Nucleotide Oligomerization Domain Proteins Research Articles

Inhibiting Peptidoglycan Hydrolase Alleviates MRSA Pneumonia Through Autolysin-Mediated MDP-NOD2 Pathway.

Methicillin-resistant Staphylococcus aureus (MRSA) is a cause of staph infection that is difficult to treat because of resistance to some antibiotics. A recent study indicated that diarylurea ZJ-2 is a novel antibacterial agent against multi-drug resistant Enterococcus faecium. In this work, we refined the bactericidal mechanism of ZJ-2 as a peptidoglycan (PG) hydrolase by affecting AtlA-mediated PG homeostasis. A wild-type strain (WT) and a mutant strain (ΔatlA) were used to investigate the effects of ZJ-2 on the cell wall, PG, and autolysin regulatory system by antimicrobial susceptibility testing, hemolytic toxin assay, microanalysis, autolysis assay, qRT-PCR, ELISA and mouse model of pneumonia. The results revealed that ZJ-2 down-regulated the expression of genes related to peptidoglycan hydrolase (PGH) (sprX, walR, atlA, and lytM), and reduced the levels of PG, muramyl dipeptide (MDP), cytokines, and hemolytic toxin, while ΔatlA interfered with the genes regulation and PG homeostasis. In the mouse MRSA pneumonia model, the same trend was observed in the nucleotide oligomerization domain protein 2 (NOD2) and relative proinflammatory factors. ZJ-2 may act as a novel inhibitor of PG hydrolyse, disrupting autolysin-mediated PG homeostasis, and reducing inflammation by down-regulating the MDP-NOD2 pathway.

Vimentin affects inflammation and neutrophil recruitment in airway epithelium during Streptococcus suis serotype 2 infection

Streptococcus suis serotype 2 (SS2) frequently colonizes the swine upper respiratory tract and can cause Streptococcal disease in swine with clinical manifestations of pneumonia, meningitis, and septicemia. Previously, we have shown that vimentin, a kind of intermediate filament protein, is involved in the penetration of SS2 through the tracheal epithelial barrier. The initiation of invasive disease is closely related to SS2-induced excessive local inflammation; however, the role of vimentin in airway epithelial inflammation remains unclear. Here, we show that vimentin deficient mice exhibit attenuated lung injury, diminished production of proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and the IL-8 homolog, keratinocyte-derived chemokine (KC), and substantially reduced neutrophils in the lungs following intranasal infection with SS2. We also found that swine tracheal epithelial cells (STEC) without vimentin show decreased transcription of IL-6, TNF-α, and IL-8. SS2 infection caused reassembly of vimentin in STEC, and pharmacological disruption of vimentin filaments prevented the transcription of those proinflammatory cytokines. Furthermore, deficiency of vimentin failed to increase the transcription of nucleotide oligomerization domain protein 2 (NOD2), which is known to interact with vimentin, and the phosphorylation of NF-κB protein p65. This study provides insights into how vimentin promotes excessive airway inflammation, thereby exacerbating airway injury and SS2-induced systemic infection.

Sodium butyrate attenuated iE-DAP induced inflammatory response in the mammary glands of dairy goats fed high-concentrate diet.

Long-term high-concentrate (HC) diet feeding increased bacterial endotoxins, which translocated into the mammary glands of dairy goats and induced inflammatory response. γ-d-Glutamyl-meso-diaminopimelic acid (iE-DAP), bacterial peptidoglycan component, triggered inflammatory response through activating nucleotide oligomerization domain protein 1 (NOD1) signaling pathway. While dietary supplemented with sodium butyrate (SB) relieved inflammatory response and improved animal health and production. To investigate the effects and the mechanisms of action of SB on the inflammatory response in the mammary glands of dairy goats fed HC diet, 12 Saanen dairy goats were randomly assigned into HC group and SB regulated (BHC) group. The results showed that SB supplementation attenuated ruminal pH decrease caused by HC diet in dairy goats resulting in a decrease of proinflammatory cytokines and iE-DAP plasma concentration and the mRNA expression of NOD1 and other inflammation-related genes. The protein levels of NOD1, NF-κB p65 and NF-κB pp65 were decreased by the SB supplementation. The expression of histone deacetylase 3 (HDAC3) was also inhibited by the SB supplementation. Meanwhile, the chromatin compaction ratios and DNA methylation levels of NOD1 and receptor-interacting protein 2 (RIP2) of BHC group were upregulated. Collectively, the SB supplementation mitigated the inflammatory response in the mammary glands of dairy goats during HC-induced subacute ruminal acidosis (SARA) by inhibiting the activation of the NOD1/NF-κB signaling pathway through the decrease of the iE-DAP concentration in the rumen fluid and plasma and HDAC3 expression. DNA methylation and chromatin remodeling also contributed to the anti-inflammatory effect of SB. © 2020 Society of Chemical Industry.

