Effects Of CD38 Deficiency On TLR2-meidiated Inflammatory Response And Its Mechanism In Mouse Macrophages

Objective:CD38 is a multifunctional ectoenzyme that catalyzes the synthesis and hydrolysis of cyclic ADP-ribose(c ADPR) from NAD+ to ADP-ribose.. CD38 gene deficiency in mice leads to significantly increase in NAD+, which results in activationof Sirt1. Our previous data showed that CD38 deficiency significantly aggravated high-fat diet-induced atherosclerotic lesions in Apo E knockout mice in, as well as the obvious invasion of macrophages in the damaged area, suggesting an pivotal role of macrophages in atherosclerosis.The macrophage can be polarized into M1-macrophages and M2-macrophages. under different microenvironments. M1-macrophages comprise immune effector cells with an acute inflammatory phenotype and the M2-macrophages exert anti-inflammatory properties. Toll like receptors(TLRs), located on the macrophage membranes, are capable of resisting exogenous invasion by the release of proinflammatory cytokines. The activation of TLR2 in macrophages posseses proinflammatory or anti-inflammatory effects, thus emphasizing the importance of CD38 deficiency-mediated TLR2 epression and its mechanism of action in macrophages.Methods and Results: 1.Culture, separation and functional analysis of primary peritoneal macrophages: Western Blot showed that CD38 deficiency significantly inhibit the expression of TLR2, Sirt1 and p65 in macrophages of mice; 2. Culture and functional analysis of RAW264.7: Real-time PCR showed that HKLM,the agonist of TLR2, can downregulate m RNA levels of IL-1β and upregulate that of IL-10; 3. Inbibition of CD38 in RAW264.7 and functional analysis: using the technology of CRISPR/d Cas9 to inhibit expression of CD38 in RAW264.7 and protein chip of Bio Ray, we observed that CD38 deficiency results in the up-reglation of MCP-1、IL-1 alpha、MIP-1 alpha、RANTESand G-CSF, and the down-regulation of Eotaxin2, Fas Ligand, Fractalkine, IL-9, LIX and IL-12 70; 4. Mechanism study of CD38 mediacted macrophage functional change : RSV, the agonist of Sirt1, can inhibit the expression of TLR2, p65 and Ac-p65, while NAM, the inhibitor of Sirt1, can promote the expression of TLR2. In addition, PDTC,the inhibitor of NF-κB can inhibit the expression of TLR2 at both m RNA and protein levels.Conclusion:1. CD38 deficiency significantly reduced TLR2, Sirt1 and p65 expression and have influence of pro-inflammatory cytokines such as IL-1 beta, and anti-inflammatory cytokines such as IL-10 in macrophage of mice, indicating that CD38-mediated activation of TLR2 may be involved in the inflammatory response of macrophages. 2. Lack of CD38 may result in the activation of Sirt1, which promotes the deacetylation of NF-κB, thus prevents the transcription of TLR2 and down-regulates its expression, ultimately affect inflammatory responses ofmacrophages.