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Blau syndrome is an autosomal dominant chronic inflammatory disease, which may begin with skin manifestations in the first months of life, alerting physicians to the diagnosis. This case reports a patient diagnosed jointly by pediatric dermatology and rheumatology consultants at two years of age.

Allergens can cross the epithelial barrier to enter the body but how this cellular passage affects protein structures and the downstream interactions with the immune system are still open questions. We sought to show the molecular details and the effects of 3 nonspecific lipid transfer proteins (nsLTPs; Mal d 3 [allergenic nsLTP1 from apple], Cor a 8 [allergenic nsLTP1 from hazelnut], and Pru p 3 [allergenic nsLTP1 from peach]) on epithelial cell uptake and transport. We used fluorescent imaging, flow cytometry, and proteomic and lipidomic screenings to identify the mechanism involved in nsLTP cellular uptake and signaling on selected epithelial and transgenic cell lines. nsLTPs are transported across the epithelium without affecting cell membrane stability or viability, and allergen uptake was largely impaired by inhibition of clathrin-mediated endocytosis. Analysis of the lipidome associated with nsLTPs showed a wide variety of lipid ligands predicted to bind inside the allergen hydrophobic cavity. Importantly, the internalization of nsLTPs was contingent on these ligands in the protein complex. nsLTPs were found to initiate cellular signaling via Toll-like receptor 2 but not the cluster of differentiation 1 protein receptor, despite neither being essential for nsLTP endocytosis. We also provide evidence that the 3 allergens induced intracellular stress signaling through activation of the NOD2 pathway. Our work consolidates the current model on nsLTP-epithelial cell interplay and adds molecular details about cell transport and signaling. In addition, we have developed a versatile toolbox to extend these investigations to other allergens and cell types. [ABSTRACT FROM AUTHOR]

Objective: Yao syndrome (YAOS) is formerly called nucleotide-binding oligomerization domain containing 2 (NOD2)-associated autoinflammatory disease.We report a large cohort of YAOS. Methods: We conducted a retrospective analysis of a cohort of adult patients with systemic autoinflammatory diseases (SAIDs). All patients underwent testing for a periodic fever syndrome gene panel. Results: A total of 194 patients carried NOD2 variants, 152 patients were diagnosed with YAOS, and 42 had mixed autoinflammatory diseases with combined variants in NOD2 and other SAID-associated genes. Demographic, clinical and molecular data were summaried. In sub-group analysis of the 194 patients, individual patients were often identified to carry two or more variants that usually included IVS8 + 158/R702W, IVS8 + 158/L1007fs, IVS8 + 158/V955I, IVS8 + 158/other, or NOD2/variants in other SAID genes. Ninety-nine patients carried single variants. Taken together, these variants contribute to the disease in combination or individually. Conclusion: This largest cohort has provided comprehensive clinical and genotyping data in YAOS. Variants in the NOD2 gene can give rise to a spectrum from inflammatory bowel disease to autoinflammatory disease.This report further raises awareness of the underdiagnosed disease in the medical community. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to characterize the phenotype and genotype of patients with Yao syndrome (YAOS), with focus on comparing to prior cohorts, identifying novel features, and describing treatment observations. Methods: A retrospective medical records review of patients with YAOS seen at Mayo Clinic was conducted to characterize clinical features, NOD2 genotypes, and therapeutic trials and responses. Results: Twenty-two patients diagnosed with YAOS were included. Eighteen patients (81.8%) were female and twenty (90.9%) were White. Mean age at symptom onset was 24.0 ± 14.8 years. Common clinical manifestations included fever (81.8% of patients), rash (95.5%), chronic gastrointestinal symptoms (100%), arthralgia/arthritis (95.5%), and sicca symptoms (68.2%). NOD2 genotypes as single variants included IVS8 + 158 in 14 patients (63.6%), R702W in 8 patients (36.4%), 1007fs in 4 (18.2%), and one patient had only a previously unreported rare variant. Eight patients (36.4%) had compound (two or more) NOD2 variants. Potential comorbidities of YAOS observed in this cohort included gastrointestinal dysmotility, autonomic dysfunction, and mast cell activation-like symptoms. Glucocorticoid responsiveness was observed in 15 of 20 patients exposed (75%). Eleven patients (50.0%) received IL-1 inhibitor therapy, and one patient (4.5%) received IL-6 inhibitor therapy with adequate disease control. Conclusion: Our findings substantiate the occurrence of fevers, arthralgia/ arthritis, rash, chronic gastrointestinal symptoms, and sicca-like symptoms described previously in patients with YAOS. Novel clinical features and one NOD2 variant not previously described were identified. Glucocorticoids, biologic IL-1 inhibitors, and IL-6 receptor inhibitors appeared to be effective for treatment of patients with YAOS. [ABSTRACT FROM AUTHOR]

[Display omitted] • Microglia are a major target of PGN in gut microbiota-brain signalling. • Physiological doses of MDP result in long-lasting transcriptional changes in microglial Nod2 signaling, cytokines and chemokines. • Increased TNF-α and CCL5 gene expression is reflected in elevated protein secretion. • In IMG cells MDP signalling via NF-κB is MAPK dependent. • IMG cells show a strong correlation in gene expression to primary microglia, making them a useful in vitro model to study microglia. Bacterial peptidoglycan (PGN) fragments are commonly studied in the context of bacterial infections. However, PGN fragments recently gained recognition as signalling molecules from the commensal gut microbiota in the healthy host. Here we focus on the minimal bioactive PGN motif muramyl dipeptide (MDP), found in both Gram-positive and Gram-negative commensal bacteria, which signals through the Nod2 receptor. MDP from the gut microbiota translocates to the brain and is associated with changes in neurodevelopment and behaviour, yet there is limited knowledge about the underlying mechanisms. In this study we demonstrate that physiologically relevant doses of MDP induce rapid changes in microglial gene expression and lead to cytokine and chemokine secretion. In immortalised microglial (IMG) cells, C–C Motif Chemokine Ligand 5 (CCL5/RANTES) expression is acutely sensitive to the lowest physiologically prevalent dose (0.1 µg/ml) of MDP. As CCL5 plays an important role in memory formation and synaptic plasticity, microglial CCL5 might be the missing link in elucidating MDP-induced alterations in synaptic gene expression. We observed that a higher physiological dose of MDP elevates the expression of cytokines TNF-α and IL-1β, indicating a transition toward a pro-inflammatory phenotype in IMG cells, which was validated in primary microglial cultures. Furthermore, MDP induces the translocation of NF-κB subunit p65 into the nucleus, which is blocked by MAPK p38 inhibitor SB202190, suggesting that an interplay of both the NF-κB and MAPK pathways is responsible for the MDP-specific microglial phenotype. These findings underscore the significance of different MDP levels in shaping microglial function in the CNS and indicate MDP as a potential mediator for early inflammatory processes in the brain. It also positions microglia as an important target in the gut microbiota-brain-axis pathway through PGN signalling. [ABSTRACT FROM AUTHOR]

