Tsc1-dependent Transcriptional Programming Of Dendritic Cell Homeostasis And Function Of Antigen Presentation

Background:Dendritic cells（DC）is known as the most potent antigen presenting cell.Tuberous sclerosis complex 1（Tsc1）is the key negative regulator of mammalian target of rapamycin complex 1（mTORC1）,which is a central integrator of metabolism and growth signal.Emerging evidence highlights important roles of tuberous sclerosis complex 1（Tsc1）in DC development and activation and DC was required to promote T-cell homeostasis and response partially through inhibiting mammalian target of rapamycin complex1（mTORC1）.However,the molecular mechanism of transcriptional regulation by which Tsc1 control DC homeostasis and function of antigen presentation remains largely unknown.Objective:This study identified the molecular mechanism of transcriptional regulation which Tsc1 control DC homeostasis and function of antigen presentation by comparing the transcriptional profiling of Tsc1-deficient DCs with wild-type DCs.Methods:（1）Homozygous mice(CD11c^CreTsc1^f/f)that specifically knocked out Tsc1in DCs were established by hybridization of Tsc1^f/f mice（The LoxP sequence is present on both ends of Tsc1 in vivo）and CD11c^Cre transgenic mice（Expression of CD11c-Cre recombinase under the control of lysozyme promoter）using the Cre-LoxP recombinase system.The knockdown of Tsc1 in DC was identified by PCR and Western blot respectively;（2）The CD11c⁺DCs of the spleen cells in Tsc1^f/f and CD11c^CreTsc1^f/f mice were highly purified with CD11c Micro Beads and flow cytometers;（3）Three experimental groups were established:Tsc1^f/f,CD11c^CreTsc1^f/f/f and treatment of CD11c^CreTsc1^f/f mouse spleen DC with rapamycin in vitro.（The evidence shows that RAPA is a specific blocker of mTORC1,so we verify that Tsc1may affect DC gene expression partially by inhibiting mTORC1 after RAPA treatment）;（4）The mitochondrial membrane potential and permeability,Ki67expression level,apoptosis and antigen presenting ability of DC in each group were detected by flow cytometry;（5）The microarray was used to detect the transcriptional profiling of DCs in three experimental groups.The pathway enrichment analyses were performed by the Kyoto Encyclopedia of Genes and Genomes（KEGG）pathway database and gene-set enrichment analysis（GSEA）;（6）Real-time fluorescence quantitative（RT-qPCR）was used to detect the differential gene expression in three experimental groups.Results:（1）Tsc1 gene were knocked out at the genetic level and protein level in DC specifically and successfully;（2）The CD11c⁺MHCII⁺DC of the spleen cells in Tsc1^f/f/f and CD11c^CreTsc1^f/f mice were highly purified with CD11c Micro Beads and flow cytometers;（3）Compared with the Tsc1^f/f mice,the mitochondrial membrane potential,the mitochondrial membrane,Ki67 expression level,apoptosis and antigen presenting ability of DC were increased in CD11c^CreTsc1^f/f mice but were decreased after RAPA treatment;（4）Tsc1 had a critical impact on the expression of a wide variety of genes that regulated metabolic process including glycolysis,oxidative phosphorylation,fatty acid metabolism,protein metabolism,amino acid metabolism,tRNA transport,amino acid transport,protein transport,phagocytosis and endocytosis;（5）Microarray results showed that the CD11c^CreTsc1^f/f DC had significantly higher expression levels of genes related to survival,proliferation,metabolism,substance transport and antigen presentation（Bub1b,Mad2l1,Ccnb2,Ccnb1,Casp6,Casp8,Prkaca,Cox6a2,Hk3,Ndufs8,Tpi1 Man1c1,Hpse,Nfe2l2,Renbp,Hexa,Lpin1,Acot2,Hexb,Xbp1 Nup35,Aaas,Sec61g,Nrp1,Lgmn,Cd4,Ctsb and Ctsl）.After treating RAPA,the results showed that Tsc1 partially affected the expression of these genes by inhibiting mTORC1.The same resμlt was obtained by RT-qPCR.Conclusion:Tsc1 programs the homeostasis and function of antigen presentation of DC through transcriptional regulation critically involved in survival,proliferation,metabolism and antigen presentation.The impacts of Tsc1 in DC genes expression were partially dependent on inhibition of mTORC1 activity.