NOD2 mRNA Research Articles

Short-peptide-based enteral nutrition affects rats MDP translocation and protects against gut-lung injury via the PepT1-NOD2-beclin-1 pathway in vivo.

Peptide transporter 1 (PepT1) transports bacterial oligopeptide products and induces inflammation of the bowel. Nutritional peptides compete for the binding of intestinal bacterial products to PepT1. We investigated the mechanism of short-peptide-based enteral nutrition (SPEN) on the damage to the gut caused by the bacterial oligopeptide product muramyl dipeptide (MDP), which is transported by PepT1. The gut-lung axis is a shared mucosal immune system, and immune responses and disorders can affect the gut-respiratory relationship. Sprague-Dawley rats were gavaged with solutions containing MDP, MDP + SPEN, MDP + intact-protein-based enteral nutrition (IPEN), glucose as a control, or glucose with GSK669 (a NOD2 antagonist). Inflammation, mitochondrial damage, autophagy, and apoptosis were explored to determine the role of the PepT1-nucleotide-binding oligomerization domain-containing protein 2 (NOD2)-beclin-1 signaling pathway in the small intestinal mucosa. MDP and proinflammatory factors of lung tissue were explored to determine that MDP can migrate to lung tissue and cause inflammation. Induction of proinflammatory cell accumulation and intestinal damage in MDP gavage rats was associated with increased NOD2 and Beclin-1 mRNA expression. IL-6 and TNF-α expression and apoptosis were increased, and mitochondrial damage was severe, as indicated by increased mtDNA in the MDP group compared with controls. MDP levels and expression of proinflammatory factors in lung tissue increased in the MDP group compared with the control group. SPEN, but not IPEN, eliminated these impacts. Gavage of MDP to rats resulted in damage to the gut-lung axis. SPEN reverses the adverse effects of MDP. The PepT1-NOD2-beclin-1 pathway plays a role in small intestinal inflammation, mitochondrial damage, autophagy, and apoptosis.

Pattern‐Recognition Receptors and Cervical Microbiome in Patients with Early Miscarriages

Early miscarriages are one of the main causes of reproductive losses. The objective of this study was to estimate Toll‐ and NOD‐like receptors mRNA expression levels and their correlation with microbiome composition in the endocervix of patients with 1st trimester miscarriages (n = 15) compared to those with healthy ongoing pregnancies (n = 15). Samples of the cervical epithelium were collected using a cytobrush. The expression of Toll‐like receptors (TLR1, TLR2, TLR4, and TLR6) and NOD‐like receptors (NOD1, NOD2, NLRP1, and NLRC4) was estimated by quantitative reverse‐transcriptase PCR. Cervical microbiome composition was assessed by 16S rRNA next‐generation sequencing. We found no differences in community state types (CSTs) and alpha‐ and beta‐diversity between the groups. The dominant microorganisms in both groups were Lactobacillus iners. Bifidobacterium breve and Aeromonas popoffii, whose abundance was significantly higher in patients with miscarriages compared to those with ongoing pregnancies. Corynebacterium callunae and Turicibacter sanguinis, on the contrary, were significantly higher in women of the control group. Only decreased expression of TLR2 was observed in patients with miscarriages. The expression of other Toll‐ and NOD‐like receptors showed no significant differences between the groups. NOD1 mRNA expression had a strong positive correlation with the abundance of L. iners, L. delbrueckii, and Aerococcus christensenii; TLR6 mRNA expression had a strong positive correlation with L. iners. The findings suggest that variations in Toll‐ and NOD‐like receptors mRNA expression and specific shifts in the cervical microbiome might play a role in the immunological landscape, potentially contributing to the pathogenesis of early miscarriages.

