Nucleotide-binding Oligomerization Domain Research Articles

Identifying functional dysregulation of NOD2 variant Q902K in patients with Yao syndrome

Background and objectivesThe study investigated the pathogenesis of Yao syndrome (YAOS), a rare systemic autoinflammatory disease associated with the nucleotide-binding oligomerization domain containing 2 (NOD2) gene variants.MethodsRNA sequencing analyses were used to detect transcriptomic profile changes. Immunoblot and immunohistochemistry were used to examine the NOD2-mediated inflammatory signaling pathways and ELISA was used to detect cytokines.ResultsTranscriptome analysis of YAOS revealed NOD-like receptor signaling pathway enrichment. Compared with HCs, P-RIP2, p-p65, p-p38, p-ERK, and p-JNK notably increased in PBMCs of a patient with YAOS. P-RIP2, p-p65, and p-p38 elevated in small intestinal mucosa tissues. P-p65 and p-p38 in synovial tissues from YAOS were higher than those in patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Serum interleukin (IL)-6 level along with tumor necrosis factor (TNF)-α and IL-6 secreted from PBMCs were markedly higher in patients with YAOS in comparison to healthy controls (HCs). The supernatants of synovial cells from a patient with YAOS showed substantially higher IL-1β and IL-6 levels than those of RA and OA. Canakinumab therapy of a Q902K heterozygous patient with YAOS resulted in notable clinical improvement.ConclusionOverproduction of pro-inflammatory cytokines and the hyperactivation of NOD2-mediated signaling pathways were found in the NOD2 variant Q902K patient with YAOS. NOD2-RIP2-MAPK pathway might play a pivotal role in the pathogenesis of YAOS. These results provide new perspectives for targeted therapies in YAOS.

Understanding a point mutation signature D54K in the caspase activation recruitment domain of NOD1 capitulating concerted immunity via atomistic simulation

Point mutation D54K in the human N-terminal caspase recruitment domain (CARD) of nucleotide-binding oligomerization domain −1 (NOD1) abrogates an imperative downstream interaction with receptor-interacting protein kinase (RIPK2) that entails combating bacterial infections and inflammatory dysfunction. Here, we addressed the molecular details concerning conformational changes and interaction patterns (monomeric-dimeric states) of D54K by signature-based molecular dynamics simulation. Initially, the sequence analysis prioritized D54K as a pathogenic mutation, among other variants, based on a sequence signature. Since the mutation is highly conserved, we derived the distant ortholog to predict the sequence and structural similarity between native and mutant. This analysis showed the utility of 33 communal core residues associated with structural-functional preservation and variations, concurrently served to infer the cryptic hotspots Cys39, Glu53, Asp54, Glu56, Ile57, Leu74, and Lys78 determining the inter helical fold forming homodimers for putative receptor interaction. Subsequently, the atomistic simulations with free energy (MM/PB(GB)SA) calculations predicted structural alteration that takes place in the N-terminal mutant CARD where coils changed to helices (45 α3- L4-α4-L6- α683) in contrast to native (45T2-L4-α4-L6-T483). Likewise, the C-terminal helices 93T1-α7105 connected to the loops distorted compared to native 93α6-L7105 may result in conformational misfolding that promotes functional regulation and activation. These structural perturbations of D54K possibly destabilize the flexible adaptation of critical homotypic NOD1CARD-CARDRIPK2 interactions (α4Asp42-Arg488α5 and α6Phe86-Lys471α4) is consistent with earlier experimental reports. Altogether, our findings unveil the conformational plasticity of mutation-dependent immunomodulatory response and may aid in functional validation exploring clinical investigation on CARD-regulated immunotherapies to prevent systemic infection and inflammation. Communicated by Ramaswamy H. Sarma

Anti-inflammatory roles of type I interferon signaling in the lung.

