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Muramyl Dipeptide Induces NOD2-Dependent Ly6Chigh Monocyte Recruitment to the Lungs and Protects Against Influenza Virus Infection  [PDF]
Fran?ois Coulombe, Stéphanie Fiola, Shizuo Akira, Yvon Cormier, Jean Gosselin
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0036734
Abstract: Bacterial peptidoglycan-derived muramyl dipeptide (MDP) and derivatives have long-recognized antiviral properties but their mechanism of action remains unclear. In recent years, the pattern-recognition receptor NOD2 has been shown to mediate innate responses to MDP. Here, we show that MDP treatment of mice infected with Influenza A virus (IAV) significantly reduces mortality, viral load and pulmonary inflammation in a NOD2-dependent manner. Importantly, the induction of type I interferon (IFN) and CCL2 chemokine was markedly increased in the lungs following MDP treatment and correlated with a NOD2-dependent enhancement in circulating monocytes. Mechanistically, the protective effect of MDP could be explained by the NOD2-dependent transient increase in recruitment of Ly6Chigh “inflammatory” monocytes and, to a lesser extent, neutrophils to the lungs. Indeed, impairment in both Ly6Chigh monocyte recruitment and survival observed in infected Nod2-/- mice treated with MDP was recapitulated in mice deficient for the chemokine receptor CCR2 required for CCL2-mediated Ly6Chigh monocyte migration from the bone marrow into the lungs. MDP-induced pulmonary monocyte recruitment occurred normally in IAV-infected and MDP-treated Ips-1-/- mice. However, IPS-1 was required for improved survival upon MDP treatment. Finally, mycobacterial N-glycolyl MDP was more potent than N-acetyl MDP expressed by most bacteria at reducing viral burden while both forms of MDP restored pulmonary function following IAV challenge. Overall, our work sheds light on the antiviral mechanism of a clinically relevant bacterial-derived compound and identifies the NOD2 pathway as a potential therapeutic target against IAV.
Down-Regulated NOD2 by Immunosuppressants in Peripheral Blood Cells in Patients with SLE Reduces the Muramyl Dipeptide-Induced IL-10 Production  [PDF]
Shui-Lian Yu, Chun-Kwok Wong, Purple Tsz-Yan Wong, Da-Peng Chen, Cheuk-Chun Szeto, Edmund K. Li, Lai-Shan Tam
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0023855
Abstract: 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 na?ve 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 na?ve patients and patients with active disease despite immunosuppressants compared with HCs. Upon MDP stimulaiton, relative induction (%) of cytokines (IL-1β) from PBMC was significantly increased in immunosuppressant na?ve 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 na?ve patients and HCs. Conclusions/Significance Bacterial exposure may increase the NOD2 expression in monocytes in immunosuppressant na?ve 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.
Nod2 Suppresses Borrelia burgdorferi Mediated Murine Lyme Arthritis and Carditis through the Induction of Tolerance  [PDF]
Tanja Petnicki-Ocwieja,Alicia S. DeFrancesco,Erin Chung,Courtney T. Darcy,Roderick T. Bronson,Koichi S. Kobayashi,Linden T. Hu
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0017414
Abstract: The internalization of Borrelia burgdorferi, the causative agent of Lyme disease, by phagocytes is essential for an effective activation of the immune response to this pathogen. The intracellular, cytosolic receptor Nod2 has been shown to play varying roles in either enhancing or attenuating inflammation in response to different infectious agents. We examined the role of Nod2 in responses to B. burgdorferi. In vitro stimulation of Nod2 deficient bone marrow derived macrophages (BMDM) resulted in decreased induction of multiple cytokines, interferons and interferon regulated genes compared with wild-type cells. However, B. burgdorferi infection of Nod2 deficient mice resulted in increased rather than decreased arthritis and carditis compared to control mice. We explored multiple potential mechanisms for the paradoxical response in in vivo versus in vitro systems and found that prolonged stimulation with a Nod2 ligand, muramyl dipeptide (MDP), resulted in tolerance to stimulation by B. burgdorferi. This tolerance was lost with stimulation of Nod2 deficient cells that cannot respond to MDP. Cytokine patterns in the tolerance model closely paralleled cytokine profiles in infected Nod2 deficient mice. We propose a model where Nod2 has an enhancing role in activating inflammation in early infection, but moderates inflammation after prolonged exposure to the organism through induction of tolerance.
