Collagen Ⅵ Antibodies Regulate Macrophages Differentiation Through FcgammaRⅡb Signal Transduction Pathway

Background:OxLDL,HSP60,β2-Glycoprotein Ⅰ and Fibronectin,etc.have been identified as atherogenic antigens.Immunization or antibodies against these antigens may represent a novel possibility for treatment or prevention of atherosclerosis.Objectives:In this study,we aim to verify that α6 chain of collagen Ⅵ is a novel atherosclero-related antigen by immunization with collagen Ⅵ related peptides,and test the hypothesis that human recombinant IgGl against collagen Ⅵ(CVI)reduces atherosclerosis in ApoE-/-mice,and clarify the underlying mechanisms of the protective effect of the therapeutic antibody against AS.Methods:1.Male ApoE-/mice(6 weeks)were immunized with collagen Ⅵ derived peptides COL6A5 or COL6A6 at 6 and 9 weeks and were fed a high-fat diet(HFD)for 15 weeks.The atherosclerotic plaque was determined by Oil Red O staining at 25 weeks.Spleenderived CD86+Dc cells and CD86+B cells were analyzed by flowcytometry.Serum cholesterol and triglyceride concentrations were measured and IgG subtypes(IgG1 and IgG2a)were determined by enzyme-linked immunosorbent assay.2.The glutamine synthetase(GS)system was used to generate collagen Ⅵ monoclonal antibody producing CHO cells.3.Male apoE-/-mice(4 weeks)were fed with HFD for 20 weeks and were subsequently transferred to chow for 1 week,then the mice were injected intraperitoneally with CVI antibodies(CVI Abs)or PBS.The injections were repeated 2 times at 1-week intervals,and the mice were sacrificed 2 weeks after the last injection and macrophage subsets were determined by flowcytometry.Atherosclerotic plaque was measured using Oil red staining.In vitro experiments,CD 14+ monocytes were exposed to oxLDL or FBS with or without pretreatment of CVI Abs,and cultured for 1 week to induced macrophage differentiation.The proportion of M1(CD68+CCR2+)and M2(CX3CR1+CD163+ or CX3CR1+CD206+)macrophages was analysed by flowcytometry.The total and phosphorylation of phosphalation of JNK,ERK,p38,SHIP,Dok and Syk was measured by Western blot.Results:1.In this study we show that immunization of apoE-/-mice with COL6A6 peptide,reduced aortic atherosclerosis compared with saline controls(30%,P<0.05).Activated B cells and Dc cells increased and serum cholesterol decreased(P<0.05).The level of serum total IgG1 increased(P=0.0559)with the absence of an increase in serum total IgG2a.2.Human collagen Ⅵ monoclonal antibody producing CHO cells were generated and the antibody productivity is 100 mg/L(after purification).3.CVI Ab reduced atherosclerosis by 40%compared to the control mice(P<0.001),and the proportion of M1 macrophages decreased(P<0.05).In vitro,CVI Ab inhibited M1,promoted M2.CVI Ab inhibited M1 through FcyRIIb signal pathway.The mechanism of the protective effect of CVI Ab against AS is that CVI Ab activates FcgammaRIIb,SHIP and Dok phosphatases,and inhibits Syk and Ras signal transduction pathway in macrophages,which are upstream kinases of MAPKs,so that CVI Ab inhibits ERK,JNK,and p38 MAPKs activation,and ensuing M1 differentiation.Conclusions:We found a new atheroprotected antigen,COL6A6.Collagen Ⅵ antibody activated FcgammaRIIb signal pathway in macrophages and reduced atherosclerosis by 40%in ApoE-/-mice.These results may provide new insights into the control of lipidinduced inflammatory reactions in large blood vessels and therapeutic methods to prevent atherosclerosis.