Deleterious Genetic Variation Across the NOD Signaling Pathway Is Associated With Reduced NFKB Signaling Transcription and Upregulation of Alternative Inflammatory Transcripts in Pediatric Inflammatory Bowel Disease.

BACKGROUND: Inflammatory bowel disease may arise with inadequate immune response to intestinal bacteria. NOD2 is an established gene in Crohn's disease pathogenesis, with deleterious variation associated with reduced NFKB signaling. We hypothesized that deleterious variation across the NOD2 signaling pathway impacts on transcription. METHODS: Treatment-naïve pediatric inflammatory bowel disease patients had ileal biopsies for targeted autoimmune RNA-sequencing and blood for whole exome sequencing collected at diagnostic endoscopy. Utilizing GenePy, a per-individual, per-gene score, genes within the NOD signaling pathway were assigned a quantitative score representing total variant burden. Where multiple genes formed complexes, GenePy scores were summed to create a "complex" score. Normalized transcript expression of 95 genes within this pathway was retrieved. Regression analysis was performed to determine the impact of genomic variation on gene transcription. RESULTS: Thirty-nine patients were included. Limited clustering of patients based on NOD signaling transcripts was related to underlying genomic variation. Patients harboring deleterious variation in NOD2 had reduced NOD2 (ß = -0.702, P = 4.3 × 10-5) and increased NFKBIA (ß = 0.486, P = .001), reflecting reduced NFKB signal activation. Deleterious variation in the NOD2-RIPK2 complex was associated with increased NLRP3 (ß = 0.8, P = 3.1475 × 10-8) and TXN (ß = -0.417, P = 8.4 × 10-5) transcription, components of the NLRP3 inflammasome. Deleterious variation in the TAK1-TAB complex resulted in reduced MAPK14 transcription (ß = -0.677, P = 1.7 × 10-5), a key signal transduction protein in the NOD2 signaling cascade and increased IFNA1 (ß = 0.479, P = .001), indicating reduced transcription of NFKB activators and alternative interferon transcription in these patients. CONCLUSIONS: Data integration identified perturbation of NOD2 signaling transcription correlated with genomic variation. A hypoimmune NFKB signaling transcription response was observed. Alternative inflammatory pathways were activated and may represent therapeutic targets in specific patients.

ISSAID/EMQN Best Practice Guidelines for the Genetic Diagnosis of Monogenic Autoinflammatory Diseases in the Next-Generation Sequencing Era.

BACKGROUND: Monogenic autoinflammatory diseases are caused by pathogenic variants in genes that regulate innate immune responses, and are characterized by sterile systemic inflammatory episodes. Since symptoms can overlap within this rapidly expanding disease category, accurate genetic diagnosis is of the utmost importance to initiate early inflammation-targeted treatment and prevent clinically significant or life-threatening complications. Initial recommendations for the genetic diagnosis of autoinflammatory diseases were limited to a gene-by-gene diagnosis strategy based on the Sanger method, and restricted to the 4 prototypic recurrent fevers (MEFV, MVK, TNFRSF1A, and NLRP3 genes). The development of best practices guidelines integrating critical recent discoveries has become essential. METHODS: The preparatory steps included 2 online surveys and pathogenicity annotation of newly recommended genes. The current guidelines were drafted by European Molecular Genetics Quality Network members, then discussed by a panel of experts of the International Society for Systemic Autoinflammatory Diseases during a consensus meeting. RESULTS: In these guidelines, we combine the diagnostic strength of next-generation sequencing and recommendations to 4 more recently identified genes (ADA2, NOD2, PSTPIP1, and TNFAIP3), nonclassical pathogenic genetic alterations, and atypical phenotypes. We present a referral-based decision tree for test scope and method (Sanger versus next-generation sequencing) and recommend on complementary explorations for mosaicism, copy-number variants, and gene dose. A genotype table based on the 5-category variant pathogenicity classification provides the clinical significance of prototypic genotypes per gene and disease. CONCLUSIONS: These guidelines will orient and assist geneticists and health practitioners in providing up-to-date and appropriate diagnosis to their patients.

