Effects Of P.gingivalis-lps On The Differentiation Of Th9 In Vitro

Objective:Th9 plays an important role in the immune inflammatory response of the body.Previous studies have found that Th9 is correlated with periodontitis.Porphyromonas gingivalis is the main pathogen causing periodontitis,and its effect on Th9 differentiation has not been reported.In this study,the effect of porphyromonas gingivalis lipopolysaccharide(P.gingivalis-LPS)on Th9 differentiation was investigated by establishing a Th9 differentiation model in vitro and administering P.gingivalis-LPS.High throughput sequencing was used to analyze the key factors of P.gingivalis-LPS regulating Th9 differentiation,which laid a theoretical foundation for the study of the correlation between Th9 and periodontitis.Methods:Peripheral blood of 4 healthy volunteers was collected,CD4~+T cells were sorted out by magnetic beads,and P.gingivalis-LPS was added under Th9 cell polarization conditions(CD3m Ab,CD28 m Ab,rh IL-2 and rh TGF-β/rh IL-4).Flow cytometry was used to detect Th9 differentiation,RT-PCR was used to detect the expression of IL-9,PU.1 and IRF4,and ELISA was used to detect the expression of IL-9 in the supernatant of cells.Three samples in the blank group,Th9 group and P.gingivalis-LPS group were sequenced by transcriptome.The differentially expressed genes were analyzed by gene function(GO)and signaling pathway significance enrichment(KEGG).The candidate genes with significant differences were verified by RT-PCR.Results:1.CD4~+T cells were sorted by immunomagnetic beads,and the sorting rate was 97%by flow cytometry.The effect of P.gingivalis-LPS on Th9 cell differentiation was as follows:(1)Flow cytometry showed that P.gingivalis-LPS could promote Th9 celldifferentiation,with the strongest effect at 1μg/m L(P<0.001);(2)RT-PCR results showed that P.gingivalis-LPS could promote the expression of IL-9,IRF4,PU.1 mRNA(P<0.01).(3)ELISA results showed that P.gingivalis-LPS could promote the expression of IL-9 protein(P<0.01).2.Transcriptome sequencing was used to analyze Th9 cell differentiation and key genes in P.gingivalis-LPS regulation of this process,and bioinformatics method was used for comparative analysis.GO enrichment and KEGG analysis were used to screen immune-related genes and signal transduction pathways involved in Th9 cell differentiation and Th9cell resistance to P.gingivalis-LPS infection.(1)Compared with the blank group,212 genes were significantly up-regulated,254 genes were significantly down-regulated,1409 transcripts were significantly up-regulated and 1168transcripts were significantly down-regulated in the Th9 group.In P.gingivalis-LPS group,155 genes were significantly up-regulated,191 genes were significantly down-regulated, 1151 transcripts were significantly up-regulated,and 1254 transcripts were significantly down-regulated.P.gingivalis-LPS significantly up-regulated 12 genes,significantly down-regulated 4 genes,significantly up-regulated 945 transcripts,and significantly down-regulated 1249 transcripts in the immune inflammatory response induced by Th9 cells.(2)GO enrichment and KEGG pathway enrichment analysis revealed that multiple immunoinflammation-related biological processes were involved in Th9 cell differentiation(immune inflammatory response,cytokine activity,chemokine activity and mediated signaling pathways,neutrophil chemotaxis,nuclease activity,etc.),suggesting that Th9 cells may play an important role in the progression of immune inflammatory diseases.3.Six differentially expressed genes,including IFN-γ,IL-7R,PDE4B,Thy-1,TGM2 and SPINT2,were selected for RT-PCR detection.The results showed that the expressions of IFN-γ,IL-7R and PDE4B in the Th9 group and P.gingivalis-LPS group were lower than those in the blank control group(P<0.0001),while the expressions of SPINT2,TGM2 and Thy-1 were higher than those in the blank control group(P<0.01).The expressions of IFN-γand TGM2 in P.gingivalis-LPS group were lower than those in Th9 group(P<0.05),while the expression of Thy-1 was higher than that in Th9 group(P<0.05).There was no significant difference in IL-7R,PDE4B and SPINT2 between P.gingivalis-LPS group and Th9 group,which was consistent with the results of transcriptome sequencing.These results indicated that these factors were closely related to Th9 cell differentiation.P.gingivalis-LPS promoted Th9 cell differentiation by regulating the expression of related factors.Conclusion:P.gingivalis-LPS can promote Th9 cell differentiation,and not only up-regulate the expression of specific factors PU.1,IRF4 and IL-9;It can also up-regulate the expression of Thy-1 and inhibit the expression of IFN-γand TGM2.Transcriptome sequencing further explored and enriched the potential genes of Th9 cell differentiation, which laid a theoretical foundation for clinical research on the correlation between Th9 cell and periodontitis.