Functional characterization of two novel 5' untranslated exons reveals a complex regulation of NOD2 protein expression
Philip Rosenstiel, Klaus Huse, Andre Franke, Jochen Hampe, Kathrin Reichwald, Cornelia Platzer, Roland G Roberts, Christopher G Mathew, Matthias Platzer, Stefan Schreiber
BMC Genomics , 2007, DOI: 10.1186/1471-2164-8-472
Abstract: We have identified two novel exons of the NOD2 gene (designated exon 1a and 1b), which are spliced to the canonical exon 2 and constitute the 5' untranslated region of two alternative transcript isoforms (i.e. exon 1a/1b/2 and exon 1a/2). The two novel transcripts are abundantly expressed and seem to comprise the majority of NOD2 transcripts under physiological conditions. We confirm the expression of the previously known canonical first exon (designated exon 1c) of the gene in unstimulated mononuclear cells. The inclusion of the second alternative exon 1b, which harbours three short upstream open reading frames (uORFs), is downregulated upon stimulation with TNF-α or under pro-inflammatory conditions in the inflamed intestinal mucosa in vivo. Using the different 5' UTR splice forms fused to a firefly luciferase (LUC) reporter we demonstrate a rapamycin-sensitive inhibitory effect of the uORFs on translation efficacy.The differential usage of two alternative promoters in the NOD2 gene leads to tissue-specific and context-dependent NOD2 transcript isoform patterns. We demonstrate for the first time that context-dependent alternative splicing is linked to uORF-mediated translational repression. The results suggest complex parallel control mechanisms that independently regulate NOD2 expression in the context of inflammatory signaling.NOD2 (CARD15, NLRC2) is a member of the family of the NACHT/LRR receptors, which are characterized by a central nucleotide-binding and oligomerization domain and a C-terminal sensor domain consisting of repeats of leucine-rich repeats (LRR) [1,2]. The LRRs of NOD2 have been described to directly or indirectly recognize intracellular muramyl-dipeptide (MDP), an abundant cell wall component of both gram-negative and gram-positive bacteria [3,4]. The recognition of MDP leads to a recruitment of the protein kinase RIP2/RICK to the N-terminal effector binding domain, which consists of two adjacent caspase-recruitment domains (CARDs). Subsequent
Evidence for the involvement of NOD2 in regulating colonic epithelial cell growth and survival  [cached]
Sheena M Cruickshank, Louise Wakenshaw, John Cardone, Peter D Howdle, Peter J Murray, Simon R Carding
World Journal of Gastroenterology , 2008,
Abstract: AIM: To investigate the function of NOD2 in colonic epithelial cells (CEC).METHODS: A combination of in vivo and in vitro analyses of epithelial cell turnover in the presence and absence of a functional NOD2 protein and, in response to enteric Salmonella typhimurium infection, were used. shRNA interference was also used to investigate the consequences of knocking down NOD2 gene expression on the growth and survival of colorectal carcinoma cell lines.RESULTS: In the colonic mucosa the highest levels of NOD2 expression were in proliferating crypt epithelial cells. Muramyl dipeptide (MDP), that is recognized by NOD2, promoted CEC growth in vitro. By contrast, the growth of NOD2-deficient CECs was impaired. In vivo CEC proliferation was also reduced and apoptosis increased in Nod2-/- mice, which were also evident following enteric Salmonella infection. Furthermore, neutralization of NOD2 mRNA expression in human colonic carcinoma cells by shRNA interference resulted in decreased survival due to increased levels of apoptosis.CONCLUSION: These findings are consistent with the involvement of NOD2 protein in promoting CEC growth and survival. Defects in proliferation by CECs in cases of CD may contribute to the underlying pathology of disrupted intestinal homeostasis and excessive inflammation.