Computational model to analyze and characterize the functional mutations of NOD2 protein causing inflammatory disorder - Blau syndrome.

Blau syndrome (BS), which affects the eyes, skin, and joints, is an autosomal dominant genetic inflammatory disorder. BS is caused by mutations in the NOD2 gene. However, there are no direct treatments, and treatment with conventional anti-inflammatory drugs such as adrenal glucocorticoids, anti-metabolites, and biological agents such as anti-TNF and infliximab have all been attempted with varying degrees of success. In this study, we tried to identify all the reported mutations in the NOD2 protein that cause BS. Collectively, 114 missense mutations were extracted from the UniProt, ClinVar, and HGMD databases. The mutations were further subjected to pathogenic, stability, and conservation analyses. According to these computational analyses, six missense mutations (R334Q, R334W, E383G, E383K, R426H, and T605P) were found to be highly deleterious, destabilizing, and positioned in the conserved position. ADP to ATP conversion plays a crucial role in switching the closed-form of NOD2 protein to the open-form, thus activating the protein. Accordingly, the mutations in the ADP binding sites have received more attention in comparison to the mutations in the non-ADP binding positions. Interestingly, the W490L mutation is positioned in the ADP binding site and exhibits highly deleterious and destabilizing properties. Additionally, W490L was also found to be conserved, with a ConSurf score of 7. Therefore, we further performed homology modeling to determine the 3D structure of native NOD2 and the W490L mutant. Molecular docking analysis was carried out to understand the change in the interaction of ADP with the NOD2 protein. We observed that ADP had a stronger interaction with the native NOD2 protein compared to the W490L mutant. Finally, ADP complexed with native NOD2 and W490L mutant were subjected to molecular dynamics simulations, and the trajectories were analyzed. In the simulations, we observed decreased deviation and fluctuations in native NOD2, whereas decreased compactness and inter- and intramolecular hydrogen bonds were observed in the W490L mutant. This study is expected to serve as a platform for developing targeted drug therapy for BS.

Familial Blau syndrome:First molecularly confirmed report from India.

Blau syndrome (BS) is a rare autoinflammatory disorder characterized by the clinical triad of arthritis, uveitis, and dermatitis due to heterozygous gain-of-function mutations in the NOD2 gene. BS can mimic juvenile idiopathic arthritis (JIA)-associated uveitis, rheumatoid arthritis, and ocular tuberculosis. We report a family comprising a mother and her two children, all presenting with uveitis and arthritis. A NOD2 mutation was confirmed in all the three patients - the first such molecularly proven case report of familial BS from India.

Flaxseed Oil Attenuates Intestinal Damage and Inflammation by Regulating Necroptosis and TLR4/NOD Signaling Pathways Following Lipopolysaccharide Challenge in a Piglet Model.

SCOPE: Flaxseed oil is a rich source of α-linolenic acid (ALA), which is the precursor of the long-chain n-3 polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). This study investigates the protective effect of flaxseed oil against intestinal injury induced by lipopolysaccharide (LPS). MATERIALS AND RESULTS: Twenty-four weaned pigs were used in a 2 × 2 factorial experiment with dietary treatment (5% corn oil vs 5% flaxseed oil) and LPS challenge (saline vs LPS). On day 21 of the experiment, pigs were administrated with LPS or saline. At 2 h and 4 h post-administration, blood samples were collected. After the blood harvest at 4 h, all piglets were slaughtered and intestinal samples were collected. Flaxseed oil supplementation led to the enrichment of ALA, EPA, and total n-3 PUFAs in intestine. Flaxseed oil improved intestinal morphology, jejunal lactase activity, and claudin-1 protein expression. Flaxseed oil downregulated the mRNA expression of intestinal necroptotic signals. Flaxseed oil also downregulated the mRNA expression of intestinal toll-like receptors 4 (TLR4) and its downstream signals myeloid differentiation factor 88 (MyD88), nuclear factor kappa B (NF-κB), and nucleotide-binding oligomerization domain proteins 1, 2 (NOD1, NOD2) and its adapter molecule, receptor-interacting protein kinase 2 (RIPK2). CONCLUSION: These results suggest that dietary addition of flaxseed oil enhances intestinal integrity and barrier function, which is involved in modulating necroptosis and TLR4/NOD signaling pathways.