Excessive or persistent inflammation may have detrimental effects on lung structure and function. Currently, our understanding of conserved host mechanisms that control the inflammatory response remains incompletely understood. In this study, we investigated the role of type I interferon signaling in the inflammatory response against diverse clinically relevant stimuli. Using mice deficient in type I interferon signaling (IFNAR1-/-), we demonstrate that the absence of interferon signaling resulted in a robust and persistent inflammatory response against Pseudomonas aeruginosa, lipopolysaccharide, and chemotherapeutic agent bleomycin. The elevated inflammatory response in IFNAR1-/- mice was manifested as elevated myeloid cells, such as macrophages and neutrophils, in the bronchoalveolar lavage. The inflammatory cell response in the IFNAR1-/- mice persisted to 14 days and there is impaired recovery and fibrotic remodeling of the lung in IFNAR1-/- mice after bleomycin injury. In the Pseudomonas infection model, the elevated inflammatory cell response led to improved bacterial clearance in IFNAR1-/- mice, although there was similar lung injury and survival. We performed RNA sequencing of lung tissue in wild-type and IFNAR1-/- mice after LPS and bleomycin injury. Our unbiased analysis identified differentially expressed genes between IFNAR1-/- and wild-type mice, including previously unknown regulation of nucleotide-binding oligomerization domain (NOD)-like receptor signaling, retinoic acid-inducible gene-I (RIG-I) signaling, and necroptosis pathway by type I interferon signaling in both models. These data provide novel insights into the conserved anti-inflammatory mechanisms of the type I interferon signaling.NEW & NOTEWORTHY Type I interferons are known for their antiviral activities. In this study, we demonstrate a conserved anti-inflammatory role of type I interferon signaling against diverse stimuli in the lung. We show that exacerbated inflammatory response in the absence of type I interferon signaling has both acute and chronic consequences in the lung including structural changes.

Isoliquiritigenin inhibits NLRP3 inflammasome activation with CAPS mutations by suppressing caspase-1 activation and mutated NLRP3 aggregation.

The nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome contributes to the development of inflammatory diseases. Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory disease caused by NLRP3 gene mutations that results in excessive IL-1β production. We previously identified isoliquiritigenin (ILG), a component of Glycyrrhiza uralensis extracts, as a potent inhibitor of the NLRP3 inflammasome. Here, we aimed to investigate whether ILG inhibits the activation of NLRP3 inflammasome caused by NLRP3 gene mutations. We demonstrated that ILG significantly inhibited NLRP3 inflammasome-mediated lactate dehydrogenase (LDH) release and IL-1β production in two CAPS model THP-1 cell lines, NLRP3-D303N and NLRP3-L353P, in a dose-dependent manner. Interestingly, the NLRP3 inhibitor MCC950 inhibited LDH release and IL-1β production in NLRP3-D303N cells, but not in NLRP3-L353P cells. Western blotting and caspase-1 activity assays showed that ILG, as well as caspase inhibitors, including Z-VAD and YVAD, suppressed caspase-1 activation. Notably, ILG prevented cryo-sensitive foci formation of NLRP3 without affecting the levels of intracellular Ca2+. We concluded that ILG effectively prevents the constitutive activation of the inflammasome associated with NLRP3 gene mutations by inhibiting the aggregation of cryo-sensitive mutated NLRP3.

Hesperitin-Copper(II) Complex Regulates the NLRP3 Pathway and Attenuates Hyperuricemia and Renal Inflammation.

