The Role Of Brd4 In Maintaining Homeostasis Of Skin T Lymphocytes And Hair Regeneration

The pathogenesis of autoimmune and inflammatory diseases is related to the abnormal activation,proliferation and differentiation of T lymphocytes.Among the diverse signals that control T cell activities,those from the T cell costimulatory molecules play a significant role.OX40 is a costimulatory molecule in the TNF receptor superfamily primarily involved in regulating T cell proliferation,survival,and memory generation.BRD4 is a member of bromodomain and extra-terminal domain（BET）protein family.BRD4 acts as a transcription and epigenetic regulator,which recognizes acetylated lysine residues,loosens chromatin structure,and regulates gene expression and cell fate decisions.Studying the roles of BRD4 on T cell homeostasis and hair regeneration in skin may provide new insights for the treatment of autoimmune skin inflammation and hair regeneration disorders.Objective:This project aims to study:（1）OX40 expression on mice immune and skin cells;（2）the role of Brd4 in the maintenance of hair follicle stem cells;（3）effect of conditional knockout of Brd4 on T lymphocytes;（4）the interaction between skin T lymphocytes and hair follicle stem cells.Methods:1.Identification of OX40 expression on mice immune and skin cellsWe constructed cell-specific Brd4 knockout(Brd4^fl/flOx40-Cre)mice to observe the development of mice skin pathology;Crossing the mice into background of Rosa26 fluorescent reporter,we detected the expression of OX40 in cells derived from bone marrow system.The knockout efficiency of Brd4 was detected by flow cytometry.With the expression of Rosa26 fluorescent protein as an indicator,the expression of OX40 in cells derived from the skin system was detected by flow cytometry.The expression of OX40 in mice skin at different ages was studied by immunofluorescence labeling.2.Investigating the role of Brd4 in the maintenance of hair follicle stem cellsThe immune deficiency Rag1^-/-mice were bred with Brd4^fl/flOx40-Cre mice to generate the Rag1^-/-Brd4^fl/flOx40-Cre mice,in which T cells and B cells were deleted to exclude the role of immune system in our mice model.In this way,the effect of conditional deletion of Brd4 in hair follicle stem cells on hair regeneration and skin pathological were observed.Through high-throughput sequencing,the differential expression of genes and the enrichment pathways related to hair regeneration as well as the enrichment direction and degree of these pathways were studied in mice at different age.The proliferation and apoptosis of mice hair follicle stem cells were detected by flow cytometry and immunohistochemistry.3.Detecting the effect of conditional knockout of Brd4 on T lymphocytesThe inflammatory cell infiltration and hair follicle integrity of Brd4^fl/flOx40-Cre mice were observed by skin section H&E staining and immunofluorescence labeling.T cells in the spleen,superficial lymph nodes and skin were isolated.The activation and proliferation of CD4,CD8 and regulatory T cells as well as their related cytokines were detected by flow cytometry.The proliferation and expression ofγδT cells as well as related cytokines were detected by flow cytometry and immunofluorescence.The effect of Brd4 conditional knockout on the function of regulatory T cells was studied in vitro by co-culture of effector T cells and regulatory T cells.The role of Brd4 in the function of regulatory T cells was further verified by cell adoptive transfer experiment.4.Exploration of the interaction between skin T lymphocytes and hair follicle stem cellsIn immune deficient Rag1^-/-mice,we studied whether T cells and other bone marrow hematopoietic cells were the initiating factors of this skin disease through cell adoptive transfer and bone marrow chimeric reconstruction.In Rag1^-/-Brd4^fl/flOx40-Cre mice with a T cells and B cells deleted background,through cell adoptive transfer experiment,we detected the activation ofγδT cells as well as their production of related cytokines under a skin pathological environment caused by the dysfunction of hair follicle stem cells.In Rag1^-/-Brd4^fl/flOx40-Cre mice with a T cells and B cells deleted background,through cell adoptive transfer experiment,we further explored the role ofγδT cells in enhancing the destruction hair follicles.Results:1.We successfully constructed Brd4 conditional knockout mice,in which Brd4 was specifically knocked out in cells that expressed Ox40.Compared with the control mice,Brd4^fl/flOx40-Cre mice didn’t show obvious abnormality within 5 weeks after birth,while progressive hair loss occurred since the end of the 6th week.At the end of the 10th week,the hair cover was almost completely lost,and inflammatory skin ulceration began to appear.By the end of the 12th week,the epidermis was obviously ulcerated on the back of mice.Using Rosa26 reporter tracing,we found that OX40 was