Disruption of XIAP-RIP2 Association Blocks NOD2-Mediated Inflammatory Signaling.

Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.

Peptidoglycan fragment release and NOD activation by commensal Neisseria species from humans and other animals.

Neisseria gonorrhoeae, a human restricted pathogen, releases inflammatory peptidoglycan (PG) fragments that contribute to the pathophysiology of pelvic inflammatory disease. The genus Neisseria is also home to multiple species of human- or animal-associated Neisseria that form part of the normal microbiota. Here we characterized PG release from the human-associated nonpathogenic species Neisseria lactamica and Neisseria mucosa and animal-associated Neisseria from macaques and wild mice. An N. mucosa strain and an N. lactamica strain were found to release limited amounts of the proinflammatory monomeric PG fragments. However, a single amino acid difference in the PG fragment permease AmpG resulted in increased PG fragment release in a second N. lactamica strain examined. Neisseria isolated from macaques also showed substantial release of PG monomers. The mouse colonizer Neisseria musculi exhibited PG fragment release similar to that seen in N. gonorrhoeae with PG monomers being the predominant fragments released. All the human-associated species were able to stimulate NOD1 and NOD2 responses. N. musculi was a poor inducer of mouse NOD1, but ldcA mutation increased this response. The ability to genetically manipulate N. musculi and examine effects of different PG fragments or differing amounts of PG fragments during mouse colonization will lead to a better understanding of the roles of PG in Neisseria infections. Overall, we found that only some nonpathogenic Neisseria have diminished release of proinflammatory PG fragments, and there are differences even within a species as to types and amounts of PG fragments released.

Activation of nucleotide-binding oligomerization domain 2 by muramyl dipeptide negatively regulates Toll-like receptor 9-mediated colonic inflammation through the induction of deubiquitinating enzyme A expression.

Mutations in nucleotide-binding oligomerization domain 2 (NOD2) are associated with Crohn's disease (CD). Although NOD2 activation contributes to the maintenance of intestinal homeostasis through the negative regulation of pro-inflammatory cytokine responses mediated by Toll-like receptors (TLRs), the effects of NOD2 activation on interferon (IFN)-α responses induced by TLR9 have been poorly defined. To explore the cross-talk between NOD2 and TLR9, human monocytes or dendritic cells (DCs) were stimulated with NOD2 and/or TLR9 ligands to measure IFN-α production. The severity of dextran sodium sulfate (DSS)-induced colitis was compared in mice treated with NOD2 and/or TLR9 ligands. Expression of IFN-α and IFN-stimulated genes (ISGs) was examined in the colonic mucosa of patients with inflammatory bowel disease (IBD). NOD2 activation reduced TLR9-induced IFN-α production by monocytes and DCs in a deubiquitinating enzyme A (DUBA)-dependent manner. Activation of DUBA induced by the co-stimulation of TLR9 and NOD2 inhibited Lys63-linked polyubiquitination of TRAF3 and suppressed TLR9-mediated IFN-α production. NOD2 activation in hematopoietic cells protected mice from TLR9-induced exacerbation of DSS-induced colitis by down-regulating IFN-α responses and up-regulating DUBA expression. Colonic mucosa of patients with active and remitted IBD phases was characterized by the enhanced and reduced expression of ISGs, respectively. Expression levels of IFN-α and IL-6 positively correlated in the active colonic mucosa of patients with ulcerative colitis and CD, whereas DUBA expression inversely correlated with that of IFN-α in patients with CD. Collectively, these data suggest that DUBA-dependent negative effect of NOD2 on TLR9-mediated IFN-α responses contributes to the maintenance of intestinal homeostasis.

Bacterial peptidoglycan signalling in microglia: Activation by MDP via the NF-κB/MAPK pathway.

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-1ß, 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.

Identification, characterization and the inflammatory regulating effect of NOD1/2 in sturgeon.

