is involved in initiating the innate immune response of which NOD1 and NOD2 are the best-characterized members. Aberrant expression of NOD1 and NOD2 has been uncovered in a number of chronic inflammatory diseases, such as inflammatory bowel disease and rheumatoid arthritis. However, the mechanism underlying NOD1/NOD2 gene expression regulation is still in its infancy. Epigenetic modifications such as DNA methylation and histone acetylation regulate the expression of genes and alterations in their patterns have been linked to many inflammatory diseases. This study investigated whether epigenetic modifying drugs affect the regulation of NOD1/NOD2 activity and expression. DNA methyltransferase inhibitors have recently been used in the treatment of myelodysplastic syndrome and as combination therapy in cancer but the full extent of their effects has not been quantified. Methods : Pharmacological inhibition of epigenetic enzymes in a human monocytic THP-1 cell line was carried out and NOD1/NOD2 expression and pro-inflammatory responses were quantified. Results : Cells primed with a DNA methyltransferase inhibitor (but not a histone deacetylase [HDAC] inhibitor) were found to be consistently more responsive to NOD1/NOD2 stimulation and had increased basal expression. Conclusion : The novel experimentation carried out here suggests for the first time that NOD1/NOD2 receptor activity and expression in monocytes are possibly regulated directly by DNA methylation.

Background: Cardiac insufficiency is a common complication of sepsis and septic shock and is the most common cause of death in critically ill patients. Recent studies have found that microRNAs (miRNAs) play a potential role in sepsis as markers, but little is known about their functional effects on sepsis-induced cardiomyopathy (SIC)., Objective: This study is designed to explore the possible role and underlying mechanisms of miR-702-3p in septic cardiomyopathy., Methods: As expected, H9c2 cells were induced with lipopolysaccharide (LPS) to construct the model of septic cardiomyopathy. The expression of miR-702-3p was detected by qRT-PCR assay and those of IL-1β, IL-6 and TNF-α by ELISA assay. The viability, proliferation and apoptosis of LPS-treated H9c2 cells were determined by CCK-8, EdU, flow cytometry and western blot assays. Moreover, Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) was predicted and confirmed as a direct target of miR-702-3p by TargetScan, miRwalk and miRDB prediction and dual-luciferase reporter gene assays., Results: While LPS can weaken the viability of H9c2 cells, miR-702-3p enhances that of LPS-treated H9c2 cells by inhibit the expressions of TNF-α, IL-6, IL-1β. We found NOD1 is a target gene of miR-702-3p, and over-expression of NOD1 restores the inhibitory effects of miR-702-3p on the LPS-treated H9c2 cells., Conclusion: MiR-702-3p played an important role in the pathogenesis of sepsis cardiomyopathy via targeting NOD1, suggesting that miR-702-3p may be a potential new target for the treatment of SIC., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Endoplasmic reticulum (ER) stress is intimately linked with inflammation in response to pathogenic infections. ER stress occurs when cells experience a buildup of misfolded or unfolded protein during times of perturbation, such as infections, which facilitates the unfolded protein response (UPR). The UPR involves multiple host pathways in an attempt to reestablish homeostasis, which oftentimes leads to inflammation and cell death if unresolved. The UPR is activated to help resolve some bacterial infections, and the IRE1α pathway is especially critical in mediating inflammation. To understand the role of the IRE1α pathway of the UPR during enteric bacterial infection, we employed Citrobacter rodentium to study host-pathogen interactions in intestinal epithelial cells and the murine gastrointestinal (GI) tract. C. rodentium is an enteric mouse pathogen that is similar to the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), for which we have limited small-animal models. Here, we demonstrate that both C. rodentium and EPEC induced the UPR in intestinal epithelial cells. UPR induction during C. rodentium infection correlated with the onset of inflammation in bone marrow-derived macrophages (BMDMs). Our previous work implicated IRE1α and NOD1/2 in ER stress-induced inflammation, which we observed were also required for proinflammatory gene induction during C. rodentium infection. C. rodentium induced IRE1α-dependent inflammation in mice, and inhibiting IRE1α led to a dysregulated inflammatory response and delayed clearance of C. rodentium. This study demonstrates that ER stress aids inflammation and clearance of C. rodentium through a mechanism involving the IRE1α-NOD1/2 axis.