Hyperuricaemia (HUA) is a disorder of purine metabolism in the body. We previously synthesized a hesperitin (Hsp)-Cu(II) complex and found that the complex possessed strong uric acid (UA)-reducing activity in vitro. In this study we further explored the complex's UA-lowering and nephroprotective effects in vivo. A mouse with HUA was used to investigate the complex's hypouricemic and nephroprotective effects via biochemical analysis, RT-PCR, and Western blot. Hsp-Cu(II) complex markedly decreased the serum UA level and restored kidney tissue damage to normal in HUA mice. Meanwhile, the complex inhibited liver adenosine deaminase (ADA) and xanthine oxidase (XO) activities to reduce UA synthesis and modulated the protein expression of urate transporters to promote UA excretion. Hsp-Cu(II) treatment significantly suppressed oxidative stress and inflammatory in the kidney, reduced the contents of cytokines and inhibited the activation of the nucleotide-binding oligomerization domain (NOD)-like receptor thermal protein domain associated protein 3 (NLRP3) inflammatory pathway. Hsp-Cu(II) complex reduced serum UA and protected kidneys from renal inflammatory damage and oxidative stress by modulating the NLRP3 pathway. Hsp-Cu(II) complex may be a promising dietary supplement or nutraceutical for the therapy of hyperuricemia.

Targeting the NLRP3 inflammasome in psoriasis.

The NLRP3 inflammasome, a complex consisting of the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing protein 3, has emerged as a critical mediator of pathological inflammation and a significant therapeutic target for various inflammatory diseases. Psoriasis, a chronic inflammatory skin condition without a definitive cure, has shown promising results in animal models through the inhibition of the NLRP3 inflammasome. This review aims to explore the development of the NLRP3 inflammasome in psoriasis and the molecular mechanisms responsible for its inhibition by natural products and small molecules currently being developed for psoriasis treatment. Furthermore, we are examining clinical trials using agents that block the NLRP3 pathway for the treatment of psoriasis. This study is timely to provide a new perspective on managing psoriasis.

Poly-(ADP-ribose)polymerases inhibitionby olaparib attenuates activities of the NLRP3 inflammasome and of NF-κB in THP-1 monocytes.

Poly-(ADP-ribose) polymerases (PARPs) are a protein family that make ADP-ribose modifications on target genes and proteins. PARP family members contribute to the pathogenesis of chronic inflammatory diseases, including atherosclerosis, in which monocytes/macrophages play important roles. PARP inhibition is protective against atherosclerosis. However, the mechanisms by which PARP inhibition exerts this beneficial effect are not well understood. Here we show that in THP-1 monocytes, inhibition of PARP by olaparib attenuated oxidized low-density lipoprotein (oxLDL)-induced protein expressions of nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing-3 (NLRP3) inflammasome components: NLRP3, apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1. Consistent with this effect, olaparib decreased oxLDL-enhanced interleukin (IL)-1β and IL-18 protein expression. Olaparib also decreased the oxLDL-mediated increase in mitochondrial reactive oxygen species. Similar to the effects of the NLRP3 inhibitor, MCC950, olaparib attenuated oxLDL-induced adhesion of monocytes to cultured human umbilical vein endothelial cells and reduced foam cell formation. Furthermore, olaparib attenuated the oxLDL-mediated activation of nuclear factor (NF)-κB through the oxLDL-mediated increase in IκBα phosphorylation and assembly of NF-κB subunits, demonstrated by co-immunoprecipitation of IκBα with RelA/p50 and RelB/p52 subunits. Moreover, PARP inhibition decreased oxLDL-mediated protein expression of a NF-κB target gene, VCAM1, encoding vascular cell adhesion molecule-1. This finding indicates an important role for NF-κB activity in PARP-mediated activation of the NLRP3 inflammasome. Thus, PARP inhibition by olaparib attenuates NF-κB and NLRP3 inflammasome activities, lessening monocyte cell adhesion and macrophage foam cell formation. These inhibitory effects of olaparib on NLRP3 activity potentially protect against atherosclerosis.

Acacetin inhibits inflammation by blocking MAPK/NF-κB pathways and NLRP3 inflammasome activation.

