Nucleotide-binding Oligomerization Domain-containing Protein Research Articles

Noninvasive Analysis of Peptidoglycan from Living Animals.

The role of the intestinal microbiota in host health is increasingly revealed in its contributions to disease states. The host-microbiome interaction is multifactorial and dynamic. One of the factors that has recently been strongly associated with host physiological responses is peptidoglycan from bacterial cell walls. Peptidoglycan from gut commensal bacteria activates peptidoglycan sensors in human cells, including the nucleotide-binding oligomerization domain-containing protein 2. When present in the gastrointestinal tract, both the polymeric form (sacculi) and depolymerized fragments can modulate host physiology, including checkpoint anticancer therapy efficacy, body temperature and appetite, and postnatal growth. To utilize this growing area of biology toward therapeutic prescriptions, it will be critical to directly analyze a key feature of the host-microbiome interaction from living hosts in a reproducible and noninvasive way. Here we show that metabolically labeled peptidoglycan/sacculi can be readily isolated from fecal samples collected from both mice and humans. Analysis of fecal samples provided a noninvasive route to probe the gut commensal community including the metabolic synchronicity with the host circadian clock. Together, these results pave the way for noninvasive diagnostic tools to interrogate the causal nature of peptidoglycan in host health and disease.

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

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

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.

A193 DECIPHERING THE INFLUENCE OF CROHN’S DISEASE-ASSOCIATED GENETIC RISK FACTOR, NOD2 IN INTESTINAL FIBRO-STENOSIS

Abstract Background Intestinal fibrosis (IF) is a common debilitating complication associated with Crohn’s disease (CD), an idiopathic inflammatory bowel disease (IBD). IF results in severe strictures that affect the entire gastrointestinal tract, often requiring surgery due to limited or no effective treatment. In general, IF is a consequence of chronic inflammation, excessive extracellular matrix (ECM) deposition, aberrant cellular functions and tissue remodeling involving the innate and adaptive immune system. At the cellular level, fibrosis has been proposed to involve the crosstalk between epithelial cells, mesenchymal stromal cells and immune cells. However, the cascade of events that establish IF is poorly understood as it comprises a dynamic interplay between host genetics, immunity, gut microbiome and aberrant environment insults. Notably, polymorphisms in nucleotide-binding oligomerization domain-containing protein 2 (NOD2; a bonafide cytosolic innate immune receptor), strong genetic risk factors for CD, also increase the incidence of IF. In this context, how NOD2 polymorphisms govern intestinal fibrostenosis in CD remains to be investigated. Aims To investigate how CD-associated risk gene, NOD2 contributes to intestinal fibrostenotic disease Methods We employed a single-cell RNA sequencing (scRNA-seq) approach to explore the cellular landscape in our CD-mouse model, where we challenged Nod2-deficient mice with a chronic inflammatory insult regime, using dextran sulfate sodium (cDSS) for 3 cycles. Subsequently, changes in inflammatory, metabolic and fibrotic markers in the gut were analyzed by quantitative real-time PCR (qRT-PCR), immunohistochemistry (IHC) or western blot analyses. Further, RNA-In-situ hybridization (RNA-ISH) and flow cytometric analyses were performed to validate cell populations contributing to, and the molecular signatures linked with intestinal fibrostenosis. Results We found that Nod2-deficient mice show increased expression of pro-inflammatory cytokines (TNF, IL6) and excessive ECM deposition (fibrosis) in the gut compared to wild-type littermate mice post-cDSS. Curiously, scRNA-seq analysis uncovered altered stromal-immune-cell crosstalk along with the identification of a novel subset of the Dpt+ interstitial stromal population with distinct gene signatures regulated by chronic inflammation. Further, we observed increased senescence (Cdkn2a) mediated trans-differentiation of stroma into fibroblast contributing to IF in Nod2-deficient mice. Conclusions The study outlined will deepen our understanding of cellular and molecular mediators as well as inter and intra-cellular networks that drive IF. This will be transformative in devising therapeutic strategies to prevent IF in progressive CD patients. Funding Agencies CCC, CIHRThe American Association of Immunologist (AAI)

Anakinra in idiopathic recurrent pericarditis: acomprehensive case series and literature review.