Background: Nod2 is involved in innate immune responses to bacteria, regulation of metabolism, and sensitivity to cancer. A Nod2 polymorphism is associated with breast cancer, but the role of Nod2 in the development and progression of breast cancer is unknown. Methods: Here, we tested the hypothesis that Nod2 protects mice from breast cancer using the 4T1 orthotopic model of mammary tumorigenesis. WT and Nod2−/− mice were injected with 4T1 mammary carcinoma cells and the development of tumors was monitored. A detailed analysis of the tumor transcriptome was performed and genes that were differentially expressed and pathways that were predicted to be altered between WT and Nod2−/− mice were identified. The activation of key signaling molecules involved in metabolism and development of cancer was studied. Results: Our data demonstrate that Nod2−/− mice had a higher incidence and larger tumors than WT mice. Nod2−/− mice had increased expression of genes that promote DNA replication and cell division, and decreased expression of genes required for lipolysis, lipogenesis, and steroid biosynthesis compared with WT mice. Nod2−/− mice also had lower expression of genes required for adipogenesis and reduced levels of lipids compared with WT mice. The tumors in Nod2−/− mice had decreased expression of genes associated with PPARα/γ signaling, increased activation of STAT3, decreased activation of STAT5, and no change in the activation of ERK compared with WT mice. Conclusions: We conclude that Nod2 protects mice from the 4T1 orthotopic breast tumor, and that tumors in Nod2−/− mice are predicted to have increased DNA replication and cell proliferation and decreased lipid metabolism compared with WT mice. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective Diagnostic pitfalls often arise in the community because of potentially misleading similarities between juvenile idiopathic arthritis (JIA) and Blau syndrome, an immune-related disorder caused by NOD2 gene mutations. It remains unclear in which population and to what extent next-generation sequencing techniques can aid in diagnosis. Methods We evaluated clinical usefulness of targeted next-generation sequencing in previously diagnosed JIA. Participants were required to have symptoms and signs suspected of Blau syndrome, including at least uveitis or cutaneous lesions in addition to arthritis. Targeted sequencing was conducted on NOD2 gene to detect diagnostic variants classified as pathogenic or likely pathogenic for Blau syndrome. We assessed the molecular diagnostic yield and clinical implications for patient care. Results Between 1 May 2008 and 1 June 2021, sequencing data were accrued from 123 previously diagnosed JIA (median age: 5 years; female: 62.6%). Targeted NOD2 sequencing yielded a positive molecular diagnosis of Blau syndrome in 21.1% (95% CI: 14.9%, 29.2%), encompassing six heterozygous missense mutations classified as pathogenic variants. Among those receiving a molecular diagnosis, changes in clinical management and treatment were considered as having occurred in 38.5%. Nine predictors were identified as being associated with a higher diagnostic yield, providing clinical clues to suspect the possibility of Blau syndrome. Conclusion Among some patients with paediatric-onset arthritis complicated with uveitis or cutaneous lesions, reassessment of the diagnosis of JIA may be warranted. Targeted NOD2 sequencing established the molecular diagnosis of Blau syndrome in nearly one-fifth of these cases and provided clinically relevant information for patient-care decisions. [ABSTRACT FROM AUTHOR]

Microbial sensors play an essential role in maintaining cellular homoeostasis. Our knowledge is limited on how microbial sensing helps in differential immune response and its link to inflammatory diseases. Recently we have confirmed that ELMO1 (Engulfment and Cell Motility Protein-1) present in cytosol is involved in pathogen sensing, engulfment, and intestinal inflammation. Here, we show that ELMO1 interacts with another sensor, NOD2 (Nucleotide-binding oligomerization domain-containing protein 2), that recognizes bacterial cell wall component muramyl dipeptide (MDP). The polymorphism of NOD2 is linked to Crohn's disease (CD) pathogenesis. Interestingly, we found that overexpression of ELMO1 and mutant NOD2 (L1007fs) were not able to clear the CD-associated adherent invasive E. coli (AIEC-LF82). The functional implications of ELMO1-NOD2 interaction in epithelial cells were evaluated by using enteroid-derived monolayers (EDMs) from ELMO1 and NOD2 KO mice. Subsequently we also assessed the immune response in J774 macrophages depleted of either ELMO1 or NOD2 or both. The infection of murine EDMs with AIEC-LF82 showed higher bacterial load in ELMO1-KO, NOD2 KO EDMs, and ELMO1 KO EDMs treated with NOD2 inhibitors. The murine macrophage cells showed that the downregulation of ELMO1 and NOD2 is associated with impaired bacterial clearance that is linked to reduce pro-inflammatory cytokines and reactive oxygen species. Our results indicated that the crosstalk between microbial sensors in enteric infection and inflammatory diseases impacts the fate of the bacterial load and disease pathogenesis.

Relevance.The recent increase in inflammatory, allergic and infectious diseases needs to update new ways of raising non-specific resistance of the organism. Innate immunity provides the first line of defense against pathogens through the activation of receptors that detect microorganisms: TLRs, NLRs and CLRs. Muramyl peptides that form the cell wall of all known bacteria are recognized by NLRs and trigger immune responses to eliminate pathogens. The aim of this study was to investigate the effect of muramyl peptides on the production of chemokines, growth factors, pro-inflammatory and anti-inflammatory cytokines by human mononuclear cells.Materials and Methods.Mononuclear cells were isolated from the peripheral blood of healthy volunteers using the Cell Separation Media Lympholyte CL 5015 reagent and cultured for 4 hours in the presence of glucosaminyl muramyl dipeptides GMDP, GMDP-OH, GMDP-Lys, GMDP-LL; an adequate amount of medium was added to the control wells. The levels of chemokines, growth factors, proinflammatory and anti-inflammatory cytokines were measured using magnetic beads with antibodies according to the manufacturer’s instructions Luminex 200, Merck (Millipore) equipment, and software (Burlington, Massachusetts, USA).Results and Discussion.It was found that muramyl peptides GMDP, GMDP-ON and GMDP-Lys enhance the production of cytokines IL-1a, IL-1b, IL-1RA, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12P40, IL-12P70, IL-15, MDC, sCD40L, IFNα2, IFN-γ, TNF-a, TNF-β, GM-CSF. GMDP-LL does not affect the production of cytokines. At the same time, muramyl peptides with the L-configuration of alanine and the D-configuration of isoglutamine (L-D muramyl peptides) did not change the values of IL-2, IL-3, IL-5, IL-9.Conclusion.The D-configuration of isoglutamine is fundamental for the implementation of the regulatory activity of muramyl peptides. Awide range of bacterial bioregulators, the source of which are microorganisms, regulate the host homeostasis and trigger immune reactions, which, depending on the context, can have opposite effects. L-D muramyl peptides activate mononuclear cells, which begin to produce proinflammatory cytokines and chemokines, as well as growth factors necessary for the destruction of pathogens. In addition, anti-inflammatory cytokines are also triggered, which have aregulatory role in the appearance of memory cells and the weakening of inflammatory reactions. Thus, normally, muramyl peptides participate in maintaining tolerance to microflora and maintaining immune homeostasis.

