Study On The Effect And Mechanism Of LILRB4 In Regulating Atherosclerosis

Atherosclerosis is the pathological basis of ischemic cardiovascular and cerebrovascular diseases such as coronary heart disease and thrombotic diseases.When plaque is ruptured,thrombus may form and impede circulation that leads to cardiovascular and cerebrovascular diseases.In China,almost 3 million people died of atherosclerosis related diseases each year.Macrophages play a crucial role in atherogenesis.Inflammation is critical for macrophage infiltration,foaming and apoptosis.Thus,study on the inflammatory response in macrophages is very important for understanding the mechanism of atherosclerosis and developing novel strategy for therapy.Our preliminary data showed that LILRB4 was up-regulated in macrophages in atherosclerosis plaques.Given that LILRB4 suppressed inflammatory response,we hypothesized that LILRB4 regulated atherogenesis by suppressing macrophage mediated inflammatory response.My project aimed to investigate the function of LILRB4 and dissect its mechanism in atherogenesis.Frist,we verified the relevance between LILRB4 protein expression and atherosclerosis.We found that the protein level of LILRB4 was markedly up-regulated in coronary arterial atherosclerosis plaques of CHD patients.We confirmed the upregulation of LILRB4 in the artery root of Apoe-/-mice fed on a high fat diet.Immunofluorescence staining showed that the increased LILRB4 expression was mainly in macrophages.Second,we discovered that LILRB4 inhibited plaque formation.We obtained Lilrb4-/-Apoe-/-mice by crossing Lilrb4-/-mice with Apoe-/-mice and fed the mice on high-fat diet.We found that LILRB4 deficiency resulted in larger atherosclerotic lesion and increased instability of plaques,which represented a worse phenotype.Bone marrow transplantation experiment showed that chimeric mice engrafted with bone marrow cells from Lilrb4-/-Apoe-/-mice exhibited a larger atherosclerotic lesion than those engrafted bone marrow cells from Apoe-/-mice.Considering that LILRB4 was up-regulated in macrophages,these results suggest that LILRB4 plays an inhibitory role in atherosclerosis by regulating macrophage function.Third,we revealed that LILRB4 inhibits NF-?B activation at least partly through SHP1.NF-?B signaling in the plaque was enhanced whereas SHP-1 phosphorylation was decreased by LILRB4 deficiency.Meanwhile,the content of pro-inflammatory factors in serum were increased,indicating that LILRB4 deficiency promoted inflammatory response in atherosclerosis plaque.Furthermore,LILRB4 interacted with SHP1,which was strengthened by Ox-LDL stimulation.In contrast,SHP1 agonist "Regorafenib" restrained NF-?B activation.These results suggest that LILRB4 inhibits NF-?B signaling through SHP-1.Finally,we dissect the mechanism by which LILRB4-SHP1 axis suppressed NF-?B signaling by inhibiting TRAF6 ubiquitination.We found that both LILRB4 and SHP1 inhibited TRAF6 ubiquitination.However,LILRB4 failed to inhibit TRAF6 ubiquitination upon SHP1 knockdown.Moreover,SHP1 interacted with coiled-coil domain of TRAF6 and LILRB4 facilitated the interaction between SHP1 and TRAF6.In contrast,LILRB4 mutant with alterations of all tyrosine sites on ITIM motif failed to recruit SHP1 and lost the ability to facilitate the interaction between SHP1 and TRAF6.These results suggest that LILRB4 recruits SHP-1 and TRAF6 complex and subsequently inhibits TRAF6 ubiquitination,which ultimately suppresses inflammatory response.In summary,as an immune inhibitory receptor,LILRB4 exhibits an inhibitory function in NF-?B signaling by recruiting SHP1.LILRB4 deficiency in macrophages increases inflammatory response in plaque and leads to atherosclerosis progressing.Our study has revealed the function of LILRB4 and its mechanism in atherosclerosis..These findings may provide experimental evidence and potential targets for developing target therapy of atherosclerosis.