primarily expressed on the surface of activated CD4 cells and regulatory T cells in the immune system.In the skin cell lines,OX40 was expressed or ever-expressed in hair follicle stem cells and their differentiated cells.The knockout efficiency of Brd4mediated by Ox40-Cre recombinase was verified by intracellular staining.Immunofluorescence staining showed the expression of OX40 in mice skin at different time points.2.Through crossing with Rag1^-/-mice,we found that the immune deficient Rag1^-/-Brd4^fl/flOx40-Cre mice began a hair loss at a significantly later time point with a prominant slower development compared with Brd4^fl/flOx40-Cre mice,while the complete hair loss could still be achieved at the end pointof observation.There was no skin inflammation throughout the observation.Skin section showed that the structure of hair follicle was abnormal,but not completely destroyed.There was no inflammatory cell infiltration despite slight hyperplasia of epidermis.Hair shave experiment showed that Rag1^-/-Brd4^fl/flOx40-Cre mice,although there was no epidermal inflammation,could not achieve hair regeneration even without the participation of immune cells.We extracted RNA from hair follicle stem cells at different mice ages.Through RNA sequencing analysis,we found that the deletion of Brd4 mainly resulted in the differential expression of genes related to hair follicle stem cell activation,thus inhibiting Wnt/β-Catenin,SHH and other key signal pathways during hair regeneration.Real-time PCR also further confirmed that the expression level of these genes was significantly inhibited.In addition,through intracellular staining of Brd U and immunohistochemistry staining for TUNEL assay,we found that knockout of Brd4 induced significantly decreased ability of proliferation of hair follicle stem cells,and these cells were more likely to reach cell apoptosis.3.Compared with the control Ox40-Cre mice,the total number of CD4⁺,CD8⁺and regulatory T cells of Brd4^fl/flOx40-Cre mice at the age of5 weeks had no significant difference,despite a slightly increase in the proportion of effector memory cells.At the 12th week of the most significant skin inflammation,skin section H&E staining and immunofluorescence staining showed a large number of inflammatory cell infiltration,severe hair follicle structure destruction and epidermal inflammatory hyperplasia in the skin of Brd4^fl/flOx40-Cre mice.In each T cell subset,there was no significant difference in the production of cytokines of CD4⁺and CD8⁺T cells,while the production of IL-17 inγδT cells was significantly increased.In vitro culture showed that the function of regulatory T cells was significantly inhibited upon the deleton of Brd4.The skin inflammation and hair loss of Brd4^fl/flOx40-Cre recipients were improved after regulatory T cells adoptive transfer compared with those of mice without the transfer,and IL-17 production ofγδT cells was also suppressed.4.T cells isolated from Brd4^fl/flOx40-Cre mice and the control Ox40-Cre mice were transferred to the immuno-deficient Rag1^-/-mice.There were no abnormalities through the observation period.Bone marrow cells isolated from the Brd4^fl/flOx40-Cre mice and the control Ox40-Cre mice were transferred to the immuno-deficient Rag1^-/-mice irradiated by lethal dose of radiation.The constructed chimeric mice again had no abnormalities.γδT cells were adoptively transferred to immune deficient Rag1^-/-Brd4^fl/flOx40-Cre and Rag1^-/-mice respectively.Through the observation period,the hair loss rate of Rag1^-/-Brd4^fl/flOx40-Cre recipients was significantly faster than that of mice without cell transfer.Skin section H&E staining showed that the degree of hair follicle destruction was increased and the skin structure was heavily damaged.The Rag1^-/-recepients remained normal.Compared with Rag1^-/-recepients,γδT cells were significantly expanded in Rag1^-/-Brd4^fl/flOx40-Cre recepients,and the production of IL-17 was significantly increased.Conclusions:1.OX40 expresses primarily on activated CD4⁺effector cells and regulatory T cells in the immune system.In skin cell lines,OX40expresses or ever expressed on hair follicle stem cells and their differentiated cells.2.The deletion of Brd4 inhibits the activation and proliferation of hair follicle stem cells,making hair follicle stem cells more prone to cell apoptosis,thus destroying the structure of hair follicle and causing hair regeneration disorder.3.While Ox40-Cre mediated Brd4 deletion does not directly affectγδT cells,the abnormal skin environment induces their significant activativation and proliferation to produce cytokine IL-17,which further destroys the structure of hair follicles and causes skin inflammation.4.The deletion of Brd4 inhibits regulatory T cell function,which hardly regulates inflammatory activation ofγδT cells.Regulatory T cells with normal function improve hair loss and skin inflammation.