Vitamin D deficiency and the pathogenesis of Crohn's disease.

Vitamin D has emerged as a key regulator of innate immune responses to pathogen threat. The hormonal form of vitamin D signals through a nuclear receptor transcription factor and regulates gene transcription. Several papers have shown that vitamin D signaling is active both upstream and downstream of pattern recognition receptors, vanguards of innate immune responses. Crohn's disease (CD) is a relapsing-recurring inflammatory bowel disease (IBD) that arises from dysregulated intestinal innate immunity. Indeed, genetic studies have identified several CD susceptibility markers linked to mechanisms of innate immune responses to infection. Interest in links between vitamin D deficiency and CD has grown substantially, particularly in the last five years. While a number of studies have consistently revealed an association between CD and vitamin D deficiency, recent experimental work has uncovered a compelling mechanistic basis for the contribution of vitamin D deficiency to the pathogenesis of the disease. Moreover, a number of intervention trials have provided generally solid evidence that robust vitamin D supplementation may be of therapeutic benefit to patients with CD. This review summarizes these laboratory and clinical findings.

Pluripotent stem cell models of Blau syndrome reveal an IFN-γ-dependent inflammatory response in macrophages.

BACKGROUND: Blau syndrome, or early-onset sarcoidosis, is a juvenile-onset systemic granulomatosis associated with a mutation in nucleotide-binding oligomerization domain 2 (NOD2). The underlying mechanisms of Blau syndrome leading to autoinflammation are still unclear, and there is currently no effective specific treatment for Blau syndrome. OBJECTIVES: To elucidate the mechanisms of autoinflammation in patients with Blau syndrome, we sought to clarify the relation between disease-associated mutant NOD2 and the inflammatory response in human samples. METHODS: Blau syndrome-specific induced pluripotent stem cell (iPSC) lines were established. The disease-associated NOD2 mutation of iPSCs was corrected by using a CRISPR-Cas9 system to precisely evaluate the in vitro phenotype of iPSC-derived cells. We also introduced the same NOD2 mutation into a control iPSC line. These isogenic iPSCs were then differentiated into monocytic cell lineages, and the statuses of nuclear factor κB pathway and proinflammatory cytokine secretion were investigated. RESULTS: IFN-γ acted as a priming signal through upregulation of NOD2. In iPSC-derived macrophages with mutant NOD2, IFN-γ treatment induced ligand-independent nuclear factor κB activation and proinflammatory cytokine production. RNA sequencing analysis revealed distinct transcriptional profiles of mutant macrophages both before and after IFN-γ treatment. Patient-derived macrophages demonstrated a similar IFN-γ-dependent inflammatory response. CONCLUSIONS: Our data support the significance of ligand-independent autoinflammation in the pathophysiology of Blau syndrome. Our comprehensive isogenic disease-specific iPSC panel provides a useful platform for probing therapeutic and diagnostic clues for the treatment of patients with Blau syndrome.

Vitamin D Promotes Pneumococcal Killing and Modulates Inflammatory Responses in Primary Human Neutrophils.