Abstract 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

Nucleotide-binding oligomerization domain containing 1 (NOD1) and NOD2 are pivotal cytoplasmic pattern-recognition receptors (PRRs) that exhibit remarkable evolutionary conservation. They possess the ability to discern specific peptidoglycan (PGN) motifs, thereby orchestrating innate immunity and contributing significantly to immune homeostasis maintenance. The comprehensive understanding of both the structure and function of NOD1 and NOD2 has been extensively elucidated. These receptors proficiently recognize an array of damage-associated molecular patterns (DAMPs) as well as pathogen-associated molecular patterns (PAMPs), subsequently mediating inflammatory responses and autophagy. In recent years, emerging evidence has highlighted the crucial roles played by NOD1 and NOD2 in regulating infectious diseases, metabolic disorders, cancer, and autoimmune conditions, among others. Perturbation in either their loss or excessive activation can detrimentally impact immune homeostasis. This review offers a comprehensive overview of the structural characteristics, subcellular localization, activation mechanisms, and significant roles of NOD1 and NOD2 in innate immunity and related disease.

Yao syndrome (YAOS) is a novel systemic autoinflammatory disease linked to the nucleotide‐binding oligomerization domain (NOD2) gene. It is characterized by periodic fevers, gastrointestinal (GI) symptoms, arthritis, and dermatitis, among other symptoms. A sparse literature exists on this disease, and little is known about its dermatological manifestations. A review of available literature was performed to characterize the cutaneous manifestations of Yao syndrome. Cutaneous manifestations were documented in 85.7% of patients, with common characteristic descriptions of erythematous patches and plaques involving the face, trunk, abdomen, and extremities. Based on our review of treatment modalities employed for Yao syndrome, prednisone is an appropriate initial approach, with oral sulfasalazine and other disease‐modifying antirheumatic drugs serving as appropriate secondary options. YAOS should be considered in the differential diagnosis of patients presenting with a dermatitic rash, especially in the context of concurrent articular symptoms, periodic fever, and GI symptoms. [ABSTRACT FROM AUTHOR]

Regulation of neuroinflammation is critical for maintaining central nervous system (CNS) homeostasis and holds therapeutic promise in autoimmune diseases such as multiple sclerosis (MS). Previous studies have highlighted the significance of selective innate signaling in triggering anti-inflammatory mechanisms, which play a protective role in an MS-like disease, experimental autoimmune encephalomyelitis (EAE). However, the individual intra-CNS administration of specific innate receptor ligands or agonists, such as for toll-like receptor 7 (TLR7) and nucleotide-binding oligomerization-domain-containing protein 2 (NOD2), failed to elicit the desired anti-inflammatory response in EAE. In this study, we investigated the potential synergistic effect of targeting both TLR7 and NOD2 simultaneously to prevent EAE progression. Our findings demonstrate that simultaneous intrathecal administration of NOD2- and TLR7-agonists led to synergistic induction of Type I IFN (IFN I) and effectively suppressed EAE in an IFN I-dependent manner. Suppression of EAE was correlated with a significant decrease in the infiltration of monocytes, granulocytes, and natural killer cells, reduced demyelination, and downregulation of IL-1β, CCL2, and IFNγ gene expression in the spinal cord. These results underscore the therapeutic promise of concurrently targeting the TLR7 and NOD2 pathways in alleviating neuroinflammation associated with MS, paving the way for novel and more efficacious treatment strategies. [ABSTRACT FROM AUTHOR]

Background: Development of sequential changes of mucous leading to gastric cancer and familial cases of gastric cancer of intestinal type is widely connected with Helicobacter pylori infections. In this study we analysed variants of genes involved in cancerogenesis and inflammatory processes of intestines in patients infected with H.pylori. Our goal was to test whether mutations in these genes predestinate to development of gastric cancer, and whether there is a genetic factor that makes it more likely for infections with H.pylori to cause gastric cancer. As infections with H. pylori are relatively common, discovering such genetic predispositions could be used for establishing risk-groups and for planning treatments. Methods: Our studies cover analysis of variants in genes involved in cancerogenesis: TP53 (rs11540652, rs587782329, COSM10771), MSH2 (rs193922376), MLH1 (rs63750217), and inflammatory processes of intestine: NOD2 (rs2066847, rs2066842), IL1A (rs1800587) and IL1B (rs1143634) from H.pylori-infected patients. Results: Mutations were more common in the group of patients with gastric cancer of intestinal type and familial cases of gastric cancer in comparison with patients with chronic gastritis, chronic atrophic gastritis, intestinal metaplasia, dysplasia or gastric cancer (p-value = 0.00824), with the prevalence of p53 mutations in patients with familial gastric cancer vs. patients with other changes of mucosa (p-value = 0.000049). Additionally, gastric cancer patients have mainly genotype TT or CT of the rs2066842 variant of the NOD2 gene. Conclusions: The lack of statistically significant changes of other interleukin genes involved in inflammatory processes may suggest the presence of H.pylori infection as a potential trigger for the development of the inflammatory process of the mucosa, leading through microbiota dysbiosis to the development of enteric gastric cancer. Mutations in analysed genes correlated with more severe mucosal changes, with a much more frequent presence of TP53 gene mutations, with a limited presence of other mutations in the familial history of gastric cancer. [ABSTRACT FROM AUTHOR]

Mycoplasma pneumoniae (M. pneumoniae, Mp) is a cell wall-deficient microorganism known to cause chronic respiratory infections in both children and adults. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor primarily responsible for identifying muramyl dipeptide (MDP) found in bacterial cell walls. Previous experiments have demonstrated that Mycoplasma ovipneumoniae induces macrophage autophagy through NOD2. In this study, we conducted RNAseq analysis on macrophages infected with M. pneumoniae and observed an up-regulation in the expression of genes associated with the NOD2 signaling pathway. Mechanistic investigations further revealed the involvement of the NOD2 signaling pathway in the inflammatory response of macrophages activated by M. pneumoniae. We utilized GST pull-down technology in conjunction with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to pinpoint the M. pneumoniae proteins that interact with NOD2. Additionally, co-immunoprecipitation (Co-IP) and immunofluorescence colocalization techniques were used to confirm the interaction between DUF16 protein and NOD2. We found that DUF16 protein can enter macrophages and induce macrophage inflammatory response through the NOD2/RIP2/NF-κB pathway. Notably, the region spanning amino acids 13-90 was identified as a critical region necessary for DUF16-induced inflammation. This research not only broadens our comprehension of the recognition process of the intracellular receptor NOD2, but also deepens our understanding of the development of M. pneumoniae infection. [ABSTRACT FROM AUTHOR]