Idiopathic recurrent pericarditis (IRP) is defined by recurring episodes of pericardial inflammation without aknown cause. This study investigates the safety and efficacy of anakinra, an interleukin‑1 inhibitor, as asuccessful therapy for IRP in cases resistant to conventional treatment. Aretrospective evaluation of patients treated at our autoinflammatory center between 2011 and 2023 was conducted. Patient files were examined for demographic, clinical, and treatment response data, including nonsteroid anti-inflammatory drugs (NSAIDs), corticosteroids, and colchicine. Monogenic autoinflammatory disease screening was performed for Mediterranean Fever (MEFV), tumor necrosis factor receptor-associated periodic syndrome (TRAPS), mevalonate kinase (MVK), nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3), and nucleotide-binding oligomerization domain-containing protein 2 (NOD2). Patients who experienced multiple episodes of pericarditis were diagnosed with recurrent pericarditis. The study evaluated anakinra treatment in IRP patients unresponsive to conventional therapy. The study included 21participants, 9(42.9%) female and 12(57.1%) male. The average age of the participants was 43.1 ± 16.5years. The MEFV mutation analysis revealed that 2(9.5%) had amutation in exon10 and4 (19.0%) had one in exon2. Out of the 16cases, 15successfully discontinued steroid treatment. Four patients (19.0%) experienced injection site reactions. C‑reactive protein (CRP) levels were measured at an average of 196 ± 67.8 mg/l before and 2.6 ± 3.15 mg/l after anakinra treatment. In conclusion, the study adds to the growing evidence for the efficacy of interleukin-1inhibitors, such as anakinra, as apromising treatment modality for IRP in cases resistant to conventional treatment.

HEMATOPOIETIC STEM CELL GENE THERAPY AS A NOVEL THERAPEUTIC APPROACH FOR SEVERE CROHN’S DISEASE ASSOCIATED WITH NOD2-DEFICIENCY

Abstract Pathogenic variants of the NOD2 (nucleotide-binding oligomerization domain containing protein 2) gene demonstrate the strongest genetic association to Crohn’s inflammatory bowel disease (CD). Mounting evidence links NOD2 deficiency with poor clinical outcome, particularly in pediatric and early onset CD. CD patients with pathogenic NOD2 variants, particularly carriers of more than one risk allele, frequently present an aggressive, fistulizing and fibrostenotic disease, requiring multiple surgical resections. NOD2-deficiency is implicated as a driver of CD pathogenesis through failure of gut innate immunity to resolve bacterial infections and by loss of tissue homeostasis within the intestinal microenvironment. Here we present preclinical data on OTL-104, an autologous haematopoietic stem cell gene therapy (HSC-GT) which aims to stably restore NOD2 expression in gut resident macrophages, to correct immune dysfunction linked to NOD2-deficient CD pathogenesis. We used in vitro and in vivo models of NOD2 deficiency to demonstrate the mechanism of action and the efficacy of OTL-104 autologous HSC-GT. NOD2KO human myeloid cells differentiated in vitro from CRISPR-generated NOD2KO CD34+ HSCs are unable to mount a proinflammatory cytokine response to MDP stimulation. Similarly, myeloid cells differentiated from CD34+ cells obtained from peripheral blood of genetically characterized NOD2-deficient CD patients, are also refractory to MDP stimulation and unable to generate a normal cytokine response profile (IL-8, TNFa, IL-6, CXCL1/2 and IL-10). In both NOD2 deficient CD34+ derived monocyte models, transduction with a lentiviral vector (LVV) expressing NOD2 fully restores NOD2-dependent cytokine and chemokine responses, restoring an immune profile that is comparable to monocytes derived from CD34+ cells from NOD2 wild-type healthy donors. Transplantation of lineage negative (Lin-) haematopoietic stem/progenitor cells (HSPCs) transduced with the OTL-104 LVV in Nod2KO mice was used as an in vivo model of gene therapy for CD. Compared to WT mice, Nod2KO mice fail to release systemic inflammatory mediators and recruit myeloid cells in response to MDP administration. Transplantation of transduced Lin- HSPCs restores MDP-induced systemic release of IL-6 and CXCL1 as well as innate mobilization of monocyte/macrophage cells and gut associated immune markers. Transplanted Nod2KO mice display normal hematopoiesis and stable vector copy numbers in hematopoietic cells. Key to our therapeutic approach, histopathological analysis of intestinal lamina propria from transplanted mice shows a normal biodistribution and physiological NOD2 gene expression in tissue resident cells (Figure 1). These results confirm the impact of NOD2 deficiency in primary immune activation and demonstrates the therapeutic potential of OTL-104 HSC-GT for long-term correction of NOD2-deficient CD.