NOD-like receptors (NLRs) are a family of pattern recognition receptors, able to respond to conserved microbial structures and endogenous danger signals. The NLR NOD1 responds to bacterial peptidoglycan, leading to recruitment of RIPK2, following activation of NFκB and MAPK pathways. In this chapter, we describe a fluorescent light microscopic approach to analyze the subcellular distribution of NOD1 upon infection with the invasive, Gram-negative bacterial pathogen Shigella flexneri. This method is based on exogenously expressed EGFP-tagged NOD1 and describes a protocol to obtain inducible cell lines with functional NOD1 signaling. The described protocol is useful to study NOD1 function, also in living cells, using live cell imaging and can be adopted for the study of other NLR proteins., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) is known to play an important role in innate immunity, while the relationship between NOD1 and inflammatory response in endometriosis remains unknown. The present study aims to investigate the effects of NOD1 on inflammatory response in endometriosis., Methods: Immunohistochemistry staining, Western blot, and qRT-PCR were conducted to investigate the levels of NOD1 and inflammatory cytokines in the endometriotic lesions. A Spearman's rank correlation analysis was conducted to determine the correlations of NOD1 and inflammatory cytokines (interleukin (IL)-6, tumor necrosis factor (TNF)-α, and monocyte chemoattractant protein (MCP)-1). Human endometrial stromal cells (HESCs) were isolated and incubated with peritoneal fluid with or without ML130. Cell viability was determined by using an MTT assay., Results: A significant elevation in NOD1 and inflammatory cytokine was observed in ectopic endometrium. Interestingly, a positive correlation between NOD1 and inflammatory cytokines was observed. In addition, treatment with ML130 significantly suppressed cell viability and the production of inflammatory cytokines in the 20% peritoneal fluid treated ectopic HESCs., Conclusions: NOD1 is related to the inflammatory response that is involved in endometriosis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The anti-inflammatory adipokine CTRP-3 might affect innate immune reactions such as NOD1. The impact of CTRP-3 on NOD1-mediated inflammation in adipocytes and monocytic cells as well as on NOD1 expression was investigated. Murine 3T3-L1 pre-adipocytes and adipocytes as well as human THP-1 monocyte-like cells were co-stimulated with the synthetic NOD1 agonist Tri-DAP and recombinant CTRP-3. Gonadal adipose tissue and primary adipocytes were obtained from a murine model carrying a knockout (KO) of CTRP-3 in adipocytes but not in stroma-vascular cells. Wildtype mice with lipopolysaccharide (LPS)-induced elevated NOD1 expression were treated with CTRP-3. Secreted inflammatory cytokines in cell supernatants were measured by ELISA and mRNA levels were quantified by RT-PCR. Pro-inflammatory chemokine and cytokine secretion (MCP-1, RANTES, TNFα) was induced by NOD1 activation in adipocytes and monocyte-like cells, and MCP-1 and RANTES release was effectively inhibited by pre-incubation of cells with CTRP-3. CTRP-3 also antagonized LPS-triggered induction of NOD1 gene expression in murine adipose tissue, whereas adipocyte CTRP-3 deficiency upregulated NOD1 expression in adipose tissue. CTRP-3 is an effective antagonist of peptidoglycan-induced, NOD1-mediated inflammation and of LPS-induced NOD1 expression. Since basal NOD1 expression is increased by adipocyte CTRP-3 deficiency, there have to be also inflammation-independent mechanisms of NOD1 expression regulation by CTRP-3., (© 2021. The Author(s).)

Purpose: Behçet's disease (BD) is an autoinflammatory disease with clinical manifestations such as mucocutaneous, ocular, vascular, gastrointestinal, musculoskeletal and central nervous system involvement. Features of innate and adaptive immunity and inflammasome pathways have been claimed in the pathogenesis of BD. We aimed to investigate the roles of NOD1, NOD2, PYDC1 and PYDC2 genes in the genetic predisposition of BD. Materials and Methods: Genetic variations of NOD1 (rs2075820 and rs2075818) and NOD2 (R334Q and R334W) genes were explored in 68 BD patients and 70 controls with PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) approach. PYDC1 and PYDC2 gene variants were investigated by Sanger sequencing. Results: The polymorphism of rs2075820 (NOD1 G/A) had a statistically significant difference between the BD and controls, AA genotype was 2.460-fold protective. When compared in terms of cardiovascular involvement in BD patients, AA genotype was increased the risk of cardiovascular involvement 4.286-fold. There was a significant difference between BD and controls in rs2075818 (NOD1 G/C) polymorphism and CC genotype increased the risk of BD by 3.780-fold. In terms of rs2075818 variants, there was a statistically significant difference between BD patients with ocular lesions, joints, cardiovascular and gastrointestinal involvement and controls. There was a significant difference between the patients with joint involvement and controls and the risk increased of 3.310-fold. Conclusion: The data shed new light on the association between polymorphisms of NOD1 gene and BD and clinicial manifestations. However, NOD2, PYDC1 and PYDC2 genes were not associated with BD in the Turkish population.

