NOD2 Ligand Muramyl Dipeptide Research Articles

IL-1α and IL-1β promote NOD2-induced immune responses by enhancing MAPK signaling

Both toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) induce a tightly regulated inflammatory response at risk of causing tissue damage, depending on the effectiveness of ensuing negative feedback regulatory mechanisms. Cross-regulation between TLRs, NLRs, and cytokine receptors has been observed. However, the cross-regulation between interleukin-1 (IL-1) receptors and NOD2 is not completely understood. In this study, we found that IL-1α/β increased NOD2-induced inflammatory response in human monocytic THP1 cells, peripheral blood mononuclear cells (PBMCs), mouse macrophage RWA264.7 cells and spleen cells, and in an in vivo experiment. IL-1α/β pre-treatment induced the production of CXC chemokines, including growth-regulated oncogene (GRO)-α, GRO-β, and IL-8, and proinflammatory cytokines, including IL-1β, IL-6, and TNFα, which are induced by the activation of NOD2, in a dose- and time-dependent manner. However, pre-treatment with the NOD2 ligand muramyl dipeptide (MDP) did not up-regulate the expression of cytokines induced by IL-1α/β re-treatment. IL-1β treatment increased the expression of A20, which is an important inhibitor of the innate immune response. However, the overexpression of A20 failed to inhibit MDP-induced cytokine production, suggesting that A20 had no effects on the NOD2-induced immune response. In addition, IL-1α/β increased the expression of NOD2 and its downstream adaptor RIP2, and IL-1α/β pre-treatment increased MDP-induced activation of mitogen-activated protein kinases (MAPKs), including ERK, JNK, and P38, which contributed to MDP-induced cytokine production. Based on these results, IL-1α/β promote the NOD2-induced immune responses by enhancing MDP-induced activation of MAPK signaling pathways.

Dual Synthetic Peptide Conjugate Vaccine Simultaneously Triggers TLR2 and NOD2 and Activates Human Dendritic Cells.

Simultaneous triggering of Toll-like receptors (TLRs) and NOD-like receptors (NLRs) has previously been shown to synergistically activate monocytes, dendritic cells, and macrophages. We applied these properties in a T-cell vaccine setting by conjugating the NOD2-ligand muramyl-dipeptide (MDP) and TLR2-ligand Pam3CSK4 to a synthetic peptide derived from a model antigen. Stimulation of human DCs with the MDP-peptide-Pam3CSK4 conjugate led to a strongly increased secretion of pro-inflammatory and Th1-type cytokines and chemokines. We further show that the conjugated ligands retain their ability to trigger their respective receptors, while even improving NOD2-triggering. Also, activation of murine DCs was enhanced by the dual triggering, ultimately leading to effective induction of vaccine-specific T cells expressing IFNγ, IL-2, and TNFα. Together, these data indicate that the dual MDP-SLP-Pam3CSK4 conjugate constitutes a chemically well-defined vaccine approach that holds promise for the use in the treatment of virus infections and cancer.

Microbial Host Interactions and Impaired Wound Healing in Mice and Humans: Defining a Role for BD14 and NOD2.

Chronic wounds cause significant patient morbidity and mortality. A key factor in their etiology is microbial infection, yet skin host-microbiota interactions during wound repair remain poorly understood. Microbiome profiles of noninfected human chronic wounds are associated with subsequent healing outcome. Furthermore, poor clinical healing outcome was associated with increased local expression of the pattern recognition receptor NOD2. To investigate NOD2 function in the context of cutaneous healing, we treated mice with the NOD2 ligand muramyl dipeptide and analyzed wound repair parameters and expression of antimicrobial peptides. Muramyl dipeptide treatment of littermate controls significantly delayed wound repair associated with reduced re-epithelialization, heightened inflammation, and up-regulation of murine β-defensins 1, 3, and particularly 14. We postulated that although murine β-defensin 14 might affect local skin microbial communities, it may further affect other healing parameters. Indeed, exogenously administered murine β-defensin 14 directly delayed mouse primary keratinocyte scratch wound closure invitro. To further explore the role of murine β-defensin 14 in wound repair, we used Defb14-/- mice and showed they had a global delay in healing invivo, associated with alterations in wound microbiota. Taken together, these studies suggest a key role for NOD2-mediated regulation of local skin microbiota, which in turn affects chronic wound etiology.

