Paeoniflorin ameliorates experimental colitis by inhibiting gram-positive bacteria-dependent MDP-NOD2 pathway.

Previous studies reported that antibiotics inhibit the growth of Gram-positive bacteria and alleviate ulcerative colitis (UC). But how Gram-positive bacteria are involved in the occurrence of inflammatory bowel disease (IBD) and which component of it causes inflammation remain unclear. This work aims to demonstrate that Gram-positive bacteria may be an underlying cause of experimental colitis in mice through the muramyl dipeptide (MDP)-nucleotide-binding oligomerization domain-containing protein-2 (NOD2) pathway and paeoniflorin inhibits the pathway above to alleviate experimental colitis. In this study, colitis mice were established by oral administration of 3% dextran sulfate sodium (DSS) and paeoniflorin (25, 50,100 mg/kg per day, ig) was administered to the mice for 10 days. Results shown that the abundance and the infiltration of Gram-positive bacteria in intestinal tissues increased in UC mice. Paeoniflorin treatment significantly alleviated DSS-induced experimental colitis mice, reduced the abundance of Gram-positive bacteria in feces and the infiltration of Gram-positive bacteria in intestinal tissues. Paeoniflorin also inhibited mRNA and protein expression of MDP-NOD2 pathway components and decreased the levels of related inflammatory cytokines. In vitro experiments showed that MDP strongly stimulated RAW264.7 cells to secrete tumor necrosis factor α (TNF-α), and induced translocation of nuclear factor-kappa B (NF-κB p65) from the cytoplasm to nucleus using immunofluorescence co-localization experiments. Overall, the results indicated that Gram-positive bacteria promote the occurrence of colitis via up-regulation of MDP-NOD2 pathway, and paeoniflorin is able to decrease the infiltration of Gram-positive bacteria in intestine and inhibit Gram-positive bacteria-dependent MDP-NOD2 pathway to alleviate mice colitis.

Screening of Cytotoxicity and Anti-Inflammatory Properties of Feijoa Extracts Using Genetically Modified Cell Models Targeting TLR2, TLR4 and NOD2 Pathways, and the Implication for Inflammatory Bowel Disease.

Feijoa has been increasingly studied in the recent decade, while investigations into its bioactivities including anti-inflammatory activity are lacking. In this article, the cytotoxicity and anti-inflammatory properties of feijoa extracts, from flesh, peel and whole fruit, from four cultivars namely APOLLO, UNIQUE, OPAL STAR and WIKI TU are presented. Three inflammatory pathways, Toll-like receptor 2 (TLR2), TLR4 and nucleotide-binding oligomerization domain-containing protein 2 (NOD2), were investigated using genetically modified cell models namely HEK-Blue™ hTLR2, HEK-Blue™ hTLR4, NOD2-WT and NOD2-G908R. Results show that feijoa peel extract induced higher cytotoxicity than flesh and whole fruit extracts, and the APOLLO cultivar was the most anti-inflammatory among the four tested cultivars. The anti-inflammatory activity of feijoa flesh was detected only through the TLR2 pathway, and the activity of feijoa peel and whole fruit was evident mainly through the TLR2 and NOD2 pathways. Most notably, feijoa anti-inflammatory activity was superior to ibuprofen particularly through the TLR2 pathway, with significantly lower secreted embryonic alkaline phosphatase IC50 concentrations (7.88, 12.81, 30.84 and 442.90 µg/mL for APOLLO flesh, peel, whole fruit extract and ibuprofen respectively). These findings indicate that feijoa has great potential to be used in the treatment and prevention of inflammation-related diseases including inflammatory bowel disease.

Discovery of Nanomolar Desmuramylpeptide Agonists of the Innate Immune Receptor Nucleotide-Binding Oligomerization Domain-Containing Protein 2 (NOD2) Possessing Immunostimulatory Properties.