OP29 Haematopoietic stem cell gene therapy as a treatment for severe Crohn’s Disease associated with pathogenic NOD2 genetic variants

Abstract Background Pathogenic variants of the NOD2 (nucleotide-binding oligomerization domain containing protein 2) gene demonstrate the strongest genetic association to Crohn’s inflammatory bowel disease (CD), with mounting evidence linking NOD2 deficiency with poor clinical outcome. CD patients with NOD2 variants, particularly carriers of more than one risk allele, frequently present an aggressive, fistulizing and fibrostenotic disease, requiring multiple surgical resections. NOD2-deficiency is implicated to drive CD pathogenesis through failure of gut innate immunity to resolve bacterial infections and by loss of tissue homeostasis within the intestinal microenvironment. Here we present preclinical data on OTL-104, an autologous haematopoietic stemcell gene therapy (HSC-GT) which aims to stably restore NOD2 expression in gut resident macrophages, to correct immune dysfunction linked to NOD2-deficient CD pathogenesis. Methods We used in vitro and in vivo models of NOD2 deficiency to demonstrate the mechanism of action and the efficacy of OTL-104 autologous HSC-GT. Results NOD2KO human myeloid cells differentiated in vitro from CRISPR-generated NOD2KO CD34+ HSCs are unable to mount a proinflammatory cytokine response to MDP stimulation. Similarly, myeloid cells differentiated from CD34+ cells obtained from peripheral blood of genetically characterized NOD2-deficient CD patients, are also refractory to MDP stimulation and unable to generate a normal cytokine response profile. In both NOD2 deficient CD34+ derived monocyte models, transduction with a lentiviral vector (LVV) expressing NOD2 fully restores NOD2-dependent cytokine and chemokine responses, restoring an immune profile that is comparable to monocytes derived from CD34+ cells from NOD2 wild-type healthy donors (Figure 1). Transplantation of lineage negative (Lin-) haematopoietic stem/progenitor cells (HSPCs) transduced with the OTL-104 LVV in NOD2KO mice was used as an in vivo model of gene therapy for CD. Compared to WT mice, NOD2KO mice fail to release systemic inflammatory mediators and recruit myeloid cells in response to MDP administration. Transplantation of transduced Lin- HSPCs restores MDP-induced systemic release of IL-6 and CXCL1 as well as innate mobilization of monocyte/macrophage cells and gut associated gene expression. Key to our therapeutic approach, histopathological analysis of intestinal lamina propria from transplanted mice shows a normal biodistribution and physiological NOD2 gene expression in tissue resident cells. Conclusion These results confirm the impact of NOD2 deficiency in primary immune activation and demonstrates the therapeutic potential of OTL-104 HSC-GT for long-term correction of NOD2-deficient CD.

P709 Role of NOD2 single nucleotide polymorphisms on ustekinumab drug durability in adult patients with Crohn’s disease

Abstract Background Ustekinumab (UST), a monoclonal antibody targeting the p19 subunit of the pro-inflammatory Interleukins 12 and 23, is a therapeutic option in patients with Crohn’s disease (CD) with high efficacy, although some patients will have to cease therapy with UST due to primary non-response, secondary loss of response, or adverse events. Single nucleotide polymorphisms (SNPs) in the Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) gene have been proposed as a prognostic factor in CD. Whether these NOD2 SNPs have an impact on UST drug durability among adult CD patients remains unclear. Methods CD patients were genotyped for the three most common NOD2 SNPs (rs2066844, rs2066845, and rs2066847). Hazard ratios (HR) for risk of NOD2 SNPs on UST durability were calculated using univariable and multivariable cox proportional hazard regression adjusting for age at first UST prescription, previous surgery, Montreal A, L, and B classification, present perianal disease, and previous anti-TNF exposure. Statistical analysis was conducted using SPSS. A two-tailed P value < 0.05 was considered statistically significant. All patients provided written informed consent. Results 92 CD patients were included in the analysis. Of those, 72 continued receiving UST until the end of the study. In univariable and multivariable analyses, NOD2 SNPs were not independent prognostic factors for UST drug durability (P>0.05 for all three NOD2 SNPs). However, age at first UST prescription (adjusted HR [aHR] 0.96, 95% confidence interval [CI] 0.93-1.00, P=0.028 for all tested NOD2 SNPs) and present perianal disease (aHR 2.65, 95% CI 1.11-6.31, P=0.028 for all tested NOD2 SNPs) were independent predictors of ustekinumab drug durability. Conclusion Among adult Crohn’s disease patients being treated with ustekinumab, the presence of NOD2 SNPs did not have a significant impact on ustekinumab drug durability.