Duck viral hepatitis (DVH) is an acute, highly lethal infectious disease of ducklings that causes huge losses in the duck industry. Duck hepatitis A virus genotype 3 (DHAV-3) has been one of the most prevalent DVH pathogen in the Asian duck industry in recent years. Here, we investigated the genetic basis of the resistance and susceptibility of ducks to DVH by comparing the genomes and transcriptomes of a resistant Pekin duck flock (Z8) and a susceptible Pekin duck flock (SZ7). Our comparative genomic and transcriptomic analyses suggested that NOD1 showed a strong signal of association with DVH susceptibility in ducks. Then, we found that NOD1 showed a significant expression difference between the livers of susceptible and resistant individuals after infection with DHAV-3, with higher expression in the SZ7 flock. Furthermore, suppression and overexpression experiments showed that the number of DHAV-3 genomic copies in primary duck hepatocytes was influenced by the expression level of NOD1 . In addition, in situ RNAscope analysis showed that the localization of NOD1 and DHAV-3 in liver cells was consistent. Altogether, our data suggested that NOD1 was likely associated with DHAV-3 susceptibility in ducks, which provides a target for future investigations of the pathogenesis of DVH., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Liang, Wang, Zhang, Feng, Tang, Xie, Huang, Zhang, Zhang and Hou.)

Background: Asthma severity has been linked to exposure to gram-negative bacteria from the environment that are recognized by NOD1 receptor and are present in house dust mite (HDM) extracts. NOD1 polymorphism has been associated with asthma., Objective: We sought to evaluate whether either host or HDM-derived microbiota may contribute to NOD1-dependent disease severity., Methods: A model of HDM-induced experimental asthma was used and the effect of NOD1 deficiency was evaluated. Contribution of host microbiota was evaluated by fecal transplantation. Contribution of HDM-derived microbiota was assessed by 16S ribosomal RNA sequencing, mass spectrometry analysis, and peptidoglycan depletion of the extracts., Results: In this model, loss of the bacterial sensor NOD1 and its adaptor RIPK2 improved asthma features. Such inhibitory effect was not related to dysbiosis caused by NOD1 deficiency, as shown by fecal transplantation of Nod1-deficient microbiota to wild-type germ-free mice. The 16S ribosomal RNA gene sequencing and mass spectrometry analysis of HDM allergen, revealed the presence of some muropeptides from gram-negative bacteria that belong to the Bartonellaceae family. While such HDM-associated muropeptides were found to activate NOD1 signaling in epithelial cells, peptidoglycan-depleted HDM had a decreased ability to instigate asthma in vivo., Conclusions: These data show that NOD1-dependent sensing of HDM-associated gram-negative bacteria aggravates the severity of experimental asthma, suggesting that inhibiting the NOD1 signaling pathway may be a therapeutic approach to treating asthma., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The cytosolic NOD1 and NOD2 pattern recognition receptors are typically known as sensors of bacterial peptidoglycan fragments. A new study in this issue links NOD1/2 activation with ER homeostasis through the bioactive metabolite sphingosine-1-phosphate (S1P)., (© 2021 The Authors.)

