The Role Of PHB2 In The Regulation Of TH17 Cells

Objective:TH17 cells not only has important protective effect on bacterial and fungal infections, also plays an important role in autoimmune inflammation. Retinoid related orphan nuclear receptor (Retinoid-related orphan receptor GammaT, RORyt) is the key transcription factor of TH17 cells, the expression determines the differentiation of TH17 cells and induce proinflammatory cytokines such as IL-17. Through the early mass spectrometry analysis found that PHB2 and RORyt has interaction, PHB2 is a highly conserved ubiquitously expressed protein and in T cell activation process is raised, the formation of phosphate complexes localized on the T cells in the inner mitochondrial membrane, to maintain mitochondrial integrity, T cell activation, differentiation and survival. This study by Co-immunoprecipitation, RNA interference technology, is expected to investigate the PHB2 how to play a role in regulating the differentiation of TH17 cells and its regulation mechanism.Methods:First of all through the PCR technique to amplify the complete sequence of PHB2, cloned into HA-pIP expression vector, Flag-RORyt and HA-PHB2 was co-transfected into HEK293T cells, and the interaction between PHB2 and RORyt was verified by Co-immunoprecipitation technique. Construction of about 600bp size of IL-17A promoter report gene, with the PEI transfection method transfect in HEK293T cells overexpressing of PHB2, RORyt and reporter gene, and explore the impact of PHB2 to RORyt function by luciferase reporter gene assay. Finally, by electroporation method in Jurket cells overexpressing PHB2, RORyt and reporter gene, explore the impact of PHB2 to RORyt function by luciferase reporter assay. Design of Two pairs for PHB2 specific shRNA, packaged shRNA expression lentiviral vector, while building a pair of overexpression of PHB2 lentiviral vectors, while building one pair PHB2 overexpression lentivirus vector plasmid, by PEI transfection method co-transfected plasmids delta 8.9, VSV-G into HEK293T cells.48 hours later, receive supernatant and Concentrated virus by the method of ultra speed centrifugation. Then by ficoll separation of PBMC from umbilical cord blood, and the NaiveCD4+T cells were selected by the kit, and the TH17 cells were induced in vitro. The PHB2 shRNA and PHB2 overexpression lentiviral direct infection of the Flag-RORyt-Jurket stably transfected cell lines and in vitro induced TH17 cells.After virus infection about a week, then was detected by Western blotting of PHB2 and RORyt protein levels and by RT-PCR analysis of PHB2 of TH17 related gene effects.Results:Through co-immunoprecipitation technique and Western blotting results can be see, Cotransfection of Flag-RORyt and HA-PHB2 group by adding anti-HA antibody with anti-Flag antibody by Western blot can see strong signal of RORyt (or adding anti-Flag after anti-HA antibody by Western blot can see strong signal of PHB2), and thus the results can be drawn that PHB2 has an interaction with RORyt. IL17A promoter luciferase reporter gene assay in HEK293T cells and Flag-Jurket cells, we found PHB2 can effectively promote RORyt mediated transcriptional activity of IL-17A and a dose-dependent manner. Using the lentivirus form over expression of PHB2 in the Flag-RORyt-Jurket stable cells, Western blot results showed that the level of RORyt protein was enhanced and the protein level of PHB2 was enhanced. In vitro successfully induced TH17 cells, and build the two shRNA of PHB2 have good silencing efficiency. In Flag-RORyt-Jurket cells and TH17 cells infected with PHB2 shRNA lentivirus and immunoblotting results analysis obtains the PHB2 protein levels were significantly decreased, at the same time the RORyt protein levels also declined. RT-PCR analysis of the results obtained PHB2 genetic level down while TH17 cell-associated gene IL-17A reduced 25%, IL-17F by 50%.Conclusion:This study preliminarily identified the interaction between PHB2 and RORyt by co-precipitation and luciferase reporter gene technique, and concluded that PHB2 could promote the transcription activity of IL-17A promoter mediated by RORyt. Finally, through RNA interference,RT-PCR and Western blot further explored the PHB2 through regulation of the protein level of RORyt thus affectting the transcriptional level of TH17 related cytokines.It can be drawn PHB2 of TH17 cells in the regulation is a positive regulatory role.