Nucleotide-binding oligomerization domain containing 2 (NOD2), located in the cell cytoplasm, is a pattern recognition receptor belonging to the innate immune receptor family. It mediates the innate immune response by identifying conserved sequences in bacterial peptide glycans and plays an essential role in maintaining immune system homeostasis. Gene mutations of NOD2 lead to the development of autoimmune diseases such as Crohn's disease and Blau syndrome. Recently, NOD2 has been shown to be associated with the pathogenesis of diabetes, cardiac-cerebral diseases, and cancers. However, the function of NOD2 in these non-communicable diseases (CNCDs) is not well summarized in reviews. Our report mainly discusses the primary function and molecular mechanism of NOD2 as well as its potential clinical significance in CNCDs. [ABSTRACT FROM AUTHOR]

Purpose: 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. Methods: 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. Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

The aim of this study was to describe the clinical and molecular genetic findings in seven individuals from three unrelated families with Blau syndrome. A complex ophthalmic and general health examination including diagnostic imaging was performed. The NOD2 mutational hot spot located in exon 4 was Sanger sequenced in all three probands. Two individuals also underwent autoinflammatory disorder gene panel screening, and in one subject, exome sequencing was performed. Blau syndrome presenting as uveitis, skin rush or arthritis was diagnosed in four cases from three families. In two individuals from one family, only camptodactyly was noted, while another member had camptodactyly in combination with non-active uveitis and angioid streaks. One proband developed two attacks of meningoencephalitis attributed to presumed neurosarcoidosis, which is a rare finding in Blau syndrome. The probands from families 1 and 2 carried pathogenic variants in NOD2 (NM_022162.3): c.1001G>A p.(Arg334Gln) and c.1000C>T p.(Arg334Trp), respectively. In family 3, two variants of unknown significance in a heterozygous state were found: c.1412G>T p.(Arg471Leu) in NOD2 and c.928C>T p.(Arg310*) in NLRC4 (NM_001199139.1). In conclusion, Blau syndrome is a phenotypically highly variable, and there is a need to raise awareness about all clinical manifestations, including neurosarcoidosis. Variants of unknown significance pose a significant challenge regarding their contribution to etiopathogenesis of autoinflammatory diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ObjectiveThe aim of this study was to characterize the phenotype and genotype of patients with Yao syndrome (YAOS), with focus on comparing to prior cohorts, identifying novel features, and describing treatment observations.MethodsA retrospective medical records review of patients with YAOS seen at Mayo Clinic was conducted to characterize clinical features, NOD2 genotypes, and therapeutic trials and responses.ResultsTwenty-two patients diagnosed with YAOS were included. Eighteen patients (81.8%) were female and twenty (90.9%) were White. Mean age at symptom onset was 24.0 ± 14.8 years. Common clinical manifestations included fever (81.8% of patients), rash (95.5%), chronic gastrointestinal symptoms (100%), arthralgia/arthritis (95.5%), and sicca symptoms (68.2%). NOD2 genotypes as single variants included IVS8 + 158 in 14 patients (63.6%), R702W in 8 patients (36.4%), 1007fs in 4 (18.2%), and one patient had only a previously unreported rare variant. Eight patients (36.4%) had compound (two or more) NOD2 variants. Potential comorbidities of YAOS observed in this cohort included gastrointestinal dysmotility, autonomic dysfunction, and mast cell activation-like symptoms. Glucocorticoid responsiveness was observed in 15 of 20 patients exposed (75%). Eleven patients (50.0%) received IL-1 inhibitor therapy, and one patient (4.5%) received IL-6 inhibitor therapy with adequate disease control.ConclusionOur findings substantiate the occurrence of fevers, arthralgia/arthritis, rash, chronic gastrointestinal symptoms, and sicca-like symptoms described previously in patients with YAOS. Novel clinical features and one NOD2 variant not previously described were identified. Glucocorticoids, biologic IL-1 inhibitors, and IL-6 receptor inhibitors appeared to be effective for treatment of patients with YAOS.

Innate immune cells show enhanced responsiveness to secondary challenges after an initial non-related stimulation (Trained Innate Immunity, TII). Acute NOD2 activation by Muramyl-Dipeptide (MDP) promotes TII inducing the secretion of pro-inflammatory mediators, while a sustained MDP-stimulation down-regulates the inflammatory response, restoring tolerance. Here we characterized in-vitro the response of murine macrophages to lipopolysaccharide (LPS) challenge under NOD2-chronic stimulation. RAW264.7 cells were trained with MDP (1 μg/ml, 48 h) and challenged with LPS (5 μg/ml, 24 h). Trained cells formed multinucleated giant cells with increased phagocytosis rates compared to untrained/challenged cells. They showed a reduced mitochondrial activity and a switch to aerobic glycolysis. TNF-α, ROS and NO were upregulated in both trained and untrained cultures (MDP+, MDP- cells, p > 0.05); while IL-10, IL-6 IL-12 and MHCII were upregulated only in trained cells after LPS challenge (MDP + LPS+, p

Background: Blau syndrome is a monogenic disease resulting from mutations in nucleotide oligomerization domain 2 (NOD2) and is phenotypically characterized by a triad of dermatitis, arthritis, and uveitis. We report Blau syndrome in a child who had been symptomatic for years, without the typical clinical triad. Clinical Description: A 7-year-old girl child presented with bilateral knee, wrist, and ankle joint swelling since the age of 2 years. She had recently developed a rash on the body. Her mother, mother’s identical twin sister, and the latter’s 5-year-old daughter had similar joint pain and swellings since childhood. In view of the strong positive family history of polyarthritis, monogenic cause of arthritis was suspected. Management and Outcome: Complete blood count was unremarkable. Erythrocyte sedimentation rate was 20 mm at C-reactive protein was 36 mg/L, antinuclear antibody and rheumatoid arthritis factor were negative. X-ray of the knee joints and wrist joints showed soft-tissue swellings around the knee and lumbar scoliosis. A heterozygous pathogenic variant in NOD2 gene confirmed the diagnosis of Blau syndrome. After receiving the genetic report, the girl was started on prednisolone and weekly methotrexate. Conclusion: Early-onset arthritis, with recurrent episodes of fever, is not to be ignored if autoimmune markers are negative. Knowledge about the clinical features of autoinflammatory diseases and the spectrum of NOD2 variants will enable physicians to carry out specific investigations, especially genetic analysis for confirmation of the diagnosis and hence administer the right treatment.

