Nucleotide Oligomerization Domain Research Articles

NLRP3 inflammasome: The rising star in cardiovascular diseases.

Cardiovascular diseases (CVDs) are the prevalent cause of mortality around the world. Activation of inflammasome contributes to the pathological progression of cardiovascular diseases, including atherosclerosis, abdominal aortic aneurysm, myocardial infarction, dilated cardiomyopathy, diabetic cardiomyopathy, heart failure, and calcific aortic valve disease. The nucleotide oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing protein 3 (NLRP3) inflammasome plays a critical role in the innate immune response, requiring priming and activation signals to provoke the inflammation. Evidence shows that NLRP3 inflammasome not only boosts the cleavage and release of IL-1 family cytokines, but also leads to a distinct cell programmed death: pyroptosis. The significance of NLRP3 inflammasome in the CVDs-related inflammation has been extensively explored. In this review, we summarized current understandings of the function of NLRP3 inflammasome in CVDs and discussed possible therapeutic options targeting the NLRP3 inflammasome.

Transcriptome Analysis of Immune Response against Streptococcus agalactiae Infection in the Nile Tilapia GIFT Strain

Streptococcus agalactiae (group B streptococcus, GBS), a broad-spectrum pathogen, causes great economic losses in fish aquaculture, especially the industry of tilapia. Until now, the knowledge of the immune response mechanism against S. agalactiae infection in tilapia has been limited. In the present study, the gill transcriptome of the tilapia from the GBS and the phosphate buffered saline (PBS) groups were sequenced. The transcriptomic analysis results presented the differentially expressed genes (DEGs) at different time points (DEGs number, 6 h: 2122, 9 h: 1851, 15 h: 1791, and 18 h: 2395) after GBS injection, and significantly enriched immune-related gene ontology (GO) terms such as the innate immune response. The significantly enriched immune pathways included the Toll-like receptor signaling pathway, the nucleotide oligomerization domain (NOD)-like receptor signaling pathway, the cytosolic-DNA sensing pathway, and the intestinal immune network for Immunoglobulin A (IgA) production. Most of the DEGs in Toll-like receptor signaling, NOD-like receptor signaling, and cytosolic-DNA sensing pathways presented upregulations at 18 h, which indicated that the innate immune pathways were activated. Two immune-related pathways (phagosome and cell adhesion molecules) were significantly enriched at all time points, suggesting that these two pathways might also play important roles in the immune response against the GBS infection. The results of HE staining showed that the gills of tilapia were damaged seriously at 9 h post-infection, which might be due to the possibility of pyroptosis resulting from the changes of DEGs in the NOD-like receptor signaling pathway. This study provided new insight into the mechanisms of gill damage in fish infected with S. agalactiae.

Epigallocatechin-3-gallate (EGCG) attenuates inflammatory responses and oxidative stress in lipopolysaccharide (LPS)-induced endometritis via silent information regulator transcript-1 (SIRT1)/nucleotide oligomerization domain (NOD)-like receptor pyrin domain-containing 3 (NLRP3) pathway.

The protective effects of epigallocatechin-3-gallate (EGCG) on lipopolysaccharide (LPS)-induced endometritis in vivo and in vitro will be explored in this study. The endometritis model was induced in female BALB/c mice uterus by perfusion with lipopolysaccharide (LPS) and EGCG were administered at 1 h before LPS induction. The primary bovine endometrial epithelial cells (BEECs) were treated with EGCG for 1 h before LPS stimulation. Uterine histopathological changes, myeloperoxidase (MPO) activity, inflammatory cytokine levels and oxidative stress markers were determined. The extent of Bax, Bcl-2, cleaved caspase-3, silent information regulator transcript-1 (SIRT1), nucleotide oligomerization domain (NOD)-like receptor pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein (ASC) and Caspase1 was detected by Western blot and real-time quantitative PCR assays. The results showed that EGCG significantly reversed the LPS-induced uterine histopathological changes, MPO activity, pro-inflammatory cytokine levels. Additionally, EGCG decreased oxidative stress and reduced cell apoptosis by upregulating SIRT1 expression, downregulating the NLRP3 inflammasome activation. These findings indicated that EGCG exerted its greatest protective effects by blocking inflammatory responses, lowering oxidative stress, and reducing apoptosis via the SIRT1/NLRP3, making its promising candidate treatment for endometritis.