Streptococcus pneumoniae is a major human pathogen and a leading cause of pneumonia, septicemia, and meningitis worldwide. Despite clinical studies linking vitamin D deficiency and pneumonia, molecular mechanisms behind these observations remain unclear. In particular, the effects of vitamin D on neutrophil responses remain unknown. Using pneumococcal strains, primary neutrophils isolated from human blood, and sera from patients with frequent respiratory tract infections (RTIs), we investigated the effects of vitamin D on neutrophil bactericidal and inflammatory responses, including pattern recognition receptors, antimicrobial peptides, and cytokine regulation. We found that vitamin D upregulated pattern recognition receptors, TLR2, and NOD2, and induced the antimicrobial human neutrophil peptides (HNP1-3) and LL-37, resulting in increased killing of pneumococci in a vitamin D receptor-dependent manner. Antibodies targeting HNP1-3 inhibited bacterial killing. Vitamin D supplementation of serum from patients with bacterial RTIs enhanced neutrophil killing. Moreover, vitamin D lowered inflammatory cytokine production by infected neutrophils via IL-4 production and the induction of suppressor of cytokine signaling (SOCS) proteins SOCS-1 and SOCS-3, leading to the suppression of NF-κB signaling. Thus, vitamin D enhances neutrophil killing of S. pneumoniae while dampening excessive inflammatory responses and apoptosis, suggesting that vitamin D could be used alongside antibiotics when treating pneumococcal infections.

Vitamin D Reduces Colitis- and Inflammation-Associated Colorectal Cancer in Mice Independent of NOD2.

Inflammatory bowel disease (IBD) patients are at increased risk of developing colorectal cancer (CRC). Vitamin D (vD) induces NOD2 gene expression, enhancing immunity, while deficiency impairs intestinal epithelial integrity, increasing inflammation. This study investigated the effect of vD on CRC in colitis, and if preventive benefits are mediated via NOD2. Inflammation-associated CRC was induced by treating C57BL/6J and Nod2-/- mice with azoxymethane (AOM) and dextran sodium sulfate (DSS) cycles (×3). vD-deficient mice displayed more severe colitis compared to vD-supplemented mice, with greater weight loss, higher colitis activity index, increased colonic weight/length ratios, and lower survival rates. Increased histological inflammation score and increased IL-6 were observed in the mucosa of vD-deficient mice. Overall incidence of colonic tumors was not significantly different between vD-deficient and vD-supplemented mice. Higher tumor multiplicity was observed in vD-deficient vs vD-supplemented groups (both mouse strains). After AOM/DSS treatment, decreased plasma 25(OH)D3 levels and downregulation of vD target genes Cyp24 and Vdr were observed in both mice strains (vD-deficient or vD-supplemented diet), compared to saline-treated controls on the vD-deficient diet. In conclusion, vD supplementation reduced colitis severity and decreased the number of inflammation-associated colorectal tumors in both C57BL/6J and Nod2-/- mice, independent of NOD2.

Brazilein inhibits neuronal inflammation induced by cerebral ischemia and oxygen-glucose deprivation through targeting NOD2 expression.

Brazilein is reported to have immunosuppressive effect on cardiovascular and cerebral-vascular diseases. The essential roles of innate immunity in cerebral ischemia are increasingly identified, but no studies concerning the influence of brazilein on the innate immunity receptors have been reported. The present study was designed to investigate the regulation of NOD2 (Nucleotide-binding oligomerization domain-containing protein 2) by brazilein for its protection of neuron in cerebral ischemia in vivo and oxygen-glucose deprivation in vitro. The results showed that brazilein could reverse the elevated expression of NOD2 and TNFα (tumor necrosis factor alpha) elicited by cerebral ischemia and reperfusion. This reduction could also be detected in normal mice and C17.2 cells, indicating that this suppressive effect of brazilein was correlated with NOD2. The results from GFP reporter plasmid assay suggested brazilein inhibited NOD2 gene transcription. In conclusion, brazilein could attenuate NOD2 and TNFα expression in cerebral ischemia and NOD2 may be one possible target of brazilein for its immune suppressive effect in neuro-inflammation.

NOD2 Mediates Odontoblast Differentiation and RANKL Expression.