Yang Yang,1,2 Zongze Yao,1 Jiazhen Zhang,1 Wei Shao,3 Bo Li,2 Huihui Wu,2 Wenjian Tang,3 Jing Zhang2 1School of Medicine, Anhui University of Science and Technology, Huainan, People’s Republic of China; 2Anhui Province Key Laboratory of Occupational Health, Anhui No.2 Provincial People’s Hospital, Hefei, People’s Republic of China; 3School of Pharmacy, Anhui Medical University, Hefei, People’s Republic of ChinaCorrespondence: Wenjian Tang; Jing Zhang, Tel +86-551-63672601, Email ahmupharm@126.com; hfzj2552@163.comBackground: Methicillin-resistant Staphylococcus aureus (MRSA) is a cause of staph infection that is difficult to treat because of resistance to some antibiotics. A recent study indicated that diarylurea ZJ-2 is a novel antibacterial agent against multi-drug resistant Enterococcus faecium. In this work, we refined the bactericidal mechanism of ZJ-2 as a peptidoglycan (PG) hydrolase by affecting AtlA-mediated PG homeostasis.Methods: A wild-type strain (WT) and a mutant strain (ΔatlA) were used to investigate the effects of ZJ-2 on the cell wall, PG, and autolysin regulatory system by antimicrobial susceptibility testing, hemolytic toxin assay, microanalysis, autolysis assay, qRT-PCR, ELISA and mouse model of pneumonia.Results: The results revealed that ZJ-2 down-regulated the expression of genes related to peptidoglycan hydrolase (PGH) (sprX, walR, atlA, and lytM), and reduced the levels of PG, muramyl dipeptide (MDP), cytokines, and hemolytic toxin, while ΔatlA interfered with the genes regulation and PG homeostasis. In the mouse MRSA pneumonia model, the same trend was observed in the nucleotide oligomerization domain protein 2 (NOD2) and relative proinflammatory factors.Conclusion: ZJ-2 may act as a novel inhibitor of PG hydrolyse, disrupting autolysin-mediated PG homeostasis, and reducing inflammation by down-regulating the MDP-NOD2 pathway. Keywords: MRSA, peptidoglycan hydrolase, homeostasis, autolysis, NOD2

Background and aims Diabetic kidney disease (DKD) is one of the most common complications of diabetes. It is reported that mesenchymal stem cells (MSCs) derived exosomes (MSCs-Exo) may have great clinical application potential for the treatment of DKD, but the underlying mechanism has not been illustrated. To clarify the effect of MSC-Exo on NOD2 signaling pathway in podocytes under high glucose (HG) and DKD, we conduct this study.Methods We co-cultured podocytes and MSCs-Exo under 30 mM HG and injected MSCs-Exo into DKD mice, then we detected the NOD2 signaling pathway by western blot, qRT-PCT, immunofluorescence, transmission electron microscopy and immunohistochemistry both in vitro and in vivo.Results In vitro, HG lead to the apoptosis, increased the ROS level and activated the NOD2 signaling pathway in podocytes, while MSCs-Exo protected podocytes from injury reduced the expression of inflammatory factors including TNF-α, IL-6, IL-1β, and IL-18 and alleviated the inflammatory response, inhibited the activation of NOD2 signaling pathway and the expression of it’s downstream protein p-P65, p-RIP2, prevented apoptosis, increased cell viability in podocytes caused by HG. In vivo, MSCs-Exo alleviated renal injury in DKD mice, protected renal function, decreased urinary albumin excretion and inhibited the activation of NOD2 signaling pathway as well as the inflammation in renal tissue.Conclusion MSCs-Exo protected the podocytes and DKD mice from inflammation by mediating NOD2 pathway, MSCs-Exo may provide a new target for the treatment of DKD.

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-kB, 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. [ABSTRACT FROM AUTHOR]

Blau syndrome is an autosomal dominant chronic inflammatory disease, which may begin with skin manifestations in the first months of life, alerting physicians to the diagnosis. This case reports a patient diagnosed jointly by pediatric dermatology and rheumatology consultants at two years of age. [ABSTRACT FROM AUTHOR]

The clinical severity of multiple sclerosis (MS), an autoimmune disorder of the central nervous system, is thought to be determined by environmental and genetic factors that have not yet been identified. In a recent genome-wide association study (GWAS), a single nucleotide polymorphism (SNP), rs10191329, has been associated with MS severity in two large independent cohorts of patients. Different approaches were followed by the authors to prioritize the genes that are transcriptionally regulated by such an SNP. It was concluded that the identified SNP regulates a group of proximal genes involved in brain resilience and cognitive abilities rather than immunity. Here, by conducting an alternative strategy for gene prioritization, we reached the opposite conclusion. According to our re-analysis, the main target of rs10191329 is N-Acetylglucosamine Kinase (NAGK), a metabolic gene recently shown to exert major immune functions via the regulation of the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) pathway. To gain more insights into the immunometabolic functions of NAGK, we analyzed the currently known list of NAGK protein partners. We observed that NAGK integrates a dense network of human proteins that are involved in glucose metabolism and are highly expressed by classical monocytes. Our findings hold potentially major implications for the understanding of MS pathophysiology. [ABSTRACT FROM AUTHOR]

In pathogen recognition, the nucleotide-binding domain (NBD) and leucine rich repeat receptors (NLRs) have noteworthy functions in the activation of the innate immune response. These receptors respond to several viral infections, among them NOD2, a very dynamic NLR, whose role in dengue virus (DENV) infection remains unclear. This research aimed to determine the role of human NOD2 in THP-1 macrophage-like cells during DENV-2 infection. NOD2 levels in DENV-2 infected THP-1 macrophage-like cells was evaluated by RT-PCR and Western blot, and an increase was observed at both mRNA and protein levels. We observed using confocal microscopy and co-immunoprecipitation assays that NOD2 interacts with the effector protein MAVS (mitochondrial antiviral signaling protein), an adaptor protein promoting antiviral activity, this occurring mainly at 12 h into the infection. After silencing NOD2, we detected increased viral loads of DENV-2 and lower levels of IFN-α in supernatants from THP-1 macrophage-like cells with NOD2 knock-down and further infected with DENV-2, compared with mock-control or cells transfected with Scramble-siRNA. Thus, NOD2 is activated in response to DENV-2 in THP-1 macrophage-like cells and participates in IFN-α production, in addition to limiting virus replication at the examined time points. [ABSTRACT FROM AUTHOR]

Vaccines such as Bacille Calmette–Guérin (BCG) can apparently defer dementia onset with an efficacy better than all drugs known to date, as initially reported by Gofrit et al. (PLoS One14, e0224433), now confirmed by other studies. Understanding how and why is of immense importance because it could represent a sea-change in how we manage patients with mild cognitive impairment through to dementia. Given that infection and/or inflammation are likely to contribute to the development of dementias such as Alzheimer's disease (Part II of this work), we provide a historical and molecular background to how vaccines, adjuvants, and their component molecules can elicit broad-spectrum protective effects against diverse agents. We review early studies in which poxvirus, herpes virus, and tuberculosis (TB) infections afford cross-protection against unrelated pathogens, a concept known as 'trained immunity'. We then focus on the attenuated TB vaccine, BCG, that was introduced to protect against the causative agent of TB, Mycobacterium tuberculosis. We trace the development of BCG in the 1920 s through to the discovery, by Freund and McDermott in the 1940 s, that extracts of mycobacteria can themselves exert potent immunostimulating (adjuvant) activity; Freund's complete adjuvant based on mycobacteria remains the most potent immunopotentiator reported to date. We then discuss whether the beneficial effects of BCG require long-term persistence of live bacteria, before focusing on the specific mycobacterial molecules, notably muramyl dipeptides, that mediate immunopotentiation, as well as the receptors involved. Part II addresses evidence that immunopotentiation by BCG and other vaccines can protect against dementia development. [ABSTRACT FROM AUTHOR]