Nod2 is required for the early innate immune clearance of Acinetobacter baumannii from the lungs

Acinetobacter baumannii (A. baumannii) is a significant cause of severe nosocomial pneumonia in immunocompromised individuals world-wide. With limited treatment options available, a better understanding of host immnity to A. baumannii infection is critical to devise alternative control strategies. Our previous study has identified that intracellular Nod1/Nod2 signaling pathway is required for the immune control of A. baumannii in airway epithelial cells in vitro. In the current study, using Nod2−/− mice and an in vivo sublethal model of pulmonary infection, we show that Nod2 contributes to the early lung defense against A. baumannii infection through reactive oxygen species (ROS)/reactive nitrogen species (RNS) production as Nod2−/− mice showed significantly reduced production of ROS/RNS in the lungs following A. baumannii infection. Consistent with the higher bacterial load, A. baumannii-induced neutrophil recruitment, cytokine/chemokine response and lung pathology was also exacerbated in Nod2−/− mice at early time points post-infection. Finally, we show that administration of Nod2 ligand muramyl dipeptide (MDP) prior to infection protected the wild- type mice from A. baumannii pulmonary challenge. Collectively, Nod2 is an important player in the early lung immunity against A. baumannii and modulating Nod2 pathway could be considered as a viable therapeutic strategy to control A. baumannii pulmonary infection.

Corrigendum to "Epicutaneous immunization with protein antigen TNP-Ig and NOD2 ligand muramyl dipeptide (MDP) reverses skin-induced suppression of contact hypersensitivity" [Pharmacol. Rep. 66 (2014) 137-142

Corrigendum to "Epicutaneous immunization with protein antigen TNP-Ig and NOD2 ligand muramyl dipeptide (MDP) reverses skin-induced suppression of contact hypersensitivity" [Pharmacol. Rep. 66 (2014) 137-142

Neutrophils counteract autophagy-mediated anti-inflammatory mechanisms in alveolar macrophage: role in posthemorrhagic shock acute lung inflammation.

Acute lung injury (ALI) is a major component of multiple organ dysfunction syndrome after hemorrhagic shock (HS) resulting from major surgery and trauma. The increased susceptibility in HS patients to the development of ALI suggests not yet fully elucidated mechanisms that enhance proinflammatory responses and/or suppress anti-inflammatory responses in the lung. Alveolar macrophages (AMϕ) are at the center of the pathogenesis of ALI after HS. We have previously reported that HS-activated polymorphonuclear neutrophils (PMNs) interact with macrophages to influence inflammation progress. In this study, we explore a novel function of PMNs regulating AMϕ anti-inflammatory mechanisms involving autophagy. Using a mouse "two-hit" model of HS/resuscitation followed by intratracheal injection of muramyl dipeptide, we demonstrate that HS initiates high mobility group box 1/TLR4 signaling, which upregulates NOD2 expression in AMϕ and sensitizes them to subsequent NOD2 ligand muramyl dipeptide to augment lung inflammation. In addition, upregulated NOD2 signaling induces autophagy in AMϕ, which negatively regulates lung inflammation through feedback suppression of NOD2-RIP2 signaling and inflammasome activation. Importantly, we further demonstrate that HS-activated PMNs that migrate in alveoli counteract the anti-inflammatory effect of autophagy in AMϕ, possibly through NAD(P)H oxidase-mediated signaling to enhance I-κB kinase γ phosphorylation, NF-κB activation, and nucleotide-binding oligomerization domain protein 3 inflammasome activation, and therefore augment post-HS lung inflammation. These findings explore a previously unidentified complexity in the mechanisms of ALI, which involves cell-cell interaction and receptor cross talk.

Early innate immunity to bacterial infection in the lung is regulated systemically by the commensal microbiota via nod-like receptor ligands.