Microbiota Composition and Immune Responses During Campylobacter Jejuni Infection in Conventionally Colonized IL-10-/- Mice Lacking Nucleotide Oligomerization Domain 2.

Host immune responses are pivotal for combating enteropathogenic infections. We here assessed the impact of the innate receptor nucleotide oligomerization domain protein 2 (NOD2) in murine Campylobacter jejuni-infection. Conventionally colonized IL-10–/– mice lacking NOD2 and IL-10–/– controls were perorally challenged with C. jejuni strain 81-176 and displayed comparable pathogenic colonization of intestines until day 14 postinfection (p.i.). Whereas overall intestinal microbiota compositions were comparable in naive mice, NOD2–/– IL-10–/– mice exhibited less fecal bifidobacteria and lactobacilli than IL-10–/– counterparts after infection. Interestingly, NOD2–/– IL-10–/– mice were clinically more compromised during the early phase of infection, whereas, conversely, IL-10–/– animals exhibited more frequently bloody feces lateron. While colonic apoptotic cell and T lymphocyte numbers were comparable in either C. jejuni-infected mice, B lymphocytes were lower in the colon of infected NOD2–/– IL-10–/– mice versus controls. At day 14 p.i., colonic TNF and IL-23p19 mRNA levels were upregulated in NOD2–/– IL-10–/– mice only. Translocation rates of intestinal commensals to mesenteric lymphnodes and extra-intestinal compartments including liver and kidney were comparable, whereas viable bacteria were more frequently detected in spleens derived from IL-10–/– as compared to NOD2–/– IL-10–/– mice. In conclusion, NOD2 is involved during C. jejuni infection in conventionally colonized IL-10–/– mice in a time-dependent manner.

Receptor-Interacting Protein Kinase-2 Inhibition by CYLD Impairs Antibacterial Immune Responses in Macrophages.

Upon infection with intracellular bacteria, nucleotide oligomerization domain protein 2 recognizes bacterial muramyl dipeptide and binds, subsequently, to receptor-interacting serine/threonine kinase 2 (RIPK2), which activates immune responses via the nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) and extracellular signal-regulated kinase (ERK) pathways. Activation of RIPK2 depends on its K63 ubiquitination by E3 ligases, whereas the deubiquitinating enzyme A20 counter regulates RIPK2 activity by cleaving K63-polyubiquitin chains from RIPK2. Here, we newly identify the deubiquitinating enzyme CYLD as a new inhibitor of RIPK2. We show that CYLD binds to and removes K63-polyubiquitin chains from RIPK2 in Listeria monocytogenes (Lm) infected murine bone marrow-derived macrophages. CYLD-mediated K63 deubiquitination of RIPK2 resulted in an impaired activation of both NF-κB and ERK1/2 pathways, reduced production of proinflammatory cytokines interleukin-6 (IL-6), IL-12, anti-listerial reactive oxygen species (ROS) and nitric oxide (NO), and, finally, impaired pathogen control. In turn, RIPK2 inhibition by siRNA prevented activation of NF-κB and ERK1/2 and completely abolished the protective effect of CYLD deficiency with respect to the production of IL-6, NO, ROS, and pathogen control. Noteworthy, CYLD also inhibited autophagy of Listeria in a RIPK2-ERK1/2-dependent manner. The protective function of CYLD deficiency was dependent on interferon gamma (IFN-γ) prestimulation of infected macrophages. Interestingly, the reduced NF-κB activation in CYLD-expressing macrophages limited the protective effect of IFN-γ by reducing NF-κB-dependent signal transducers and activators of transcription-1 (STAT1) activation. Taken together, our study identifies CYLD as an important inhibitor of RIPK2-dependent antibacterial immune responses in macrophages.

