Regulatory Role And Mechanisms Of The Methylation Modification Of NLRC5 In Macrophage Phenotypic Modulation

Macrophages are essential to innate immune response and derived from circulating monocytes, when monocytes migrate from the circulation and extravasate through the endothelium, they can further differentiate into a range of tissue macrophages. In parallel to the Th1/Th2 paradigm, two well-established polarized phenotypes are often referred to as tissue M1 and M2 macrophages. The M1 macrophages were reserved for classically activated macrophages, which are induced by interferon(IFN-γ) alone or in combination with lipopolysaccharide(LPS, endotoxin) and the M2 macrophages for alternatively activated macrophages, which are induced by exposure to a variety of signals including the cytokines IL-4 and/or IL-13 and immune complexes. In general, M1 activated macrophages express interleukin(IL)-12 high, IL-23 high, IL-10low; metabolize arginine, produce high levels of inducible nitric oxide synthase(i NOS); secrete inflammatory cytokines such as IL-1β, IL-6, and TNF- a. In contrast, M2 activated macrophages express IL-12 low, IL-23 low, and IL-10high; produce arginase in the place of arginine. Moreover, macrophages have diverse activities, and the hallmarks of many of them have mutual contradiction: pro-inflammatory activities and anti-inflammatory activities, pro-tumor activities and anti-tumor activities, immunogenic and tolerogenic activities, and tissue-destructiveand tissue-restorative activities.These changes, also termed as macrophage phenotypic modulation. The polarization macrophage is an important contributor to many diseases,such as liver diseases, tumor, obesity and so on. Targeting macrophage polarization and skewing their phenotype to adapt to the micro-environment might hold great promise for these diseases.But the phenotypic modulation of macrophages and its underlying molecular mechanism is still not clear at present. The NOD-like receptor family, one of the pattern recognition receptors in the immune system, has also been implicated in regulation of inflammasome signaling and MHC class I transcription. It was shown by our previous experiments in our laboratory that NOD-, LRR- and CARD-domain containing5(NLRC5), the member of NLRs family, could participate in regulating cytokine secretion and liver fibrosis by NF-κB signaling pathway partly. According to effectively inhibiting the innate immunity and inflammatory of NLRC5, we further study the effect and potential mechanism of NLRC5 in macrophage polarization, so this research mainly divided into the following several parts:1. Introduction of M1/M2 phenotypic modulation of RAW264.7 cellsWe chose RAW264.7 as the object of our research, M1-like macrophage induced by LPS 1000 ng/ml and IFN-γ 10 ng/ml 12 h and the activation of M2-like macrophage is stimulated by IL-4 15 ng/ml 48 h.2. Expression level of NLRC5 in M1- and M2-type macrophageThe m RNA and protein expressions of NLRC5 in M1 and M2 were measured by q RT-PCR and Western Blotting. These results suggest that the expression of NLRC5 decreased in M1-like macrophage and increased in M2-like macrophage.3. The regulation of NLRC5 downstream in macrophage phenotypic modulationIt was reported that Notch signaling pathway play an important role in M1 macrophage polarization. In this paper, through LipofectamineTM 2000 liposome transfected NLRC5 RNAi and p EGFP-C2-NLRC5 and observed the transfectionefficiency by inverted fluorescence microscope, then we detected the expression of Notch1 and downstream Hes1 after the silence and over-expression of NLRC5. These results suggested NLRC5 regulated M1 macrophage may be through the Notch signaling pathway.4. The regulation of NLRC5 upstream in macrophage phenotypic modulationTreatment of M1-type macrophage with the DNA methylation inhibitor 5-aza-2’-deoxycytidine(5-azad C) could markedly reduce aberrant promoter hypermethylation of NLRC5 and elevate the expression of NLRC5, and decrease the secretion of M1 markers, Inducible Nitric oxide synthase(i NOS) and Tumor Necrosis Factor-α(TNF-α). Then we detected NLRC5 methylation state in the promoter region by pyrophosphate sequencing, further proveed the methylation of NLRC5 in M1 macrophages. And next knockdown of DNMT1 by transfecting si RNA not only attenuated the NLRC5 gene promoter methylation but also up-regulated the expression of NLRC5 in M1-like macrophage. Our results implied that NLRC5 might play a pivotal role in regulating macrophage phenotypic modulation, DNMT1-mediated NLRC5 hypermethylation caused the loss of NLRC5 expression results in negative regulation of the shift of M1-type in RAW264.7 by Notch1 signaling.