Nucleotide-binding Oligomerization Domain-containing Protein Research Articles

Profiling of Potential Anti-Diabetic Active Compounds in White Tea: An Integrated Study of Polyphenol-Targeted Metabolomics, Network Pharmacology, and Computer Simulation.

Diabetes remains a critical global public health challenge, posing a growing threat to human health and well-being. White tea is a lightly fermented tea and one of the six traditional tea categories in China. Owing to its rich content of bioactive compounds such as catechins and alkaloids, it has demonstrated potential anti-diabetic properties. However, its precise bioactive components, mechanisms of action, and relevant molecular targets require further investigation. In this study, an integrated approach combining polyphenol-targeted metabolomics, in vitro antioxidant assays, α-glucosidase inhibition tests, network pharmacology analysis, GEO database exploration, molecular docking, and molecular dynamics simulations was employed to identify the potential anti-diabetic compounds, targets, and mechanisms of white tea. The findings revealed that white tea is particularly abundant in 10 bioactive compounds, including epigallocatechin gallate, epicatechin gallate, and catechin, all of which exhibit significant anti-diabetic potential. These compounds were found to exert their effects by interacting with core molecular targets, namely cathepsin V (CTSV) and nucleotide-binding oligomerization domain-containing protein 1 (NOD1), and engaging in pathways related to signal transduction, apoptosis, and immune responses. This study establishes a strong theoretical basis for advancing white tea research and underscores new opportunities for applying natural products in diabetes therapy.

Linker Chemistry and Connectivity Fine-Tune the Immune Response and Kinetic Solubility of Conjugated NOD2/TLR7 Agonists.

There is a growing interest in developing novel immune potentiators capable of eliciting a cellular immune response. We tackle this challenge by harnessing the synergistic cross-activation between two innate immune receptors─the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and Toll-like receptor 7 (TLR7). Herein, we investigate the structure-activity relationship of a series of novel conjugated NOD2/TLR7 agonists incorporating a variety of flexible aliphatic, poly(ethylene glycol)-based and triazole-featuring linkers. Our findings reveal potent immune-enhancing properties of conjugates in human primary peripheral blood mononuclear cells, characterized by a Th1/Th17 polarized cytokine response. Importantly, we demonstrate that both the chemistry of the linker and the site of linkage affect the immune fingerprint and the kinetic solubility of these conjugated agonists. These results shed further light on the immunostimulatory potential of NOD2/TLR7 cross-activation and provide insights for designing innovative immune potentiators.

Systemic Multifunctional Nanovaccines for Potent Personalized Immunotherapy of Acute Myeloid Leukemia.

Hematological malignancies (HM) like acute myeloid leukemia (AML) are often intractable. Cancer vaccines possibly inducing robust and broad anti-tumor immune responses may be a promising treatment option for HM. Few effective vaccines against blood cancers are, however, developed to date partly owing to insufficient stimulation of dendritic cells (DCs) in the body and lacking appropriate tumor antigens (Ags). Here it is found that systemic multifunctional nanovaccines consisting of nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and Toll-like receptor 9 (TLR9) agonists - muramyl dipeptide (MDP) and CpG, and tumor cell lysate (TCL) as Ags (MCA-NV) induce potent and broad immunity against AML. MCA-NV show complementary stimulation of DCs and prime homing to lymphoid organs following systemic administration. Of note, in orthotopic AML mouse models, intravenous infusion of different vaccine formulations elicits substantially higher anti-AML efficacies than subcutaneous administration. Systemic MCA-NV cure 78% of AML mice and elicit long-term immune memory with 100% protection from rechallenging AML cells. Systemic MCA-NV can also serve as prophylactic vaccines against the same AML. These systemic nanovaccines utilizing patient TCL as Ags and dual adjuvants to elicit strong, durable, and broad immune responses can provide a personalized immunotherapeutic strategy against AML and other HM.

Distinctive Immune Signatures Driven by Structural Alterations in Desmuramylpeptide NOD2 Agonists.

Herein we report on the design, synthesis and biological evaluation of a series of nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) desmuramylpeptide agonists. The structural prerequisites that shape both physicochemical and immunomodulatory profiles of desmuramylpeptide NOD2 agonists have been delineated. Within this context, we identified 3, a butyrylated desmuramylpeptide, as a potent in vitro NOD2 agonist (EC50 = 4.6 nM), exhibiting an almost 17-fold enhancement in potency compared to its unsubstituted counterpart 1 (EC50 = 77.0 nM). The novel set of desmuramylpeptides demonstrate unique in vitro immunomodulatory activities. They elicited cytokine production in peripheral blood mononuclear cells (PBMCs), both alone and in conjunction with lipopolysaccharide (LPS). The spermine-decorated 32 also stimulated the LPS-induced cytotoxic activity (2.95-fold) of PBMCs against K562 cancer cells. Notably, the cholesterol-conjugate 26 displayed anti-inflammatory actions, highlighted by its capacity to convert the inflammatory monocyte subset into an anti-inflammatory phenotype. Finally, the eicosapentaenoylated derivative 23 augmented antigen presentation by mouse bone marrow-derived dendritic cells (BMDCs), thus highlighting its potential as a vaccine adjuvant.