Objective: Our preliminary research indicates that acacetin modulates the nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) inflammasome, providing protection against Alzheimer's Disease (AD) and cerebral ischemic reperfusion injury. The mechanisms of acacetin to inhibit the activation of the NLRP3 inflammasome remain fully elucidated. This study aims to investigate the effects and potential mechanisms of acacetin on various agonists induced NLRP3 inflammasome activation. Methods: A model for the NLRP3 inflammasome activation was established in mouse bone marrow-derived macrophages (BMDMs) using Monosodium Urate (MSU), Nigericin, Adenosine Triphosphate (ATP), and Pam3CSK4, separately. Western blot analysis (WB) was employed to detect Pro-caspase-1, Pro-Interleukin-1β (Pro-IL-1β) in cell lysates, and caspase-1, IL-1β in supernatants. Enzyme-Linked Immunosorbent Assay (ELISA) was used to measured the release of IL-1β, IL-18, and Tumor Necrosis Factor-alpha (TNF-α) in cell supernatants to assess the impact of acacetin on NLRP3 inflammasome activation. The lactate dehydrogenase (LDH) release was also assessed. The Nuclear Factor Kappa B (NF-κB) and Mitogen-Activated Protein Kinase (MAPK) signaling pathways related proteins were evaluated by WB, and NF-κB nuclear translocation was observed via laser scanning confocal microscopy (LSCM). Disuccinimidyl Suberate (DSS) cross-linking was employed to detect oligomerization of Apoptosis-associated Speck-like protein containing a Caspase Recruitment Domain (ASC), and LSCM was also used to observe Reactive Oxygen Species (ROS) production. Inductively Coupled Plasma (ICP) and N-(6-methoxyquinolyl) acetoethyl ester (MQAE) assays were utilized to determined the effects of acacetin on the efflux of potassium (K+) and chloride (Cl-) ions. Results: Acacetin inhibited NLRP3 inflammasome activation induced by various agonists, reducing the release of TNF-α, IL-1β, IL-18, and LDH. It suppressed the expression of Lipopolysaccharides (LPS)-activated Phosphorylated ERK (p-ERK), p-JNK, and p-p38, inhibited NF-κB p65 phosphorylation and nuclear translocation. Acacetin also reduced ROS production and inhibited ASC aggregation, thus suppressing NLRP3 inflammasome activation. Notably, acacetin did not affect K+ and Cl-ions efflux during the activation process. Conclusion: Acacetin shows inhibitory effects on both the priming and assembly processes of the NLRP3 inflammasome, positioning it as a promising new candidate for the treatment of NLRP3 inflammasome-related diseases.

Cryptotanshinone Inhibits Bladder Cancer Cell Malignant Progression in a Lipopolysaccharide-Induced Inflammatory Microenvironment through NLRP3 Inhibition.

Bladder cancer (BC) is one of the most common malignancies of the urogenital system. This study assessed the nucleotide-binding oligomerization domain and leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) in BC as well as the effects of cryptotanshinone on changes in BC malignant behaviors and NLRP3 expression under a lipopolysaccharide (LPS)-induced inflammatory microenvironment. BC tissue specimens from 62 patients were collected for immunohistochemical detection of NLRP3 protein. BC and normal urothelial cell lines were cultured for the detection of NLRP3 mRNA and protein. Then, BC cells were pretreated with LPS to mimic the inflammatory tumor microenvironment. Next, these cells were incubated with a low or high dose of cryptotanshinone to assess its effects on tumor cell malignant behaviors as well as transfected with NLRP3 cDNA to confirm the role of NLRP3 in BC cells in vitro. High NLRP3 expression was associated with larger tumor diameters (>2 cm), muscle invasion, and metastasis. The levels of NLRP3 mRNA and protein were greater in BC cells than in normal urothelial cells. LPS pretreatment significantly promoted NLRP3 and inflammatory cytokine expression in BC cells, and induced cell viability, migration, and invasion. However, cryptotanshinone was able to reduce the LPS-induced increase of NLRP3 and inflammatory cytokine expression as well as the BC cell malignant progression. NLRP3 overexpression using NLRP3 cDNA further promoted BC cell malignant progression after LPS stimulation and reversed cryptotanshinone-reduced LPS-induced BC cell malignant behaviors. NLRP3 might possess oncogenic activity in BC, and the antitumor activity of cryptotanshinone in BC in vitro might be related to its inhibition of NLRP3 expression.

