Immune-regulatory Effect Of Novel MTOR Inhibitor AZD8055 And Its Corresponding Mechanism

Objects1?To interpret the immune-modulatory effects of novel mTOR inhibitor AZD8055 in murine IBD model in vitro and in vivo,to further enhance the understanding of the pathogenesis of IBD,and to seek new therapeutic approaches or intervention targets for clinical treatment of autoimmune diseases and transplant rejection.2?To explore the effects of novel mTOR inhibitor AZD8055 on the differentiation,maturation of murine dendritic cell in vitro and in vivo.To perform a series of functional studies on AZD8055-treated DC in order to conduct a preliminary exploration of the mechanisms on immune tolerance induced by the intervention of mTOR signaling pathway in dendritic cells.3?To enhance the understanding of the biological mechanisms of tolerogenic dendritic cells induced by novel mTOR inhibitor AZD8055 and pave a road for future work.MethodsPart I1)In vivo studies:DSS-induced experimental colitis was established in C57BL/6 mice,which were treated with AZD8055.We evaluated the protective role of AZD8055 in DSS-induced colitis by monitoring clinical manifestations,assessing disease activity scores and analyzing pathology of colon tissues.Meanwhile,we used FCM to analyze the percentage of Th17?Th1?Treg cells in MLN and to examine the percentages of CD4+?CD8+ T cells in spleen?LN?MLNs.2)In vitro studies:We attained CD4+ T cell from the spleen,lymph nodes of C57BL/6 by magnetic sorting,and activated by anti-CD3 and CD28 with gradient AZD8055 for intervention.We then used Western-blot to detect the phosphorylation of key proteins in mTOR signaling pathway in CD4+ T cells;spleenocytes and magnetic sorted Naive T(CD4+CD62L+)were labeled with CFSE,then activated by anti-CD3 and CD28 or cultured in Th17,Treg,Th1 skewing conditions with gradient AZD8055 for intervention.We used FCM to detect the proliferation of CD4+ T helper cells and CD8+ T cells and the proliferation and differentiation of Th1,Th17,Treg cells.Part II1)In vivo studies: We gave intraperitoneal injection of the AZD8055(10mg/kg/day×7days)with DMSO as a control,and sacrificed C57BL/6 mice after 7 days to obtain the spleen and lymph nodes.We then assessed the proportion of CD11c+CD8+ and the plasmacytoid DC(p DC)subsets;We then used bone marrow precursor cells to differentiate into DC in vitro,and detect their expression of costimulatory surface molecules and CD11 c at the day of 5,7,9.2)In vitro studies: We treated DC with a gradient of AZD8055,then stimulated them with LPS after 1 h.Western-blot were performed to detect the phosphorylation of key proteins in mTOR signaling pathway in CD11c+ DC;We cultured the bone marrow precursor cells with AZD8055 in the process of cytokine-inducing dendritic cells differentiation.We then co-cultured these induced DC with allogeneic CD4+ T cells.FCM were performed to detect DC phenotype,proliferation of allogenic CD4+ T lymphocyte,and the proportion of Treg.Part IIIWe performed a RNA-seq to study the transcriptome of AZD-treated DC in comparison to WT DC and stimulated WT DC(LPS-treated DC),which represented as the mature and activated DCs.These three kinds of dendritic cells reflect three different functional states of cells.Using DEG-seq to calculate and identify a number of significant differences genes for subsequent trend analysis,GO and KEGG analysis.ResultsPart I1)AZD8055 can significantly improve the condition of IBD mice by decreased body weight loss,disease activity scores and histopathology scores.2)AZD8055 can reduce the proportion of Th1,Th17 cells of MLN while increase the proportion of Treg cells of MLN in IBD mice.3)AZD8055 can reduce the proportion of CD4+,CD8+T cell of spleen,lymph nodes and peripheral blood in IBD mice.4)AZD8055 can inhibit the mTOR signaling pathway in CD4+ T cells in vitro.5)AZD8055 can inhibit CD4+ and CD8+ T cell proliferation in vitro.6)AZD8055 can inhibit differentiation of naive T lymphocytes into Th1?Th17 and decrease proliferation of Th1?Th17 while promoting Treg cell differentiation and proliferation.Part II1)AZD8055 can reduce the proportion of CD11c+ DC of bone marrow,spleen,peripheral and mesenteric lymph nodes in vivo.2)AZD8055 have little effect on the differentiation of bone marrow precursor cells into dendritic cells,but significantly inhibited the maturation of DC.3)AZD8055 can significantly