22: NOD2 immunity protects against obesity-induced inflammation and insulin resistance

Aims Obesity is associated with immune cell infiltration and inflammation in metabolic tissues that contributes to insulin resistance. Pattern recognition receptors (PRRs) link obesity-induced inflammation and insulin resistance. Nucleotide oligomerization domain protein 2 (NOD2) is a PRR that detects muramyl dipeptide (MDP) present in the cell walls of all bacteria. NOD2 mutations are associated with inflammatory bowel disease, and NOD2 activation with MDP can ameliorate the inflammatory effects of experimental colitis in mice. Our lab has linked NOD2 immunity with metabolism and found that NOD2 deficiency exacerbates obesity-induced inflammation and insulin resistance. We sought to determine if NOD2 activation with MDP confers anti-inflammatory effects during acute bacterial or chronic dietary stress. We have found that MDP has anti-inflammatory and insulin sensitizing effects in mice during bacterial stress and diet-induced obesity. Methods Mice were fed a chow or high fat diet (HFD; 60% kcal from fat) for 5 weeks. MDP (100 ug) or LPS (0.2 mg/kg) was administered i.p. as indicated. Glucose and insulin tolerance tests, and hyperinsulinemic-euglycemic clamps were performed in 6 h-fasted conscious mice. Tissues were immediately collected and frozen in liquid nitrogen. Gene expression was determined by RT-PCR with TaqMan assays. Results Mice that received MDP prior to LPS challenge had increased glucose tolerance, reduced glucose-stimulated insulin secretion, and increased suppression of hepatic glucose production during hyperinsulinemic-euglycemic clamps. Mice that received MDP throughout a HFD were more glucose and insulin tolerant, experienced less hepatic insulin resistance during clamps, and had lower expression of many inflammatory markers in adipose and liver tissues. Conclusions NOD2 activation improves glucose homeostasis during acute bacterial stress and reduces inflammation and metabolic defects associated with diet-induced obesity.

NOD1 Activators Link Innate Immunity to Insulin Resistance

OBJECTIVEInsulin resistance associates with chronic inflammation, and participatory elements of the immune system are emerging. We hypothesized that bacterial elements acting on distinct intracellular pattern recognition receptors of the innate immune system, such as bacterial peptidoglycan (PGN) acting on nucleotide oligomerization domain (NOD) proteins, contribute to insulin resistance.RESEARCH DESIGN AND METHODSMetabolic and inflammatory properties were assessed in wild-type (WT) and NOD1/2−/− double knockout mice fed a high-fat diet (HFD) for 16 weeks. Insulin resistance was measured by hyperinsulinemic euglycemic clamps in mice injected with mimetics of meso-diaminopimelic acid–containing PGN or the minimal bioactive PGN motif, which activate NOD1 and NOD2, respectively. Systemic and tissue-specific inflammation was assessed using enzyme-linked immunosorbent assays in NOD ligand–injected mice. Cytokine secretion, glucose uptake, and insulin signaling were assessed in adipocytes and primary hepatocytes exposed to NOD ligands in vitro.RESULTSNOD1/2−/− mice were protected from HFD-induced inflammation, lipid accumulation, and peripheral insulin intolerance. Conversely, direct activation of NOD1 protein caused insulin resistance. NOD1 ligands induced peripheral and hepatic insulin resistance within 6 h in WT, but not NOD1−/−, mice. NOD2 ligands only modestly reduced peripheral glucose disposal. NOD1 ligand elicited minor changes in circulating proinflammatory mediators, yet caused adipose tissue inflammation and insulin resistance of muscle AS160 and liver FOXO1. Ex vivo, NOD1 ligand caused proinflammatory cytokine secretion and impaired insulin-stimulated glucose uptake directly in adipocytes. NOD1 ligand also caused inflammation and insulin resistance directly in primary hepatocytes from WT, but not NOD1−/−, mice.CONCLUSIONSWe identify NOD proteins as innate immune components that are involved in diet-induced inflammation and insulin intolerance. Acute activation of NOD proteins by mimetics of bacterial PGNs causes whole-body insulin resistance, bolstering the concept that innate immune responses to distinctive bacterial cues directly lead to insulin resistance. Hence, NOD1 is a plausible, new link between innate immunity and metabolism.

The Role of Intracellular Receptor NODs for Cytokine Production by Macrophages Infected withMycobacterium leprae

The nucleotide-oligomerization domain (NOD) proteins are members of the NOD-like receptor (NLR) family, which are intracellular and cytoplasmic receptors. We analyzed the role of NODs for cytokine production by macrophages infected with intracellular pathogen M. leprae, the causative agent of leprosy. Production of pro-inflammatory cytokines such as IL-1β and TNF-α was inhibited in the presence of cytochalasin D, an agent blocking phagocytosis, suggesting that intracellular signaling was, partially, required for macrophage activation to M. leprae infection. Next, we investigated the role of NOD1 and NOD2 proteins on NF-κB activation and cytokine expression. Treatment with M. leprae significantly increased NF-κB activation and expression of TNF-α and IL-1β in NOD1- and NOD2-transfected cells. Interestingly, their activation and expression were inhibited by cytochalasin D, suggesting that stimulation of NOD proteins may be associated with the enhancement of cytokine production in host to M. leprae.