Dietary tryptophan alleviates intestinal inflammation caused by long photoperiod via gut microbiota derived tryptophan metabolites-NLRP3 pathway in broiler chickens

Light pollution is a potential risk factor for intestinal health. Tryptophan plays an important role in the inhibition of intestinal inflammation. However, the mechanism of tryptophan in alleviating intestinal inflammation caused by long photoperiod is still unclear. This study investigated the anti-inflammatory effect of dietary tryptophan on intestinal inflammatory damage induced by long photoperiod and its potential mechanism in broiler chickens. We found that dietary tryptophan mitigated long photoperiod-induced intestinal tissue inflammatory damage and inhibited the activation of Nucleotide-Binding Oligomerization Domain, Leucine-Rich Repeat and Pyrin Domain-Containing 3 inflammasome. Moreover, dietary tryptophan significantly increased the relative abundance of Faecalibacterium, Enterococcus, and Lachnospiraceae_NC2004_group were significantly decreased the relative abundance of Ruminococcus_torques_group and norank_f_UCG-010 under the condition of long photoperiod (P < 0.05). The results of tryptophan targeted metabolomics show that tryptophan significantly increased indole-3-acetic acid (IAA) and indole-3 lactic acid (ILA), and significantly decreased xanthurenic acid (XA) under long photoperiod (P < 0.05). In conclusion, the results indicated that dietary tryptophan alleviates intestinal inflammatory damage caused by long photoperiod via the inhibition of Nucleotide-Binding Oligomerization Domain, Leucine-Rich Repeat and Pyrin Domain-Containing 3 inflammasome activation, which was mediated by tryptophan metabolites. Therefore, tryptophan supplementation could be a promising way to protect the intestine health under the condition of long photoperiod.

Homomultivalent Polymeric Nanotraps Disturb Lipid Metabolism Homeostasis and Tune Pyroptosis in Cancer.

Genetic manipulations and pharmaceutical interventions to disturb lipid metabolism homeostasis have emerged as an attractive approach for the management of cancer. However, the research on the utilization of bioactive materials to modulate lipid metabolism homeostasis remains constrained. In this study, heptakis (2,3,6-tri-O-methyl)-β-cyclodextrin (TMCD) is utilized to fabricate homomultivalent polymeric nanotraps, and surprisingly, its unprecedented ability to perturb lipid metabolism homeostasis and induce pyroptosis in tumor cells is found. Through modulation of the density of TMCD arrayed on the polymers, one top-performing nanotrap, PTMCD4, exhibits the most powerful cholesterol-trapping and depletion capacity, thus achieving prominent cytotoxicity toward different types of tumor cells and encouraging antitumor effects in vivo. The interactions between PTMCD4 and biomembranes of tumor cells effectively enable the reduction of cellular phosphatidylcholine and cholesterol levels, thus provoking damage to the biomembrane integrity and perturbation of lipid metabolism homeostasis. Additionally, the interplays between PTMCD4 and lysosomes also induce lysosomal stress, activate the nucleotide-binding oligomerization domain-like receptor protein 3 inflammasomes, and subsequently trigger tumor cell pyroptosis. To sum up, this study first introduces dendronized bioactive polymers to manipulate lipid metabolism and has shed light on another innovative insight for cancer therapy.

Role of biomarkers and molecular signaling pathways in acute lung injury.