Role of the nucleotide-binding oligomerization domain-containing protein 1 pathway in the development of periodontitis

ObjectivesDuring innate immune defense, host pattern recognition receptors, including toll-like receptors and nucleotide-binding oligomerization domain-like receptors (NLRs), can activate downstream pathways by recognizing pathogen-associated molecular patterns produced by microorganisms, triggering immune responses. NOD1, an important cell membrane protein in the NLR-like receptor protein family, exerts anti-infective effects through γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) recognition. Oral epithelial cells resist bacterial invasion through iE-DAP-induced interleukin (IL)-8 production, recruiting neutrophils to sites of inflammation in response to bacterial threats to periodontal tissues. To date, the regulatory mechanisms of iE-DAP in gingival epithelial cells (GECs) are poorly understood. This study was conducted to investigate the role of the NOD1 pathway in the development of periodontitis by examining the effect of iE-DAP on IL-8 production in Ca9-22 cells. MethodsIL-8 production by iE-DAP-stimulated-Ca9-22 cells was assessed using an enzyme-linked immunosorbent assay. Phosphorylation levels of intracellular signaling molecules were evaluated using western blot analyses.Results: iE-DAP induced NOD1 receptor expression in Ca9-22 cells. Additionally, iE-DAP induced expression of pro-IL-1β protein without extracellular secretion. Our results suggest that iE-DAP regulates IL-8 production by activating p38 mitogen-activated protein kinase (MAPK) and ERK1/2 signaling pathways. iE-DAP also promoted nuclear factor kappa-B p65 phosphorylation, facilitating its nuclear translocation. Notably, p38 MAPK and ERK1/2 inhibitors suppressed iE-DAP-stimulated IL-8 production, suggesting that JNK is not involved in this mechanism. ConclusionsOur results indicate that p38 MAPK and ERK1/2, but not JNK, are involved in innate immune responses in GECs.

281 Effects of Corynebacterium Glutamicum Cell Mass Supplementation on Modulation of Mucosa-Associated Microbiota to Intestinal Health of Nursery Pigs

Abstract The objectives were to evaluate the effects of Corynebacterium glutamicum cell mass (CGCM) supplementation, replacing blood plasma in diets, on the gene expressions of biomarkers associated with peptidoglycan and their correlation with intestinal health in nursery pigs. Weaned pigs (n = 32) were allotted to 4 dietary treatments based on the randomized complete block design with initial BW as blocks. The diets with supplementation of CGCM (0, 0.7, 1.4, and 2.1%) replacing blood plasma up to 1.5% were fed for 21 d. Pigs were euthanized on d 21 to collect proximal and distal jejunal mucosa to measure intestinal health markers. Gene expression of receptors associated with peptidoglycan (PGN) and intestinal health were evaluated by PCR. Correlations between gene expressions of receptors associated with PGN and intestinal health markers in jejunal mucosa were assessed by Pearson’s analysis. Data were analyzed using the Mixed procedure of SAS 9.4. Increasing CGCM supplementation affected (quadratic) the relative gene expression of peptidoglycan recognition protein (PGLYRP) 1 (P = 0.080, max: 1.85% at 1.1% CGCM), PGLYRP4 (P < 0.05, max: 3.24% at 1.2% CGCM), and interferon-γ (P = 0.088, max: 3.25% at 1.1% CGCM). Increasing CGCM supplementation did not affect the relative gene expression of PGLYRP2A, PGLYRP2B, PGLYRP3, Toll-like receptor (TLR) 2, TLR4, Nucleotide-binding oligomerization domain-containing protein (NOD) 1, NOD2, cluster of differentiation (CD) 14, and nuclear factor-kappa B. In proximal jejunal mucosa, PGLYRP1 was positively correlated with immunoglobulin A (IgA) and interleukin 8 (r = 0.50 and 0.45, respectively; P < 0.05). PGLYRP4 was positively correlated with protein carbonyl and IgA (r = 0.65 and 0.60, respectively; P < 0.05). Interferon-γ was positively correlated with Campylobacter coli (r = 0.61; P < 0.05) and IgA (r = 0.52; P < 0.05). PGLYRP2A was negatively correlated (r = -0.44; P < 0.05) with Pseudomonadaceae. TLR2 was positively correlated with Corynebacteriaceae, Pasteurellaceae, and Propionibacteriaceae (r = 0.55, 0.51, and 0.52, respectively; P < 0.05). NOD1 was positively correlated (r = 0.50; P < 0.05) with Propionibacteriaceae. NOD2 were positively correlated with Pasteurellaceae and Propionibacteriaceae (r = 0.73 and 0.53, respectively; P < 0.05). CD14 was negatively correlated (r = -0.47; P < 0.05) with IgA. Campylobacteraceae was positively correlated with tumor necrosis factor-alpha (r = 0.50; P < 0.05). Pseudomonadaceae was negatively correlated with IgG (r = -0.39; P < 0.05). In conclusion, supplementing 1.1% to 1.2% CGCM, replacing 0.8 to 0.9% blood plasma, increased the relative gene expressions of PGLYRP1, PGLYRP4, and interferon-γ, which were related to the modulation of the mucosa-associated microbiota, immune response, and oxidative stress markers caused by CGCM supplementation. Supplementing 2.1% CGCM showed to have a similar expression of receptors associated with PGN and intestinal health by promoting the growth of less inflammatory microbiota.