Background: Helicobacter pylori is detected by pathogen recognition receptors including toll-like receptors (TLR) and nucleotide-binding oligomerization domain (NOD)-like receptors, eliciting an innate immune response against this bacteria. The aim of this study was to assess if polymorphisms of TLR2, TLR4, TLR5, NOD1 and NOD2 genes are associated with gastric cancer, in particular in individuals infected with H. pylori., Results: A case-control study of 297 gastric cancer patients and 300 controls was performed to assess the association of 17 polymorphisms. Analyses performed under the allele model did not find association with gastric cancer. However, NOD1 rs2075820 (p.E266K) showed association with intestinal-type gastric cancer among H. pylori infected subjects (OR = 2.69, 95% CI 1.41-5.13, p = 0.0026). The association was not statistically significant in diffuse-type gastric cancer cases (OR = 1.26, 95% CI 0.63-2.52, p = 0.51). When the analyses were performed in patients carrying H. pylori strains harboring the cag pathogenicity island (cagPAI), we noticed significant association with NOD1 rs2075820 (OR = 4.90, 95% CI 1.80-3.36, p = 0.0019), in particular for intestinal-type gastric cancer cases (OR = 7.16, 95% CI 2.40-21.33, p = 4.1 × 10 - 4 ) but not among diffuse-type gastric cancer cases (OR = 3.39, 95% CI 1.13-0.10, p = 0.03)., Conclusions: NOD1 rs2075820 increases the risk of intestinal-type gastric cancer among individuals infected with H. pylori, particularly in those harboring the cagPAI.

Increasing evidence suggests that NODs are involved in liver diseases; however, the underlying mechanisms remain obscure. In the present study, we analyzed the effect of NOD1 agonist pretreatment on acute liver failure induced by lipopolysaccharide (LPS) in D-galactosamine (D-GalN)-sensitized mice. We found that pretreatment with the NOD1 agonist markedly reduced LPS/D-GalN-induced mortality, elevation of serum ALT levels, and hepatocyte apoptosis. The protective effect of NOD1 agonist was independent of tumor necrosis factor (TNF)-α inhibition. NOD1 agonist pretreatment also attenuated TNF-α/D-GalN-induced apoptotic liver damage. The anti-apoptotic protein A20 expression was more pronounced in NOD1 agonist pretreated mice than in controls, and knockdown of A20 abrogated the protective effect of NOD1 agonist on LPS/D-GalN-induced liver injury and hepatocyte apoptosis. Further experiments showed that NOD1 agonist-induced A20 upregulation required the presence of kupffer cells and TNF-α. Taken together, our data strongly indicate that NOD1 is involved in the regulation of liver injury and could be a potential therapeutic target for liver diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jia, Deng, Xu, Li, Wang, Hu, Ren, Tong and Yin.)

As a lower vertebrate, the immune defense mechanism of fish mainly depends on the innate immune system. Nucleotide-binding oligomerization domain-like receptors (NLRs) are an important class of pattern recognition receptors in the innate immune system. In this study, NOD1 gene was cloned and characterized in Nile tilapia (Oreochromis niloticus). The ORF of Nile tilapia NOD1 gene was 2826 bp long and encoded 941 amino acid residues with a structure of CARD-NACHT-LRRs that was similar to the other counterparts in mammals and fishes. Phylogenetic and synteny analysis showed that NOD1 was conserved among different fishes and existed at least in the early stage of fish evolution. Expression pattern revealed that NOD1 mRNA was constitutively expressed in the tested tissues, while had high expression level in main immune organs and mucosal immune tissues (liver, head kidney, spleen, blood, gill, and intestine). Following Streptococcus agalactiae challenge, Nile tilapia NOD1 mRNA expression levels were altered in immune organs (liver, head kidney, spleen, blood), and the expression pattern was similar in liver, spleen and blood. Furthermore, the ligand recognition and signaling pathway of Nile tilapia NOD1 were also analyzed, it showed that NOD1 could recognize Tri-DAP intracellularly and activated NF-κB signaling pathway. In summary, our results indicated that the Nile tilapia NOD1 may play an important role in innate immune system and provided a basis for the functional study of NOD1 in teleost., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Effects of supplementation of dried alkaline (referred to as MVP1) and aqueous (referred to as PBD1) extracts of Kappaphycus alvarezii, were evaluated in broiler (Vencobb 400) chickens (1-35 days post-hatch). In experiment I, each of the seven diets (basal diet with three levels (0.5, 1.5 or 5.0 g kg -1 diet) of MVP1 or PBD1 and a negative control was fed to 12 pen replicates containing five birds in each. In experiment II, each of three diets [a negative