Activation of the pattern recognition receptor NOD1 in periodontitis impairs the osteogenic capacity of human periodontal ligament stem cells via p38/MAPK signalling.

Nucleotide oligomerization domain receptor 1 (NOD1) mediates host recognition of pathogenic bacteria in periodontium. However, the specific role of NOD1 in regulating osteogenesis is unclear. Therefore, this study focused on the activation status of NOD1 in periodontitis and its effect on the osteogenic capacity of human periodontal ligament stem cells (hPDLSCs) as well as the underlying mechanism. Histological staining and Western blot were utilized to assess NOD1 expression in the periodontium of people with or without periodontitis. HPDLSCs were cultured under NOD1 agonist or antagonist treatment. Q-PCR and Western blot were employed to assess the expression of osteogenic marker genes and proteins. Alizarin red staining and alkaline phosphatase staining were used to determine the osteogenic capability of hPDLSCs. The activation of downstream signalling was determined and specific inhibitors were utilized to confirm the signalling pathway in NOD1-regulated osteogenesis. NOD1 expression is significantly elevated in periodontitis. With NOD1 activated by particular agonist tri-DAP, the osteogenic potential of hPDLSCs was impaired. NOD1 antagonist co-incubation partially restored the decreased osteogenesis in hPDLSCs. P38/MAPK was phosphorylated in tri-DAP-induced NOD1 activation. The inhibitor of p38 rescued the suppression of osteogenesis induced by tri-DAP in hPDLSCs. Our study revealed the expression status of NOD1 in periodontitis. Its activation greatly decreased the osteogenic capacity of hPDLSCs which was mediated by the phosphorylation of p38 downstream signalling.

Spatiotemporal evolution of pyroptosis and canonical inflammasome pathway in hSOD1G93A ALS mouse model

BackgroundEvidences indicate that inflammasome compounds participate in amyotrophic lateral sclerosis (ALS), a fatal progressive motoneuron degenerative disease. Researchers have observed the expressions of nucleotide oligomerization domain (NOD)-like receptor protein 3 (NLRP3) related inflammasome components in specific regions of the central nervous system in different ALS models, but the cellular spatiotemporal evolution of this canonical inflammasome pathway and pyroptosis during ALS progression are unclear.MethodsThe spinal cords of hSOD1G93A mice (ALS mice) and age-matched littermates (CON mice) were dissected at pre-symptomatic stage (60 d), early- symptomatic stage (95 d), symptomatic stage (108 d) and late-symptomatic stage (122 d) of the disease. By using Nissl staining, double immunofluorescence labelling, qRT-PCR or western blot, we detected morphology change and the expression, cellular location of GSDMD, NLRP3, caspase-1 and IL-1β in the ventral horn of lumbar spinal cords over the course of disease.ResultsNeural morphology changes and GSDMD+/NeuN+ double positive cells were observed in ventral horn from ALS mice even at 60 d of age, even though there were no changes of GSDMD mRNA and protein expressions at this stage compared with CON mice. With disease progression, compared with age-matched CON mice, increased expressions of GSDMD, NLRP3, activated caspase-1 and IL-1β were detected. Double immunofluorescence labeling revealed that NLRP3, caspase-1, IL-1β positive signals mainly localized in ventral horn neurons at pre- and early-symptomatic stages. From symptomatic stage to late-symptomatic stage, robust positive signals were co-expressed in reactive astrocytes and microglia.ConclusionsEarly activation of the canonical NLRP3 inflammasome induced pyroptosis in ventral horn neurons, which may participate in motor neuron degeneration and initiate neuroinflammatory processes during ALS progression.