NOD-like receptors in pathogenesis of missed and spontaneous abortions

Nucleotide-binding oligomerization domain-like receptors (NOD-like receptors) are cytosolic signaling receptors of innate immune cells recognizing ligands derived from bacteria, viruses, fungi and protozoa. They can initiate apoptosis and pro-inflammatory cytokines production. Meanwhile, the role of decidual NOD-like receptors in pathogenesis of early miscarriages remains unknown. Aim: to study NOD-like receptor (NOD1, NOD2, NLRP1, NLRP3, NLRC4) messenger ribonucleic acid (mRNA) expression in decidual tissue from patients with missedand spontaneous abortions compared to progressive pregnancy.Materials and Methods. NOD1, NOD2, NLRP1, NLRP3, NLRC4 and pathway protein receptorinteracting-serine/threonine-protein kinase 2 (RIP-2) mRNA expression in decidua from 34 patients with missed abortions (group I), 34 patients with spontaneous abortions (group II) and 57 women with progressive pregnancy admitted for artificial abortion (group III, control group) were analyzed by reverse transcription quantitative polymerase chain reaction (PCR) at gestational age of 6–10 weeks. Exclusion criteria were as follows: endocrine disorders, severe extragenital diseases, antiphospholipid syndrome, inherited thrombophilia, uterine malformations and fetal chromosomal abnormalities. Samples were collected by uterine abrasion. Results. It was found that mRNA expression of NOD2 was significantly higher in decidua from patients with missed and spontaneous abortions, whereas for RIP-2 (related to relevant signaling pathway) – in women with missed abortions. A moderate positive correlation between gestational age and mRNA expression for NOD2 (R = 0.48; p = 0.01) and RIP-2 (R = 0.41; p = 0.007) was observed in subjects with progressive pregnancy. In contrast, women with missed abortions showed a moderate negative correlation between body weight and mRNA expression for NOD2 (R = –0.46; p = 0.03) and RIP-2 (R = –0.51; p = 0.02) whereas spontaneous abortions was associated with moderate negative correlation between RIP-2 mRNA expression and body weight (R= –0.47; p=0.04) as well as body mass index (R= –0.48; p = 0.04) along with moderate positive correlation with age of menarche (R = 0.46; p = 0.04). However, compared with progressive pregnancy no significant differences were found in expression level form NOD1, NLRP1, NLRP3 and NLRC4 mRNA in decidua from patients with missed and spontaneous abortions. Conclusion. Elevated NOD2 mRNA expression was observed in decidua from patients with missed and spontaneous abortions compared to progressive pregnancy paralleled with upregulated RIP-2 mRNA expression in missed abortions. Finally, it was found that NOD1, NLRP1, NLRP3 and NLRC4 were not involved in pathogenesis of miscarriages.

Modulation of Nod-like Receptor Expression in the Thymus during Early Pregnancy in Ewes.

Nucleotide-binding oligomerization domain receptors (NOD-like receptors, NLRs) are involved in modulating the innate immune responses of the trophoblast and the placenta in normal pregnancy. The thymus participates in regulation of innate and adaptive immune responses. However, it is unclear whether expression of NLR is modulated in the maternal thymus during early pregnancy. In this study, thymuses were sampled at day 16 of the estrous cycle, and at days 13, 16 and 25 of gestation (n = 6 for each group) from ewes after slaughter. Different stages were chosen because the maternal thymus was under the different effects of interferon-tau and/or progesterone or not. RT-qPCR, Western blot and immunohistochemistry analysis were used to analyze the expression of the NLR family, including NOD1; NOD2; major histocompatibility complex class II transactivator (CIITA); NLR family apoptosis inhibitory protein (NAIP); nucleotide-binding oligomerization domain and Leucine-rich repeat and Pyrin domain containing protein 1 (NLRP1), NLRP3 and NLRP7. The results showed that expression level of NOD1 was changed with the pregnancy stages, and expression levels of NOD2, CIITA, NAIP, NLRP1, NLRP3 and NLRP7 mRNA and proteins were peaked at day 13 of pregnancy. The levels of NOD2 and CIITA were increased during early pregnancy. The stainings for NOD2 and NLRP7 proteins were located in epithelial reticular cells, capillaries and thymic corpuscles. In summary, pregnancy stages changed expression of NLR family in the maternal thymus, which may be related to the modulation of maternal thymic immune responses, and beneficial for normal pregnancy in sheep.

FTO promotes innate immunity by controlling NOD1 expression via m6A-YTHDF2 manner in teleost.

N6-methyladenosine (m6A), the most abundant internal mRNA modification in eukaryotes, plays a vital role in regulating innate immunity. However, its underlying mechanism remains largely unknown, especially in lower vertebrates. The results of the present study show that fat-mass- and obesity-associated protein (FTO), also known as a m6A demethylase, improved the innate immunity and prevented Siniperca chuatsi rhabdo virus and Vibrio anguillarum infection in miiuy croaker. Significantly, FTO-promoted immunity was dependent on its m6A demethylase activity. In terms of mechanism, NOD1 has abundant methylation modification in its CDS and 3'UTR regions, and FTO can reduce its methylation level, thus avoiding its degradation by YTHDF2. In summary, our results indicate that the FTO-mediated m6A modification in NOD1 mRNA promotes innate immunity by activating the NOD-like receptor pathway, which provides a molecular mechanism for the regulation of immune response via RNA methylation in teleost.