As an ancient species with both conservation and commercial value, Sturgeon's inflammatory regulation mechanism is a research point. Nucleotide-binding and oligomerization domain-containing proteins 1 and 2 (NOD1/2) are classical intracellular pattern recognition receptors (PRRs) in immunity of anti-bacterial infection. However, the characterization and function of NOD1/2 in Sturgeon are still unclear. In this study, we analyzed the synteny relationship of NOD1/2 genes between Acipenser ruthenus and representative fishes at the genome-level. Results showed that the ArNOD2 collinear genes pair was present in all representative fishes. The duplicated ArNOD1/2 genes were under purifying selection during evolution as indicated by their Ka/Ks values. To explore the function of NOD1/2, we further investigated their expression patterns and the effects of pathogenic infection, PAMPs treatment, and siRNA interference in Acipenser baerii, the sibling species of A. ruthenus. Results showed that both AbNOD1/2 were expressed at early developmental stages and in different tissues. Pathogenic infection in vivo and PAMPs treatment in vitro demonstrated that AbNOD1/2 could respond to pathogen stimulation. siRNA interference with AbNOD1/2 inhibited expression levels of RIPK2 and inflammatory cytokines compared to the control group after iE-DAP or MDP treatment. This study hinted that the AbNOD1/2 could stimulate the inflammatory cytokines response during evolutionary processes.

Clinical outcomes and perioperative morbidity and mortality following segmental resections of the colon for Crohn's colitis.

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.

Cell membrane-camouflaged bufalin targets NOD2 and overcomes multidrug resistance in pancreatic cancer.

AIMS: Multidrug resistance in pancreatic cancer poses a significant challenge in clinical treatment. Bufalin (BA), a compound found in secretions from the glands of toads, may help overcome this problem. However, severe cardiotoxicity thus far has hindered its clinical application. Hence, the present study aimed to develop a cell membrane-camouflaged and BA-loaded polylactic-co-glycolic acid nanoparticle (CBAP) and assess its potential to counter chemoresistance in pancreatic cancer. METHODS: The toxicity of CBAP was evaluated by electrocardiogram, body weight, distress score, and nesting behavior of mice. In addition, the anticarcinoma activity and underlying mechanism were investigated both in vitro and in vivo. RESULTS: CBAP significantly mitigated BA-mediated acute cardiotoxicity and enhanced the sensitivity of pancreatic cancer to several clinical drugs, such as gemcitabine, 5-fluorouracil, and FOLFIRINOX. Mechanistically, CBAP directly bound to nucleotide-binding and oligomerization domain containing protein 2 (NOD2) and inhibited the expression of nuclear factor kappa-light-chain-enhancer of activated B cells. This inhibits the expression of ATP-binding cassette transporters, which are responsible for chemoresistance in cancer cells. CONCLUSIONS: Our findings indicate that CBAP directly inhibits NOD2. Combining CBAP with standard-of-care chemotherapeutics represents a safe and efficient strategy for the treatment of pancreatic cancer.

Mutations in Helicobacter pylori infected patients with chronic gastritis, intestinal type of gastric cancer and familial gastric cancer.

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.

The Immunometabolic Gene N-Acetylglucosamine Kinase Is Uniquely Involved in the Heritability of Multiple Sclerosis Severity.

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.

Shikonin attenuates cerebral ischemia/reperfusion injury via inhibiting NOD2/RIP2/NF-κB-mediated microglia polarization and neuroinflammation.

OBJECTIVES: Microglia-mediated neuroinflammation plays a crucial role in the pathophysiological process of multiple neurological disorders such as ischemic stroke, which still lacks effective therapeutic agents. Shikonin possesses anti-inflammatory and neuroprotective properties. However, its underlying mechanism remains elusive. This study aimed to investigate whether Shikonin confers protection against cerebral ischemia/reperfusion (I/R) injury by modulating microglial polarization and elucidate the associated mechanisms. METHODS: This study employed an oxygen-glucose deprivation and reoxygenation (OGD/R) BV2 microglial cellular model and a middle cerebral artery occlusion/reperfusion (MCAO/R) animal model to investigate the protection and underlying mechanism of Shikonin against ischemic stroke. RESULTS: The results demonstrated that Shikonin treatment significantly reduced brain infarction volume and improved neurological function in MCAO/R rats. Simultaneously, Shikonin treatment significantly reduced microglial proinflammatory phenotype and levels of proinflammatory markers (inducible-NO synthase (iNOS), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and IL-6), increased microglial anti-inflammatory phenotype and levels of anti-inflammatory markers (Arginase-1 (Arg1), transforming growth factor-beta (TGF-ß), and IL-10), reversed the expression of Nucleotide-binding oligomerization domain 2 (NOD2) and phosphorylation receptor interacting protein 2 (p-RIP2), and suppressed nuclear factor kappa-B (NF-κB) signaling activation in the ischemic penumbra regions. These effects of Shikonin were further corroborated in OGD/R-treated BV2 cells. Furthermore, overexpression of NOD2 markedly attenuated the neuroprotective effects of Shikonin treatment in MCAO/R rats. NOD2 overexpression also attenuated the regulatory effects of Shikonin on neuroinflammation, microglial polarization, and NF-κB signaling activation. CONCLUSION: This study illustrates that Shikonin mitigates inflammation mediated by microglial proinflammatory polarization by inhibiting the NOD2/RIP2/NF-κB signaling pathway, thereby exerting a protective role. The findings uncover a potential molecular mechanism for Shikonin in treating ischemic stroke.