It is not NOD2 — genetic and clinical risk factors for postoperative complications following ileocolic resection in Crohn’s disease

PurposeTo evaluate the role of the nucleotide oligomerization domain 2 (NOD2) mutation status and other risk factors for the incidence of postoperative complications after ileocolic resection for Crohn’s disease (CD).MethodsData of 138 patients consecutively undergoing ileocolic resection for CD at a tertiary academic referral center were retrospectively analyzed including single nucleotide polymorphism (SNP) data of the NOD2 gene. Uni- and multivariate regression analysis was performed to identify factors associated with increased risk of severe postoperative complications.ResultsFrom 114 patients (83%), the NOD2 mutation status was available. Of these, 60 (53%) had a NOD2 wildtype, whereas eleven (10%) were homozygous for the high risk p.Leu1007fsX1008 (rs2066847) variant. Major postoperative complications occurred in 28 patients (20%). Twenty-seven of these (96%) were intraabdominal septic complications such as anastomotic leakage or abscess. Male gender (P = 0.029; OR 3.052, the duration of CD (time [months] from initial diagnosis of CD to surgery; P = 0.001; OR 1.009), previous abdominal surgery for CD (P = 0.017; OR 3.49), and the presence of enteric fistulas (P = 0.023; OR 3.21) were identified as independent risk factors for major postoperative complications. Homozygosity for the NOD2 high-risk variant p.Leu1007fsX1008 did not show increased postoperative morbidity in the short and long-term outcome.ConclusionsWe could detect independent risk factors for major postoperative complications after ileocolic resection for Crohn’s disease. However, patients with the high-risk variant p.Leu1007fsX1008 of the NOD2 gene did not show increased postoperative morbidity.

Essential Role of the Innate Immune Adaptor RIP2 in the Response to Otitis Media.

Intracellular nucleotide binding and oligomerization domain (NOD) and Toll-like (TLR) receptors have emerged as pivotal sensors of infection. Both Nod1 and Nod2 contain a caspase activation and recruitment domain (CARD) that interacts with the adaptor protein RIP2 (receptor-interaction protein-2). This leads to ubiquitination of RIP2 and in turn to the activation of NFκB and MAPK transcription factors, to command the host defensive response against pathogenic infections. RIP2 is also activated by TLRs 2 and 4, although the mechanism of this activation is less. The role of RIP2 in otitis media (OM) pathogenesis has yet to be examined. Herein, we used in vivo animal models including C57BL/6 wild-type (WT) and RIP2−/− knockout mice inoculated in the middle ear (ME) with non-typeable Haemophilus influenzae (NTHi), a common human OM pathogen, to evaluate the expression of RIP2 and its signaling genes at the cellular level to determine the role of RIP2 in OM pathogenesis and recovery. The Nod1, Nod2, and Ripk2 genes are minimally expressed in the normal ME. However, they are strongly upregulated during acute OM, as are many genes related to RIP2 signaling. However, while signaling genes were expressed by various ME cell types, only mucosal epithelial and stromal cells expressed the NODs, RIP2, and signaling genes required for the activation of the host defensive response. Whereas WT mice clear ME bacteria and recover from OM within 5 days after infection, RIP2-deficient mice show persistent ME bacterial carriage and inflammation to at least 15 days. This includes significantly prolonged mucosal hyperplasia and ME leukocytic infiltration. Recruitment of macrophages is also delayed in comparison to WT mice. Thus, RIP2 is required to elicit a robust innate immune response that promotes bacterial clearance and increases host innate resistance. The results also identify the structural cells of the ME mucosa, as opposed to leukocytes, as the primary sites of NOD/RIP2 activity in the infected ME.