Early Pregnancy Modulates Expression of the Nod-like Receptor Family in Lymph Nodes of Ewes.

NOD receptors (NLRs) mediate adaptive immune responses and immune tolerance. Nevertheless, it is not clear if gestation modulates the NLR signaling pathway in lymph nodes of ewes. In this study, lymph nodes of ewes were collected at day 16 of the estrous cycle, and at days 13, 16 and 25 of gestation (n = 6 for each group). RT-qPCR, Western blot and immunohistochemistry analysis were used to analyze the expression of the NLR family, including NOD1, NOD2, CIITA, NAIP, NLRP1, NLRP3 and NLRP7. The data showed that early gestation enhanced expression of NOD1, CIITA, NLRP1, NLRP3 and NLRP7 mRNA, as well as proteins at day 16 of gestation, and the expression levels of NOD2, CIITA, NLRP1 and NLRP7 were higher at days 13 and 25 of gestation than day 16 of the estrous cycle. However, NOD1 expression was lower on days 13 and 25 of gestation compared to day 16 of the estrous cycle, and early gestation suppressed NAIP expression. In summary, early pregnancy modulated expression of the NLR family in ovine lymph nodes, which participates in immune regulation, and this modulation may be necessary for pregnancy establishment in ewes.

Borrelia burgdorferi is strong inducer of IFN-γ production by human primary NK cells.

Natural Killer (NK) cells belong to the innate lymphoid lineage and are highly present in the human skin. NK cells can produce a range of pro-inflammatory mediators, including cytokines and chemokines. The role of NK(-T) cells in the immune response towards Borrelia burgdorferi infection was studied. The production of interleukin (IL)-6, IL-1β and interferon-gamma (IFN-γ) by human primary peripheral blood mononuclear cells (PBMCs) exposed to B. burgdorferi was assessed. Interestingly, CD56+ (NK+NK-T) cells were the only cells within the PBMC-fraction that produced IFN-γ during the first 24h of stimulation. Within the NK(-T) cell fraction, NK cells seemed to be responsible for the IFN-γ production. Since it was previously demonstrated that both TLR2 and NOD2 receptors are involved in the recognition of B. burgdorferi, the expression of both TLR2 and NOD2 mRNA on NK cells was determined. In contrast to TLR2, NOD2 mRNA was upregulated on CD56+ (NK+NK-T) cells after Borrelia exposure. Finally, to unravel the mechanisms underlying erythema migrans (EM) development, crosstalk between CD56+ (NK+NK-T) cells and keratinocytes was investigated. CD56+ (NK+NK-T) cells activated by B. burgdorferi produced soluble mediators strongly inducing the expression of antimicrobial peptides, such as β-defensin-2 and psoriasin in human keratinocytes. In conclusion, CD56+ (NK+NK-T) cells produced IFN-γ shortly after exposure to B. burgdorferi and released soluble mediators that were able to activate keratinocytes. These observations underscore the important role of CD56+ (NK+NK-T) cells during early host defence when Borrelia burgdorferi enters the human skin during a tick bite.

METTL3 regulates LPS-induced inflammatory response via the NOD1 signaling pathway

N6-methyladenosine (m6A) is a prevalent mRNA modification that plays a crucial function in multiple biological processes. Methyltransferase-like 3 (METTL3), an m6A methyltransferase, is essential for the m6A modification. Recently, the effect of METTL3 on the immune response has been reported. However, the effect is unclear, and the results are contradictory. In the present study, the total m6A and the expression of METTL3 decreased in LPS-stimulated macrophages. METTL3 knockdown significantly upregulated expression of proinflammatory cytokines, including TNF-α, IL-6 and NO. RNA sequencing analysis showed that the upregulated genes were enriched in inflammation-related signaling pathways and that the NOD-like receptor signaling pathway might be the target molecules of METTL3. METTL3 depletion resulted in upregulation of the NOD1 pathway without impacting NOD2. Moreover, the increase in proinflammatory cytokines induced by METTL3 knockdown was reversed by blocking the NOD1 pathway using specific inhibitors. Mechanistically, METTL3 knockdown promoted the mRNA expression and stability of NOD1 and RIPK2, and the same results were detected in m6A-binding protein YTHDF1- or YTHDF2-silenced cells. All findings suggested that METTL3 depletion inhibits the degradation of NOD1 and RIPK2 mRNA mediated by YTHDF1 and YTHDF2, which upregulate the NOD1 pathway and subsequently promote the LPS-induced inflammatory response in macrophages.

