The Role Of PKC Signaling Pathway In The Dendritic Cell-mediated Inflammatory Immune Response In Atherogenesis Complicated By Diabetes Mellitus And Its Mechanism

With the changes of diet structure and aging of the population, diabetes has been taking up a larger proportion of the disease spectrum over the recent years. Studies have revealed that the risk of coronary artery disease in diabetic patients is2to6times of that in non-diabetic patients, thus high blood glucose level, as an independent risk factor, not only affect the atherogenetic process, but also play an important role in the recovery of heart function after PCI. Diabetes, as a coronary artery disease equivalent, produces a series of features in atherogenesis, such as multivessel disease, diffusion of the atheromatic plaque, small vessel disease and highly stenosis. These features indicated that diabetes exerted its unique effect on atherogenetic process, which is a hot research field worldwide.In our study,8weeks after the establishment of the diabetic models by STZ injection intraperitoneally in the ApoE-/-mice, the atherogenesis of the aortic root was accelerated, and the instability of the plaque was increased as shown in decrease in the collagen content and increase in the invasion of macrophages and dendritic cells. We also found that the expressions of inflammatory factors, such as TNFa and IFNy, were elevated peripherally in the diabetic ApoE-/-mice, and the expressions of ICAM and VCAM on the aortic artery were also elevated indicating low-grade chronic inflammation, which are consistent with the results of previous studies. Besides, we revealed that dendritic cells, the most powerful antigen presenting cells, accumulated locally in the atheromatic plaque; and the expression of the maturity phenotype related molecules, such as CD83, on the dendritic cells in spleen was increased, which indicated higher antigen presenting potential in the dendritic cells. The results we presented above suggested that the inflammatory response mediated by dendritic cells might involve in the atherogenesis in diabetes.To explore the possible mechanism involved in the DCs maturation in diabetic atherosclerotic plaque, we used GM-CSF and IL4to induce the differentiation of bone marrow cells of mice into dendritic cells in vitro, and treated cells with the oxLDL+AGEs for48hours simulating the hyperglycemic and hyperlipidemic internal enviroment of diabetic atherogenesis (with a control group treated with oxLDL alone simulating the hyperlipidemic internal enviroment of ApoE-/-mice). It was found that the immune maturation of DCs could be further induced by AGEs on the basis of oxLDL treatment, as indicated by the up-regulation of the maturity marker CD83, the co-stimulatory molecule CD86, the expressions of TNFa, IFNy and the cytokines, such as interleukins and chemokines, and that the proliferation of T lymphocytes was promoted. Furthermore, AGEs could induce high expression of RAGE on dendritic cells, as revealed by our pervious study. In the present study, we found that AGEs could not only induce high expression of RAGE on DCs on the basis of oxLDL, but also induce the phosphorelation and membrane transferring of PKCa/(31/p2, activating the TLR4signaling pathway, promoting the nuclear transferring of NF-κB and inducing the transcription of inflammatory factors.Then, we treated the DCs stimulated by oxLDL and AGEs with the RAGE neutralizing antibody and TLR4inhibitor, and found that the phosphorylation of PKCβ1, as well as the immune maturity of DCs were inhibited. To explore the role of PKC signaling pathway in DCs immune maturity, we treated the DCs with PKCa inhibitor, PKCβ1inhibitor, PKC(32inhibitor and PKC stimulator PMA. We found that PMA could promote the immune maturity of DCs; PKCβ1inhibitor could suppress the immune maturity of DCs; however, PKCa inhibitor and PKCβ2inhibitor did not display similar effects. We also found that the expressions of RAGE and TLR4were not affected by PKCβ1inhibition; houwever, the phosphorylation of IRAK downstream of TLR4was significantly inhibited by PKCβ1inhibition, indicating the phosphorylation of PKCβ1was associated with the IRAK downstream of TLR4. Furthermore, we treated with RAGE neutralizing antibody, TLR4inhibitor and PKC(31inhibitor respectively, and found that the phosphorylation of NF-κB and IκB were significantly down-regulated, suggesting oxLDL+AGEs could induce DCs maturation via the RAGE-TLR4-pPKCβ1signaling pathway.To investigate the relationship among RAGE, TLR4and PKCβ1on the cellular membrane, we