Inhibition of RIP2's tyrosine kinase activity limits NOD2-driven cytokine responses

Upon intracellular bacterial exposure, the Crohn's disease and sarcoidosis susceptibility protein NOD2 (nucleotide oligomerization domain protein 2) binds to the protein kinase RIP2 (receptor-interacting protein 2) to coordinate NF-κB (nuclear factor κ B)-mediated cytokine responses. While RIP2 clearly has kinase activity, the function of its kinase domain has been enigmatic. Although originally classified as a serine-threonine kinase based on homology scans, we find that RIP2 also has tyrosine kinase activity. RIP2 undergoes autophosphorylation on Tyr 474 (Y474). This phosphorylation event is necessary for effective NOD2 signaling and does not occur in the presence of the most common Crohn's disease-associated NOD2 allele. Given this tyrosine kinase activity, a small-molecule inhibitor screen designed to identify pharmacologic agents that inhibit RIP2's tyrosine kinase activity was performed. At nanomolar concentrations, the EGFR (epidermal growth factor receptor) tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) were found to inhibit both RIP2 tyrosine phosphorylation and MDP (muramyl dipeptide)-induced cytokine release in a variety of NOD2 hyperactivation states. This effect is specific for RIP2 and does not depend on EGFR. The finding that RIP2 has tyrosine kinase activity and the finding that gefitinib and erlotinib, two agents already used clinically for cancer chemotherapy, can inhibit this activity suggest that RIP2's tyrosine kinase activity could be targeted specifically in the treatment of inflammatory diseases.

Toll-like receptor 4 gene polymorphism modulates phenotypic expression in patients with hereditary hemochromatosis

Clinical penetrance of hereditary hemochromatosis is highly variable. We hypothesized that it might be modified by factors involved in the cellular immune response, such as toll-like receptors (TLRs) or nucleotide oligomerization domain proteins (NODs). Clinical expression of hemochromatosis was assessed as a function of TLR4, TLR9, and NOD2 polymorphisms in 99 homozygous carriers of the HFE C282Y mutation with mild-to-severe iron overload. Thirteen (13%) of the 99 hemochromatosis patients were heterozygous for a TLR4 Asp299Gly polymorphism and 86 (87%) were TLR4 wild-type-only carriers. Clinical expression of hemochromatosis was observed more frequently in carriers of the TLR4 polymorphism (100%) than in TLR4 wild-type carriers (56%, P = 0.002). This was based on higher prevalences of liver disease (92 vs. 45%, P = 0.002) and arthropathy of metacarpophalangeal joints (69 vs. 35%, P = 0.018) in TLR4 polymorphism carriers. The finding was strengthened by the strong association of TLR4 polymorphism with liver fibrosis in the subgroup of 52 patients who underwent a liver biopsy (P = 0.011). The TLR4 polymorphism did, however, not correlate with body iron overload. The study results remained significant in multiple regression analyses after excluding possible confounding effects, such as age, sex, alcohol, or meat intake, and in the subgroup of 84 patients presenting as the first members of their families. TLR4 Asp299Gly polymorphism modulates clinical expression in patients with hereditary hemochromatosis. The polymorphism does not correlate with iron overload suggesting that TLR4 plays a role in an inflammatory process arising from toxic effects of iron accumulation.

Role of pattern-recognition receptors in cardiovascular health and disease

A role for PRRs (pattern-recognition receptors) in immune cell function is now well established. In macrophages and other immune cells, activation of TLRs (Toll-like receptors) and cytosolic NLRs [NOD (nucleotide oligomerization domain) proteins containing a leucine-rich repeat] results in the induction of genes and release of imunoregulator hormones including cytokines and NO (nitric oxide). In addition to immune cells, structural cells of the cardiovascular system including endothelial cells, vascular smooth muscle and cardiac myocytes express functional PRRs and sense PAMPs (pathogen-associated molecular patterns). Furthermore, bacteria and PAMPs activate the coagulation system and platelets. TLRs are now implicated in a range of cardiovascular diseases and syndromes including atherosclerosis and sepsis. Our group is working on the hypotheses that differences exist in how tissues of the cardiovascular system, including vessels, endothelium, heart and blood, sense pathogens compared with immune cells (principally macrophages) and that identifying such differences will reveal new therapeutic targets for the treatment of cardiovascular disease. We have identified examples of similarities and differences in how cardiovascular tissues and macrophages sense PAMPs. These findings will be discussed together with our interpretation of how this information may lead to new treatments.