Indirectly Immunomodulatory Effects Of DCs By Ganoderma Atrum Polysaccharide In Intestine Based On RNA-seq

Ganoderma atrum(G.atrum)is a kind of rare medicinal and edible fungi,which has been available throughout history in China,Japan,South Korea and other eastern countries.Polysaccharide is considered to be one of the main active ingredients of G.atrum,with a variety of bio-activities including immune regulation,anti-tumor,anti-oxidation,anti-aging.In previous studies,G.atrum polysaccharide(PSG-1),extracted and purified from the cultivated G.atrum in southern Jiangxi Province,can activate the immune response of dendritic cells(DCs)by directly contacting with its surface receptors.However,as a kind of macromolecule compound,PSG-1 cannot directly stimulate DCs via passing through the intestine epithelial cells.In this study,a Caco-2/DCs co-culture model was established,and then the PSG-1-treated or untreated Caco-2 and DCs transcripts were globally screened by RNA-seq.According to the RNA-seq analysis,the effect of PSG-1 on the immune function of DCs under intestinal epithelial barrier was explored,as well as the influence of PSG-1 on "dialogue" between Caco-2 and DCs.The main conclusions are summarized as follows:1.RNA-seq analysis found out 2293 differentially expressed genes(DEGs)of DCs between PSG-1 group and control group,including 1180 up-regulated genes and1113 down-regulated genes.GO enrichment analysis was performed on the DEGs obtained,most of the significantly enriched GO terms were related to "immunity",which indicated that PSG-1 still affect the immune function of DCs in Caco-2/DCs co-culture model.KEGG enrichment analysis identified 109 significant enrichment pathways,among which 10 pathways were related to "environmental information processing",including "cytokine-cytokine receptor interaction","NF-?B signaling pathway" and "MAPK signaling pathway".2.PSG-1 indirectly stimulate the immune function of DCs in Caco-2/DCs co-culture model.The expression of cytokines(TNF-?,IL-1?,IL-6,IL-10)and chemokines(RANTES,MCP-1,MIP-1?)of DCS was significantly increased after the indirect stimulation of PSG-1,the activation of NF-?B and MAPK pathways was found in DCs indirectly stimulated by PSG-1.These results suggested that PSG-1could indirectly promote inflammatory factors secretion of DCs in Caco-2/DCs co-culture model,and further regulate the immune function of DCs,among which the activation of NF-?B and MAPK pathways in DCs played an important role.3.RNA-seq analysis found out 452 DEGs of DCs between PSG-1 group and control group,including 256 up-regulated genes and 196 down-regulated genes.The first three significant enriched GO terms were all related to "cytokines",indicating the important role of cytokines in the effect of PSG-1 on DCs through Caco-2.KEGG enrichment identified 53 significant enrichment pathways,among which 5 pathways were related to "environmental information processing",including "cytokine-cytokine receptor interaction","TNF signaling pathway" and "NF-?B signaling pathway".4.In the Caco-2/ DCs co-culture model,PSG-1 indirectly regulates the immune function of DCS through TNF-? and NF-?B signaling pathways of Caco-2.After treatment of Caco-2 with TNF-? and NF-?B inhibitors,the secretion of cytokines(TNF-?,IL-1?,IL-6,IL-10)and chemokines(RANTES,MCP-1,MIP-1?)on DCs which indirectly stimulated by PSG-1were reduced,and the activation of NF-?B and MAPK pathways on DCs indirectly mediated by PSG-1 were inhibited.