control, and PBD1 at two levels (1.0 or 1.5 g kg -1 diet)] was fed to 16 pen replicates of five chicks in each., Results: Concentrations of total phenolics, phycobillins and free radical scavenging activity were higher (P < 0.01) whereas carrageenan was lower in PBD1 than in MVP1. In the experiment I, PBD1 at 1.5 g kg -1 diet improved (P < 0.05) body weight (BW) (7.11% higher). In the experiment II, both the treatments improved (P < 0.01) BW (9.18% and 8.47%, respectively) compared to the control. The group fed with PBD1@ 1.0 g kg -1 had higher (P < 0.05) haemagglutination inhibition titre, expression of intestinal claudin 2, TLR2A, NOD1, avian beta defensin 4, interleukin 2 and interleukin 6 genes than control. Treatments did not influence feed efficiency or levels of most of the antioxidant enzymes. Villus width and crypt depth were significantly higher in the group fed with 1.5 g kg -1 of PBD1., Conclusion: Supplementing dried aqueous extract of K. alvarezii at 1 g kg -1 diet may be an effective strategy to increase growth and immunity in broiler chickens. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

Objectives: To assess the correlation between a number of genetic variations of CYP2C19, TNF-α, NOD1, NOD2, and PPARγ genes with the severity of Helicobacter pylori (H. pylori) infections and peptic ulcers (PU)., Methods: A retrospective cross-sectional design was used in this study. Formalin-fixed paraffin-embedded (FFPE) tissue was used to extract genomic DNA that was collected from Jordanian patients who visited endoscopy clinics between 2014 to 2018 at the King Abdullah University Hospital (KAUH), Irbid, Jordan. Genotyping of the studied single nucleotide polymorphisms (SNPs) were applied using the sequencing protocol. Results:  A total of 251 patients (mean age: 42.12 ± 16.09 years) and healthy controls (mean age: 52.76 ± 19.45 years) were enrolled in this study. This study showed no significant association between patients and the studied polymorphisms except for rs2075820 of the NOD1 (p=0.0046). It is hypothesized that the heterozygous genotype (TC); 44.8% in patients versus 61.3% in controls has a decreased risk of peptic ulcers (OR:  0.49). The alleles frequency association was insignificant in all studied SNPs with a p-value more than 0.05., Conclusion: This study provided evidence regarding the association of the rs2075820 with H. pylori infections. The other studied SNPs were not statistically significant.

Increasing evidence shows that the long noncoding RNA (lncRNA) is a major regulator and participates in the regulation of various physiological and pathological processes, such as cell proliferation, differentiation, metastasis, and apoptosis. Unlike mammals, however, the study of lncRNA in lower invertebrates is just beginning and the extent of lncRNA-mediate regulation remains unclear. Here, we for the first time identify an lncRNA, termed nucleotide oligomerization domain 1 (NOD1) antibacterial and antiviral-related lncRNA (NARL), as a key regulator for innate immunity in teleost fish. We found that NOD1 plays an important role in the antibacterial and antiviral process in fish and that the microRNA miR-217-5p inhibits NOD1 expression and thus weakens the NF-κB and the IRF3-driven signaling pathway. Furthermore, our results indicated that NARL functions as a competing endogenous RNA (ceRNA) for miR-217-5p to regulate protein abundance of NOD1; thus, invading microorganisms are eliminated and immune responses are promoted. Our study also demonstrates the regulation mechanism that lncRNA NARL can competitive adsorption miR-217-5p to regulate the miR-217-5p/NOD1 axis is widespread in teleost fish. Taken together, our results reveal that NARL in fish is a critical positive regulator of innate immune responses to viral and bacterial infection by suppressing a feedback to NOD1-NF-κB/IRF3-mediated signaling., Competing Interests: Conflict of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Activation of innate immune components promotes cell autonomous inflammation in adipocytes. Oxidative stress links pattern recognition receptor-mediated detection of inflammatory ligands and the immune response. Reactive oxygen species (ROS) may mediate the effect of nucleotide-binding oligomerization domain protein-1 (NOD1) activation on inflammation in adipocytes. Here, we define the potential role of NADPH oxidase (NOX)-derived ROS in NOD1-mediated inflammatory response in adipocytes. Differentiated 3T3-L1 adipocytes were treated with NOD1 activating ligand D-gamma-Glu-meso-diaminopimelic acid (iE-DAP) to evaluate the oxidative stress and contribution of NOX as source of intracellular ROS. NOD1 activation potently induced ROS generation in 3T3-L1 adipocytes. Of the NOX family members, expression of NOX1 and NOX4 was increased