Changes in immune genes expression, immune response, digestive enzymes -antioxidant status, and growth of catla (Catla catla) fed with Astragalus polysaccharides against edwardsiellosis disease

The effect of four level of Astragalus polysaccharides (APs) supplementation diets, (CD: control diet and three experiment diet (E), EA: 100 mg kg−1 APs; EB: 200 mg kg-1 APs; EC: 300 mg kg−1 APs) on growth, changes in haemato-biochemical parameters and metabolic-digestive enzymes, enhancement of antioxidant activity, innate-adaptive immune response, and cytokine gene expression were studied in catla (Catla catla) against Edwardsiella tarda. The healthy and challenged groups fed the CD displayed no mortality, while fish fed EA or EC revealed 10% mortality, but the mortality was only 5% in diet EB. Fish fed diet EB and EC revealed significantly better growth rates and high RBC count during the experimental period. Albumin and globulin levels were significant improved when fish were fed the diet EB and EC from weeks 6–8. The superoxide dismutase (SOD) was significant ameliorated by EB feeding from weeks 4–8. In contrast, serum myeloperoxidase (MPO), catalase (CAT), malondialdehyde (MDA)/lipid peroxidation (LPO), glutathione peroxidase (GPx), respiratory burst activity (RBA), bactericidal action (BCA), serum lysozyme activity (SLA), nitric oxide synthase (NOS), head kidney leukocytes response proliferation (HKLP), hemolytic action (HLA), hydrogen peroxides (H2O2), and immunoglobulin (Ig) were significantly improved from week 6–8. Groups fed the APs enriched diets had significant ameliorated interleukin (IL)-1β and interferon (IFN)-γ mRNA expression after 6 and 8 weeks of feeding. However, IL-10 and major histocompatibility complex (MHC)-1 mRNA expressions were significant enhanced in catla fed all APs diets on week 8. APs enriched diets revealed significant improved tumor necrosis factor (TNF)-α and TNF receptor-associated factor-6 (TRAF6) mRNA expression on week 4, but toll-like receptor-2 (TLR2) and TLR4 mRNA expression were significant enhanced by diet EB and EC after weeks 6 and 8. Similarly, the lysozyme (Lyz)-C and Lyz-G mRNA levels in the head kidney (HK) increased by APs feeding on weeks 6 and 8, whereas the EB diet, the expression of nucleotide binding oligomerization domain-1 (NOD1) was significantly improved on weeks 6 and 8, but NOD2 mRNA expression was only significant enhanced after 8 weeks of diet EB. By feeding healthy catla and E. tarda challenged fish fed diet EB, resulted in significantly increased growth, haemato-biochemical indices, metabolic-digestive enzymes, antioxidant activities, innate-adaptive immune responses, and cytokine gene expression mainly between 6 and 8 weeks.

NOD2 signalling in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is associated with dysregulated immune responses including altered expression of cytokines, chemokines, and antimicrobial peptides and proteins (AMPs). To evaluate the expression of nucleotide-binding oligomerization domain-containing (NOD)2 and related factors in HS skin samples and keratinocyte cultures. We performed real-time PCR for NOD2, receptor-interacting serine/threonine-protein kinase (RIP)2, cyclic amine resistance locus (CARL), skin-derived antileukoproteinase (SKALP)/elafin, human β-defensin (hBD)2, LL37, psoriasin and RNAse7 in lesional and nonlesional skin of 19 patients with HS and in keratinocyte cultures [unstimulated, muramyl dipeptide (MDP)-stimulated or Pam2CSK4 (Pam2)-stimulated] from and nonlesional skin. We observed significantly elevated mRNA expression for NOD2 (P < 0.01), hBD2 (P = 0.02), RNase7 (P < 0.001), psoriasin (P < 0.01) and SKALP/elafin (P = 0.02) in lesional compared with nonlesional skin. We found a significant correlation between NOD2 mRNA and hBD2 (r = 46; P = 0.04), psoriasin (r = 0.67; P < 0.01) and SKALP/elafin (r = 0.65; P < 0.01). In unstimulated, Pam2-stimulated and MDP-stimulated normal keratinocytes, NOD2, RIP2, CARL and SKALP/elafin expression significantly (P < 0.05) increased from 6 to 48 h, whereas in unstimulated, Pam2-stimulated and MDP-stimulated HS keratinocytes, RIP2, CARL and SKALP/elafin expression significantly (P < 0.05) declined from 6 to 48 h. mRNA expression of NOD2 (unstimulated, Pam2-stimulated, MDP-stimulated), CARL (unstimulated, Pam2-stimulated, MDP-stimulated) and SKALP/elafin (unstimulated, Pam2-stimulated) at 6 h was significantly increased in HS compared with normal keratinocytes. We have shown for the first time that NOD2 signalling is activated in HS and might contribute to the pathogenesis via induction of AMPs and activation of other pathways such as nuclear factor κB signalling.