Multifaceted roles and regulation of nucleotide-binding oligomerization domain containing proteins.

Nucleotide-binding oligomerization domain-containing proteins, NOD1 and NOD2, are cytosolic receptors that recognize dipeptides and tripeptides derived from the bacterial cell wall component peptidoglycan (PGN). During the past two decades, studies have revealed several roles for NODs beyond detecting PGN fragments, including activation of an innate immune anti-viral response, NOD-mediated autophagy, and ER stress induced inflammation. Recent studies have also clarified the dynamic regulation of NODs at cellular membranes to generate specific and balanced immune responses. This review will describe how NOD1 and NOD2 detect microbes and cellular stress and detail the molecular mechanisms that regulate activation and signaling while highlighting new evidence and the impact on inflammatory disease pathogenesis.

Heteroantigen-assembled nanovaccine enhances the polyfunctionality of TILs against tumor growth and metastasis

The dysfunction of tumor infiltrating lymphocytes (TILs) directly correlates with out of control of tumor growth and metastasis. New approaches and insightful clarity for rescuing TILs dysfunction are urgently needed. Here, we design two heterogenous antigens based on MHC-I epitope and MHC-II epitope from tumor, and assemble heterogenous antigens by electrostatic interactions and π-π stacking into heteroantigen-assembled nanovaccine (HANV). HANV not only significantly increases the abundance of CD8+ and CD4+ TILs, but also elicits stronger polyfunctionality of CD8+ and CD4+ TILs in vivo. Enhanced polyfunctionality of CD8+ and CD4+ TILs positively correlate to suppression of tumor growth and metastasis in melanoma-bearing mouse models. We also validate that nucleotide-binding oligomerization domain-containing protein 2 (NOD2) dominantly enhances anti-tumor capacity of TILs in a temporal immunoregulation manner. This work presents a new insight in developing HANV as a rational strategy to shape TILs polyfunctionality for tumor growth and metastasis.

The Protective Role of TLR4 in Intestinal Epithelial Cells through the Regulation of the Gut Microbiota in DSS-Induced Colitis in Mice.

The cause of ulcerative colitis (UC) is not yet fully understood. Previous research has pointed towards a potential role for mutations in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in promoting the onset and progression of inflammatory bowel disease (IBD) by altering the microbiota of the gut. However, the relationship between toll-like receptor 4 (TLR4) and gut microbiota in IBD is not well understood. To shed light on this, the interaction between TLR4 and gut microbiota was studied using a mouse model of IBD. To examine the function of TLR4 signaling in intestinal injury repair, researchers developed Dextran Sulfate Sodium Salt (DSS)-induced colitis and injury models in both wild-type (WT) mice and TLR4 knockout (TLR4-KO) mice. To assess changes in the gut microbiota, 16S rRNA sequencing was conducted on fecal samples from both the TLR4-KO and WT enteritis mouse models. The data obtained depicted a protective function of TLR4 against DSS-induced colitis. The gut microbiota composition was found to vary considerably between the WT and TLR4-KO mice groups as indicated by β-diversity analysis and operational taxonomic units (OTUs) cluster. Statistical analysis of microbial multivariate variables depicted an elevated abundance of Escherichia coli/Shigella, Gammaproteobacteria, Tenerlcutes, Deferribacteres, Enterobacteria, Rikenellaceae, and Proteobacteria in the gut microbiota of TLR4-KO mice, whereas there was a considerable reduction in Bacteroidetes at five different levels of the phylogenetic hierarchy including phylum, class, order, family, and genus in comparison with the WT control. TLR4 may protect intestinal epithelial cells from damage in response to DSS-induced injury by controlling the microbiota in the gut.