upon NOD1 activation, in a PKCδ-dependent manner. siRNA-mediated down-regulation of NOX1 or NOX4 inhibited NOD1-mediated ROS production and increased the expression of antioxidant defense enzyme catalase and superoxide dismutase (SOD). siRNA-mediated lowering of NOX1 or NOX4 also suppressed NOD1-mediated activation of JNK1/2 and NF-κB, and consequent activation of inflammatory response in 3T3-L1 adipocytes. In summary, our findings demonstrate that NOD1 activation provokes oxidative stress in adipocytes via NOX1/4 and that oxidative stress, at least in part, contributes to induction of inflammatory response. Defining the source of ROS after immune response engagement may lead to new therapeutic strategies for adipose tissue inflammation., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Nucleotide-binding oligomerization domain (NOD)-like receptor 1 (NOD1) is a cytosolic pattern recognition receptor with a crucial role in the innate immune response of cells triggered by the presence of compounds such as gamma-d-glutamyl-meso-diaminopimelic acid (iE-DAP) present in the peptidoglycan of all gram-negative and certain gram-positive bacteria. Methionine (Met) and arginine (Arg) are functional AA with immunomodulatory properties. In the present study, we aimed to assess the effect of increased Met and Arg supply on mRNA abundance of genes associated with innate immune response, antioxidant function, and AA metabolism during iE-DAP challenge in bovine mammary epithelial cells (BMEC). Primary BMEC (n = 4 per treatment) were precultured in modified medium for 12 h with the following AA formulations: ideal profile of AA (control), increased Met supply (incMet), increased Arg supply (incArg), or increased supply of Met plus Arg (incMetArg). Subsequently, cells were challenged with or without iE-DAP (10 μg/mL) for 6 h. Data were analyzed as a 2 × 2 × 2 factorial using the MIXED procedure of SAS 9.4. Greater mRNA abundance of NOD1, the antioxidant enzyme SOD1, and AA transporters (SLC7A1 and SLC3A2) was observed in the incMet cells after iE-DAP stimulation. Although increased Met alone had no effect, incMetArg led to greater abundance of the inflammatory cytokine IL-6, and the antioxidant enzyme GPX1 after iE-DAP stimulation. The increased Arg alone downregulated NOD1 after iE-DAP stimulation, coupled with a downregulation in the AA transporters mRNA abundance (SLC7A1, SLC7A5, SLC3A2, and SLC38A9), and upregulation in GSS and KEAP1 mRNA abundance. Overall, the data indicated that increased supply of both Met and Arg in the culture medium were more effective in modulating the innate immune response and antioxidant capacity of BMEC during in vitro iE-DAP stimulation., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Cerebral ischemia-reperfusion (CIR) can regulate multiple transcription factors to enhance or attenuate injury. Nucleotide-binding oligomerization domain protein 1 (NOD1) has been reported to be involved in autophagy and endoplasmic reticulum (ER) stress. Moreover, autophagy and ER stress play important roles in CIR injury. Hence, the function of NOD1 in CIR injury was explored in this study. Primary rat cortical neurons were treated with oxygen-glucose deprivation and reperfusion (OGD/R) in vitro. NOD1 level was measured using immunofluorescence, real-time quantitative PCR and western blotting and its ubiquitination using co-immunoprecipitation. Results showed that OGD/R up-regulated NOD1 level but inhibited NOD1 ubiquitination. Then the effect of NOD1 on OGD/R-induced changes in cell viability, apoptosis, autophagy and ER stress was evaluated by methyl thiazolyl tetrazolium assay, lactate dehydrogenase release, Hoechst staining, detection of autophagy and ER stress-related proteins using western blotting and infection with GFP-LC3 lentiviruses. OGD/R decreased cell viability and increased cell apoptosis. NOD1 up-regulation promoted these changes, but NOD1 down-regulation reversed these changes. Moreover, OGD/R triggered autophagy and ER stress and NOD1 silencing reversed OGD/R-induced changes in autophagy and ER stress. To validate the role of autophagy in OGD/R injury, autophagy inducer rapamycin was used. Rapamycin promoted OGD/R-induced decrease in cell viability and counteracted NOD1 silencing-induced increase in cell viability. In addition, ER stress inducer tunicamycin was used to investigate the relationship between ER stress and autophagy. Tunicamycin promoted OGD/R-induced decrease in cell viability and reversed NOD1 silencing-induced increase in cell viability. Tunicamycin also enhanced OGD/R-induced autophagy and reversed NOD1 silencing-induced inhibition in autophagy. The results indicated that NOD1 promoted OGD/R injury in cortical neurons through activating ER stress-mediated autophagy. This study provides new