The SNPs of three immunity genes in striped catfish, Pangasianodon hypophthalmus are associated with resistance against infection of Aeromonas hydrophila

Three important immunity-related genes, NLRC3 (nucleotide-oligomerization domain (NOD)-like receptor subfamily C), IL4R (interleukin 4 receptor), and C4 (complement component 4), can respond to infection by Aeromonas hydrophila in the spleen of striped catfish, Pangasianodon hypophthalmus. However, their association with resistance against infection by A. hydrophila are unknown. In this study, the expression of NLRC3, IL4R, and C4 in heart, liver, spleen, head kidney, trunk kidney, and gill of striped catfish after A. hydrophila infection was determined using quantitative real-time PCR. Single nucleotide polymorphism (SNP) identification and genotyping were performed by Sanger sequencing. We then analyzed the correlation between these polymorphisms and resistance to A. hydrophila infection. NLRC3, IL4R, and C4 were strongly induced by A. hydrophila infection in the liver, spleen, trunk kidney, and gill that peaked in the liver at 12 h post-infection. Three SNPs from NLRC3, two SNPs from IL4R, and two SNPs from C4 were identified by direct sequencing, in which NLRC3-CM018541.1-359661A/T, NLRC3-CM018541.1-359672G/T, NLRC3-CM018541.1-359815G/C, IL4R-CM018563.1-17094599G > A, and C4-CM018554.1-20401796A > T were associated with the incidence of resistant and susceptible phenotypes in striped catfish. Additionally, three SNP loci (NLRC3-CM018541.1-359661A/T, NLRC3-CM018541.1-359672G/T, and NLRC3-CM018541.1-359815G/C) showed strong pairwise linkage disequilibrium, and the haplotype AGC generated with these three SNPs was associated with resistance to A. hydrophila in striped catfish. These findings suggest strongly a relationship between the NLRC3, IL4R, C4 polymorphisms and A. hydrophila resistance in striped catfish, and that the NLRC3 gene could be used as a candidate marker for genetic selection of A. hydrophila-resistant striped catfish.

Nucleotide oligomerization domain polymorphism confers no risk to Guillain-Barré syndrome.

Nucleotide oligomerization domain (NOD) proteins are cytoplasmic receptors that play important roles in host innate immune responses to pathogens by recognizing self or non-self-molecules and have been implicated in many autoimmune diseases including Guillain-Barré syndrome (GBS). The current study investigated whether NOD polymorphisms (NOD1-Glu266Lys, rs2075820, and NOD2- [Arg702Trp, rs2066844 and Gly908Arg, rs2066845]) contribute to ligand sensing and thus affect the susceptibility and/or severity of GBS. We determined single nucleotide polymorphisms (SNPs) of NOD gene (NOD1-Glu266Lys and NOD2-[Arg702Trp; Gly908Ar]) in 303 patients with GBS and 303 healthy controls from Bangladesh by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and Sanger sequencing. Genotypes and allele frequencies were compared by performing chi-squared or Fisher's exact test with Yates' continuity correction. Serology for Campylobacter jejuni and anti-GM1 antibodies were determined by enzyme-linked immunosorbent assay (ELISA) techniques. NOD variants (NOD1-Glu266Lys and NOD2- [Arg702Trp; Gly908Arg]) were not associated with susceptibility and severity of GBS when compared with healthy controls and mild or severe form of disease. Moreover, NOD2 polymorphisms showed wild-type NOD2 C2104 and NOD2 G2722, respectively, with homozygous Arg/Arg genotype of NOD2 (Arg702Trp) polymorphism and homozygous Gly/Gly genotype of NOD2 (Gly908Arg) for all study subjects in Bangladesh. Homogenous distribution of NOD1 genotypes was observed in patients with axonal and demyelinating form of GBS. NOD variants confer no risk to the susceptibility and severity of GBS. Moreover, NOD2 polymorphism is rare or absent in patients with GBS as well as in the healthy individuals of Bangladesh.