Introduction: Crohn's disease (CD) is a chronic inflammatory bowel disease of a multifactorial pathogenesis. Recently numerous genetic variants linked to an aggressive phenotype were identified, leading to a progress in therapeutic options, resulting in a decreased necessity for surgery. Nevertheless, surgery is often inevitable. The aim of the study was to evaluate possible risk factors for postoperative complications and disease recurrence specifically after colonic resections for CD. Patients and methods: A total of 241 patients who underwent colonic and ileocaecal resections for CD at our instiution between 2008 and 2018 were included. All data was extracted from clinical charts. Results: Major complications occurred in 23.8% of all patients. Patients after colonic resections showed a significantly higher rate of major postoperative complications compared to patients after ICR (p = < 0.0001). The most common complications after colonic resections were postoperative bleeding (22.2%), the need for revision surgery (27.4%) and ICU (17.2%) or hospital readmission (15%). As risk factors for the latter, we identified time interval between admission and surgery (p = 0.015) and the duration of the surgery (p = 0.001). Isolated distal resections had a higher risk for revision surgery and a secondary stoma (p = 0.019). Within the total study population, previous bowel resections (p = 0.037) were identified as independent risk factors for major perioperative complications. Conclusion: The results indicate that both a complex surgical site and a complex surgical procedure lead to a higher perioperative morbidity in colonic resections for Crohn's colitis. [ABSTRACT FROM AUTHOR]

Loss-of-function variants in the FLG gene have been identified as the strongest cause of susceptibility to atopic dermatitis (AD) in Europeans and Asians. However, very little is known about the genetic etiology behind AD in African populations, where the prevalence of AD is notably high. We sought to investigate the genetic origins of AD by performing whole-genome sequencing in an Ethiopian family with 12 individuals and several affected in different generations. We identified 2 variants within FLG2 (p.D13Y) and NOD2 (p.A918S) genes cosegregating with AD in the affected individuals. Further genotyping analyses in both Ethiopian and Swedish AD cases and controls revealed a significant association with the FLG2 variant (p.D13Y, P < .0013) only in the Ethiopian cohort. However, the NOD2 variant (p.A918S) did not show any association in our Ethiopian cohort. Instead, 2 previously recognized NOD2 variants (p.A849V, P < .0085 and p.G908R, P < .0036) were significantly associated with AD in our Ethiopian cohort. Our study indicates that the FLG2 and NOD2 genes might be important in the etiology of AD in Ethiopians. Additional genetic and functional studies are needed to confirm the role of these genes and the associated variants into the development of AD.

Blau syndrome (BS), is an autoinflammatory granulomatosis disease characterized by a distinct triad of skin, joint, and eye disorders similar to those of sarcoidosis, but the lung involvement frequently observed in sarcoidosis are rare. Granulomas from patients with BS displayed a distinct morphology indicating an exuberant chronic inflammatory response. Patients with BS may have granulomatous lung lesions, which require early diagnosis. To determine whether therapeutic intervention is needed for lung lesions, examining transbronchial lung cryobiopsy specimens and accumulating cases of BS with lung involvement could be contributed to improving BS management in the future.

Enterococcus faecium is a microbiota species in humans that can modulate host immunity (Griffin and Hang, 2022), but has also acquired antibiotic resistance and is a major cause of hospital-associated infections (Van Tyne and Gilmore, 2014). Notably, diverse strains of E. faecium produce SagA, a highly conserved peptidoglycan hydrolase that is sufficient to promote intestinal immunity (Rangan et al., 2016; Pedicord et al., 2016; Kim et al., 2019) and immune checkpoint inhibitor antitumor activity (Griffin et al., 2021). However, the functions of SagA in E. faecium were unknown. Here, we report that deletion of sagA impaired E. faecium growth and resulted in bulged and clustered enterococci due to defective peptidoglycan cleavage and cell separation. Moreover, ΔsagA showed increased antibiotic sensitivity, yielded lower levels of active muropeptides, displayed reduced activation of the peptidoglycan pattern-recognition receptor NOD2, and failed to promote cancer immunotherapy. Importantly, the plasmid-based expression of SagA, but not its catalytically inactive mutant, restored ΔsagA growth, production of active muropeptides, and NOD2 activation. SagA is, therefore, essential for E. faecium growth, stress resistance, and activation of host immunity.

Abstract Objective Synovial 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. Design We 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. Results Expression 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. Conclusions Supported 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.

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.

This document provides detailed information on the synthesis and characterization of lipophilic DMPs, which are derivatives of muramyl dipeptide (MDP) with immunologic adjuvant activity. The article discusses a method for synthesizing these compounds and highlights their potential as vaccine adjuvants for bacterial and viral infections. The document also includes chemical data for various compounds, including their molecular structures, physical properties, and spectroscopic data. This information can be valuable for researchers studying these compounds and their properties. [Extracted from the article]

Objective: To assess the impact conferred by NOD2 variants on the clinical spectrum of patients with systemic autoinflammatory diseases (SAIDs) in Greece. Methods: Consecutive patients (n=167) with confirmed SAIDs who underwent screening by next generation sequencing (NGS) targeting 26 SAID-associated genes, and carried at least one NOD2 gene variant, were retrospectively studied. The demographic, clinical and laboratory parameters were recorded. Results: In total, 24 rare NOD2 variants in 23/167 patients (14%) were detected. Notably, 18 patients had at least one co-existing variant in 13 genes other than NOD2. Nine patients had juvenile- and 14 adult-onset disease. All patients presented with symptoms potentially induced by the NOD2 variants. In particular, the candidate clinical diagnosis was Yao syndrome (YAOS) in 12 patients (7% of the whole SAID cohort). The clinical spectrum of patients with YAOS (mean episode duration 8 days) was fever (n=12/12), articular symptoms (n=8), gastrointestinal symptoms (n=7; abdominal pain/bloating in 7; diarrhea in 4; oral ulcers in 3), serositis (n=7), and rash (n=5), while the inflammatory markers were elevated in all but one patient. Most of these patients showed a poor response to nonsteroidal anti-inflammatory drugs (n=7/9), colchicine (n=6/8) and/or anti-TNF treatment (n=3/4), while a complete response was observed in 6/10 patients receiving steroids and 3/5 on anti-IL1 treatment. Another 8 patients were diagnosed with either FMF (n=6) or PFAPA syndrome (n=2) presenting with prominent diarrhea (n=7), oral ulcers (n=2), periorbital swelling and sicca-like symptoms (n=1), or maculopapular rash (n=1). One patient had a clinically undefined SAID, albeit characterized by oral ulcers and diarrhea. Finally, one patient presented with chronic relapsing urticaria with periorbital edema and inflammatory markers, and another one had a Crohn-like syndrome with good response to anti-IL-1 but refractory to anti-TNF treatment. Conclusion: NOD2 variants were detected in 1 out of 7 SAID patients and seem to have an impact on disease phenotype and treatment response. Further studies should validate combined molecular and clinical data to better understand these distinct nosological entities. [ABSTRACT FROM AUTHOR]

