探討巨噬細胞NOD2在Toll-Like Receptor 4的訊息傳遞及誘發的發炎基因表現中所扮演的角色

Abstract

NOD2 of NLR family has an important role in innate immunity as the cytosolic sensor of microbial components derived from bacterial peptidoglycan. Recent reports indicated that mutations in the leucine-rich repeat (LRR) domain of NOD2 have been implicated in the pathogenesis of Crohn’s disease (CD), yet the function of NOD2 and regulation of the NOD2 pathway remain unclear. Moreover, Toll-like receptor (TLR) is a major family of pattern recognition receptors (PRRs) and plays a crucial role in innate immune system. Recently, TLR4 is strongly up-regulated in both ulcerative colitis and CD. TLR4 polymorphisms (Asp299Gly and Thr399Ile) have been described in CD. Molecular interaction between TLR- and NOD2-signaling pathways is still controversial. Herein, we investigated the role of NOD2 in TLR-mediated signaling and gene regulation in murine macrophages. In murine RAW 264.7 macrophages, MDP (NOD2 ligand) can further increase LPS-induced pro-inflammatory cytokines and mediators, including TNFα, IL-1β, iNOS and COX-2. Accordingly we found the ability of TLR4 to induce NOD2 mRNA expression. We also found that MDP treatment could not affect TLR4-mediated activation of MAPK and IKK or IκBα degradation in RAW 264.7 macrophages, but it could up-regulate NF-κB activation. It is suggested that NOD2 is a positive regulator of TLR4-mediated signaling upon MDP treatment. Interestingly, NOD2 silencing cells lead to a further increase of LPS-induced inflammatory responses, but a decrease of LPS tolerance. We proposed that NOD2 could play an important role in negative regulation of TLR4-mediated signaling pathway in the absence of MDP treatment. Further, we demonstrated that both CARD and LRR domains of NOD2 protein were responsible for negative regulating function of TLR4. Besides, we also found that NOD2 could participate in down-regulation of macrophage bactericidal activity and TLR4-induced LC3 activation (an autophagy marker protein). In summary, NOD2 could have dual effects on TLR4 signaling. We proposed that the constitutively expressed NOD2 or infection up-regulated NOD2 without ligand stimulation plays roles to inhibit TLR4-mediated signaling, increase TLR4 tolerance and decrease bactericidal activity of macrophages. Nevertheless, activated NOD2 upon binding to pathogen-derived ligands can integrate with TLR4 signaling to enhance inflammatory response. How to interpret these two distinct roles together which possibly concomitantly happen in pathogenesis of infectious diseases and control the outcomes of infectious severity, e.g. the development of CD in patients whose NOD2 is functional mutant, still needs more investigation.