insights for the target of CIR injury treatment., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Seasonal influenza is an acute viral infection caused by influenza virus, which is often prevalent in the summer and winter. The influenza virus can infect pigs and poultry. Some literature reports that the influenza virus has an outbreak in summer. The summer climate is characterized by a high humidity and high temperature environment, which is the same as many animal feeding and growing environments. We established a flu animal model under a high temperature and humidity environment during the day to observe the impact of high humidity and high temperature environment on the mice after contracting the influenza virus. Our results indicate that the intestinal flora of 16 s rDNA cultured in High humidity and high temperature environment changes, the intestinal mucosal permeability increases, the expression of pIgR, sIgA, and IgA in the intestinal mucosal immune system decreases, and the NLR immune recognition signaling pathway NOD1 is activated. The expression of related genes such as NOD2, NF-κB, and pIgR increases, which leads to the increase of related inflammatory factors in the vicinity of the intestines, aggravating local inflammation. High humidity and high temperature environment can cause the expression of inflammatory cytokines in the body to rise, causing Th17/Treg immune imbalance, inhibiting Treg maturation and differentiation, and increasing the expression of IL-2, IL-6, and other cytokines, while the expression of IFN-γ and IL-17A decreases. This condition worsens after infection with the influenza virus. Overall, our study found that High humidity and high temperature environment affect the intestinal flora and the body's immune status, thereby aggravating the status of influenza virus infection., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Risk of cardiovascular disease (CVD) increases considerably as renal function declines in chronic kidney disease (CKD). Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) has emerged as a novel innate immune receptor involved in both CVD and CKD. Following activation, NOD1 undergoes a conformational change that allows the activation of the receptor-interacting serine/threonine protein kinase 2 (RIP2), promoting an inflammatory response. We evaluated whether the genetic deficiency of Nod1 or Rip2 in mice could prevent cardiac Ca 2+ mishandling induced by sixth nephrectomy (Nx), a model of CKD. We examined intracellular Ca 2+ dynamics in cardiomyocytes from Wild-type ( Wt ), Nod1 -/- and Rip2 -/- sham-operated or nephrectomized mice. Compared with Wt cardiomyocytes, Wt -Nx cells showed an impairment in the properties and kinetics of the intracellular Ca 2+ transients, a reduction in both cell shortening and sarcoplasmic reticulum Ca 2+ load, together with an increase in diastolic Ca 2+ leak. Cardiomyocytes from Nod1 -/- -Nx and Rip2 -/- -Nx mice showed a significant amelioration in Ca 2+ mishandling without modifying the kidney impairment induced by Nx. In conclusion, Nod1 and Rip2 deficiency prevents the intracellular Ca 2+ mishandling induced by experimental CKD, unveiling new innate immune targets for the development of innovative therapeutic strategies to reduce cardiac complications in patients with CKD.

The human pathogen Pseudomonas aeruginosa can rapidly induce fatal sepsis, even in previously healthy infants or children treated with appropriate antibiotics. To reduce antibiotic overuse, exploring novel complementary therapies, such as probiotics that reportedly protect patients against P. aeruginosa infection, would be particularly beneficial. However, the major mechanism underlying the clinical effects is not completely understood. We thus aimed to investigate how probiotics affect IL-8 and human beta-defensin 2 (hBD-2) in P. aeruginosa -infected intestinal epithelial cells (IECs). We infected SW480 IECs with wild type PAO1 P. aeruginosa following probiotic treatment with Lactobacillus rhamnosus GG or Bifidobacterium longum spp. infantis S12, and analysed the mRNA expression and secreted protein of IL-8 and hBD-2, Akt signalling and NOD1 receptor protein expression. We observed that probiotics enhanced hBD-2 expression but suppressed IL-8 responses when administered before infection. They also enhanced P. aeruginosa -induced membranous NOD1 protein expression and Akt activation. The siRNA-mediated Akt or NOD1 knockdown counteracted P. aeruginosa -induced IL-8 or hBD-2 expression, indicating regulatory effects of these probiotics. In conclusion, these data suggest that probiotics exert reciprocal regulation of inflammation and antimicrobial peptides in P. aeruginosa -infected IECs and provide supporting evidence for applying probiotics to reduce antibiotic overuse.

