Skin Microbiome Enhance Wound Induce Skin And Hair Follicle Regeneration

Background and objectiveWound induce hair Neogenesis(WIHN)is a rare post-wounding skin and hair follicle regeneration model in mammals.By creating a large full-thickness skin wound on the back of mice or rabbits,after the wound is healed,the center of the scar will regenerate hair follicles with complete structure and hair cycle and induce the generation of adipocytes around it.So that the skin in the center of the scar will have normal skin structure and function.Studying this hair follicle regeneration model can bring new insights to human skin regenerative medicine.As an important participant of wounding,the effect of skin microbes on skin regeneration is still unknown.Our work used different gradients of skin microbial levels to study its effects on the regeneration of skin and hair follicles,and reveals its internal biological mechanisms,providing new treatments for regeneration of wounding and baldness.Methods1.The effect of skin microbiome on the skin and hair follicles regeneration after wounding in mouseMice with different skin microbial gradients:①germ free(GF)mice,②Specific-pathogen-free(SPF)mice,③mice were treated with topical antibiotics and④mice with exogenous bacteria added were used to test the effect of microbiome on skin regeneration.The α-diversity,β-diversity and taxonomy of mouse skin microbiome was detected by 16SrDNA sequencing to screen out the microbiome with the strongest ability to induce regeneration.2.The effect of skin microbiome on the skin immune microenvironment after wounding in mouseFlow cytometry was used to detect the infiltration of immune cells in the wound bed of mice with different skin microbial gradients,especially the γδT cells and M2 macrophages for skin regeneration.Immune signals Myd88 and IL17 were tested to determine how microbiome regulate skin and hair follicle regeneration through the immune microenvironment.3.The biological mechanism of skin microbiome induce skin and hair follicle regeneration after wounding in mouseBy screening microbiome immune receptor signals:IL1R,IL36R,TLR2,and TLR3 to determine the essential receptors for skin microbes promoting regeneration.By screening MyD88,IL1a,IL1b,and IL17A immune genes to determine the upstream and downstream signals that promote regeneration.4.The effect of skin microbiome on human wound healingCreate small wounds on the skin of the left and right popliteal fossa of 6 volunteers and use antibiotics on one side and Vaseline on the other side for control.Detect the healing rate of wounds,microbial changes and the expression of stem cell markers and regeneration signals on both sides.Results1.The effect of skin microbiome on the skin and hair follicles regeneration after wounding in mouseCompared with SPF mice,GF mice have the least of hair follicle regeneration(fold=-17.9,n=13,p=1.9×10-6),mice were treated with topical antibiotics have the second least of hair follicle regeneration(fold=-7.9,n=10,p=8.4×10-5),and mice with exogenously added of Staphylococcus aureus have the most hair follicle regeneration(fold=3.3,n=12,p=7.5×10-5).2.The effect of skin microbiome on the skin immune microenvironment after wounding in mouseThe increase in the abundance and diversity of skin microbiome increase the infiltration of γδT cells and M2 macrophages in the mice wound bed,and induce the expression of immune-related genes.Myd88 and IL17 signals promote the infiltration of immune cells,but IL17 signal is not essential for skin regeneration.3.The biological mechanism of skin microbiome induce skin and hair follicle regeneration after wounding in mouseKeratinocyte-dependent IL1R gene knockout mice,IL-1β gene knockout mice,and Myd88 gene knockout mice have lower skin and hair follicle regeneration,and their regenerative capacity cannot be induced by exogenous bacteria.4.The effect of skin microorganisms on the healing of human skinThe human wound healing was slowed down by antibiotic treated.The expression of regeneration-related signals in the wound bed was decreased after antibiotic treated.Conclusions1.Skin microbiome can promote complete skin and hair regeneration in mice.2.Skin microbiome promote the infiltration of immune cells in the wound bed.3.Skin microbiome promote regeneration through IL-1β,keratinocyte-dependent IL1R-Myd88 signal.4.For small non-infected wounds,using antibiotics will slow the wound healing.