Research About The Mechanism Of IL-6/STAT3/TGF-β Axis In Human Peripheral Monocytes Against Cryptococcus Neoformans

Part 1 The expression of IL-6 in human peripheral monocytes stimulated with heat-killed Cryptococcus Neoformans was significantly increasedIn the first part,we aim to screen the cytokines that differentially expressed in human peripheral monocytes immune response against heat-killed Cryptococcus Neoformans.Firstly,we accquired peripheral blood mononuclear cells by Ficoll Density Gradient Centrifugation.And then,using CD14 microbeads kit separated and cultured monocytes.Using heat-killed Cryptococcus Neoformans stimulated monocytes for 12 hours,then the supernatant and sediments were collected respectively.RNA was extracted from the cell deposits,and RNA need to translate into cDNA by reverse transcript ion.Using quantitative real time PCR to identify the expression of some cytokines,in cluding TNF-α,IFN-β,IL-4,MCP-1,IL-6.We find that there were differences in the ex pression of cytokines after monocyte were stimulated with Cryptococcus Neoformans.Compared with unstimulated group,the above cytokines expressed higher after mono cyte were stimulated with Cryptococcus Neoformans,the most obvious is the rise of IL-6.Meantime,contents of cytokines including TNF-α,IFN-β,IL-4,MCP-1,IL-6 were also indentified by Enzyme-linked Immunosorbent Assay.Compared with unstimulat ed group,the above cytokines expressed higher in stimulated group,the mostobvious is also the rise of IL-6.We can see by this part that IL-6 was involved in human peripheral monocytes immune response against heat-killed Cryptococcus Neoformans,and then relevant with pathogenesis of Cryptococcal associated disease.Part 2 IL-6 can function as a positive regulator for theexpression of STAT3 and TGF-β during monocytes immune response against heat-killed Cryptococcus NeoformansIn this part,we aim to explore IL-6 can function as a positive regulator for the expression of STAT3 and TGF-β during monocytes immune response against heat-killed Cryptococcus Neoformans.By Ficoll Density Gradient Centrifugation and CD14 microbeads kit,we separated and cultured peripheral blood mononuclear cells.Monocytes were stimulated with heat-killed Cryptococcus Neoformans for 12 hours,then the supernatant and sediments were collected respectively.Using quantitative real time PCR to explore the expression of STAT family protein,and upstream regulator,such as IL-12,IL-4,TGF-β and so on,involved in T helper cell differentiation were also mesured by ELISA.Compared with control group,STATs expression down regulate in the group that IL-6 was neutralized,and the most obvious was the reduce of STAT3.And in the group that IL-6 stimulated,STATs expression up regulate,the most obvious was the increase of STAT3.The expression of cytokines in supernatant were measured by ELISA.we found TGF-β dropped most obviously in the group that IL-6 was neutralized,and it also increased in the IL-6 stimulated group.Thus,we can think that IL-6 function as a positive regulator for the expression of STAT3 and TGF-β during monocytes immune response against heat-killed Cryptococcus Neoformans.Part 3 Monocytes stimulated with heat-killed Cryptococcus Neoformans can promotes Th17 cell development by IL-6/STAT3/TGF-β axisIn this part,we aim to explore that monocytes stimulated with heat-killed Cryptococcus Neoformans can promote Th17 cell development by IL-6/STAT3/TGF-β axis.Jurkat T cell were coincubated with culture supernatant for 12 hours which we collected after monocytes were stimulated with heat-killed Cryptococcus Neoformans for12 hours.Using Flow Cytometric to measure the differentiation of Th17.Compared with control group,the differentiation of Th17 cells increased notably in the experime ntal group.And in the group that IL-6 was neutralized,the differentiation of Th17 was decreased.From this,we can see that monocytes stimulated with heat-killed Cryptococcus Neoformans can promotes Th17 cell development by IL-6/STAT3/TGF-β axis.