Muramyl dipeptide (MDP), a fragment of bacterial peptidoglycan, has long been known as the smallest fragment possessing adjuvant activity, on the basis of its agonistic action on the nucleotide-binding oligomerization domain-containing protein 2 (NOD2). There is a pressing need for novel adjuvants, and NOD2 agonists provide an untapped source of potential candidates. Here, we report the design, synthesis, and characterization of a series of novel acyl tripeptides. A pivotal structural element for molecular recognition by NOD2 has been identified, culminating in the discovery of compound 9, the most potent desmuramylpeptide NOD2 agonist to date. Compound 9 augmented pro-inflammatory cytokine release from human peripheral blood mononuclear cells in synergy with lipopolysaccharide. Furthermore, it was able to induce ovalbumin-specific IgG titers in a mouse model of adjuvancy. These findings provide deeper insights into the structural requirements of desmuramylpeptides for NOD2-activation and highlight the potential use of NOD2 agonists as adjuvants for vaccines.

Nod2-mediated recognition of the microbiota is critical for mucosal adjuvant activity of cholera toxin.

Cholera toxin (CT) is a potent adjuvant for inducing mucosal immune responses. However, the mechanism by which CT induces adjuvant activity remains unclear. Here we show that the microbiota is critical for inducing antigen-specific IgG production after intranasal immunization. After mucosal vaccination with CT, both antibiotic-treated and germ-free (GF) mice had reduced amounts of antigen-specific IgG, smaller recall-stimulated cytokine responses, impaired follicular helper T (TFH) cell responses and reduced numbers of plasma cells. Recognition of symbiotic bacteria via the nucleotide-binding oligomerization domain containing 2 (Nod2) sensor in cells that express the integrin CD11c (encoded by Itgax) was required for the adjuvanticity of CT. Reconstitution of GF mice with a Nod2 agonist or monocolonization with Staphylococcus sciuri, which has high Nod2-stimulatory activity, was sufficient to promote robust CT adjuvant activity, whereas bacteria with low Nod2-stimulatory activity did not. Mechanistically, CT enhanced Nod2-mediated cytokine production in dendritic cells via intracellular cyclic AMP. These results show a role for the microbiota and the intracellular receptor Nod2 in promoting the mucosal adjuvant activity of CT.

Down-regulated NOD2 by immunosuppressants in peripheral blood cells in patients with SLE reduces the muramyl dipeptide-induced IL-10 production.

BACKGROUND: Pattern recognition receptors (PRRs) such as Toll-like receptors are aberrantly expressed of peripheral blood mononuclear cells (PBMCs) in systemic lupus erythematosus (SLE) patients, for playing immunopathological roles. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the expression and function of the PRR nucleotide-binding oligomerization domain (NOD2) in SLE. NOD2 expression in T, B lymphocytes, monocytes, myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) was assessed in SLE patients and healthy controls (HCs) using flow cytometric analysis. Ex vivo production of cytokines from PBMCs upon NOD2 agonist muramyl dipeptide (MDP) stimulation was assessed using Cytometric Bead Array. Over-expression of NOD2 in monocytes was observed in immunosuppressant naïve SLE patients, and was positively associated with longer disease duration. Immunosuppressive therapy was an independent explanatory variable for downregulating NOD2 expression in CD8+ T, monocytes, mDCs and pDCs. Ex vivo basal productions of cytokines (IL-6, IL-8 and IL-10) were significantly increased in immunosuppressant naïve patients and patients with active disease despite immunosuppressants compared with HCs. Upon MDP stimulaiton, relative induction (%) of cytokines (IL-1ß) from PBMC was significantly increased in immunosuppressant naïve patients with inactive disease, and patients with active disease despite immunosuppressant treatment compared with HCs. Immunosuppressant usage was associated with a decreased basal production and MDP induced relative induction (%) of IL-10 in patients with inactive disease compared with immunosuppressant naïve patients and HCs. CONCLUSIONS/SIGNIFICANCE: Bacterial exposure may increase the NOD2 expression in monocytes in immunosuppressant naïve SLE patients which can subsequently lead to aberrant activation of PBMCs to produce proinflammatory cytokines, implicating the innate immune response for extracellular pathogens in the immunopathological mechanisms in SLE. Immunosuppressant therapy may downregulate NOD2 expression in CD8+ T lymphocytes, monocytes, and DCs in SLE patients which subsequently IL-10 reduction, contributing towards the regulation of immunopathological mechanisms of SLE, at the expense of increasing risk of bacterial infection.