1322-P: Role of Macrophage NOD1 in Fat-Induced ß-Cell Dysfunction

In individuals with obesity, free fatty acids (FFA) are chronically raised resulting in ß-cell dysfunction, leading to type 2 diabetes (T2D) . Recent data implicate the role of inflammation in developing ß-cell dysfunction, which is in part mediated by nucleotide-oligomerization domain 1 (NOD1) , an intracellular pattern recognition receptor. NOD1 is activated by peptidoglycans of bacterial cell walls; however, in vitro studies have documented that NOD1 can also be activated by saturated FFA. Previous data from our lab have shown that palmitate-induced ß-cell dysfunction was prevented in whole body and ß-cell specific NOD1 null mice. We have seen earlier reports on increased macrophage infiltration in islets after elevation in circulating palmitate, and much higher expression of NOD1 in macrophages than ß-cells. Hence, we hypothesized that macrophage NOD1 receptors play a pivotal role in FFA-induced ß-cell dysfunction. We have generated myeloid specific NOD1 null mice to target macrophage NOD1 receptors by crossing NOD1 floxed mice with Lyz2 Cre+ mice. Both knockouts and their controls (floxed and Cre) were infused for 48 h with ethylpalmitate (hydrolyzed to palmitate and ethanol in plasma) , to achieve elevated circulating FFA in a non-toxic manner. Ethanol vehicle was infused as control treatment. To assess in vivo ß-cell function, hyperglycemic clamp was performed following the 48 h infusion, and the disposition index (DI) was calculated. Ethylpalmitate resulted in elevated plasma FFA compared to vehicle infusion. Ethylpalmitate caused marked reduction in DI in control mice, while knockout mice displayed no significant changes in DI between ethylpalmitate and vehicle infused groups. These results suggest that macrophage NOD1 plays a causal role in palmitate-induced ß-cell dysfunction in vivo. Overall, our data reveal NOD1 as a key factor and a potential therapeutic target for obesity-associated diabetes. Disclosure S.Rahman: None. J.Yung: None. Y.Zhang: None. A.Giacca: None.

A narrative review of research progress on the role of NLRP3 inflammasome in acne vulgaris.

Background and ObjectiveAcne vulgaris is a common skin disease around the world which affects the appearance of patients, as well as their physical and mental health. Cutibacterium acnes plays a vital role in the occurrence and development of acne vulgaris. Pattern recognition receptors (PRRs) are the first line of defense against external pathogens. The nucleotide oligomerization domain (NOD)-like receptor family pyrin containing 3 (NLRP3) inflammasome has recently been shown to contribute to the pathogenesis of acne vulgaris. The purpose of this review is to clarify the underlying mechanisms of NLRP3 inflammasome in the pathogenesis of acne vulgaris, and its potential as a therapeutic target for the condition.MethodsThe PubMed database was searched for relevant articles published in English between January 2003 to December 2021 using keywords “acne vulgaris”, “NLRP3 inflammasome”, and “Cutibacterium acnes”. The reference lists of retrieved articles were also reviewed to identify relevant articles.Key Content and FindingsCutibacterium acnes infection can lead to a series of inflammatory reactions and the production of inflammatory factors such as interleukin (IL)-1β. In vitro and in vivo studies demonstrated that the NLRP3 inflammasome plays essential roles in acne vulgaris. Further, innate immunity and adaptive immunity pervade the entire pathogenesis of acne vulgaris.ConclusionsThe NLRP3 inflammasome may be a potential therapeutic target for acne vulgaris. Future studies are needed to investigate the potential therapeutic effects of NLRP3 inhibitors on acne vulgaris.

Giardia lamblia regulates the production of proinflammatory cytokines through activating the NOD2–Rip2–ROS signaling pathway in mouse macrophages

Giardia lamblia is a zoonotic protozoan that causes the diarrheal illness giardiasis, with the highest prevalence reported in the tropics and subtropics. Giardia is currently the most frequently identified pathogen in waterborne outbreaks in the United States. Nucleotide oligomerization domain (NOD) 1 and NOD2, intracellular NOD-like receptors, recognize pathogens to induce proinflammatory and antimicrobial responses. However, the roles of NOD1 and NOD2 signaling in Giardia infection have not yet been investigated. In the present study, the activation of NOD1 and NOD2 signaling pathways and the production of proinflammatory cytokines, reactive oxygen species (ROS) and nitric oxide in mouse macrophages stimulated with G. lamblia or parasite excretory-secretory products (ESPs) were examined. The results showed that G. lamblia and ESPs activated NOD2 and its downstream adaptor protein kinase, Receptor-interacting protein 2 (Rip2), in mouse macrophages. Blocking NOD2-Rip2 signaling significantly reduced the production of ROS and subsequently decreased the phosphorylation of nuclear factor-κB p65 and extracellular signal-regulated kinase, which in turn inhibited the production of four proinflammatory cytokines, namely, interleukin (IL)-1β, IL-6, IL-12p40 and tumor necrosis factor-α. In summary, our results indicate that the NOD2-Rip2 signal, which is activated by G. lamblia, contributes to the production of proinflammatory cytokines and ROS in mouse macrophages.