The precise regulation of odontoblast differentiation and osteoclastogenic cytokine expression in human dental pulp cells (HDPCs) is crucial for the pathology of bacteria-related pulpitis. Although the up-regulation of nucleotide-binding oligomerization domain-containing protein 2 (NOD2) has been reported in inflamed human dental pulps, the role of NOD2 in the differentiation of HDPCs remains unclear. Here, we show the involvement of NOD2 in odontoblast differentiation together with osteoclastogenic cytokine expression in HDPCs. Treatment with muramyl dipeptide (MDP), a known NOD2-agonist, significantly inhibited odontoblast differentiation of HDPCs, as revealed by reduced ALP activity, osteoblast/odontoblast marker expression, and mineralized nodule formation. Importantly, the forced down-regulation of NOD2 by small interfering RNA (siRNA) recovered MDP-down-regulated odontoblast differentiation. MDP-elicited suppression of odontoblast differentiation resulted from the increased expression of MKP-1 protein and the subsequent decline of MAPKs phosphorylation, which is a prerequisite for odontoblast differentiation. Furthermore, we found that MDP treatment elevated the expression of osteoclastogenic cytokines in HDPCs, which was also reversed by NOD2 silencing. Analysis of these data, taken together, suggests that the regulation of NOD2 expression upon MDP challenge might serve as an intrinsic mechanism that underlies the hindered dentin formation and accelerated dentin resorption in bacterial infection-mediated pulpitis.

Repeated exclusive enteral nutrition in the treatment of paediatric Crohn's disease: predictors of efficacy and outcome.

BACKGROUND: Exclusive enteral nutrition (EEN) induces remission and mucosal healing in children with active Crohn's disease (CD). AIM: To compare short- and long-term outcomes of the first vs. second courses of EEN, and to identify predictors of sustained remission. METHODS: Retrospective single centre analysis of all patients with CD (6-18 years) treated with EEN over 7.5 years. Patients were excluded if exposed to anti-TNFα or corticosteroids 3 months prior to EEN. Data included disease phenotype, activity, NOD2 genotype, laboratory indices and anthropometrics. Remission and relapse were defined by mathematically weighted Paediatric Crohn's Disease Activity Index (wPCDAI) with 1-year follow-up. RESULTS: Of 94 patients treated with EEN, 52 fulfilled inclusion criteria (31 male, mean age 13.2 years). Azathioprine was started within the first month in 33/52 patients; 26/52 received a second EEN course. First compared to second EEN revealed higher wPCDAI at start (59 vs. 40, P < 0.0001), tended to higher remission rates after 3 months (92% vs. 77%, n.s.), but showed comparable 1-year relapse rates (67% vs. 70%, median time 231 vs. 145 days, n.s.). Disease activity, weight gain and inflammatory markers showed better improvement with first EEN. Faecal calprotectin >200 µg/g during EEN was associated with shorter remission (median time 157 vs. 287 days, n.s.). Certain NOD2 genotypes were related to higher relapse rates (92% R702W or G908R vs. 50% 1007fs vs. 60% wild-type, P < 0.01). CONCLUSIONS: Exclusive enteral nutrition induces remission in active Crohn's disease, but efficacy tends to decrease with the second course. Despite early azathioprine use, 1-year relapse rates are high, but may be related to NOD2 genotype.

Genetic susceptibility to increased bacterial translocation influences the response to biological therapy in patients with Crohn's disease.

OBJECTIVE: The aetiology of Crohn's disease (CD) has been related to nucleotide-binding oligomerisation domain containing 2 (NOD2) and ATG16L1 gene variants. The observation of bacterial DNA translocation in patients with CD led us to hypothesise that this process may be facilitated in patients with NOD2/ATG16L1-variant genotypes, affecting the efficacy of anti-tumour necrosis factor (TNF) therapies. DESIGN: 179 patients with Crohn's disease were included. CD-related NOD2 and ATG16L1 variants were genotyped. Phagocytic and bactericidal activities were evaluated in blood neutrophils. Bacterial DNA, TNFα, IFNγ, IL-12p40, free serum infliximab/adalimumab levels and antidrug antibodies were measured. RESULTS: Bacterial DNA was found in 44% of patients with active disease versus 23% of patients with remitting disease (p=0.01). A NOD2-variant or ATG16L1-variant genotype was associated with bacterial DNA presence (OR 4.8; 95% CI 1.1 to 13.2; p=0.001; and OR 2.4; 95% CI 1.4 to 4.7; p=0.01, respectively). This OR was 12.6 (95% CI 4.2 to 37.8; p=0.001) for patients with a double-variant genotype. Bacterial DNA was associated with disease activity (OR 2.6; 95% CI 1.3 to 5.4; p=0.005). Single and double-gene variants were not associated with disease activity (p=0.19). Patients with a NOD2-variant genotype showed decreased phagocytic and bactericidal activities in blood neutrophils, increased TNFα levels in response to bacterial DNA and decreased trough levels of free anti-TNFα. The proportion of patients on an intensified biological therapy was significantly higher in the NOD2-variant groups. CONCLUSIONS: Our results characterise a subgroup of patients with CD who may require a more aggressive therapy to reduce the extent of inflammation and the risk of relapse.