have done the IP testing of RAGE and phospho-PKCβ1with TLR4antibody, and revealed the combination of RAGE and p-PKC(31with TLR4with some fixed pattern, which was further enhanced by AGEs on the basis of oxLDL. When we done the IP testing of RAGE with phospho-PKCβ1antibody, we also found the combination of RAGE with p-PKCβ1. These results supporting the RAGE-TLR4-PKCβ1trinity model on the membrane and the interaction among these three, as we hypothesized before.Finally, we treated the diabetic ApoE-/-mice with PKCβ inhibitor LY333531, and found that LY333531could significantly down-regulate the peripheral expressions of TNFa and IFNy, and the immune maturity of splenic DCs, reversing the atherogenesis of aortic root.To sum up, we proved that the activation of the PKCβ1signaling pathway played a key role in the low-grade chronic inflammatory response mediated by DCs in diabetes; AGEs could activate TLR4signaling pathway by combining with the RAGE receptor, recruite IRAK accompanied by the phosphorylation and membrane transferring of PKC(31which in turn phsphorylated IRAK, and induce the phosphorylation and nuclear transferring of NF-κB, and promot the immune maturity of DCs; PKCβ inhibitor could significantly suppress the mild chronic inflammatory response in diabetes, showing its clinical significance in the treatment of atherosclerosis in diabetes and contributing to the further clinical drug trials. Part1Exacerbation of aortic atherosclerosis accompanied by low-grade chronic inflammation and dendritic cells activation in diabetic ApoE-/-miceObjective To investigate the enhancement of inflammatory immune response and exacerbation of aortic atherosclerosis in diabetic ApoE-/-miceMethod168-week old ApoE-/-mice were divided into2groups, the control group and the DM group. Mice in the DM group were injected with STZ to create the DM models and observed for8weeks. Blood glucose and lipid levels and body weight were measured. The volumes of the atheromatic plaques were assessed on the oil red o stained frozen sections of the aortic root. Quantification of the collagen content in the plaques was performed by Masson staining. Invasions of macrophages and DCs were quantified by immunofluorescent staining of CD68and CD11c. The peripheral plasma concentrations of TNFa and IFNy were measured by ELISA. The expressions of ICAM and VCAM were assessed by qPCR. Furthermore, splenic DCs were isolated by magnetic bead selection of C11c. C83and CD86were detected by flow cytometry. The expressions of cytokines and chemokines were detected by qPCR.Results Increase in blood glucose level, decrease in body weight but no changes in blood lipid levels were observed in diabetic ApoE-/-mice. The plaque volumes of DM group were larger than those of the control group. The collagen contents were decreased in DM group, but the numbers of macrophages and DCs, the peripheral TNFa and IFNy concentrations, the expressions of ICAM and VCAM on the aortic root and the expressions of CD83and C86on the splenic DCs were increased in the DM group, when compared with the control group. The expressions of cytokines such as IL12b, IL4and IL6, and the chemokines including CCL4, CCR7and CXCR4were significantly up-regulated. Conclusion Mild chronic inflammatory response could be induced by the activation of DCs in diabetes, and was associated with the volume and stability of atheromatic plaque. Part2Induction of immune maturity of DCs and activation of typical PKC signaling pathway by AGEs on the basis of oxLDLObjective To investigate the induction of immune maturity of DCs treated with oxLDL by AGEs, and its possible mechanism.Method Bone marrow cells from C57mice were differentiated into DCs with GM-CSF and IL4. DCs were treated with oxLDL and oxLDL+AGEs for48hours. CD83and CD86were detected by flow cytometry. The transcriptions of cytokines and chemokines were quantified by qPCR. The TNFa and IFNy in the cell culture supernate were detected by ELISA. The phosphorylations of NF-κB and IκB were detected by Western blot, demonstrating by theAllogeneic T lymphocyte proliferation. The phosphorylation of typical PKC and the expressions of RAGE and TLR4were detected by western blot.Result AGEs could further up-regulate the maturity marker CD83and co-stimulatory molecule CD86on DCs on the basis of oxLDL. The expressions of cytokines and chemokines, and the phosphorylation of NF-κB and IκB were up-regulated as revealed by ELISA and qPCR. The expression of RAGE, the phosphorylation of PKCa/β1/β2and the activation of TLR4signaling pathway were further