Caspase-8 Promotes Pulmonary Hypertension by Activating Macrophage-Associated Inflammation and IL-1β (Interleukin 1β) Production.

Macrophages are involved in the pathogenesis of pulmonary arterial hypertension (PAH). Caspase-8, an apical component of cell death pathways, is significantly upregulated in macrophages of PAH animal models. However, its role in PAH remains unclear. Caspase-8 plays a critical role in regulating inflammatory responses via inflammasome activation, cell death, and cytokine induction. This study investigated the mechanism of regulation of IL-1β (interleukin 1β) activation in macrophages by caspase-8. A hypoxia + SU5416-induced PAH mouse model and monocrotaline-induced rat model of PAH were constructed and the role of caspase-8 was analyzed. Caspase-8 and cleaved-caspase-8 were significantly upregulated in the lung tissues of SU5416 and hypoxia-treated PAH mice and monocrotaline-treated rats. Pharmacological inhibition of caspase-8 alleviated PAH compared with wild-type mice, observed as a significant reduction in right ventricular systolic pressure, ratio of right ventricular wall to left ventricular wall plus ventricular septum, pulmonary vascular media thickness, and pulmonary vascular muscularization; caspase-8 ablated mice also showed significant remission. Mechanistically, increased proliferation of pulmonary arterial smooth muscle cellss is closely associated with activation of the NLRP3 (NOD [nucleotide oligomerization domain]-, LRR [leucine-rich repeat]-, and PYD [pyrin domain]-containing protein 3) inflammasome and the IL-1β signaling pathway. Although caspase-8 did not affect extracellular matrix synthesis, it promoted inflammatory cell infiltration and pulmonary arterial smooth muscle cell proliferation via NLRP3/IL-1β activation during the development stage of PAH. Taken together, our study suggests that macrophage-derived IL-1β via caspase-8-dependent canonical inflammasome is required for macrophages to play a pathogenic role in pulmonary perivascular inflammation.

Genome-wide identification and characterization of NLR genes in lamprey (Lethenteron reissneri) and their responses to lipopolysaccharide/poly(I:C) challenge

The nucleotide oligomerization domain (NOD)-like receptor (NLR) is a relatively conserved receptor family involved in natural immunity that plays a key role in the resistance to pathogen invasion and regulation of the innate immune response. Lethenteron reissneri (lamprey) is a representative species of existing ancient jawless vertebrates. Studies of the evolutionary relationship of immune system-related molecules in lampreys can provide an important reference for the origin and evolution of innate immunity. However, the characterization and evolutionary patterns of the NLR family remain unclear in the lamprey genome. Based on the genome database of L. reissneri, we identified nine NLR genes, characterized their functional domains and chromosomal positions, and constructed a network comprising the results of gene structure and gene-collinearity analyses. Comparative genomics studies suggest that Lr-NODa and Lr-NODb most likely share the common ancestor of NOD1 and NOD2 in jawed vertebrates, and that Lr-NODb may have been generated by lamprey-specific tandem duplication of Lr-NODa. Additionally, phylogenetic analysis of the NLRC subfamily suggests that Lr-NLRC3a has ancestral traits and may be derived from the common ancestor of another vertebrate NLRC subfamily. Further analysis of the formation of the NLRC subfamily has shown that exon shuffling, domain recombination, and chromosome rearrangement play important roles in its structural evolution. Furthermore, real-time quantitative polymerase chain reaction shows that most NLR genes in lamprey are highly expressed in the immune tissues of the heart, gill, and supraneural body, with these genes also showing significant responses to polyinosinic-polycytidylic acid infection. These results indicate that NLR genes are involved in the immune protection of L. reissneri and provide an important theoretical foundation for studies of the functional evolution of vertebrate NLRs involved in the innate immune system.