Macrophage RIPK3 triggers inflammation and cell death via the XBP1–Foxo1 axis in liver ischaemia–reperfusion injury

Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) is a central player in triggering necroptotic cell death. However, whether macrophage RIPK3 may regulate NOD1-dependent inflammation and calcineurin/transient receptor potential cation channel subfamily M member 7 (TRPM7)-induced hepatocyte death in oxidative stress-induced liver inflammatory injury remains elusive. A mouse model of hepatic ischaemia-reperfusion (IR) injury, the primary hepatocytes, and bone marrow-derived macrophages were used in the myeloid-specific RIPK3 knockout (RIPK3M-KO) and RIPK3-proficient (RIPK3FL/FL) mice. RIPK3M-KO diminished IR stress-induced liver damage with reduced serum alanine aminotransferase/aspartate aminotransferase levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators compared with the RIPK3FL/FL controls. IR stress activated RIPK3, inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α), x-box binding protein 1 (XBP1), nucleotide-binding oligomerisation domain-containing protein 1 (NOD1), NF-κB, forkhead box O1 (Foxo1), calcineurin A, and TRPM7 in ischaemic livers. Conversely, RIPK3M-KO depressed IRE1α, XBP1, NOD1, calcineurin A, and TRPM7 activation with reduced serum tumour necrosis factor α (TNF-α) levels. Moreover, Foxo1M-KO alleviated IR-induced liver injury with reduced NOD1 and TRPM7 expression. Interestingly, chromatin immunoprecipitation coupled with massively parallel sequencing revealed that macrophage Foxo1 colocalised with XBP1 and activated its target gene Zc3h15 (zinc finger CCCH domain-containing protein 15). Activating macrophage XBP1 enhanced Zc3h15, NOD1, and NF-κB activity. However, disruption of macrophage Zc3h15 inhibited NOD1 and hepatocyte calcineurin/TRPM7 activation, with reduced reactive oxygen species production and lactate dehydrogenase release after macrophage/hepatocyte coculture. Furthermore, adoptive transfer of Zc3h15-expressing macrophages in RIPK3M-KO mice augmented IR-triggered liver inflammation and cell death. Macrophage RIPK3 activates the IRE1α-XBP1 pathway and Foxo1 signalling in IR-stress livers. The XBP1-Foxo1 interaction is essential for modulating target gene Zc3h15 function, which is crucial for the control of NOD1 and calcineurin-mediated TRPM7 activation. XBP1 functions as a transcriptional coactivator of Foxo1 in regulating NOD1-driven liver inflammation and calcineurin/TRPM7-induced cell death. Our findings underscore a novel role of macrophage RIPK3 in stress-induced liver inflammation and cell death, implying the potential therapeutic targets in liver inflammatory diseases. Macrophage RIPK3 promotes NOD1-dependent inflammation and calcineurin/TRPM7-induced cell death cascade by triggering the XBP1-Foxo1 axis and its target gene Zc3h15, which is crucial for activating NOD1 and calcineurin/TRPM7 function, implying the potential therapeutic targets in stress-induced liver inflammatory injury.

The Synergistic Potential of Combining PD-1/PD-L1 Immune Checkpoint Inhibitors with NOD2 Agonists in Alzheimer's Disease Treatment.

Our research over the past decade has compellingly demonstrated the potential of Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) receptor agonists in Alzheimer's disease (AD) treatment. These agonists facilitate the conversation of pro-inflammatory monocytes into patrolling monocytes, leading to the efficient clearance of amyloid-β (Aβ) in the AD-affected cerebrovascular system. This approach surpasses the efficacy of targeting Aβ formation, marking a significant shift in therapeutic strategies. Simultaneously, inhibitors of PD-1/PD-L1 immune check point or glycogen synthase kinase 3 beta (GSK3β), which modulates PD-1, have emerged as potent AD treatment modalities. PD-1 inhibitor exhibits a profound potential in monocytes' recruitment to the AD-afflicted brain. Recent evidence suggests that an integrated approach, combining the modulation of NOD2 and PD-1, could yield superior outcomes. This innovative combinatorial therapeutic approach leverages the potential of MDP to act as a catalyst for the conversion of inflammatory monocytes into patrolling monocytes, with the subsequent recruitment of these patrolling monocytes into the brain being stimulated by the PD-1 inhibitor. These therapeutic interventions are currently under preclinical investigation by pharmaceutical entities, underscoring the promise they hold. This research advocates for the modulation, rather than suppression, of the innate immune system as a promising pharmacological strategy in AD.