The commensal microbiota is a major regulator of the immune system. The majority of commensal bacteria inhabit the gastrointestinal tract and are known to regulate local mucosal defenses against intestinal pathogens. There is growing appreciation that the commensal microbiota also regulates immune responses at extraintestinal sites. Currently, however, it is unclear how this influences host defenses against bacterial infection outside the intestine. Microbiota depletion caused significant defects in the early innate response to lung infection by the major human pathogen Klebsiella pneumoniae. After microbiota depletion, early clearance of K. pneumoniae was impaired, and this could be rescued by administration of bacterial Nod-like receptor (NLR) ligands (the NOD1 ligand MurNAcTri(DAP) and NOD2 ligand muramyl dipeptide [MDP]) but not bacterial Toll-like receptor (TLR) ligands. Importantly, NLR ligands from the gastrointestinal, but not upper respiratory, tract rescued host defenses in the lung. Defects in early innate immunity were found to be due to reduced reactive oxygen species-mediated killing of bacteria by alveolar macrophages. These data show that bacterial signals from the intestine have a profound influence on establishing the levels of antibacterial defenses in distal tissues.

NOD1 expression is increased in the adipose tissue of women with gestational diabetes.

Maternal peripheral insulin resistance and increased inflammation are two features of pregnancies, complicated by gestational diabetes mellitus (GDM). The nucleotide-binding oligomerisation domain (NOD) intracellular molecules recognise a wide range of microbial products, as well as other intracellular danger signals, thereby initiating inflammation through activation of nuclear factor κB (NFκB). The aim of this study was to determine whether levels of NOD1 and NOD2 are increased in adipose tissue of women with GDM. The effect of NOD1 and NOD2 activation on inflammation and the insulin signalling pathway was also assessed. NOD1, but not NOD2, expression was higher in omental and subcutaneous adipose tissues obtained from women with GDM when compared with those from women with normal glucose tolerance (NGT). In both omental and subcutaneous adipose tissues from NGT and GDM women, the NOD1 ligand g-d-glutamyl-meso-diaminopimelic acid (iE-DAP) significantly induced the expression and secretion of the pro-inflammatory cytokine interleukin 6 (IL6) and chemokine IL8; COX2 (PTGS2) gene expression and subsequent prostaglandin production; the expression and secretion of the extracellular matrix remodelling enzyme matrix metalloproteinase 9 (MMP9) and the gene expression and secretion of the adhesion molecules ICAM1 and VCAM1. There was no effect of the NOD2 ligand muramyl dipeptide on any of the endpoints tested. The effects of the NOD1 ligand iE-DAP were mediated via NFκB, as the NFκB inhibitor BAY 11-7082 significantly attenuated iE-DAP-induced expression and secretion of pro-inflammatory cytokines, COX2 gene expression and subsequent prostaglandin production, MMP9 expression and secretion and ICAM1 and VCAM1 gene expression and secretion. In conclusion, the present findings describe an important role for NOD1 in the development of insulin resistance and inflammation in pregnancies complicated by GDM.

Epicutaneous immunization with protein antigen TNP-Ig and NOD2 ligand muramyl dipeptide (MDP) reverses skin-induced suppression of contact hypersensitivity

BackgroundEpicutaneous (EC) immunization offers a new method of a needle-free and self-administrable immunization by using a topically applied patch to deliver vaccine.We have previously shown that EC immunization with hapten-conjugated protein antigen TNP-Ig prior to hapten sensitization inhibits Th1-mediated contact hypersensitivity (CHS) in mice. Our further work showed that EC immunization with TNP-Ig and Toll-like receptor (TLR) ligands prior to hapten sensitization reverses skin-induced suppression. MethodsAnimal model of contact hypersensitivity was used to study reversal of skin-induced suppression. ResultsCurrent work showed that EC immunization with protein antigen TNP-Ig and MDP NOD2 agonist – muramyldipeptide (L isoform) reverses skin-induced suppression of CHS. On the other hand L18-MDP NOD2 agonist – muramyldipeptide with a C18 fatty acid chain and MDP control – negative control for MDP – muramyldipeptide (D isoform, inactive) did not reverse skin-induced suppression. “Transfer in” experiment showed that reversal of skin-induced suppression can be adoptively transferred with lymphoid cells isolated from donors EC treated with TNP-Ig and MDP NOD2 agonist. Moreover, experiment employing two non-cross-reacting antigens TNP-Ig and OX-Ig proved that reversal of skin-induced suppression is antigen specific. Additionally, lymph node cells isolated from mice EC immunized with TNP-Ig and MDP NOD2 agonist produced increased level of IFN-γ suggesting that this cytokine might be involved in reversal of skin-induced suppression. ConclusionThis work shows that EC immunization with protein antigen plus NOD2 ligand MDP may be a potential tool to increase the immunogenicity of weekly immunogenic antigens.