RIP2 Knockdown Attenuates Vascular Smooth Muscle Cells Activation via Negative Regulating Myocardin Expression.

RIP2 is an adaptor protein contributing to the activation of nuclear factor-κB induced by TNF receptor-associated factor (TRAF) and nucleotide oligomerization domain (NOD)-dependent signaling implicated in innate and adaptive immune response. Beyond regulation of immunity, we aimed to elucidate the role of RIP2 in vascular smooth muscle cell (VSMC) phenotypic modulation. In the current study, we observed that RIP2 showed an increased expression in VSMCs with PDGF-BB stimulation in a dose-dependent manner. Knockdown of RIP2 expression mediated by adenovirus dramatically accelerated the expression of VSMC-specific differentiation genes induced by PDGF-BB. Silencing of RIP2 inhibited proliferative and migratory ability of VSMCs. Additionally, we demonstrated that RIP2 knockdown can promoted myocardin expression. Furthermore, RIP2 inhibition also can attenuate the formation of intimal hyperplasia. These findings suggested that RIP2 played an important role in regulation of VSMCs differentiation, migration, and proliferation that may due to affect myocardin expression. Our results indicated that RIP2 may be a novel therapeutic target for intimal hyperplasia.

Peptidoglycan-based immunomodulation.

Peptidoglycan (PGN) is a unique component in the cytoderm of prokaryotes which can be recognized by different pathogen-associated molecular patterns (PAMPs) in eukaryotes, followed by a cascade of immune responses via different pathways. This review outlined the basic structure of PGN, its immunologic functions. The immunomodulation pathways mediated by PGN were elaborated. PGN induces specific immunity through stimulating different cytokine release and Th1/Th2-dominated immune responses during humoral/cellular immune response. The nonspecific immunity activation by PGN involves immunomodulation by different pattern recognition receptors (PRRs) including PGN recognition proteins (PGRPs), nucleotide oligomerization domain (NOD)-like receptors (NLRs), Toll-like receptors (TLRs), and C-type lectin receptors (CLRs). The sources and classification of PGRPs were summarized. In view of the stimulating activities of PGN and its monomers, the potential application of PGN as vaccine or adjuvant was prospected. This review provides systematic information on PGN functionalities from the point of immunoregulation, which might be useful in the deep exploitation of PGN.Key points. The immunological functions of PGN were illustrated. Cellular and humoral immunomodulation by PGN were outlined. The use of PGN as vaccine or adjuvant was prospected.

Probing Inflammasome Activation in Atherosclerosis.

Atherosclerosis is driven by chronic inflammation in all stages of the disease. Inflammation is fueled by elevated levels of pro-inflammatory cytokines. Interleukins (IL) are cytokines of particular importance in atherosclerosis, due to their key involvement in various pro-atherogenic processes, including infiltration of immune cells to the lesion, stimulation of the production of other pro-inflammatory mediators by other sources, and generation of lipid laden foam cells, all of which contribute to plaque development and progression. Various stimuli that are abundant in atherosclerotic plaques, including oxidized low-density lipoprotein, cholesterol crystals and reactive oxygen species can trigger inflammasome activation. Importantly, activation of the nucleotide oligomerization domain leucine-rich repeat and pyrin domain containing protein 3 (NLRP3) inflammasome activates the caspase-1 protease and results in the generation and release of potent pro-inflammatory cytokines, IL-1β and IL-18. Both cytokines are influential in driving chronic inflammation and atherogenesis. This chapter describes the use of enzyme-linked immunosorbent assay (ELISA) and Western blot to quantify these cytokines in cell supernatant and lysate respectively, after stimulating inflammasome activation in cultured cells.