Recognition of MDP and activation of NF-κB by cytosolic sensor NOD2 in Oreochromis niloticus

The NOD-like receptors (NLRs) are an important part of the innate immune system, capable of recognizing or sensing pathogens or cell homeostasis imbalance, and play an important role in pathogen removal and body protection. NOD2 is one of the important members in NLR family. In present study, NOD2 was cloned and identified from Nile tilapia (Oreochromis niloticus). The Nile tilapia NOD2 ORF was 2961 bp in length encoding a peptide of 986 amino acid residues with a typical structure of CARD-CARD-NACHT-LRRs. Phylogenetic and synteny analysis illustrated that NOD2 was highly conserved between different species. Tissue distribution of NOD2 mRNA showed that it was widely expressed and highly expressed in immune-related tissues (blood, head kidney, liver and spleen). Post Streptococcus agalactiae challenge, NOD2 mRNA expression levels were altered in immune organs or tissues (liver, head kidney, spleen and blood). Subcellular localization of Nile tilapia NOD2 illuminated that it was an intracellular PRR like mammalian counterparts, most of which was co-localized with mitochondria, and a few of which was co-localized with endosome and endoplasmic reticulum. In addition, Nile tilapia NOD2-mediated signaling pathway and ligand recognition were also studied, it illuminated that Nile tilapia NOD2 could recognize MDP intracellularly and activate NF-κB signaling pathway. In conclusion, this study indicated that the Nile tilapia NOD2 was conservative and had a significant role in innate immune defense against bacterial infection.

Recognition of DAP and activation of NF-κB by cytosolic sensor NOD1 in Oreochromis niloticus

As a lower vertebrate, the immune defense mechanism of fish mainly depends on the innate immune system. Nucleotide-binding oligomerization domain-like receptors (NLRs) are an important class of pattern recognition receptors in the innate immune system. In this study, NOD1 gene was cloned and characterized in Nile tilapia (Oreochromis niloticus). The ORF of Nile tilapia NOD1 gene was 2826 bp long and encoded 941 amino acid residues with a structure of CARD-NACHT-LRRs that was similar to the other counterparts in mammals and fishes. Phylogenetic and synteny analysis showed that NOD1 was conserved among different fishes and existed at least in the early stage of fish evolution. Expression pattern revealed that NOD1 mRNA was constitutively expressed in the tested tissues, while had high expression level in main immune organs and mucosal immune tissues (liver, head kidney, spleen, blood, gill, and intestine). Following Streptococcus agalactiae challenge, Nile tilapia NOD1 mRNA expression levels were altered in immune organs (liver, head kidney, spleen, blood), and the expression pattern was similar in liver, spleen and blood. Furthermore, the ligand recognition and signaling pathway of Nile tilapia NOD1 were also analyzed, it showed that NOD1 could recognize Tri-DAP intracellularly and activated NF-κB signaling pathway. In summary, our results indicated that the Nile tilapia NOD1 may play an important role in innate immune system and provided a basis for the functional study of NOD1 in teleost.

Expression of nucleotide-binding oligomerization domain 1 and 2 in oral lichen planus

Background/purposeOral lichen planus (OLP) is a chronic inflammatory disease of oral mucosa. The present study investigated the expression of nucleotide-binding oligomerization domain (NOD), a pivotal sensor protein of the innate immune system, in OLP. Materials and methodsOral mucosal biopsies were collected from 20 patients with OLP and 6 individuals with normal oral mucosa (NOM). The expression of NOD1 and NOD2 was determined using RT-PCR and immunohistochemistry in OLP and NOM samples. ResultsThe mRNA expression of NOD1 and NOD2 was significantly higher in the OLP group than in the NOM group. The protein expression of NOD1 was marginally upregulated in all mucosal layers in the OLP group compared with that of the NOM group; however, the differences were not significant. The expression of NOD2 was elevated in infiltrating lymphocytes of the submucosal layer in the OLP group compared with the NOM group, but was undetected in other inflammatory disease, inflammatory fibrous hyperplasia (IFH). This study revealed the upregulation of NOD2 mRNA and protein in the OLP group, but not in the NOM group. ConclusionThese findings suggest that NOD2 may play an important role in the pathogenesis of OLP and represents a new diagnostic and treatment target.