NOD1 and NOD2 in inflammatory and infectious diseases.

It has been long recognized that NOD1 and NOD2 are critical players in the host immune response, primarily by their sensing bacterial peptidoglycan-conserved motifs. Significant advances have been made from efforts that characterize their upstream activators, assembly of signaling complexes, and activation of downstream signaling pathways. Disruption in NOD1 and NOD2 signaling has also been associated with impaired host defense and resistance to the development of inflammatory diseases. In this review, we will describe how NOD1 and NOD2 sense microbes and cellular stress to regulate host responses that can affect disease pathogenesis and outcomes.

Crosstalk between NOD2 and TLR2 suppresses the development of TLR2-mediated experimental colitis.

Nucleotide-binding oligomerization domain 2 (NOD2) is an intracellular sensor for muramyl dipeptide (MDP), a degradation product of bacterial cell wall peptidoglycan (PGN). PGN stimulates cell-surface Toll-like receptor 2 (TLR2) independently of NOD2, indicating the presence of crosstalk between extracellular TLR2 and intracellular NOD2 upon exposure to PGN. NOD2-deficient mice were sensitive, while TLR2-deficient mice were resistant to experimental colitis induced by intrarectal administration of PGN. Severe colitis in NOD2-deficient mice was accompanied by increased expression of nuclear factor-kappa B-dependent cytokines and decreased expression of autophagy-related 16-like 1 (ATG16L1). MDP activation of NOD2 enhanced autophagy mediated by TLR2 in human dendritic cells. mRNA expression of TLR2 tended to be higher in the colonic mucosa of patients with active ulcerative colitis compared to that of those in remission. Induction of remission was associated with increased mRNA expression of ATG16L1 in both ulcerative colitis and Crohn's disease patients. Conversely, mRNA expression of receptor-interacting serine/threonine-protein kinase 2 was higher in the inflammatory colonic mucosa of patients with active disease than in the non-inflamed mucosa of patients in remission, in both ulcerative colitis and Crohn's disease. These findings highlight the role of NOD2-TLR2 crosstalk in the immunopathogenesis of colitis.

RIPK2 NODs to XIAP and IBD.

The receptor-interacting protein kinases (RIPKs) are key regulators of inflammatory signalling and cell death pathways triggered by innate immune receptors, and RIPKs have emerged as promising therapeutic targets for treatment of immune-related disorders. RIPK2 mediates signalling responses initiated by the bacterial-sensing pattern recognition receptors nucleotide-binding oligomerization domain-containing proteins 1 and 2 (NOD1/2), which play a key role in regulation of intestinal immunity and inflammation. Modification of RIPK2 by non-degradative ubiquitin chains generated by the E3 ubiquitin ligase XIAP and other ligases govern NOD1/2 signalling. Recent advances suggest that the interaction between RIPK2 and XIAP is a druggable protein-protein interaction to modulate NOD1/2-dependent immune responses. Here, we discuss the mechanistic function of RIPK2 in immune signalling, its clinical relevance, and the on-going efforts to target RIPK2 in inflammatory bowel disease and beyond.

Distinct NOD2 mutations reported in three families with Blau syndrome (BS) from a single center in India - Case series and review of literature.

OBJECTIVE: Blau syndrome (BS), considered a rare pediatric autoinflammatory disease, is characterised by a triad of granulomatous arthritis, dermatitis and uveitis. Here we present a tale of three families visited in our outpatient department in the last two years (2020-2022) where more than one member was affected with either skin, ophthalmological and joint involvement with either biopsy-proven granuloma or genetic mutation at NOD2 gene suggesting the diagnosis of BS. CASE SERIES: The first family had three affected members where the mother and her two children had skin changes, polyarthritis and a pathogenic mutation in NOD2 gene (exon 4, c.1000C > T, p.Arg334Trp) suggesting BS. The second family had two affected members where both mother and her son had uveitis, skin changes with NOD2 mutation at exon 4 with c.1147G > A (p Glu 383 Lys) variant. The son also had polyarthritis and his skin biopsy was suggestive of granulomatous inflammation. In the third family with two affected members, we found a mutation in NOD2 on exon 4 (c 1324C > T, p.Lys 442 Phe) which was described as pathogenic with only one report published till date. CONCLUSION: These three cases presented to us within the last two years and led to a diagnosis of BS in three other family members with discrete mutations (commonest to rarest) on the NOD2 gene in the three families.