Proinflammatory effects of muramyldipeptide on human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Because human gingival fibroblasts (HGFs) are the predominant cells in periodontal tissues, we hypothesized that HGFs are contributed to receptors for components of bacteria. In this study, we focused on expression and function of nucleotide binding oligomerization domain 2 (NOD2) in HGFs, which is a mammalian cytosolic pathogen recognition molecule. MATERIAL AND METHODS: Expression of NOD2 in HGFs was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry. Production of interleukin (IL)-6, IL-8, cc chemokine ligand2, cxc chemokine ligand10 (CXCL10) and CXCL11 from HGFs was examined by enzyme-linked immunosorbent assay (ELISA). We used RT-PCR and immunohistochemistry to detect the NOD2 expression in human gingival tissues. RESULTS: We found clear NOD2 expression in HGFs. Upon stimulation with NOD2 agonist, muramyldipeptide (MDP), production of proinflammatory cytokines was enhanced. Moreover, MDP-induced production of proinflammatory cytokines was inhibited in a different manner by mitogen-activated protein kinase inhibitors and phosphatidylinositol 3-kinase inhibitor. Furthermore, MDP enhanced CXCL10 and CXCL11 productions by tumor necrosis factor-alpha (TNF-alpha)- or interferon-gamma (IFN-gamma)-stimulated HGFs, although MDP alone did not induce these chemokines. TNF-alpha and IFN-gamma increased NOD2 expression in HGFs. In addition, we detected NOD2 expression in mononuclear cells and HGFs in periodontally diseased tissues. CONCLUSION: These findings indicate that MDP which induces production of cytokines and chemokines from HGFs is related to the pathogenesis of periodontal disease.

Higher monocyte expression of TLR2 and TLR4, and enhanced pro-inflammatory synergy of TLR2 with NOD2 stimulation in sarcoidosis.

INTRODUCTION: Sarcoidosis is an inflammatory disease of unknown etiology. However, an infectious cause has been proposed suggesting a role for pattern-recognition receptors, such as Toll-like receptors (TLRs) and nucleotide-binding domain, leucin-rich repeat containing family proteins (NLRs), in the pathogenesis. OBJECTIVE: Our aim was to investigate whether differences in TLR2 and TLR4 expression, and the response to TLR2, TLR4, and NOD2 stimulation, are associated with sarcoidosis. MATERIALS AND METHODS: Blood mononuclear cells from sarcoidosis patients (n = 24) and healthy subjects (n = 19) were incubated with the TLR2 ligands PGN and Pam3CSK4, the TLR4 ligand LPS, the NOD2 ligand MDP, or medium alone. After 16 h, monocyte TLR2 and TLR4 expression and cytokine secretion, including TNFalpha, IL-1 beta, IL-6, IL-8, IL-10, and IL-12p70, were measured using flow cytometry and cytometric bead array. RESULTS: TLR2 and TLR4 expression at baseline was significantly higher in patients. Combined TLR2 and NOD2 stimulation induced a four-fold higher secretion of TNFalpha and a 13-fold higher secretion of IL-1 beta in patients. Additionally, there was a synergistic effect of TLR2 with NOD2 stimulation on induction of IL-1 beta in patients, whereas IL-10 was synergistically induced in healthy subjects. CONCLUSION: Increased TLR expression and enhanced secretion of pro-inflammatory cytokines after combined TLR2 and NOD2 stimulation may be related to the pathogenesis of sarcoidosis.

Synthesis of peptidoglycan fragments and evaluation of their biological activity.

The peptidoglycan (PG) bacterial cell wall glycoconjugate has been well known as a strong immunopotentiator. Partial structures of PG were chemically synthesized for elucidation of precise biological activities. Effective construction of distinct repeating glycans of PG was accomplished by the coupling of a key disaccharide glucosaminyl-beta(1-4)-muramic acid unit. Stereoselective glycosylation of disaccharide units was achieved by neighboring group participation of the N-Troc (Troc = 2,2,2-trichloroethoxycarbonyl) group and appropriate reactivity of N-Troc-glucosaminyl trichloroacetimidate. By using an efficient synthetic strategy, mono-, di-, tetra- and octasaccharide fragments of PG were synthesized in high yields. The biological activity of synthetic fragments of PG was evaluated by induction of tumor necrosis factor-alpha (TNF-alpha) from human monocytes, and toll-like receptor 2 (TLR2) and Nod2 dependencies by using transfected HEK293 cells, respectively. Here we reveal that TLR2 was not stimulated by the series of synthetic PG partial structures, whereas Nod2 recognizes the partial structures containing the MDP moiety.