Pivotal role of NOD2 in inflammatory processes affecting atherosclerosis and periodontal bone loss

The purpose of this study was to elucidate the role of nucleotide binding oligomerization domain-containing protein 2 (NOD2) signaling in atherosclerosis and periodontal bone loss using an Apolipoprotein E^−/− (ApoE^−/−) mouse model based on the proposed role of NOD2 in inflammation. NOD2^−/−ApoE^−/− and ApoE^−/− mice fed a standard chow diet were given an oral gavage of Porphyromonas gingivalis for 15 wk. NOD2^−/−ApoE^−/− mice exhibited significant increases in inflammatory cytokines, alveolar bone loss, cholesterol, and atherosclerotic lesions in the aorta and the heart compared with ApoE^−/− mice. In contrast, ApoE^−/− mice injected i.p. with Muramyl DiPeptide (MDP) to stimulate NOD2 and given an oral gavage of P. gingivalis displayed a reduction of serum inflammatory cytokines, alveolar bone loss, cholesterol, and atherosclerotic lesions in the aorta and aortic sinus compared with ApoE^−/− mice orally challenged but injected with saline. A reduction in body weight gain was observed in ApoE^−/− mice fed a high-fat diet (HFD) and injected with MDP compared with ApoE^−/− mice fed a high-fat diet but injected with saline. MDP treatment of bone marrow-derived macrophages incubated with P. gingivalis increased mRNA expressions of NOD2, Toll-like receptor 2, myeloid differentiation primary response gene 88, and receptor-interacting protein-2 but reduced the expressions of inhibitor of NF-κB kinase-β, NF-κB, c-Jun N-terminal kinase 3, and TNF-α protein levels compared with saline control, highlighting pathways involved in MDP antiinflammatory effects. MDP activation of NOD2 should be considered in the treatment of inflammatory processes affecting atherosclerosis, periodontal bone loss ,and possibly, diet-induced weight gain.The nucleotide binding and oligomerization domain 2 protein (NOD2) is an intracellular protein containing leucine-rich repeats similar to the repeats found in Toll-like receptors (TLRs) that are capable of sensing bacteria-derived muramyl dipeptide (MDP), and it was initially described as a susceptibility gene for Crohn disease and intestinal inflammatory diseases (1–3). NOD2 is expressed in various cell subsets, including myeloid cells (particularly macrophages, neutrophils, and dendritic cells), as well as Paneth cells in the small intestine (4), and it was found to process inflammatory signals (5). Immune cells express receptors that recognize a broad range of molecular patterns foreign to the mammalian host but commonly found on pathogens. These molecules trigger immune responses through interactions with members of the toll-like receptor family (TLRs) at the cell membrane and NACHT, neuronal apoptosis inhibitor protein (NAIP), CIITA, HET-E and TP-1 domain–Leucine-rich repeat (LRR) proteins (NLRs) in the cytosol (6, 7). Cells expressing NOD2 can activate NF-κB after intracellular recognition of MDP (8, 9). The recognition of MDP is mediated through the LRR domain of NOD2, leading to downstream signaling through interaction between the caspase recruitment domain (CARD) of Receptor-interacting serine/threonine-protein kinase 2 (RIP2) and the CARDs of NOD2. In vitro, NOD2 has been found to be involved in bacterial clearance (10). NOD2-deficient mice display increased susceptibility to Staphylococcus aureus because of, in part, defective neutrophil phagocytosis, elevated serum levels of Th1 cytokines, and a higher bacterial tissue burden (11). However, stimulation of NOD2 with MDP was found to enhance host antibacterial function in vitro (12).Atherosclerosis is a chronic inflammatory condition that can lead to an acute clinical event by plaque rupture and thrombosis. It is a multifactorial disease characterized by the accumulation of cells from both the innate and acquired immune system within the intima of the arterial wall (13). Triglyceride-rich lipoproteins and free fatty acids are important factors involved in fatty streak formation and advanced atherosclerosis (14). Microorganisms have also been implicated as aggravating factors in atherosclerosis (15). In atherosclerosis, normal homeostatic functions of the endothelium are altered, promoting an inflammatory response that results in an increased expression of adhesion molecules. This increased expression leads to the recruitment of leukocytes, including monocytes, that penetrate the intima, predisposing the vessel wall to lipid deposits (13). Reportedly, mast cells also contribute to coronary plaque progression and diet-induced obesity and diabetes through the secretion of vasoactive mediators, cytokines, and proteinases (16).Evidence is accumulating that distant bacterial infection is involved in the pathophysiology of local chronic inflammatory processes underlying atherosclerosis (17). The transfer of bacteria into the blood or lymph system from barrier organ surfaces has been suggested as a possible mechanism of atherosclerosis. Advanced gum infection (periodontitis) is known to induce local inflammation, often leading to gingival ulcerations and local vascular changes, which have the potential to increase the incidence and severity of transient bacteremia. Porphyromonas gingivalis, an important microorganism associated with periodontitis, has been identified in atherosclerotic plaques of patients suffering from atherosclerosis, suggesting that this pathogen might be critical for atheroma formation (18). Indeed, we showed that endothelial dysfunction associated with repetitive exposure by P. gingivalis can exacerbate the development of atherosclerosis (19).NOD2 expression and unique functions have also been described in other cell types, including adipocytes, gingival, pulp and periodontal fibroblasts, oral epithelial cells, and vascular endothelial cells (20–25). However, the precise role of NOD2 in chronic inflammatory diseases remains unclear.We showed previously that, in Apolipoprotein E^+/− (ApoE^+/−) mice, TLR2 deficiency reduces pathogen-associated atherosclerosis (26). In this report, we tested the role of NOD2 in two chronic inflammatory diseases, atherosclerosis and alveolar bone loss, by capitalizing on our model of P. gingivalis-associated atherosclerosis in ApoE^−/− mice.