p62/SQSTM1 Enhances NOD2-Mediated Signaling and Cytokine Production through Stabilizing NOD2 Oligomerization  [PDF]
Sangwook Park, Soon-Duck Ha, Macon Coleman, Shahab Meshkibaf, Sung Ouk Kim
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0057138
Abstract: NOD2 is a cytosolic pattern-recognition receptor that senses muramyl dipeptide of peptidoglycan that constitutes the bacterial cell wall, and plays an important role in maintaining immunological homeostasis in the intestine. To date, multiple molecules have shown to be involved in regulating NOD2 signaling cascades. p62 (sequestosome-1; SQSTM1) is a multifaceted scaffolding protein involved in trafficking molecules to autophagy, and regulating signal cascades activated by Toll-like receptors, inflammasomes and several cytokine receptors. Here, we show that p62 positively regulates NOD2-induced NF-κB activation and p38 MAPK, and subsequent production of cytokines IL-1β and TNF-α. p62 associated with the nucleotide binding domain of NOD2 through a bi-directional interaction mediated by either TRAF6-binding or ubiquitin-associated domains. NOD2 formed a large complex with p62 in an electron-dense area of the cytoplasm, which increased its signaling cascade likely through preventing its degradation. This study for the first time demonstrates a novel role of p62 in enhancing NOD2 signaling effects.
Rapid generation of an anthrax immunotherapeutic from goats using a novel non-toxic muramyl dipeptide adjuvant  [cached]
Kelly Cassandra D,O'Loughlin Chris,Gelder Frank B,Peterson Johnny W
Journal of Immune Based Therapies and Vaccines , 2007, DOI: 10.1186/1476-8518-5-11
Abstract: Background There is a clear need for vaccines and therapeutics for potential biological weapons of mass destruction and emerging diseases. Anthrax, caused by the bacterium Bacillus anthracis, has been used as both a biological warfare agent and bioterrorist weapon previously. Although antibiotic therapy is effective in the early stages of anthrax infection, it does not have any effect once exposed individuals become symptomatic due to B. anthracis exotoxin accumulation. The bipartite exotoxins are the major contributing factors to the morbidity and mortality observed in acute anthrax infections. Methods Using recombinant B. anthracis protective antigen (PA83), covalently coupled to a novel non-toxic muramyl dipeptide (NT-MDP) derivative we hyper-immunized goats three times over the course of 14 weeks. Goats were plasmapheresed and the IgG fraction (not affinity purified) and F(ab')2 derivatives were characterized in vitro and in vivo for protection against lethal toxin mediated intoxication. Results Anti-PA83 IgG conferred 100% protection at 7.5 μg in a cell toxin neutralization assay. Mice exposed to 5 LD50 of Bacillus anthracis Ames spores by intranares inoculation demonstrated 60% survival 14 d post-infection when administered a single bolus dose (32 mg/kg body weight) of anti-PA83 IgG at 24 h post spore challenge. Anti-PA83 F(ab')2 fragments retained similar neutralization and protection levels both in vitro and in vivo. Conclusion The protection afforded by these GMP-grade caprine immunotherapeutics post-exposure in the pilot murine model suggests they could be used effectively to treat post-exposure, symptomatic human anthrax patients following a bioterrorism event. These results also indicate that recombinant PA83 coupled to NT-MDP is a potent inducer of neutralizing antibodies and suggest it would be a promising vaccine candidate for anthrax. The ease of production, ease of covalent attachment, and immunostimulatory activity of the NT-MDP indicate it would be a superior adjuvant to alum or other traditional adjuvants in vaccine formulations.
T Cell Intrinsic NOD2 Is Dispensable for CD8 T Cell Immunity  [PDF]
Gloria H. Y. Lin, Michael E. Wortzman, Stephen E. Girardin, Dana J. Philpott, Tania H. Watts
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0056014
Abstract: NOD2 is an intracellular pattern recognition receptor that provides innate sensing of bacterial muramyl dipeptide by host cells, such as dendritic cells, macrophages and epithelial cells. While NOD2's role as an innate pathogen sensor is well established, NOD2 is also expressed at low levels in T cells and there are conflicting data as to whether NOD2 plays an intrinsic role in T cell function. Here we show that following adoptive transfer into WT hosts, NOD2?/? OT-I T cells show a small decrease in the number of OVA-specific CD8 T cells recovered at the peak of the response to respiratory influenza virus infection. On the other hand, no such defect was observed upon intranasal immunization with a replication defective adenovirus carrying the OVA epitope recognized by OT-I, or when OVA was delivered with LPS subcutaneously, or when influenza-OVA was delivered intraperitoneally. Thus we observed a selective defect in NOD2-deficient T cell responses only during a live viral infection. Moreover, there was no apparent defect when NOD2?/? OT-I T cells were stimulated in vitro. Finally, this selective defect in recovery of NOD2-deficient CD8 T cells was not observed in a non-transgenic respiratory infection model in which mixed bone marrow chimeras were used such that the NOD2?/? T cells were allowed to develop and respond in a NOD2-sufficient host. Taken together our data indicate that T cell intrinsic NOD2 is not required for CD8 T cell responses to antigen delivered under a variety of conditions in vitro and in vivo. However, CD8 T cells that have developed in the absence of NOD2 show a selective and modest impairment in their response to live respiratory influenza infection.