inhibit the mTOR signaling pathway at a concentration of 10 n M in DC.4)AZD8055 do not affect the DC differentiation in vitro.5)AZD8055 can reduce the expression of costimulatory molecules induced by LPS and affect DC maturation.6)DC induced by AZD8055 can significantly inhibited proliferation of allogeneic CD4+T lymphocytes in MLR in vitro.7)DC induced by AZD8055 can promote Treg differentiation in allogeneic CD4+ T lymphocytes in vitro.8)DC induced by AZD8055 can induce increased apoptosis of allogeneic CD4+ T lymphocytes.9)DC induced by AZD8055 can promote more Treg proliferation compared with non-Treg in MLR.Part III1)We have identified 430,1172 and 1436 genes which were differentially expressed between WT and AZD DCs,WT and LPS DCs,and AZD and LPS DCs,respectively.2)Cluster analysis showed that AZD DCs transcriptomeis similar to WT DCs,However,LPS-DCs transcriptome was largely distinguished from those of WT and AZD-DCs.3)we classified these genes into 8 possible dynamic expression patterns based on vari ous DC maturation stages.Among these groups,group 1,4,5 were significantly enriched.The minimal P-value was in the group I.4)The results of GO analysis: we found that the GO categories of “immune response” and “response to wounding” were highly enriched in all DEGs.The DEGs between AZD and WT DCs were highly enriched in GO categories of “chemokine activity”,“chemokine receptor binding”,“homeostatic process” and “carbohydrate binding”,which were not identified in those DEGs between LPS and WT DCs.we found that the FDR values are relatively higher among the differentially expressed genes between AZD and WT.FDR values are relatively lower in up-regulated genes.Genes in group I are closely related to translation.5)The results of KEGG analysis: Three groups of differentially expressed genes are focused on these signaling pathways,such as NF-kappa B signaling pathway,chemokine signaling pathway,Cytokine-cytokine receptor interaction,MAPK signaling pathway,Toll-like receptor signaling pathway,apoptosis,etc.Group I is closely related to the ribosome and PPAR signaling pathway.6)AZD-DC were significantly different with WT-DC,LPS-DC on the four shear mode.7)Wayne combined trend analysis found that the expression level of these genes such as MGL2,Cadherin-1,4-1BB,Rho B,PDPN raised or lowered during the process from immature tolerogenic to a mature immune status.ConclusionsPart I1)AZD8055 can decrease the proliferation of effector lymphocyte?inhibit the Th1 and Th17 type reaction and enhance the proportion of regulatory T cells(Treg),therefore,it exerted its protective role in DSS-induced colitis.2)The protective effect of AZD8055 in a murine IBD model may be related to its inhibition on differentiation of naive T lymphocytes into Th1,Th17,and promote Treg cell differentiation and proliferation,thereby regulating the balance of Treg and Th17.3)AZD8055 can play a important role in regulating the differentiation and function of T helper cell subtype,which provides a new way of thinking and direction for further understanding of the immune regulatory role and mechanism of the mTOR signaling pathway on T lymphocytes.mTOR could be considered as a novel target in IBD therapy,the protective mechanism of which in IBD required further investigation.Part II1)mTOR signaling pathway plays an important role in the maintenance of the number and maturation of DC in mice.2)AZD8055 may induce tolerance by influencing DC maturation.3)Tolerogenic DC induced by AZD8055 plays its role in the induction of immune tolerance probably through inducing T cell anergy or incompetent,therefore increasing the proportion of Treg?inducing T cell apoptosis,etc.4)AZD8055 play a very important role in regulating differentiation,maturation,and function of dendritic cell,which provides a new way of thinking and direction for further understanding of the immune regulatory role and mechanism of the mTOR signaling pathway on dendritic cell.mTOR could be considered as a novel target of inducing tolerogenic DC.Part III1)p38MAPK and NF-?B signaling pathway are involved in the process of differentiation,and maturation of DC.2)The activation of PPAR and ribosome signaling pathway are involved in the mechanisms of tolerogenic DC induced by AZD8055.3)MGL2,Cadherin-1,4-1BB,Rho B and Pdpn genes play important roles in the mechanisms of tolerogenic DC induced by AZD8055.