Protein Tyrosine Phosphatase Non-Receptor Type 22 Modulates NOD2-Induced Cytokine Release and Autophagy

BackgroundVariations within the gene locus encoding protein tyrosine phosphatase non-receptor type 22 (PTPN22) are associated with the risk to develop inflammatory bowel disease (IBD). PTPN22 is involved in the regulation of T- and B-cell receptor signaling, but although it is highly expressed in innate immune cells, its function in other signaling pathways is less clear. Here, we study whether loss of PTPN22 controls muramyl-dipeptide (MDP)-induced signaling and effects in immune cells.Material & MethodsStable knockdown of PTPN22 was induced in THP-1 cells by shRNA transduction prior to stimulation with the NOD2 ligand MDP. Cells were analyzed for signaling protein activation and mRNA expression by Western blot and quantitative PCR; cytokine secretion was assessed by ELISA, autophagosome induction by Western blot and immunofluorescence staining. Bone marrow derived dendritic cells (BMDC) were obtained from PTPN22 knockout mice or wild-type animals.ResultsMDP-treatment induced PTPN22 expression and activity in human and mouse cells. Knockdown of PTPN22 enhanced MDP-induced activation of mitogen-activated protein kinase (MAPK)-isoforms p38 and c-Jun N-terminal kinase as well as canonical NF-κB signaling molecules in THP-1 cells and BMDC derived from PTPN22 knockout mice. Loss of PTPN22 enhanced mRNA levels and secretion of interleukin (IL)-6, IL-8 and TNF in THP-1 cells and PTPN22 knockout BMDC. Additionally, loss of PTPN22 resulted in increased, MDP-mediated autophagy in human and mouse cells.ConclusionsOur data demonstrate that PTPN22 controls NOD2 signaling, and loss of PTPN22 renders monocytes more reactive towards bacterial products, what might explain the association of PTPN22 variants with IBD pathogenesis.

NOD-like receptors mediated activation of eosinophils interacting with bronchial epithelial cells: a link between innate immunity and allergic asthma

Key intracytosolic pattern recognition receptors of innate immunity against bacterial infections are nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). We elucidated the NOD1 and NOD2-mediated activation of human eosinophils, the principal effector cells for allergic inflammation, upon interacting with human bronchial epithelial BEAS-2B cells in allergic asthma. Eosinophils constitutively expressed NOD1,2 but exhibited nonsignificant responses to release chemokines upon the stimulation by NOD1 ligand γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) and NOD2 ligand muramyl dipeptide (MDP). However, iE-DAP and MDP could significantly upregulate cell surface expression of CD18 and intercellular adhesion molecule (ICAM)-1 on eosinophils and ICAM-1 on BEAS-2B cells, as well as induce chemokines CCL2 and CXCL8 release in the coculture system (all P<0.05). Both eosinophils and BEAS-2B cells were the main source for CXCL8 and CCL2 release in the coculture system upon iE-DAP or MDP stimulation. Direct interaction between eosinophils and BEAS-2B cells is responsible for CCL2 release, and soluble mediators are implicated in CXCL8 release. ERK and NF-κB play regulatory roles for the expression of adhesion molecules and chemokines in coculture. Treatment with NOD1,2 ligand could induce the subepithelial fibrosis and significantly enhance the serum concentration of total IgE, chemokine CCL5 for eosinophils and T helper type 2 (Th2) cells and asthma Th2 cytokine IL-13 in bronchoalveolar lavage fluid of ovalbumin-sensitized allergic asthmatic mice (all P<0.05). This study provides further evidence of bacterial infection-mediated activation of NOD1,2 in triggering allergic asthma via the activation of eosinophils interacting with bronchial epithelial cells at inflammatory airway.