Fish oil enhances intestinal integrity and inhibits TLR4 and NOD2 signaling pathways in weaned pigs after LPS challenge.

Long-chain (n-3) PUFA exert beneficial effects on inflammatory bowel diseases in animal models and clinical trials. In addition, pattern recognition receptors such as toll-like receptors (TLR) and nucleotide-binding oligomerization domain proteins (NOD) play a critical role in intestinal inflammation. We hypothesized that fish oil could alleviate Escherichia coli LPS-induced intestinal injury via modulation of TLR4 and NOD signaling pathways. Twenty-four weaned piglets were used in a 2 × 2 factorial design and the main factors included a dietary treatment (5% corn oil or 5% fish oil) and immunological challenge (LPS or saline). After feeding fish oil or corn oil diets for 21 d, pigs were injected with LPS or saline. At 4 h postinjection, blood samples were collected and pigs were killed. EPA, DHA, and total (n-3) PUFA were enriched in intestinal mucosa through fish supplementation. Fish oil improved intestinal morphology, indicated by greater villus height and villus height:crypt depth ratio, and intestinal barrier function, indicated by decreased plasma diamine oxidase (DAO) activity and increased mucosal DAO activity as well as enhanced protein expression of intestinal tight junction proteins including occludin and claudin-1. Moreover, fish oil decreased intestinal TNFα and PGE(2) concentrations and caspase-3 and heat shock protein 70 protein expression. Finally, fish oil downregulated the mRNA expression of intestinal TLR4 and its downstream signals myeloid differentiation factor 88, IL-1 receptor-associated kinase 1, TNFα receptor-associated factor 6, and NOD2, and its adaptor molecule, receptor-interacting serine/threonine-protein kinase 2. Fish oil decreased the protein expression of intestinal NFκB p65. These results indicate that fish oil supplementation is associated with inhibition of TLR4 and NOD2 signaling pathways and concomitant improvement of intestinal integrity under an inflammatory condition.

[Effects of muramyl dipeptide on the differentiation of human dental pulp cells].

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

Direct and indirect induction by 1,25-dihydroxyvitamin D3 of the NOD2/CARD15-defensin beta2 innate immune pathway defective in Crohn disease.

Vitamin D signaling through its nuclear vitamin D receptor has emerged as a key regulator of innate immunity in humans. Here we show that hormonal vitamin D, 1,25-dihydroxyvitamin D(3), robustly stimulates expression of pattern recognition receptor NOD2/CARD15/IBD1 gene and protein in primary human monocytic and epithelial cells. The vitamin D receptor signals through distal enhancers in the NOD2 gene, whose function was validated by chromatin immunoprecipitation and chromatin conformation capture assays. A key downstream signaling consequence of NOD2 activation by agonist muramyl dipeptide is stimulation of NF-kappaB transcription factor function, which induces expression of the gene encoding antimicrobial peptide defensin beta2 (DEFB2/HBD2). Pretreatment with 1,25-dihydroxyvitamin D(3) synergistically induced NF-kappaB function and expression of genes encoding DEFB2/HBD2 and antimicrobial peptide cathelicidin in the presence of muramyl dipeptide. Importantly, this synergistic response was also seen in macrophages from a donor wild type for NOD2 but was absent in macrophages from patients with Crohn disease homozygous for non-functional NOD2 variants. These studies provide strong molecular links between vitamin D deficiency and the genetics of Crohn disease, a chronic incurable inflammatory bowel condition, as Crohn's pathogenesis is associated with attenuated NOD2 or DEFB2/HBD2 function.