Acute lung injury (ALI) is caused by bacterial, fungal, and viral infections. When pathogens invade the lungs, the immune system responds by producing cytokines, chemokines, and interferons to promote the infiltration of phagocytic cells, which are essential for pathogen clearance. Their excess production causes an overactive immune response and a pathological hyper-inflammatory state, which leads to ALI. Until now, there is no particular pharmaceutical treatment available for ALI despite known inflammatory mediators like neutrophil extracellular traps (NETs) and reactive oxygen species (ROS). Therefore, the primary objective of this review is to provide the clear overview on the mechanisms controlling NETs, ROS formation, and other relevant processes during the pathogenesis of ALI. In addition, we have discussed the significance of epithelial and endothelial damage indicators and several molecular signaling pathways associated with ALI. The literature review was done from Web of Science, Scopus, PubMed, and Google Scholar for ALI, NETs, ROS, inflammation, biomarkers, Toll- and nucleotide-binding oligomerization domain (NOD)-like receptors, alveolar damage, pro-inflammatory cytokines, and epithelial/endothelial damage alone or in combination. This review summarized the main clinical signs of ALI, including the regulation and distinct function of epithelial and endothelial biomarkers, NETs, ROS, and pattern recognition receptors (PRRs). However, no particular drugs including vaccine for ALI has been established. Furthermore, there is a lack of validated diagnostic tools and a poor predictive rationality of current therapeutic biomarkers. Hence, extensive and precise research is required to speed up the process of drug testing and development by the application of artificial intelligence technologies, structure-based drug design, in-silico approaches, and drug repurposing.

In vitro evaluation of the immunomodulatory and wakame assimilation properties of Lactiplantibacillus plantarum strains from swine milk.

The emergence and spread of antibiotic resistance threat forced to explore alternative strategies for improving the resistance to pathogens in livestock production. Probiotic lactic acid bacteria represent an alternative for this objective. In this study, seven Lactiplantibacillus plantarum strains from porcine colostrum and milk were isolated, identified and characterized in terms of their abilities to modulate immunity in porcine intestinal epithelial (PIE) cells. Then, two potential immunoregulatory strains were studied in terms of their ability to utilize and grow in wakame (Undaria pinnafida). Isolates were identified by 16S rRNA gene and evaluated by studying their interaction with PIE cells. The expressions of peptidoglycan recognition proteins (PGRPs), nucleotide-binding oligomerization domain (NODs), host defense peptides (pBD), and type I interferons (IFNs) were evaluated by RT-qPCR. The strain 4M4417 showed a remarkable capacity to differentially regulate the expression of PGRP1, PGRP3, NOD1, NOD2, and pBD1 in PIE cells. On the other hand, the strain 4M4326 was the most efficient to improve the expression of IFN-α and IFN-β in PIE cells challenged with poly (I:C). Both L. plantarum 4M4326 and 4M4417 were characterized in terms of their ability to utilize wakame. Results demonstrated that both strains efficiently grew in wakame-based broth. Our results suggest that L. planatrum 4M4326 and 4M4417 are interesting candidates to develop immunomodulatory feeds based on wakame utilization. These new immunosynbiotic feeds could help to reduce severity of intestinal infections and improve immune health status in pigs.

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.

Effect of up-regulation of micro-ribonucleic acid-22 and NLRP3 inhibition on viability of malignant melanoma cells

Purpose: To determine the effect of up-regulation of micro-ribonucleic acid-22 (miR-22) targeting nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) on the proliferation and invasiveness of malignant melanoma cells, as well as the underlying mechanisms.&#x0D; Methods: Mouse-derived melanoma B16 cells were subjected to thawing, sub-culture and transfection. The cells were assigned to groups (A1 - A5) in line with different transfections viz: A1 (miR-22 mimic overexpression-transfected), A2 (miR-22 inhibitor-transfected), A3 (miR-22 mimic+siNLRP3-transfected), A4 (miR-22 mimic NC sequence-transfected), and A5 (un-transfected).&#x0D; Results: The miR-22 expression was significantly up-regulated in A1, relative to A2, A3, A4, and A5 groups, but significantly lower, relative to A3, A4, and A5. The NLRP3 mRNA and protein levels were significantly lower in A1 than in other groups, but significantly up-regulated in A2, relative to A3, A4, and A5 (p &lt; 0.05). Cell proliferation rate and colony formation rate were significantly lower in A1 group but were significantly up-regulated in A2, relative to A3, A4, and A5.&#x0D; Conclusion: Targeting NLRP3 inhibition by up-regulating miR-22 expression level significantly reduces the proliferation, invasiveness, and matrix metalloproteinase levels of melanoma B16 cells, thereby reducing the occurrence and development of malignant melanoma. The results provide some useful reference data for the treatment of malignant melanoma at the gene level.