Lentiviral Gene Transfer Corrects Immune Abnormalities in XIAP Deficiency.

BACKGROUND: X-linked inhibitor of apoptosis protein (XIAP) deficiency is a severe immunodeficiency with clinical features including hemophagocytic lymphohistiocytosis (HLH) and inflammatory bowel disease (IBD) due to defective NOD2 responses. Management includes immunomodulatory therapies and hematopoietic stem cell transplant (HSCT). However, this cohort is particularly susceptible to the chemotherapeutic regimens and acutely affected by graft-vs-host disease (GvHD), driving poor long-term survival in transplanted patients. Autologous HSC gene therapy could offer an alternative treatment option and would abrogate the risks of alloreactivity. METHODS: Hematopoietic progenitor (Lin-ve) cells from XIAPy/- mice were transduced with a lentiviral vector encoding human XIAP cDNA before transplantation into irradiated XIAP y/- recipients. After 12 weeks animals were challenged with the dectin-1 ligand curdlan and recovery of innate immune function was evaluated though analysis of inflammatory cytokines, body weight, and splenomegaly. XIAP patient-derived CD14+ monocytes were transduced with the same vector and functional recovery was demonstrated using in vitro L18-MDP/NOD2 assays. RESULTS: In treated XIAPy/- mice, ~40% engraftment of gene-corrected Lin-ve cells led to significant recovery of weight loss, splenomegaly, and inflammatory cytokine responses to curdlan, comparable to wild-type mice. Serum IL-6, IL-10, MCP-1, and TNF were significantly reduced 2-h post-curdlan administration in non-corrected XIAPy/- mice compared to wild-type and gene-corrected animals. Appropriate reduction of inflammatory responses was observed in gene-corrected mice, whereas non-corrected mice developed an inflammatory profile 9 days post-curdlan challenge. In gene-corrected patient CD14+ monocytes, TNF responses were restored following NOD2 activation with L18-MDP. CONCLUSION: Gene correction of HSCs recovers XIAP-dependent immune defects and could offer a treatment option for patients with XIAP deficiency.

Molecular diagnostic yield for Blau syndrome in previously diagnosed juvenile idiopathic arthritis with uveitis or cutaneous lesions.

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 which 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 on patient care. RESULTS: Between May 1, 2008, and June 1, 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 to be associated with a higher diagnostic yield, providing clinical clues to suspect the possibility of Blau syndrome. CONCLUSION: Among some patients with pediatric-onset arthritis complicated with uveitis or cutaneous lesions, reassessing their 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.

Susceptibility of nucleotide-binding oligomerization domain 2 mutations to Whipple's disease.

OBJECTIVES: Whipple's disease (WD) results from infection of the bacteria Tropheryma whipplei (TW). This disease is characterized by macrophage infiltration of intestinal mucosa and primarily affects Caucasian males. Genetic studies of host susceptibility are scarce. Nucleotide-binding oligomerization domain containing protein 2 (NOD2) is an innate immune sensor, resides mainly in monocytes/macrophages and contributes to defense against infection and inflammatory regulation. NOD2 mutations are associated with autoinflammatory diseases. We report the association of NOD2 mutations with TW and WD for the first time. METHODS: A multicenter, retrospective study of three patients with WD was conducted. Patients received extensive multidisciplinary evaluations and were cared for by the authors. NOD2 and its association with infection and inflammation were schematically represented. RESULTS: All patients were Caucasian men and presented with years of autoinflammatory phenotypes, including recurrent fever, rash, inflammatory arthritis, gastrointestinal symptoms, and elevated inflammatory markers. All patients underwent molecular testing using a gene panel for periodic fever syndromes and were identified to carry NOD2 mutations associated with NOD2-associated autoinflammatory disease. Despite initially negative gastrointestinal evaluations, repeat endoscopy with duodenal tissue biopsy ultimately confirmed WD. After initial ceftriaxone and maintenance with doxycycline and/or hydroxychloroquine, symptoms were largely controlled, though mild relapses occurred in follow up. CONCLUSION: Both NOD2 and TW/WD are intensively involved in monocytes/macrophages. WD is regarded as a macrophage disease. NOD2 leucin rich repeat-associated mutations in monocytes/macrophages cause functional impairment of these cells and consequently may make the host susceptible for TW infection and WD, especially in the setting of immunosuppression.