Licochalcone B specifically inhibits the NLRP3 inflammasome by disrupting NEK7-NLRP3 interaction.

The activation of the nucleotide oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome is related to the pathogenesis of a wide range of inflammatory diseases, but drugs targeting the NLRP3 inflammasome are still scarce. In the present study, we demonstrated that Licochalcone B (LicoB), a main component of the traditional medicinal herb licorice, is a specific inhibitor of the NLRP3 inflammasome. LicoB inhibits the activation of the NLRP3 inflammasome in macrophages but has no effect on the activation of AIM2 or NLRC4 inflammasome. Mechanistically, LicoB directly binds to NEK7 and inhibits the interaction between NLRP3 and NEK7, thus suppressing NLRP3 inflammasome activation. Furthermore, LicoB exhibits protective effects in mouse models of NLRP3 inflammasome-mediated diseases, including lipopolysaccharide (LPS)-induced septic shock, MSU-induced peritonitis and non-alcoholic steatohepatitis (NASH). Our findings indicate that LicoB is a specific NLRP3 inhibitor and a promising candidate for treating NLRP3 inflammasome-related diseases.

Activation of RIPK2-mediated NOD1 signaling promotes proliferation and invasion of ovarian cancer cells via NF-κB pathway.

The goal of this study was to investigate the role and mechanism of action of nucleotide oligomerization domain receptor 1 (NOD1) in ovarian cancer. Results showed that the expressions of NOD1 and receptor interacting serine/threonine kinase 2 (RIPK2) were notably upregulated in non-metastatic and metastatic ovarian tumors compared with matched non-tumor tissues, and their expression in metastatic tumor tissues was higher than that in non-metastatic tumors. Overexpression of NOD1 facilitated the expression of proliferation-related proteins (PCNA and Ki67) and proliferation and invasion of ovarian cancer cells. Overexpression of NOD1 promoted NF-κB expression and phosphorylation. Importantly, NOD1 bound with RIPK2, and silencing of RIPK2 partly rescued the promotion of NOD1 to NF-κB expression and its phosphorylation. The promotion of NOD1 to ovarian cancer cell proliferation and invasion was partly reversed by RIPK2 silencing. Results from our in vivo study indicate that overexpression of NOD1 accelerated the growth of ovarian cancer tumors, expression of proliferation-related proteins, and activation of NF-κB. However, silencing of NOD1 suppressed tumor growth. In summary, NOD1 facilitates ovarian cancer progression by activating NF-κB signaling by binding to RIPK2. We suggest a new strategy for the treatment of ovarian cancer.

NOD2 Genotypes Affect the Symptoms and Mortality in the Porcine Circovirus 2-Spreading Pig Population.

The nucleotide oligomerization domain (NOD)-like receptor 2 (NOD2) is an intracellular pattern recognition receptor that detects components of peptidoglycans from bacterial cell walls. NOD2 regulates bowel microorganisms, provides resistance against infections such as diarrhea, and reduces the risk of inflammatory bowel diseases in humans and mice. We previously demonstrated that a specific porcine NOD2 polymorphism (NOD2-2197A > C) augments the recognition of peptidoglycan components. In this study, the relationships between porcine NOD2-2197A/C genotypes affecting molecular functions and symptoms in a porcine circovirus 2b (PCV2b)-spreading Duroc pig population were investigated. The NOD2 allele (NOD2-2197A) with reduced recognition of the peptidoglycan components augmented the mortality of pigs at the growing stage in the PCV2b-spreading population. Comparison of NOD2 allele frequencies in the piglets before and after invasion of PCV2b indicated that the ratio of NOD2-2197A decreased in the population after the PCV2b epidemic. This data indicated that functional differences caused by NOD2-2197 polymorphisms have a marked impact on pig health and livestock productivity. We suggest that NOD2-2197CC is a PCV2 disease resistant polymorphism, which is useful for selective breeding by reducing mortality and increasing productivity.