NOD2/CARD15: geographic differences in the Spanish population and clinical applications in Crohn's disease
Barreiro-de-Acosta,M.; Mendoza,J. L.; Lana,R.; Domínguez-Mu?oz,J. E.; Díaz-Rubio,M.;
Revista Espa?ola de Enfermedades Digestivas , 2010, DOI: 10.4321/S1130-01082010000500006
Abstract: crohn's disease (cd) is a genetically complex disease in which both genetic susceptibility and environmental factors play key roles in the development of the disorder. nod2/card15 mutations are associated with cd. nod2 encodes for a protein that is an intracellular receptor for a bacterial product (muramyl dipeptide), though the exact functional consequences of these mutations remain the subject of debate. nod2/card15 mutations are associated with ileal cd, with stricturing behavior, and possibly with a more complicated course of cd. nod2/card15 mutations associated with cd have demonstrated heterogeneity across ethnicities and populations throughout the world, with regional variations across europe and spain. however, "nod2/card15 testing" is not yet ready for use in the clinical setting. one of the reasons is that we know that these genetic variants increase the risk of disease only marginally, and many healthy individuals carry the risk alleles, at present it is not recommended to screen first-degree relatives, because we do not have the ability to prevent the disease at the present time.
Identification of Benzimidazole Diamides as Selective Inhibitors of the Nucleotide-Binding Oligomerization Domain 2 (NOD2) Signaling Pathway  [PDF]
David J. Rickard, Clark A. Sehon, Viera Kasparcova, Lorena A. Kallal, Xin Zeng, Monica N. Montoute, Tushar Chordia, Derek D. Poore, Hu Li, Zining Wu, Patrick M. Eidam, Pamela A. Haile, Jong Yu, John G. Emery, Robert W. Marquis, Peter J. Gough, John Bertin
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0069619
Abstract: NOD2 is an intracellular pattern recognition receptor that assembles with receptor-interacting protein (RIP)-2 kinase in response to the presence of bacterial muramyl dipeptide (MDP) in the host cell cytoplasm, thereby inducing signals leading to the production of pro-inflammatory cytokines. The dysregulation of NOD2 signaling has been associated with various inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. To identify inhibitors of the NOD2 signaling pathway, we utilized a cell-based screening approach and identified a benzimidazole diamide compound designated GSK669 that selectively inhibited an MDP-stimulated, NOD2-mediated IL-8 response without directly inhibiting RIP2 kinase activity. Moreover, GSK669 failed to inhibit cytokine production in response to the activation of Toll-like receptor (TLR)-2, tumor necrosis factor receptor (TNFR)-1 and closely related NOD1, all of which share common downstream components with the NOD2 signaling pathway. While the inhibitors blocked MDP-induced NOD2 responses, they failed to block signaling induced by NOD2 over-expression or single stranded RNA, suggesting specificity for the MDP-induced signaling complex and activator-dependent differences in NOD2 signaling. Investigation of structure-activity relationship allowed the identification of more potent analogs that maintained NOD2 selectivity. The largest boost in activity was achieved by N-methylation of the C2-ethyl amide group. These findings demonstrate that the NOD2 signaling pathway is amenable to modulation by small molecules that do not target RIP2 kinase activity. The compounds we identified should prove useful tools to investigate the importance of NOD2 in various inflammatory processes and may have potential clinical utility.
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