High-Concentrate Feeding to Dairy Cows Induces Apoptosis via the NOD1/Caspase-8 Pathway in Mammary Epithelial Cells

(1) Background: The effects of a high-concentrate (HC) diet in inducing mammary epithelial cell apoptosis in dairy cows via the NOD1/Caspase-8 pathway have never been investigated before the current study. (2) Methods: Twelve Holstein Frisian cows at mid-lactation were selected to conduct this research. The animals were randomly allocated to two groups (n = 6), and both groups received one of two diets: a low-concentrate (LC) (forage: concentrate 6:4) or a high-concentrate (HC) (forage: concentrate 4:6) diet. Furthermore, an enzyme activity assay, tunnel cell assay, RT-qPCR, western blotting, and an immunofluorescence antibody (IFA) assay were performed to elucidate the effect of an HC diet in the mammary gland of dairy cows. (3) Results: The tunnel cell assay revealed a significant number of apoptotic cells in HC group, and the concentration of Caspase-3, and Caspase-8 was higher in the HC group than in the LC group. NOD1, Rip-2, Caspase-3, Caspase-8, Caspase-9, and Bax mRNA expressions, and NOD1, Caspase-3, Caspase-8, and Bax protein expressions, in the HC group were markedly higher than those in the LC group. Furthermore, Bcl-2 mRNA and protein expressions were markedly decreased in the HC compared to those in the LC group. (4) Conclusions: A HC diet fed to dairy cows incites subacute ruminal acidosis (SARA), which increases the iE-DAP concentration and induces apoptosis in the mammary gland via the NOD1/Caspase-8 pathway.

Nod2 Protects the Gut From Experimental Colitis Spreading to Small Intestine.

Nucleotide oligomerization domain 2 [NOD2] mutations are key risk factors for Crohn's disease [CD]. NOD2 contributes to intestinal homeostasis by regulating innate and adaptive immunity together with intestinal epithelial function. However, the exact roles of NOD2 in CD and other NOD2-associated disorders remain poorly known. We initially observed that NOD2 expression was increased in epithelial cells away from inflamed areas in CD patients. To explore this finding, Nod2 mRNA expression, inflammation, and cytokines expression were examined in the small bowel of wild-type [WT], Nod2 knockout and Nod2 mutant mice after rectal instillation of 2,4,6-trinitrobenzene sulphonic acid [TNBS]. In WT mice, Nod2 upregulation upstream to rectal injury was associated with pro-inflammatory cytokine expression but no overt histological inflammatory lesions. Conversely, in Nod2-deficient mice the inflammation spread from colitis to ileum and duodenum. Nod2 protects the gut from colitis spreading to small intestine.

P5565S100A8/A9 serum levels as a diagnostic biomarker and its potential connection to NOD2-NLRP3 in patients with a recent onset of myocarditis