Discovery of a novel RIPK2 inhibitor for the treatment of inflammatory bowel disease

Nucleotide-binding oligomerization domain-containing protein 1 and 2 (NOD 1/2) are important cytosolic pattern recognition receptors that initiate host immune response. The dysregulation of NOD signaling is highly associated with inflammatory bowel disease (IBD) that needs novel treatment options. Receptor-interacting protein kinase 2 (RIPK2) is a critical mediator of NOD signaling and considered a promising therapeutic target for IBD treatment. However, there are currently no RIPK2 inhibitors available for clinical use. Here, we report the discovery and characterization of Zharp2-1 as a novel and potent RIPK2 inhibitor that effectively blocks RIPK2 kinase function and NOD-mediated NF-κB/MAPK activation in both human and mouse cell lines. Zharp2-1 exhibits significantly superior solubility compared to the non-prodrug form of the advanced RIPK2 inhibitor prodrug GSK2983559. The improved solubility combined with favorable in vitro metabolic stability translated to excellent in vivo pharmacokinetic profiles for Zharp2-1. In addition, Zharp2-1 demonstrates better effects than GSK2983559 in inhibiting the muramyl dipeptide (MDP)-induced production of pro-inflammatory cytokines in human peripheral blood mononuclear cells (PBMCs) and MDP-induced peritonitis in mice. Furthermore, Zharp2-1 markedly reduces Listeria monocytogenes infection-induced cytokines release in both human and mouse cells. Importantly, Zharp2-1 significantly ameliorates DNBS-induced colitis in rats and suppressed pro-inflammatory cytokine release in intestinal specimens from IBD patients. Collectively, our findings indicate that Zharp2-1 is a promising RIPK2 inhibitor with the potential to be further developed for IBD therapy.

IL-10 Modulates the Expression and Activation of Pattern Recognition Receptors in Mast Cells.

Mast cells (MCs) are involved in several immune-related responses, including those in bacterial infections, autoimmune diseases, inflammatory bowel diseases, and cancer, among others. MCs identify microorganisms by pattern recognition receptors (PRRs), activating a secretory response. Interleukin (IL)-10 has been described as an important modulator of MC responses; however, its role in PRR-mediated activation of MC is not fully understood. We analyzed the activation of TLR2, TLR4, TLR7 and Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in mucosal-like MCs (MLMCs) and peritoneum-derived cultured MCs (PCMCs) from IL-10-/- and wild-type (WT) mice. IL-10-/- mice showed a reduced expression of TLR4 and NOD2 at week 6 and TLR7 at week 20 in MLMC. In MLMC and PCMC, TLR2 activation induced a reduced secretion of IL-6 and TNFα in IL-10-/- MCs. TLR4- and TLR7-mediated secretion of IL-6 and TNFα was not detected in PCMCs. Finally, no cytokine release was induced by NOD2 ligand, and responses to TLR2 and TLR4 were lower in MCs at 20 weeks. These findings indicate that PRR activation in MCs depends on the phenotype, ligand, age, and IL-10.

Risk factors and predictors of urogenous sepsis after percutaneous nephrolithotomy for idiopathic calcium oxalate nephrolithiasis.

The aim of this study was to use bioinformatics approaches to screen and identify the key genes of idiopathic calcium oxalate nephrolithiasis, and explore its potential molecular mechanism. The GSE73680 kidney stone data set was downloaded from the Gene Expression Omnibus (GEO). R software (The R Foundation for Statistical Computing) was used to screen differentially expressed genes. GeneMANIA and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) databases were used to analyze related genes interacting with crucial genes, and a protein-protein interaction (PPI) network was constructed. The differential genes were then subjected to the Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) database. The clinical data of 156 patients who received percutaneous nephrolithotomy (PCNL) therapy at our facility between January 2013 and December 2017 were retrospectively analyzed. The various parameters associated with postoperative urogenous sepsis were identified using multivariable logistic regression analysis. The study discovered one differentially expressed gene was nucleotide-binding oligomerization domain-containing protein 2 (NOD2). GO and KEGG analysis showed that NOD2 might affect the occurrence of idiopathic calcium oxalate kidney stones by affecting inflammation, receptor expression, immune environment, necrosis, apoptosis, and other pathways. The clinical parameter of patients who participated in the study, including preoperative urinary white blood cell (WBC) count, preoperative urinary nitrite, stone diameter, operation time, WBC count, and WBC D values, were statistically different between the systemic inflammatory response syndrome (SIRS) group and the urosepsis group. According to multivariate logistic regression analysis, the preoperative urine nitrite, calculus diameter, blood WBC, and NOD2 expression 3 hours after surgery were all independently associated with the urosepsis development. Preoperative urinary nitrite positive status, postoperative WBC count ≥2.98×109/L 3 hours after operation, stone diameter >6 cm, and low expression of NOD2 in renal papillary tissue are more likely to cause the urinary source of idiopathic calcium oxalate nephrolithiasis after PCNL urogenous sepsis. These parameters also offer a viable treatment paradigm for the perioperative management of PCNL in treating idiopathic calcium oxalate kidney stones.