Introduction: Diabetic ulcers have become one of the major complications of diabetes mellitus (DM) and are a leading cause of death and disabling disease. However, current therapies are not effective enough to meet clinical needs. A traditional Chinese medicine (TCM) formula, Pien Tze Huang (PZH), is known as a medicine that is used to treat diabetic ulcers. Methods: In this study, PZH (0.05 g/cm2 and 0.15 g/cm2) and the positive drugrhEGF were topically administered in a high-fat diet (HFD) and streptozotocin (STZ)-induced diabetic full-thickness incisional wounds, respectively. Wound healing was assessed by wound closure rate, two-photon microscope (SHG), staining with Hematoxylin and eosin (H&E), and Masson's trichrome (MTC). Then, RNA sequencing (RNA-seq) analysis, Enzyme-linked immunosorbent assay (ELISA), western blotting, and immunofluorescence (IF), network analysis, were performed. Results and discussion: The results showed that PZH significantly accelerated wound healing, as well as enhanced the expression of collagen. RNA-seq analysis showed that PZH has functions on various biological processes, one of the key biological processes is inflammatory response. Tlr9, Klrk1, Nod2, Tlr2, and Ifng were identified as vital targets and the NF-κB signaling pathway was identified as the vital pathway. Additionally, PZH profoundly reduced the levels of Cleaved caspase-3 and promoted the expression of CD31 and TGF-ß1. Mechanically, PZH significantly decreased expression of NKG2-D, NOD2, and TLR2, and further inhibited the activation of downstream NF-κB signaling pathway and inhibited expression of inflammatory factors (IFN-γ and IL-1ß). Importantly, we found that several active ingredients may play a significant role in diabetic wound healing, including Notoginsenoside R1, Deoxycorticosterone, Ursolic acid, and 4-Methoxyphenol. In summary, our study sheds light on the complicated mechanisms underlying the promising anti-diabetic wounds of PZH and provides the discovery of agents treating diabetic ulcers. [ABSTRACT FROM AUTHOR]

Nucleotide-binding oligomerization domain-like receptors (NOD-like receptors) are cytosolic signaling receptors of innate immune cells recognizing ligands derived from bacteria, viruses, fungi and protozoa. They can initiate apoptosis and pro-inflammatory cytokines production. Meanwhile, the role of decidual NOD-like receptors in pathogenesis of early miscarriages remains unknown. Aim: to study NOD-like receptor (NOD1, NOD2, NLRP1, NLRP3, NLRC4) messenger ribonucleic acid (mRNA) expression in decidual tissue from patients with missedand spontaneous abortions compared to progressive pregnancy.Materials and Methods. NOD1, NOD2, NLRP1, NLRP3, NLRC4 and pathway protein receptorinteracting-serine/threonine-protein kinase 2 (RIP-2) mRNA expression in decidua from 34 patients with missed abortions (group I), 34 patients with spontaneous abortions (group II) and 57 women with progressive pregnancy admitted for artificial abortion (group III, control group) were analyzed by reverse transcription quantitative polymerase chain reaction (PCR) at gestational age of 6–10 weeks. Exclusion criteria were as follows: endocrine disorders, severe extragenital diseases, antiphospholipid syndrome, inherited thrombophilia, uterine malformations and fetal chromosomal abnormalities. Samples were collected by uterine abrasion. Results. It was found that mRNA expression of NOD2 was significantly higher in decidua from patients with missed and spontaneous abortions, whereas for RIP-2 (related to relevant signaling pathway) – in women with missed abortions. A moderate positive correlation between gestational age and mRNA expression for NOD2 (R = 0.48; p = 0.01) and RIP-2 (R = 0.41; p = 0.007) was observed in subjects with progressive pregnancy. In contrast, women with missed abortions showed a moderate negative correlation between body weight and mRNA expression for NOD2 (R = –0.46; p = 0.03) and RIP-2 (R = –0.51; p = 0.02) whereas spontaneous abortions was associated with moderate negative correlation between RIP-2 mRNA expression and body weight (R= –0.47; p=0.04) as well as body mass index (R= –0.48; p = 0.04) along with moderate positive correlation with age of menarche (R = 0.46; p = 0.04). However, compared with progressive pregnancy no significant differences were found in expression level form NOD1, NLRP1, NLRP3 and NLRC4 mRNA in decidua from patients with missed and spontaneous abortions. Conclusion. Elevated NOD2 mRNA expression was observed in decidua from patients with missed and spontaneous abortions compared to progressive pregnancy paralleled with upregulated RIP-2 mRNA expression in missed abortions. Finally, it was found that NOD1, NLRP1, NLRP3 and NLRC4 were not involved in pathogenesis of miscarriages.

Abstract Background Identification of rare variants involved in complex, polygenic diseases like Crohn’s disease (CD) has accelerated with the introduction of whole exome/genome sequencing association studies. Rare variants can be used in both diagnostic and therapeutic assessments; however, since they are likely to be restricted to specific ancestry groups, their contributions to risk assessment need to be evaluated outside the discovery population. Prior studies implied that the three known rare variants in NOD2 are absent in West African and Asian populations and only contribute in African Americans via admixture. Methods Whole genome sequencing (WGS) data from 3418 African American individuals, 1774 inflammatory bowel disease (IBD) cases, and 1644 controls were used to assess odds ratios and allele frequencies (AF), as well as haplotype-specific ancestral origins of European-derived CD variants discovered in a large exome-wide association study. Local and global ancestry was performed to assess the contribution of admixture to IBD contrasting European and African American cohorts. Results Twenty-five rare variants associated with CD in European discovery cohorts are typically five-fold lower frequency in African Americans. Correspondingly, where comparisons could be made, the rare variants were found to have a predicted four-fold reduced burden for IBD in African Americans, when compared to European individuals. Almost all of the rare CD European variants were found on European haplotypes in the African American cohort, implying that they contribute to disease risk in African Americans primarily due to recent admixture. In addition, proportion of European ancestry correlates the number of rare CD European variants each African American individual carry, as well as their polygenic risk of disease. Similar findings were observed for 23 mutations affecting 10 other common complex diseases for which the rare variants were discovered in European cohorts. Conclusions European-derived Crohn’s disease rare variants are even more rare in African Americans and contribute to disease risk mainly due to admixture, which needs to be accounted for when performing cross-ancestry genetic assessments.