enhanced by AGEs on the basis of oxLDL.Conclusion The immune maturity of DCs treated with oxLDL could be further induced by AGEs, which might involve the RAGE-TLR4-PKC signaling pathway. Part3RAGE-TLR4-pPKCβ1signaling pathway involved in the immune maturity of DCs induced by AGEs on the basis of oxLDLObjective To investigate the possible molecular mechanism in the immune maturity of DCs induced by AGEs on the basis of oxLDL.Method Bone marrow cells from C57mice were differentiated into DCs with GM-CSF and IL4. DCs treated with oxLDL+AGEs were interfered with RAGE neutralizing antibody, TLR4inhibitor, PKCa inhibitor, PKCβ1inhibitor, PKCβ2inhibitor and PMA on the sixth day. CD83and CD86were detected by flow cytometry. The TNFa and IFNγ in the cell culture supernate were detected by ELISA. The TLR4-NK-KB signaling pathway were detected by western blot. Phospho-PKCβ1and RAGE were co-Immunoprecipitated with TLR4antibody and RAGE were co-Immunoprecipitated with Phospho-PKCβ1antibody.Result RAGE neutralizing antibody, TLR4inhibitor and PKCβ1inhibitor, rather than PKCa inhibitor and PKCβ2inhibitor, could significantly suppress the immune maturity induced by AGEs on the basis of oxLDL. PMA, the PKC agonist, could promote the immune maturity of DCs, which was attenuated by PKC(31inhibitor. The combination of RAGE-TLR4-pPKCβ1on membrane and the interaction among these three were demonstrated by IP.Conclusion RAGE-TLR4-pPKCβ1-NF-κB signaling pathway might be the possible molecular mechanism involved in the immune maturity of DCs induced by AGEs on the basis of oxLDL. Part4The inhibition of DCs immune maturation and the amelioration of aortic atherosclerosis in diabetic ApoE-/-by LY333531Objective To investigate whether PKCβ inhibitor LY333531could ameliorate the progress of diabetic atherosclerosis.Method168-week old ApoE-/-diabetic models induced by STZ were randomly divided into the control group and the LY333531group treated with LY333531(3mg.kg-1.d-1). The blood glucose and lipid levels and body weight were measured after8weeks’treatment. Splenic DCs were isolated by magnetic bead selection of C11c. CD83and CD86were detected by flow cytometry. The expressions of cytokines and chemokines were quantified by qPCR. The peripheral plasma concentrations of TNFa and IFNy were measured by ELISA. The expressions of ICAM and VCAM were assessed by qPCR. The plaque volumes were measured on the oil red o stained frozen sections of the aortic root. Quantification of the collagen content in the plaques was performed by Massion staining. Invasions of macrophages and DCs were quantified by immunofluorescent staining of CD86and CD11c.Results No changes of blood glucose and lipid levels but increase in body weight were observed in LY333531group. The expressions of CD83and C86on the splenic DCs and the secretions of cytokines and chemokines were significantly decreased. The peripheral TNFa and IFNy concentrations and the expressions of ICAM and VCAM on the aortic root were decreased in the LY333531group. The plaque volumes of LY333531group were decreased when compared with those of the control group. The collagen contents were increased, whereas the numbers of macrophages and DCs were decreased.Conclusion LY333531could inhibit the immune maturity of DCs in diabetic atheroslceotic enviroment independently of the blood glucose and lipid changes, attenuate the mild chronic inflammatory response, reverse and stabilize the atheromatic plaques. Conclusion1Mild chronic inflammatory response could be induced by the activation of DCs in diabetes, and was associated with the volume and stability of atheromatic plaque.2The immune maturity of DCs treated with oxLDL could be further induced by AGEs, which might involve the RAGE-TLR4-pPKC(31-NF-KB signaling pathway.3LY333531could inhibit the immune maturity of DCs in diabetes, attenuate the mild chronic inflammatory response, reverse and stabilize the atheromatic plaques.Innovation and significance1PKCP was found to play a role in the pathogenesis of atherosclerosis for the first time. The RAGE-TLR4-pPKCpi-NF-KB signaling pathway was introduced as the possible molecular mechanism involved in the immune maturity of DCs in atherosclerosis, which emphasized the anti-atherosclerotic potential of PKC[3and provided a brandnew way for the clinical treatment and prevention of atherosclerosis in diabetes.2The role of PKCβ specific inhibitor in prevention and reversion of atheromatic plaque formation in diabetes were investigated for the first time, providing the theoretical basis for future clinical trials.