Chronic Inflammatory Intestinal Disorders in Hidradenitis Suppurativa

Background: Intestinal symptoms are common in patients with hidradenitis suppurativa (HS). HS patients may experience a broad spectrum of chronic inflammatory intestinal disorders (CIID), not exclusive to inflammatory bowel diseases, which are diagnosed by colonoscopy and intestinal biopsies. The frequency of CIID in patients with HS has not been investigated. Objective: The objectives of this study were to determine the occurrence of CIID in HS and characterize this clinical population. Furthermore, the feasibility of using faecal calprotectin (FC) test or anti-Saccharomyces cerevisiae antibody (ASCA) levels to assess the colonic inflammation of CIID in HS patients was investigated. Methods: All newly diagnosed and untreated HS patients (n = 74) were referred to a gastroenterologist for FC followed by colonoscopy after informed consent. C-reactive protein (CRP), white blood cell count, nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) polymorphism, and ASCA levels were measured. Patients were divided into HS-only and HS with CIID (HS + CIID) groups, based on the absence or presence of CIID. Laboratory and clinical parameters (age, gender, HS onset, clinical stage, family history, body mass index (BMI), smoking) were compared between the groups. Results: Thirteen patients complained gastrointestinal symptoms prior to any examination, including 11 in the HS + CIID group. The CIID frequency in HS was 28.4% (n = 21/74), based on colonoscopy and histology. Significantly more patients had severe disease state in the HS + CIID group compared with the HS-only group, and BMI was significantly lower in the HS + CIID group (28.20 ± 5.58 vs. 32.74 ± 6.45, p = 0.006). FC positivity occurred significantly more in HS + CIID patients compared with HS-only patients (90.48% vs. 3.77%, p < 0.001), and ASCA IgG levels were significantly elevated in HS + CIID patients (22.08 ± 23.07 vs. 8.41 ± 10.94 U/mL, p = 0.001). The FC test identified HS + CIID patients with 96.23% specificity and 91.3% sensitivity, while ASCA displayed 77.8% sensitivity and 76.3% specificity. Blood count, CRP, and the presence of NOD2 polymorphisms were indifferent between the two groups. Conclusion: A high frequency of CIID was detected in the examined HS population. The noninvasive FC test has high sensitivity and specificity for diagnosing CIID in HS patients. Concomitant CIID and HS may indicate the need for an early-start for biological treatment.

NOD2 Agonism Counter-Regulates Human Type 2 T Cell Functions in Peripheral Blood Mononuclear Cell Cultures: Implications for Atopic Dermatitis.

Atopic dermatitis (AD) is known as a skin disease; however, T cell immunopathology found in blood is associated with its severity. Skin Staphylococcus aureus (S. aureus) and associated host-pathogen dynamics are important to chronic T helper 2 (Th2)-dominated inflammation in AD, yet they remain poorly understood. This study sought to investigate the effects of S. aureus-derived molecules and skin alarmins on human peripheral blood mononuclear cells, specifically testing Th2-type cells, cytokines, and chemokines known to be associated with AD. We first show that six significantly elevated Th2-related chemokine biomarkers distinguish blood from adult AD patients compared to healthy controls ex vivo; in addition, TARC/CCL17, LDH, and PDGF-AA/AB correlated significantly with disease severity. We then demonstrate that these robust AD-associated biomarkers, as well as associated type 2 T cell functions, are readily reproduced from healthy blood mononuclear cells exposed to the alarmin TSLP and the S. aureus superantigen SEB in a human in vitro model, including IL-13, IL-5, and TARC secretion as well as OX-40-expressing activated memory T cells. We further show that the agonism of nucleotide-binding oligomerization domain-containing protein (NOD)2 inhibits this IL-13 secretion and memory Th2 and Tc2 cell functional activation while inducing significantly increased pSTAT3 and IL-6, both critical for Th17 cell responses. These findings identify NOD2 as a potential regulator of type 2 immune responses in humans and highlight its role as an endogenous inhibitor of pathogenic IL-13 that may open avenues for its therapeutic targeting in AD.