The NOD2 defect in Blau syndrome does not result in excess interleukin-1 activity.

OBJECTIVE: Blau syndrome is a rare, autosomal-dominant, autoinflammatory disorder characterized by granulomatous arthritis, uveitis, and dermatitis. Genetics studies have shown that the disease is caused by single nonsynonymous substitutions in NOD-2, a member of the NOD-like receptor or NACHT-leucine-rich repeat (NLR) family of intracellular proteins. Several NLRs function in the innate immune system as sensors of pathogen components and participate in immune-mediated cellular responses via the caspase 1 inflammasome. Mutations in a gene related to NOD-2, NLRP3, are responsible for excess caspase 1-dependent interleukin-1beta (IL-1beta) in cryopyrinopathies such as Muckle-Wells syndrome. Furthermore, functional studies demonstrate that caspase 1-mediated release of IL-1beta also involves NOD-2. The aim of this study was to test the hypothesis that IL-1beta may mediate the inflammation seen in patients with Blau syndrome. METHODS: IL-1beta release was measured in peripheral blood mononuclear cells cultured in vitro, obtained from 5 Blau syndrome individuals with a NOD2 (CARD15) mutation. RESULTS: We observed no evidence for increased IL-1beta production in cells obtained from subjects with Blau syndrome compared with healthy control subjects. Furthermore, we presented 2 cases of Blau syndrome in which recombinant human IL-1 receptor antagonist (anakinra) was ineffective treatment. CONCLUSION: Taken together, these data suggest that in contrast to related IL-1beta-dependent autoinflammatory cryopyrinopathies, Blau syndrome is not mediated by excess IL-1beta or other IL-1 activity.

Clinical and molecular characteristics of isolated colonic Crohn's disease.

BACKGROUND: Clinical, serological, and molecular data support the existence of discrete subsets of Crohn's disease (CD) defined by location of disease. Little is known about the epidemiology and natural history of isolated CD of the colon (Montreal Classification L2) because most studies have not accurately distinguished it from ileocolonic disease. Our objectives were to describe the clinical features and natural history of isolated colonic CD in a rigorously characterized patient cohort and to investigate the association of polymorphisms in a number of genes with colonic location of disease and disease behavior. METHODS: Patients with L2 disease were identified from a database of 675 CD patients. Only patients with a normal small bowel enema (70%), ileoscopy alone (30%), or both (20%) were included. Genotyping was performed using PCR-SSP or the iPLEX platform. RESULTS: In all, 135 patients were classified with L2 disease. L2 disease was more common in women (74.0% versus 58.0%; P = 0.0004; odds ratio [OR] = 2.11, 95% confidence interval [CI] 1.36-3.26) and in never smokers (48.9% versus 36.9%; P = 0.008; OR = 1.64, 95% CI 1.09-2.45); 20.7% underwent colonic resection for severe disease. We confirmed that carriage of the HLA-DRB1*0103 allele is strongly associated with isolated colonic CD (14.9% versus 4.0%; P = 0.000016; OR 4.6, 95% CI 2.25-9.47) and report the novel association of this allele with time to first surgical event (log rank P = 0.001). There was no association with any of the known CD susceptibility loci (NOD2, IBD5, NOD1, IL23R, ATG16L1) and isolated colonic CD. A nonsynonymous polymorphism in MEKK1 (rs832582) was associated with CD susceptibility overall (15% versus 19%; P = 0.0083; OR = 1.28, 95% CI 1.07-1.54). The association was strongest in those patients not carrying a NOD2 mutation and had no effect on disease location. CONCLUSIONS: This study describes the clinical features of isolated colonic CD and demonstrates the importance of the HLA region in determining the molecular basis of colonic inflammation.