Abstract Background The alarmin S100A8/A9 has been shown to be of importance in several inflammatory cardiovascular disorders. We recently demonstrated the pivotal role of cardiac S100A8/A9 in human and experimental Coxsackievirus B3 (CVB3)-induced myocarditis (MC). Purpose We aimed to evaluate whether serum S100A8/A9 levels are a marker in patients with a recent onset of MC Methods Serum S100A8/A9, hsCRP, and NT pro-BNP levels were analyzed in patients with a recent onset of MC (≤30 days (d), n=29; ejection fraction (EF): 44.3%±13%), dilated cardiomyopathy patients with inflammation (DCMi: n=112; EF: 28.8%±12%) or without inflammation (DCM: n=58; EF: 26.7%±9%), and controls (co: n=25; EF: 68.5%±5%). Blood samples and endomyocardial biopsies (EMB) were collected at time point (T1). In a subgroup, S100A8/A9 serum levels and EMB were available at T1 (n=10) and follow-up (T2, n=10, mean follow-up 8 months). Results MC ≤30 d patients showed a 4.5-fold (p&lt;0.0001), 19.3-fold (p&lt;0.0001), and 4.0-fold (p&lt;0.0001) increase in S100A8/A9, hsCRP, NT pro-BNP levels vs co, respectively. S100A8/A9 levels correlated with the disease activity, displayed by EMB counts of inflammatory cells (CD3: r=0.464, p=0.0128, XY pairs=28, LFA-1: r=0.551, p=0.002, XY pairs=28, Mac-1: r=0.418, p=0.026, XY pairs=28), and the EF (r=0.545, p=0.0027, XY pairs=28). MC ≤30 d patients showed an association between serum S100A8/A9 levels and EMB S100A8 (r=0.482, p=0.060, XY pairs=16), S100A9 (r=0.441, p=0.088, XY pairs=16), nucleotide-binding oligomerization domain containing-protein 2 (NOD2, r=0.55, p=0.035, XY=15), and Nod-like receptor family, pyrin domain-containing 3 protein (NLRP3, r=0.52, p=0.048, XY=15) mRNA levels. Also EMB S100A8 and S100A9 mRNA levels showed a significant correlation with EMB NOD2 and NLRP3 mRNA expression. Serum S100A8/A9 levels were increased by 3.0-fold (p&lt;0.0001) and 1.8-fold (p=0.0005) in DCMi (n=112), and DCM (n=58) patients vs co, respectively. However, the S100A8/A9 levels of DCMi and DCM patients were 1.5-fold (p=0.07) and 2.5-fold (p&lt;0.0001) lower vs MC ≤30 d patients. ROC analyses of S100A8/A9 in MC ≤30 d provided a cut-off of 583 ng/ml with a specificity=92%, sensitivity=86.2%, a PPV=92.6%, a NPV=85.2%, and an AUC=0.934 vs co, which was superior to hsCRP (cut-off=5 mg/l): specificity=95.8%, sensitivity=58.6%, a PPV=94.4%, a NPV=65.7%, AUC=0.885. In the subgroup, S100A8/A9 levels decreased after heart failure medication (T1: 2454±1931 ng/ml vs T2: 934.4±552 ng/ml; p=0.002), reflected by a decrease of EMB inflammatory cells. Baseline serum S100A8/A9 levels predicted the change in EMB CD3 and Mac-1. Conclusions These results support an additional value for S100A8/A9 serum levels as a potential diagnostic biomarker and as a tool to monitor the course of the disease. We provide first evidence that S100A8/A9 is connected to the NOD2-NLRP3 pathway in these patients. Acknowledgement/Funding Novartis

Neovascular glaucoma regulation by arylsulfonyl indoline-benzamide (ASIB) through targeting NF-kB signalling pathway.

The present study investigated the effect of arylsulfonyl indoline-benzamide (ASIB) on neovascular glaucoma in the mice model in vivo. In the mice model of glaucoma, ASIB treatment significantly (P < 0.05) increased PDGF-B-positive cell count in the corneal tissues. ASIB treatment at 5, 10, 15 and 20mg/kg doses raised the level of PDGF-B mRNA in the mice cornea by 2.3-, 3.8-, 5.4- and 5.5-fold, respectively. Pre-treatment of the glaucoma mice with ASIB leads to inhibition of TNF-α and IL-6 production. In the glaucoma mice, treatment with ASIB leads to a marked decrease in the level of NOD2 mRNA and protein. ASIB treatment caused a significant decrease in the glaucoma-induced up-regulation of NF-κB p65 activation. The phosphorylation of NF-κB p65 was almost completely inhibited in the glaucoma mice on treatment with 15mg/kg dose of ASIB. ASIB exhibited inhibitory effect on glaucoma-induced inflammatory cytokine and oxidative factor damage in the mice. It caused up-regulation of PDGF expression and down-regulated NF-κB activation. Therefore, ASIB can be of therapeutic significance for neovascular glaucoma treatment. However, more studies need to be performed to fully understand the molecular mechanism of ASIB in glaucoma treatment.

NOD1 and NOD2 of the innate immune system is differently expressed in human clear cell renal cell carcinoma, corresponding healthy renal tissue, its vasculature and primary isolated renal tubular epithelial cells.

NOD1 and NOD2 (nucleotide-binding oligomerization domain)-receptors are intracellular receptors and belong to the family of pattern recognition receptors being present in both human and murine renal tubular cells. Besides, NOD1 has been proved to promote apoptosis, upon its overexpression. Hence, we aimed to investigate NOD1 and NOD2 expression in human clear cell renal cell carcinoma (ccRCC). Tumor and corresponding adjacent healthy tissues from 41 patients with histopathological diagnosis of ccRCC as well as primary isolated renal tubular epithelial cells (TECs) and tumor tissue from a murine xenograft model using CAKI-1 ccRCC cells were analyzed. NOD1 and NOD2 mRNA was constitutively expressed in both tumor and adjacent healthy renal tissue, with NOD1 being significantly lower and in contrast NOD2 significantly higher expressed in tumor tissue compared to healthy tissues. Immunohistochemically, NOD1 was located not only in the cytoplasm, but also in the nucleus in ccRCC tissue whereas NOD2 was solely localized in the cytoplasm in both human ccRCC as well as in the healthy tubular system. Focusing on the vasculature, NOD2 displayed broader expression than NOD1. In primary TECs as well as CAKI-1 cells NOD1 and NOD2 was constitutively expressed and increasable upon LPS stimulation. In the mouse xenograft model, human NOD1 mRNA was significantly higher expressed compared to NOD2. In contrast hereto, we observed a shift towards lower mouse NOD1 compared to NOD2 mRNA expression. In view of reduced apoptosis-associated NOD1 expression in ccRCC tissue opposed to higher expression of NOD2 in tumor vasculature, inducibility of NOD expression in TECs as well as the detected shift of NOD1 and NOD2 expression in the mouse xenograft model, modulation of NOD receptors might, therefore, provide a molecular therapeutic approach in ccRCC.