Topical N-phosphonacetyl-l-aspartate is a dual action candidate for treating non-melanoma skin cancer.

Each year, 3.3 million Americans are diagnosed with non-melanoma skin cancers (NMSC) and an additional 40 million individuals undergo treatment of precancerous actinic keratosis lesions. The most effective treatments of NMSC (surgical excision and Mohs surgery) are invasive, expensive and require specialised training. More readily accessible topical therapies currently are 5-fluorouracil (a chemotherapeutic agent) and imiquimod (an immune modulator), but these can have significant side effects which limit their efficacy. Therefore, more effective and accessible treatments are needed for non-melanoma cancers and precancers. Our previous work demonstrated that the small molecule N-phosphonacetyl-L-aspartate (PALA) both inhibits pyrimidine nucleotide synthesis and activates pattern recognition receptor nucleotide-binding oligomerization domain 2. We propose that topical application of PALA would be an effective NMSC therapy, by combining the chemotherapeutic and immune modulatory features of 5-fluorouracil and imiquimod. Daily topical application of PALA to mouse skin was well tolerated and resulted in less irritation, fewer histopathological changes, and less inflammation than caused by either 5-fluorouracil or imiquimod. In an ultraviolet light-induced NMSC mouse model, topical PALA treatment substantially reduced the numbers, areas and grades of tumours, compared to vehicle controls. This anti-neoplastic activity was associated with increased expression of the antimicrobial peptide cathelicidin and increased recruitment of CD8+ T cells and F4/80+ macrophages to the tumours, demonstrating both immunomodulatory and anti-proliferative effects. These findings indicate that topical PALA is an excellent candidate as an effective alternative to current standard-of-care NMSC therapies.

Design, synthesis and biological evaluation of novel lipophilic 2, 5-disubstituted tetrazole analogues of muramyl dipeptide as NOD2 agonists.

A focused library of six new 2, 5-disubstituted tetrazole (2, 5-DST) analogues of N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP) as potential immunomodulators were synthesized by the bioisosteric replacement of α-amide of d-isoglutamine with 5-substituted tetrazole (5-ST). Another parameter 'lipophilicity' was also considered to improve the pharmacological properties of MDP through the alkylation of 5-substituted tetrazole during synthesis. In total, six 2, 5-DST analogues of MDP were synthesized and bio-evaluated for the study of human NOD2 stimulation activity in the innate immune response. Interestingly, among the varied lengths of the alkyl chain in 2, 5-disubstituted tetrazole derivatives, the tetrazole analogues 12b bearing the -Butyl (C4) and 12c having -Octyl (C8) chain showed the best NOD2 stimulation potency equivalent with reference compound MDP. These analogues were evaluated for their adjuvanticity against dengue antigen and analogues 12b and 12c have elicited a potent humoral and cell mediated response.

NOD2 modulates immune tolerance via the GM-CSF-dependent generation of CD103+ dendritic cells.

Four decades ago, it was identified that muramyl dipeptide (MDP), a peptidoglycan-derived bacterial cell wall component, could display immunosuppressive functions in animals through mechanisms that remain unexplored. We sought to revisit these pioneering observations because mutations in NOD2, the gene encoding the host sensor of MDP, are associated with increased risk of developing the inflammatory bowel disease Crohn's disease, thus suggesting that the loss of the immunomodulatory functions of NOD2 could contribute to the development of inflammatory disease. Here, we demonstrate that intraperitoneal (i.p.) administration of MDP triggered regulatory T cells and the accumulation of a population of tolerogenic CD103+ dendritic cells (DCs) in the spleen. This was found to occur not through direct sensing of MDP by DCs themselves, but rather via the production of the cytokine GM-CSF, another factor with an established regulatory role in Crohn's disease pathogenesis. Moreover, we demonstrate that populations of CD103-expressing DCs in the gut lamina propria are enhanced by the activation of NOD2, indicating that MDP sensing plays a critical role in shaping the immune response to intestinal antigens by promoting a tolerogenic environment via manipulation of DC populations.