Mettl3‑mediated m6A RNA methylation regulates osteolysis induced by titanium particles.

Peri‑prosthetic osteolysis (PPO) induced by wear particles is considered the primary cause of titanium prosthesis failure and revision surgery. The specific molecular mechanisms involve titanium particles inducing multiple intracellular pathways, which impact disease prevention and the targeted therapy of PPO. Notably, N6‑methyladenosine (m6A) serves critical roles in epigenetic regulation, particularly in bone metabolism and inflammatory responses. Thus, the present study aimed to determine the role of RNA methylation in titanium particle‑induced osteolysis. Results of reverse transcription‑quantitative PCR (RT‑qPCR), western blotting, ELISA and RNA dot blot assays revealed that titanium particles induced osteogenic inhibition and proinflammatory responses, accompanied by the reduced expression of methyltransferase‑like (Mettl) 3, a key component of m6A methyltransferase. Specific lentiviruses vectors were employed for Mettl3 knockdown and overexpression experiments. RT‑qPCR, western blotting and ELISA revealed that the knockdown of Mettl3 induced osteogenic inhibition and proinflammatory responses comparable with that induced by titanium particle, while Mettl3 overexpression attenuated titanium particle‑induced cellular reactions. Methylated RNA immunoprecipitation‑qPCR results revealed that titanium particles mediated the methylation of two inhibitory molecules, namely Smad7 and SMAD specific E3 ubiquitin protein ligase 1, via Mettl3 in bone morphogenetic protein signaling, leading to osteogenic inhibition. Furthermore, titanium particles induced activation of the nucleotide binding oligomerization domain 1 signaling pathway through methylation regulation, and the subsequent activation of the MAPK and NF‑κB pathways. Collectively, the results of the present study indicated that titanium particles utilized Mettl3 as an upstream regulatory molecule to induce osteogenic inhibition and inflammatory responses. Thus, the present study may provide novel insights into potential therapeutic targets for aseptic loosening in titanium prostheses.

Regulation and functions of the NLRP3 inflammasome in RNA virus infection.

Virus infection is one of the greatest threats to human life and health. In response to viral infection, the host's innate immune system triggers an antiviral immune response mostly mediated by inflammatory processes. Among the many pathways involved, the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome has received wide attention in the context of viral infection. The NLRP3 inflammasome is an intracellular sensor composed of three components, including the innate immune receptor NLRP3, adaptor apoptosis-associated speck-like protein containing CARD (ASC), and the cysteine protease caspase-1. After being assembled, the NLRP3 inflammasome can trigger caspase-1 to induce gasdermin D (GSDMD)-dependent pyroptosis, promoting the maturation and secretion of proinflammatory cytokines such as interleukin-1 (IL-1β) and interleukin-18 (IL-18). Recent studies have revealed that a variety of viruses activate or inhibit the NLRP3 inflammasome via viral particles, proteins, and nucleic acids. In this review, we present a variety of regulatory mechanisms and functions of the NLRP3 inflammasome upon RNA viral infection and demonstrate multiple therapeutic strategies that target the NLRP3 inflammasome for anti-inflammatory effects in viral infection.