The present study was aimed to investigate the relationship between NOD1/CARD4 and NOD2/CARD15 gene polymorphisms and osteoporosis in the Turkish population. The first time we thought that the functional polymorphisms in NOD1/CARD4 and NOD2/CARD15 genes might have triggered the development of osteoporosis. The objective of our study was to determine the relationship between NOD1/CARD4 and NOD2/CARD15 SNPs and osteoporosis. The NOD1/CARD4 (rs5743336) and NOD2/CARD15 (rs2066847) SNPs were analyzed by PCR restriction fragment length polymorphism (PCR-RFLP) in 94 healthy controls and 164 subjects with osteoporosis. PCR products were digested with restriction enzymes AvaI for NOD1/CARD4 and ApaI for NOD2/CARD15. We found that NOD1/CARD4 genotype distribution of AA, GA and GG were 15, 44 and 41% for patients and 17, 46 and 37% for controls, respectively. NOD2/CARD15 mutation was found only in three patients (1.8%) as heterozygote. The results did not show any statistical difference between NOD1/CARD4 and NOD2/CARD15 genotype distribution of patients and healthy groups (χ2 = 1.740, P=0.187; χ2 = 1.311, P=0.519). However, the most frequent AG genotype (46%) of NOD1/CARD4 was observed in healthy controls, GG genotype (44%) of NOD1/CARD4 was observed as the most frequent in osteoporotic patients. NOD2/CARD15 WT/WT genotype, the most frequent genotype, was observed in both groups. Statistical analysis revealed that NOD1/CARD4 and NOD2/CARD15 polymorphisms are not associated with osteoporosis. However, a definite judgement is difficult to be made due to restricted number of patients and small size of control group. Further research is sorely warranted in this direction., (© 2020 The Author(s).)

Prompt recognition of microbes by cells is critical to eliminate invading pathogens. Some cell-associated pattern recognition receptors (PRRs) recognize and respond to microbial ligands. However, others can respond to cellular perturbations, such as damage-associated molecular patterns (DAMPs). Nucleotide oligomerization domains 1 and 2 (NOD1/2) are PRRs that recognize and respond to multiple stimuli of microbial and cellular origin, such as bacterial peptidoglycan, viral infections, parasitic infections, activated Rho GTPases, and endoplasmic reticulum (ER) stress. How NOD1/2 are stimulated by such diverse stimuli is not fully understood but may partly rely on cellular changes during infection that result in ER stress. NOD1/2 are ER stress sensors that facilitate proinflammatory responses for pathogen clearance; thus, NOD1/2 may help mount broad antimicrobial responses through detection of ER stress, which is often induced during a variety of infections. Some pathogens may subvert this response to promote infection through manipulation of NOD1/2 responses to ER stress that lead to apoptosis. Here, we review NOD1/2 stimuli and cellular responses. Furthermore, we discuss pathogen-induced ER stress and how it might potentiate NOD1/2 signaling., (Copyright © 2020 American Society for Microbiology.)

The inflammatory response to Chlamydia infection is likely to be multifactorial and involve a variety of ligand-dependent and -independent recognition pathways. We previously reported the presence of NOD1/NOD2-dependent endoplasmic reticulum (ER) stress-induced inflammation during Chlamydia muridarum infection in vitro , but the relevance of this finding to an in vivo context is unclear. Here, we examined the ER stress response to in vivo Chlamydia infection. The induction of interleukin 6 (IL-6) production after systemic Chlamydia infection correlated with expression of ER stress response genes. Furthermore, when tauroursodeoxycholate (TUDCA) was used to inhibit the ER stress response, an increased bacterial burden was detected, suggesting that ER stress-driven inflammation can contribute to systemic bacterial clearance. Mice lacking both NOD1 and NOD2 or RIP2 exhibited slightly higher systemic bacterial burdens after infection with Chlamydia Overall, these data suggest a model where RIP2 and NOD1/NOD2 proteins link ER stress responses with the induction of Chlamydia -specific inflammatory responses. IMPORTANCE Understanding the initiation of the inflammatory response during Chlamydia infection is of public health importance given the impact of this disease on young women in the United States. Many young women are chronically infected with Chlamydia but are asymptomatic and therefore do not seek treatment, leaving them at risk of long-term reproductive harm due to inflammation in response to infection. Our manuscript explores the role of the endoplasmic reticulum stress response pathway initiated by an innate receptor in the development of this inflammation., (Copyright © 2020 Pham et al.)