The nucleotide-binding oligomerization domain-containing-2 ligand muramyl dipeptide enhances phagocytosis and intracellular killing of Escherichia coli K1 by Toll-like receptor agonists in microglial cells

Increasing the phagocytic activity of microglia could improve the resistance of immunocompromised patients to CNS infections. We studied the microglial responses upon stimulation with the Nod2 ligand muramyl dipeptide (MDP) alone or in combination with a TLR1/2, 3 or 4 agonist. MDP caused a mild release of NO, but induced neither a significant release of pro-inflammatory cytokines nor an expression of molecules associated with professional antigen presentation. Using the Escherichia coli K1 model, microglial pre-stimulation with MDP enhanced bacterial phagocytosis which was strengthened on TLR-pre-stimulated cells. Dual pre-stimulation of Nod2 and TLR1/2 or 4 caused maximal phagocytosis and intracellular killing.

Yersinia pseudotuberculosis Effector YopJ Subverts the Nod2/RICK/TAK1 Pathway and Activates Caspase-1 to Induce Intestinal Barrier Dysfunction

Yersinia pseudotuberculosis is an enteropathogenic bacteria that disrupts the intestinal barrier and invades its host through gut-associated lymphoid tissue and Peyer's patches (PP). We show that the Y.pseudotuberculosis effector YopJ induces intestinal barrier dysfunction by subverting signaling of the innate immune receptor Nod2, a phenotype that can be reversed by pretreating with the Nod2 ligand muramyl-dipeptide. YopJ, but not the catalytically inactive mutant YopJ(C172A), acetylates critical sites in the activation loops of the RICK and TAK1 kinases, which are central mediators of Nod2 signaling, and decreases the affinity of Nod2 for RICK. Concomitantly, Nod2 interacts with and activates caspase-1, resulting in increased levels of IL-1β. Finally, IL-1β within PP plays an essential role in inducing intestinal barrier dysfunction. Thus, YopJ alters intestinal permeability and promotes the dissemination of Yersinia as well as commensal bacteria by exploiting the mucosal inflammatory response.

Morphine Inhibits Murine Dendritic Cell IL-23 Production by Modulating Toll-like Receptor 2 and Nod2 Signaling

IL-23, produced by dendritic cells (DCs) and macrophages, plays a critical role in innate immunity against bacterial infection. Our previous studies show that morphine disrupts the IL-23/IL-17 mediated pulmonary mucosal host defense and increases susceptibility to Streptococcus pneumoniae lung infection. To determine the mechanism by which morphine modulates IL-23 production, mouse bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs) were treated with morphine, and infected with S. pneumoniae or stimulated with Toll-like receptor (TLR) and Nod2 ligands. We found that a significant increase in IL-23 protein production was observed in S. pneumoniae, TLR2 ligand lipoteichoic acid (LTA), and TLR4 ligand pneumolysin (PLY) stimulated BMDCs and BMDMs. Interestingly, although Nod2 ligand muramyldipeptide (MDP) alone had no effect on IL-23 production, it potentiated LTA induced IL-23 production to the same level as that observed following S. pneumoniae infection, suggesting that S. pneumoniae induced IL-23 production in DCs involves activation of both TLR2 and Nod2 signaling mechanisms. Furthermore, pretreatment of DCs with MyD88 (myeloid differentiation primary response gene 88) and IL-1 receptor-associated kinase (IRAK) 1/4 inhibitors, or TLR2 antibody diminished the S. pneumoniae induced IL-23 and abolished the inhibitory effects of morphine, indicating that S. pneumoniae induced IL-23 production depends on activation of the TLR2-MyD88-IRAK1/4 signaling pathway. Moreover, morphine decreased S. pneumoniae induced phosphorylation of interferon regulatory factor 3 (IRF3) and activating transcription factor 2 in DCs. Taken together, our study shows that morphine impairs S. pneumoniae induced IL-23 production through MyD88-IRAK1/4-dependent TLR2 and Nod2 signaling in DCs.