Autophagy dysfunction contributes to NLRP1 inflammasome-linked depressive-like behaviors in mice

BackgroundMajor depressive disorder (MDD) is a common but severe psychiatric illness characterized by depressive mood and diminished interest. Both nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 1 (NLRP1) inflammasome and autophagy have been reported to implicate in the pathological processes of depression. However, the mechanistic interplay between NLRP1 inflammasome, autophagy, and depression is still poorly known.MethodsAnimal model of depression was established by chronic social defeat stress (CSDS). Depressive-like behaviors were determined by social interaction test (SIT), sucrose preference test (SPT), open field test (OFT), forced swim test (FST), and tail-suspension test (TST). The protein expression levels of NLRP1 inflammasome complexes, pro-inflammatory cytokines, phosphorylated-phosphatidylinositol 3-kinase (p-PI3K)/PI3K, phosphorylated-AKT (p-AKT)/AKT, phosphorylated-mechanistic target of rapamycin (p-mTOR)/mTOR, brain-derived neurotrophic factor (BDNF), phosphorylated-tyrosine kinase receptor B (p-TrkB)/TrkB, Bcl-2-associated X protein (Bax)/B-cell lymphoma-2 (Bcl2) and cleaved cysteinyl aspartate-specific proteinase-3 (caspase-3) were examined by western blotting. The mRNA expression levels of pro-inflammatory cytokines were tested by quantitative real-time PCR. The interaction between proteins was detected by immunofluorescence and coimmunoprecipitation. Neuronal injury was assessed by Nissl staining. The autophagosomes were visualized by transmission electron microscopy. Nlrp1a knockdown was performed using an adeno-associated virus (AAV) vector containing Nlrp1a–shRNA–eGFP infusion.ResultsCSDS exposure caused a bidirectional change in hippocampal autophagy function, which was activated in the initial period but impaired at the later stage. In addition, CSDS exposure increased the expression levels of hippocampal NLRP1 inflammasome complexes, pro-inflammatory cytokines, p-PI3K, p-AKT and p-mTOR in a time-dependent manner. Interestingly, NLRP1 is immunoprecipitated with mTOR but not PI3K/AKT and CSDS exposure facilitated the immunoprecipitation between them. Hippocampal Nlrp1a knockdown inhibited the activity of PI3K/AKT/mTOR signaling, rescued the impaired autophagy and ameliorated depressive-like behavior induced by CSDS. In addition, rapamycin, an autophagy inducer, abolished NLRP1 inflammasome-driven inflammatory reactions, alleviated depressive-like behavior and exerted a neuroprotective effect.ConclusionsAutophagy dysfunction contributes to NLRP1 inflammasome-linked depressive-like behavior in mice and the regulation of autophagy could be a valuable therapeutic strategy for the management of depression.

Inter-relationships of galectin-3 and NLR family pyrin domain containing 3 inflammasomes with oral lichen planus: a preliminary cross-sectional in vitro study

BackgroundThe nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome has been reported to be highly expressed in oral lesions with the potential for malignant development such as oral lichen planus (OLP). And the NLRP3 inflammasome can be activated by galectin-3 (Gal-3) in immune-mediated chronic inflammatory diseases. This study aimed to explore the inter-relationships among Gal-3, NLRP3 inflammasome, and OLP.MethodsA cross-sectional analysis of oral biopsy specimens from 30 patients with Erosive OLP and 30 healthy controls was performed. Immunohistochemical staining was used to evaluate the expression of Gal-3 and NLRP3 inflammasome. Two-sample t-test and Pearson correlation test were applied to analyze the data.Results Erosive OLP patients had significantly higher Gal-3 levels compared with controls (p < 0.0001). A similar pattern emerged for NLRP3 inflammasome. In the overall sample, a positive correlation was observed between Gal-3 and NLRP3 (r = 0.92, p < 0.01).ConclusionsPatients with Erosive OLP lesions showed increased protein expression levels of Gal-3. A positive correlation was observed between Gal-3 and NLRP3 inflammasome.