Gene Variants, mRNA and NOD1/2 Protein Levels in Tunisian Childhood Asthma.

Asthma is a common respiratory childhood disease that results from an interaction between genetic, environmental and immunologic factors. The implication of nucleotide-binding and oligomerization domain 1 and 2 (NOD1/CARD4, NOD2/CARD15) was highlighted in many inflammatory diseases. In this case-control study, we analyzed the association of three NOD2 polymorphisms and one NOD1 variant, in 338 Tunisian asthmatic children and 425 healthy Controls, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. We also assessed NOD1 and NOD2 mRNA and protein levels by qRT-PCR and ELISA techniques. The homozygous AA genotype of rs2075820 was a risk factor for asthma (OR 2.39). The influence of the E266K variant in the presence of the heterozygous AG genotype was higher in male than female groups. The homozygous AA genotype was a risk factor associated with asthma, for patients aged between 6 and 18years OR 2.39, IC95% (1.04-5.49) p < 0.01. The mRNA expression of NOD1, but not NOD2, was enhanced in asthma patients compared to Controls. We noted a significant difference between asthmatics and healthy controls in NOD1 protein expression (asthma patients : 31.18 ± 10.9pg/ml, Controls: 20.10 ± 2.58pg/ml; p < 0.001). The NOD1 rs2075820 variant was associated with a higher childhood asthma risk and the NOD1 expression at mRNA and protein levels was significantly increased in asthma patients.

Effect of Herb-Partitioned Moxibustion on Autophagy and Immune-Associated Gene Expression Profiles in a Rat Model of Crohn's Disease.

Objective To investigate the immune regulation mechanism of herb-partitioned moxibustion in rats with Crohn's disease (CD) focusing on autophagy. Methods Rats were randomly divided into normal (N) group, CD model (M) group, CD model with herb-partitioned moxibustion (MM) group, normal with herb-partitioned moxibustion (NM) group, CD model with mesalazine (western medicine, Med ) group, and normal saline (NS) group, with 10 rats in each group. The CD model rats were prepared by trinitrobenzene sulphonic expect for the N group and NM group. After the CD rats model were established, the rats in the MM and NM groups were treated with herb-partitioned moxibustion at Tianshu (ST25) and Qihai (CV6) acupoints once daily for 7 days, and rats in the Med and NS groups were respectively treated with mesalazine enteric coated tablet and normal saline once daily for 7 days. After intervention, hematoxylin-eosin staining was used to observe the histological changes of colon; RNA sequencing was used to observe the changes in autophagy- and immune-associated gene expression profiles. In addition, autophagy- and immune-associated cytokines and signaling pathways in CD rats were also screened. Results HPM significantly increased the body weight of CD rats (P<0.01) and improved the pathological injury of colon in CD rats (P<0.01). HPM also changed the expression of many autophagy- and immune-associated genes, especially downregulating the expression of autophagy-associated Nod2, Irgm genes as well as the receptor of immune-associated Il12b, Il22 (Il12rb1, Il22ra2) genes in the colon of CD rats. HPM also changed the enrichment levels of differentially expressed genes in the human T-cell leukemia virus type-1 infection pathway, the Epstein-Barr virus infection pathway, and the cell adhesion molecule pathway. In addition, the expression levels of Nod2, Irgm, IL-12b, and IL-22 mRNA were increased (all P< 0.01) in the M group compared to the N group, while the expression levels of Nod2, Irgm, IL-12b, and IL-22 mRNA were decreased (P<0.05 or P<0.01) in the MM and Med groups compared to the M group. Conclusion Herb-partitioned moxibustion may effectively attenuate intestinal inflammation and promote the repair of colon mucosal injury of CD rats through the regulation of autophagy- and immune-associated gene expression and signaling pathways.