Effects Of TFAM On Homeostasis And Function Of Epidermal Langerhans Cells

Background and Objectives:Skin,as the first line of defense against external stimuli,has the function of protecting the body from various physical,chenmical,microbial and other factors.Increased susceptibility to skin infections and increased incidence of skin tumors have been observed in the older age group,which is closely related to the decline of skin immunity with age.Langerhans cells(LCs),highly expressing CD207,are professional antigen-presenting cells on the epidermis,which form the first immune barrier and play a crucial role in maintaining skin homeostasis.As the skin ages,the number and maturity of LCs decrease significantly,which is closely related to the skin diseases associated with aging.However,the reason why LCs change with age is not clear.Mitochondria are the organelles of biosynthesis and biological energy,and the main driving force for regulating cell homeostasis and function.The decline in mitochondrial quality and activity has long been known to be associated with normal ageing,as well as with age-related diseases.For example,mitochondrial dysfunction can lead to UV-induced skin aging,photoaged skin and skin cancer,among others.Age-related reduction in epidermal LCs are associated with ultraviolet exposure,which may play an important role in skin cancer development.mitochondrial transcription factor A(TFAM)is A nuclear encoded transcription factor that binds to mtDNA to control its replication,packaging,and transcription,and stable mtDNA is critical for cell adaptability and function.A large number of studies have shown that TFAM-mediated mitochondrial function is essential for the maintenance and function of T cells and Macrophages,but the regulation and mechanism of LCs homeostasis and function are still unclear.The objective of this study was to elucidate the effects of TFAM-mediated mitochondrial stability on homeostasis and function of cutaneous Langerhans cells.This objective of this study was to elucidate the effects of TFAM-mediated mitochondrial stability on homeostasis and function of cutaneous Langerhans cells.Methods:1.Tfamfl/fl mice were hybridized with CD11ccre mice to generate CD11ccre Tfamfl/fl cKOconditional knockout mice.The absence of TFAM in the epidermal LCs of KO mice was detected by qRT-PCR,and the expression of TFAM decreased with age.Flow cytometry and immunofluorescence techniques were used to determine that mitochondrial dysfunction of LCs might be related to the decreased expression of TFAM.2.The kinetics of LCs reaction after TFAM absence was evaluated.For better experiment,4-week-old mice were selected for experiment.The changes of proliferation and apoptosis were detected by Ki67 and AnnexinV staining.After 72h of fresh and in vitro epidermal culture,the frequency and expression levels of mature markers of LCs were evaluated..The phagocytic function of LCs was determined by phagocytic function test.3.The morphological changes of mitochondria of LCs were observed by electron microscopy,and were evaluated by FACS analysis of mito Tracker,mito TMRM and mito SOX staining.4.Through bulk RNA-sequence analysis,the regulatory mechanism of TFAM deletion on the homeostasis and function of LCs and the changes of signal pathway.It was further suggested that mitochondrial disorder medisted by TFAM deletion leads to enhanced apoptosis of LCs.Analysis of LCs expression,apoptosis,and inflammation in old and young skin through human skin single-cell RNA sequencing.Langerhans cells of young and old skin were stained by immunofluorescence staining.Results:1.Compared with Tfamfl/fl mice,TFAM was absent in the epidermal LCs of CD11ccre Tfamfl/fl cKO mice(6-8 weeks of age),and the expression of TFAM in the epidermal LCs of mice decreased with age.2.There was no significant difference in the proliferation function of LCs after TFAM deletion,but apoptosis increased significantly.After the loss of TFAM,the maturity of LCs decreased significantly,but the phagocytosis function was enhanced.3.Mitochondrial abnormalities appeared in epidermal LCs after TFAM deletion,including morphology,mitochondrial membrane permeability.Calcium ion homeostasis and autophagy related MAPK signaling pathway related gene enrichment were significantly down-regulated,while inflammatory response related enrichment pathway was up-regulated.Conclusions:The absence of TFAM leads to mitochondrial morphology and membrane permeability of LCs in the epidermis,as well as defects in autophagy mediated by kinase cascades mediated by extracellular signals of MAPK and autophagy regulated by cellular calcium homeostasis,resulting in increased apoptosis,reduced maturation,enhanced phagocytosis,and inflammation.Therefore,TFAM-mediated mitochondrial stability is essential for postnatal epidermal LCs maintenance and function.