NOD2 controls the nature of the inflammatory response and subsequent fate of Mycobacterium tuberculosis and M. bovis BCG in human macrophages

Mycobacterium tuberculosis (M.tb), which causes tuberculosis, is a host-adapted intracellular pathogen of macrophages. Intracellular pattern recognition receptors in macrophages such as nucleotide-binding oligomerization domain (NOD) proteins regulate pro-inflammatory cytokine production. NOD2-mediated signalling pathways in response to M.tb have been studied primarily in mouse models and cell lines but not in primary human macrophages. Thus we sought to determine the role of NOD2 in regulating cytokine production and growth of virulent M.tb and attenuated Mycobacterium bovis BCG (BCG) in human macrophages. We examined NOD2 expression during monocyte differentiation and observed a marked increase in NOD2 transcript and protein following 2-3 days in culture. Pre-treatment of human monocyte-derived and alveolar macrophages with the NOD2 ligand muramyl dipeptide enhanced production of TNF-α and IL-1β in response to M.tb and BCG in a RIP2-dependent fashion. The NOD2-mediated cytokine response was significantly reduced following knock-down of NOD2 expression by using small interfering RNA (siRNA) in human macrophages. Finally, NOD2 controlled the growth of both M.tb and BCG in human macrophages, whereas controlling only BCG growth in murine macrophages. Together, our results provide evidence that NOD2 is an important intracellular receptor in regulating the host response to M.tb and BCG infection in human macrophages.

Recognition of commensal microbes: if innate responses are NOD in balance

Evaluation of: Petnicki-Ocwieja T, Hrncir T, Liu YJ et al. Nod2 is required for the regulation of commensal microbiota in the intestine. Proc. Natl Acad. Sci. USA 106, 15813–15818 (2009).Nod2 is an intracellular pattern recognition receptor binding bacterial wall muramyl dipeptide (MDP) and inducing NF-κB activation via the Rip2 pathway. NOD2 variants have been found to be the most important genetic susceptibility factor for the development of Crohn’s disease. Nod2 is expressed in the Paneth cells of the small intestine and in antigen-presenting cells of the mucosal innate immune system. Furthermore, Nod2 is a regulator of a number of secreted antimicrobial proteins such as defensins. Subsequently, the question was raised whether Nod2 activity (or a loss of activity in Nod2 variants) influences the composition of the intestinal commensal flora. The Nod2 ligand MDP is able to induce bacterial killing when intestinal crypts are incubated with MDP and the supernatants are used for microbial killing activity assays, thus indicating that upregulation of Nod2 activity induces antimicrobial activity. In Nod2-deficient crypts, the ability to kill bacteria is reduced. Consequently, the commensal bacterial flora in Nod2-deficient mice differs from that seen in wild-type mice and, in general, is found to be increased in load. Nod2 activity influences the colonization of the mucosal wall by potentially pathogenic bacteria; the commensal flora, on the other hand, influence the expression of Nod2. This indicates a balanced system in which commensal bacteria and Nod2-mediated pathways influence each other to achieve a steady state without inflammation. The controlled balance between Nod2 and the commensal flora creates a negative-feedback loop in which the bacteria positively regulate Nod2 and associated signaling molecules, which in turn regulate the commensal flora. This pathway is likely to be one of the regulative loops that exist between the intestinal flora and the gut immune system.

Stimulation of the Intracellular Bacterial Sensor NOD2 Programs Dendritic Cells to Promote Interleukin-17 Production in Human Memory T Cells

How the development of antibacterial T helper 17 (Th17) cells is selectively promoted by antigen-presenting dendritic cells (DCs) is unclear. We showed that bacteria, but not viruses, primed human DCs to promote IL-17 production in memory Th cells through the nucleotide oligomerization domain 2 (NOD2)-ligand muramyldipeptide (MDP), a derivative of bacterial peptidoglycan. MDP enhanced obligate bacterial Toll-like receptor (TLR) agonist induction of IL-23 and IL-1, which promoted IL-17 expression in T cells. The role of NOD2 in this IL-23-IL-1-IL-17 axis could be confirmed in NOD2-deficient DCs, such as DCs from selected Crohn's disease patients. Thus, antibacterial Th17-mediated immunity in humans is orchestrated by DCs upon sensing bacterial NOD2-ligand MDP.