Regulation of neuroinflammation is critical for maintaining central nervous system (CNS) homeostasis and holds therapeutic promise in autoimmune diseases such as multiple sclerosis (MS). Previous studies have highlighted the significance of selective innate signaling in triggering anti-inflammatory mechanisms, which play a protective role in an MS-like disease, experimental autoimmune encephalomyelitis (EAE). However, the individual intra-CNS administration of specific innate receptor ligands or agonists, such as for toll-like receptor 7 (TLR7) and nucleotide-binding oligomerization-domain-containing protein 2 (NOD2), failed to elicit the desired anti-inflammatory response in EAE. In this study, we investigated the potential synergistic effect of targeting both TLR7 and NOD2 simultaneously to prevent EAE progression. Our findings demonstrate that simultaneous intrathecal administration of NOD2- and TLR7-agonists led to synergistic induction of Type I IFN (IFN I) and effectively suppressed EAE in an IFN I-dependent manner. Suppression of EAE was correlated with a significant decrease in the infiltration of monocytes, granulocytes, and natural killer cells, reduced demyelination, and downregulation of IL-1β, CCL2, and IFNγ gene expression in the spinal cord. These results underscore the therapeutic promise of concurrently targeting the TLR7 and NOD2 pathways in alleviating neuroinflammation associated with MS, paving the way for novel and more efficacious treatment strategies.

Abstract Background Previous studies has shown that nucleotide-binding and oligomerization domain-containing protein 2 (NOD2) is expressed in Fibroblast-like synoviocytes (FLSs) of rheumatoid arthritis (RA) patients which is stimulated by muramyl dipeptide (MDP) present in the joint environment and induces inflammation via the NF-κB pathway. Also, other studies have shown that curcumin inhibits proliferation, migration, invasion, and Inflammation and on the other hand increases the apoptosis of RA FLSs. In this study, we aim to evaluate the effect of curcumin, a natural anti-inflammatory micronutrient, on the expression of NOD2 and inflammatory cytokines. Methods Synovial membranes were collected from ten patients diagnosed with RA and ten individuals with traumatic injuries scheduled for knee surgery. The FLSs were isolated and treated with 40 μM curcumin alone or in combination with 20.3 μM MDP for 24 h. mRNA was extracted, and real-time PCR was performed to quantitatively measure gene expression levels of NOD2, p65, IL-6, TNF-α, and IL-1β. Results The study findings indicate that administering MDP alone can significantly increase the mRNA expression levels of IL-6 and IL-1β in the trauma group and TNF-α in the RA group. Conversely, administering curcumin alone or in combination whit MDP can significantly reduce mRNA expression levels of P65 and IL-6 in FLSs of both groups. Moreover, in FLSs of RA patients, a single curcumin treatment leads to a significant reduction in NOD2 gene expression. Conclusion This study provides preliminary in vitro evidence of the potential benefits of curcumin as a nutritional supplement for RA patients. Despite the limitations of the study being an investigation of the FLSs of RA patients, the results demonstrate that curcumin has an anti-inflammatory effect on NOD2 and NF-κB genes. These findings suggest that curcumin could be a promising approach to relieve symptoms of RA.

ObjectiveTo assess the impact conferred by NOD2 variants on the clinical spectrum of patients with systemic autoinflammatory diseases (SAIDs) in Greece.MethodsConsecutive patients (n=167) with confirmed SAIDs who underwent screening by next generation sequencing (NGS) targeting 26 SAID-associated genes, and carried at least one NOD2 gene variant, were retrospectively studied. The demographic, clinical and laboratory parameters were recorded.ResultsIn total, 24 rare NOD2 variants in 23/167 patients (14%) were detected. Notably, 18 patients had at least one co-existing variant in 13 genes other than NOD2. Nine patients had juvenile- and 14 adult-onset disease. All patients presented with symptoms potentially induced by the NOD2 variants. In particular, the candidate clinical diagnosis was Yao syndrome (YAOS) in 12 patients (7% of the whole SAID cohort). The clinical spectrum of patients with YAOS (mean episode duration 8 days) was fever (n=12/12), articular symptoms (n=8), gastrointestinal symptoms (n=7; abdominal pain/bloating in 7; diarrhea in 4; oral ulcers in 3), serositis (n=7), and rash (n=5), while the inflammatory markers were elevated in all but one patient. Most of these patients showed a poor response to nonsteroidal anti-inflammatory drugs (n=7/9), colchicine (n=6/8) and/or anti-TNF treatment (n=3/4), while a complete response was observed in 6/10 patients receiving steroids and 3/5 on anti-IL1 treatment. Another 8 patients were diagnosed with either FMF (n=6) or PFAPA syndrome (n=2) presenting with prominent diarrhea (n=7), oral ulcers (n=2), periorbital swelling and sicca-like symptoms (n=1), or maculopapular rash (n=1). One patient had a clinically undefined SAID, albeit characterized by oral ulcers and diarrhea. Finally, one patient presented with chronic relapsing urticaria with periorbital edema and inflammatory markers, and another one had a Crohn-like syndrome with good response to anti-IL-1 but refractory to anti-TNF treatment.ConclusionNOD2 variants were detected in 1 out of 7 SAID patients and seem to have an impact on disease phenotype and treatment response. Further studies should validate combined molecular and clinical data to better understand these distinct nosological entities.

To identify candidate pathogenic genes of early-stage Crohn's disease (CD) and predict potential roles of genetic factors in CD, we performed whole exome sequencing on a child with early-stage Crohn's disease (CD) and her parents (core family), found that the patient carried heterozygous variants of 4 genes: NOD2 c. 2257 C > T, IL10RA c. 301 C > T, PLA2G6 c. 2029 C > T, COL7A1 c. 3190 G > A. Heterozygous variants of NOD2, IL10RA, PLA2G6 and COL7A1, intestinal inflammatory response is triggered, normal intestinal wall tissue damage, leading to CD phenotype.

ABSTRACTMicrobial sensors play an essential role in maintaining cellular homoeostasis. Our knowledge is limited on how microbial sensing helps in differential immune response and its link to inflammatory diseases. Recently we have confirmed that ELMO1 (Engulfment and Cell Motility Protein-1) present in cytosol is involved in pathogen sensing, engulfment, and intestinal inflammation. Here, we show that ELMO1 interacts with another sensor, NOD2 (Nucleotide-binding oligomerization domain-containing protein 2), that recognizes bacterial cell wall component muramyl dipeptide (MDP). The polymorphism of NOD2 is linked to Crohn’s disease (CD) pathogenesis. Interestingly, we found that overexpression of ELMO1 and mutant NOD2 (L1007fs) were not able to clear the CD-associated adherent invasive E. coli (AIEC-LF82). The functional implications of ELMO1-NOD2 interaction in epithelial cells were evaluated by using enteroid-derived monolayers (EDMs) from ELMO1 and NOD2 KO mice. Subsequently we also assessed the immune response in J774 macrophages depleted of either ELMO1 or NOD2 or both. The infection of murine EDMs with AIEC-LF82 showed higher bacterial load in ELMO1-KO, NOD2 KO EDMs, and ELMO1 KO EDMs treated with NOD2 inhibitors. The murine macrophage cells showed that the downregulation of ELMO1 and NOD2 is associated with impaired bacterial clearance that is linked to reduce pro-inflammatory cytokines and reactive oxygen species. Our results indicated that the crosstalk between microbial sensors in enteric infection and inflammatory diseases impacts the fate of the bacterial load and disease pathogenesis.