Tocilizumab unfolds colo-protective and immunomodulatory effect in experimentally induced ulcerative colitis via mitigating autophagy and ER stress signaling

Ulcerative colitis (UC) is an idiopathic, chronic, relapsing inflammatory bowel disease (IBD), characterized by chronic inflammation of the gastrointestinal tract. The pathophysiology of UC is complicated and involves several factors including immune, genetic, and environmental factors. Recently, a huge amount of research has concentrated on the role of interleukins including interleukin-6 (IL-6) in its pathophysiology. Thus, this study aims to examine the colo-protective and immunomodulatory effect of Tocilizumab (TCZ) in an experimental model of dextran sulfate sodium (DSS) induced UC. In the current study, we analyzed the inflammatory, immunomodulatory, apoptotic, autophagy, and endoplasmic reticulum (ER) stress markers and other clinical features including stool consistency, rectal bleeding, and edema markers in rats. Our results showed that induction of colitis caused bloody diarrhea and increased IL-6 levels. Treatment with TCZ significantly ameliorated DSS-induced injury via decreasing inflammatory markers of colon injury (IL-6), signal transducer and activator of transcription-3 (STAT-3), and C-reactive protein (CRP). Furthermore, TCZ attenuated the apoptotic marker (caspase-3), and down-regulated endoplasmic reticulum stress sensor proteins (inositol- requiring transmembrane kinase endonuclease-1 (IRE-1) and activated transcription factor-6 (ATF-6)) and autophagy proteins (autophagy-related 16-like protein 1 (ATG16L1) and nucleotide-binding oligomerization domain-containing protein-2 (NOD2)), as compared to DSS group. Altogether, the current data suggest TCZ to be a promising protective therapy against UC.Graphical

Synergistic Targeting of Innate Receptors TLR7 and NOD2 for Therapeutic Intervention in Multiple Sclerosis.

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

Identification of Mycoplasma pneumoniae proteins interacting with NOD2 and their role in macrophage inflammatory response.

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

Commensal Bacillus pumilus SE5-Derived Peptidoglycan and Lipoteichoic Acid Showed Synergistic Effects in Improving Growth, Immunity, and Intestinal Health of Grouper (Epinephelus coioides).

Commensal-derived peptidoglycan (PG) or lipoteichoic acid (LTA) can improve the growth, immunity, and intestinal health of fish, but it is not clear whether the two components have synergistic effects. To clarify this, grouper (Epinephelus coioides) was fed basal diet (CG) or diets containing 1.0 × 108CFU/g heat-inactivated SE5 (HIB), PG (21.30mg/kg), LTA (6.70mg/kg), mixture (PL1) of PG (10.65mg/kg) and LTA (3.35mg/kg), and mixture (PL2) of PG (21.30mg/kg) and LTA (6.70mg/kg). Improved growth performance and feed utilization were observed in groups PG, LTA, PL1, and PL2, and the optimum growth performance was recorded in group PL1. Furthermore, improved serum alkaline phosphatase (AKP) activity and immunoglobulin M (IgM) and complement C3 (C3) contents were observed in all treatments, and the AKP activity in group PL1 was significantly superior to that of groups PG and LTA. Although PG and LTA alone or in combination exert comparable effects on intestinal microbiota and physical structure, obviously enhanced intestinal protease activity was observed in group PL1. The combined efficacy of PL1 could further potentiate the immune response by modulating the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and upregulating the expression of antimicrobial peptides (epinecidin-1, hepcidin-1, and β-defensin) as well as IgM. At the same time, group PL1 could further mitigate intestinal inflammation by downregulating pro-inflammatory cytokines and upregulating anti-inflammatory cytokines. In conclusion, probiotic B. pumilus SE5-derived PG and LTA mixture (10.65mg/kg PG and 3.35mg/kg LTA) exhibits better potential for improving the growth performance, intestinal health, and immune function compared to another mixture (21.30mg/kg PG and 6.70mg/kg LTA) and PG or LTA alone in grouper.

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.

Di-mannosylation enhances the adjuvant properties of adamantane-containing desmuramyl peptides in vivo.

Muramyl dipeptide (MDP) is the smallest essential peptidoglycan substructure capable of promoting both innate and adaptive immune responses. Herein, we report on the design, synthesis, and in vivo study of the adjuvant properties of two novel MDP analogs containing an achiral adamantyl moiety attached to the desmuramyl dipeptide (DMP) pharmacophore and additionally modified by one mannosyl subunit (derivative 7) or two mannosyl subunits (derivative 11). Mannose substructures were introduced in order to assess how the degree of mannosylation affects the immune response and nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) binding affinity, compared to the reference compound ManAdDMP. Both mannosylated MDP analogs showed improved immunomodulating properties, while the di-mannosylated derivative 11 displayed the highest, statistically significant increase in anti-OVA IgG production. In this study, for the first time, the di-mannosylated DMP derivative was synthesized and immunologically evaluated. Derivative 11 stimulates a Th-2-polarized type of immune reaction, similar to the reference compound ManAdDMP and MDP. Molecular dynamics (MD) simulations demonstrate that 11 has a higher NOD2 binding affinity than 7, indicating that introducing the second mannose significantly contributes to the binding affinity. Mannose interacts with key amino acid residues from the LRR hydrophobic pocket of the NOD2 receptor and loop 2.

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.