| Part One:The effect ofγδT cells on skin graft rejection and the underlying mechanismsBackgroud:γδT cells are the minor group of T cells compared withαβT cells.The proportation ofγδT cells is low in peripheral circulation and secondary lymphoid organs,while it is pretty high in skin.γδT cells occupy more than 90%in murine epidermal T cells and nearly half of dermal T cells.Vγ1,Vγ4 and Vγ5 T cells are the major subsets ofγδT cells which reside in murine skin,but they have dinstinct biological properties.Vγ5 T cells only reside in epidermis and exhibit dendrites under steady state,therefore they are also called as dendritic epidermal T cells（DETC）.DETC can secrete IGF-1 and KGF to sustain the homeostasis of skin and facilitate wound healing.Vγ1 T cells and Vγ4 T cells not only exist in pheripheral circulation,but are also distributed in skin dermis.They participate in skin autoimmune diseases,anti-tumour immunity and infection immunity by producing IL-17A and IFN-γ.However,whether Vγ1 T cells and Vγ4 T cells take part in skin graft rejection is not clear.IL-17A as one of powerful proinflammatory cytokines,can recruit neutrophils and macrophages,and play an important role in the initial stage of inflammatory cascade reactions.IL-17A is majorly orginated form Th17 cells andγδT cells.γδT cells secrete IL-17A in early stage of immune responses and participate in heart allogenic rejection and autoimmune diseases.But whetherγδT cell-derived IL-17A play a role in skin graft rejection is unclear.Vγ4 T cells have some similar charaters with Th17 cells,such as expressing IL-23 receptor,CCR6 and RORγ.Th17 cells can be recruited to inflammatory sites and produce a large amout of IL-17A to amplify local inflammatory responses.CCL20is in the charge of recruiting CCR6+Th17 cells to inflammatory sites.It is unknown whetherγδT cells infiltrating into the sites of skin grafts dependens on chemokine-chemokine receptor CCL20-CCR6 pathway.Besides,IL-1βand IL-23 can stimulateγδT cells to produce IL-17A which is essential to launch and amplify inflammatory reactions.Therefore,it is necessary to explore the roles of IL-1βand IL-23 inγδT cell-mediated skin graft rejection.It has been reported that host allogenicαβT cells actuate skin graft rejection.Inflammatory responses caused by allognic graft rejection strongly promote the maturity of local dendritic cells（DC）.Matured DC migrate to draining lymph nodes（DLN）to activate specific antigen responsiveαβT cells.It is needed to further illustrate whetherγδT cell-derived IL-17A accelerate matured DC to accumulate in DLN,and regulate the effector function ofαβT cells.We investiged the roles of Vγ1 T cells and Vγ4 T cells in skin graft rejection,mainly including the following four questions:1.Do Vγ1 T cells or/and Vγ4 T cells participate in graft rejection?2.How do Vγ1 T cells or/and Vγ4 T cells influence graft rejection?3.How do IL-1βand IL-23 affect the expression of IL-17A by Vγ1 T cells or/and Vγ4T cells?4.What are the effects of Vγ1 T cells or/and Vγ4 T cells on the activation and migration of DC thus to regulateαβT cells?Results:1.Vγ4 T cells played a role in skin graft rejection and mainly participated in the early stage of acute rejection.The skin of the WT mice was transplanted to the back of Tcrδ-/-mice,and the survival time of the graft was significantly prolonged.However,when Tcrδ-/-mouse skin was transplanted to the back of WT mice,the survival time of the graft did not significantly change,suggesting thatγδT cells in the host skin,rather than the skin grafts,were involved in the rejection of skin transplantation.To investigate the specific role of Vγ1 T cells or Vγ4T cells on skin graft rejection,WT mice were cleared of Vγ1 T cells or Vγ4 T cells,or Vγ1T cells and Vγ4 T cells were respectively transferred to the wound beds of Tcrδ-/-mice,we found that the promoting effect of Vγ4 T cells on graft rejection was more significant than that of Vγ1 T cells.Three days before the transplantation or 10 days after the transplantation,the female WT host was cleared of Vγ4 T cells.It was shown that the effect of Vγ4 T cell-depletion on prolonging graft survival before transplantation was more obvious than that of after transplantation,suggesting that Vγ4 T cells played a role mainly in the early stage of acute rejection.2.Vγ4 T cells secreted IL-17A to promote skin rejection and infiltrated into the epidermis through the CCR6-CCL20 pathway.2.1 Vγ4 T cells accelerated skin rejection depending on IL-17A.We injected neutralizing antibodies of IFN-γor IL-17A at the margin of grafts,or transplanted skin grafts of wild male mice to the backs of female Ifn-γ-/-and Il-17a-/-mice at the same age,and observed that IL-17A had a more significant effect on graft rejection than IFN-γ.Ifn-γ-/-and Il-17a-/-Vγ4 T cells were transferred to the wound beds,and Ifn-γ-/-Vγ4 T cells significantly accelerated rejection,while Il-17a-/-Vγ4 T cells had no significant effect.WB and immunohistochemistry were used to detect the expression of IL-17A in the graft and host skin around grafts between Vγ4 T cell scavenge group and control group.The results showed that the expression of IL-17A was decreased in the graft and host skin around grafts of Vγ4 T cell depletion group compared to control.Therefore,we regarded that Vγ4 T cells mainly depend on IL-17A to facilitate skin graft rejection.2.2 Vγ4 T cells migrated from dermis to epidermis through CCR6-CCL20 during skin graft rejection.On the third day after transplantation,the skin graft and the epidermal tissue around the graft were collected,and we detected that Vγ4 T cells were infiltrated into epidermis from dermis and Vγ4 T cells highly expressed CCR6.Moreover,the expression of CCL20was significantly increased in the skin graft and host epidermis around grafts.After the neutralization of CCL20 surrounding the skin graft of WT mice,the survival time of the skin graft was significantly prolonged,and the number of Vγ4 T cells and the expression of IL-17A were sharply reduced.The above results indicated that after transplantation,Vγ4 T cells infiltrated into the epidermis through the CCR6-CCL20 pathway.3.The expression of IL-17A in epidermal infiltrated Vγ4 T cells was induced by IL-1βand IL-23.By blocking the expression of IL-1βand IL-23 in the recipents,we discovered that the expression of IL-17A in the graft and host epidermis around grafts was significantly reduced and graft rejection was markedly delayed.However,depleting Vγ4 T cells before neutralizing IL-1βand IL-23 did not further delay graft rejection.These results suggested that IL-1βand IL-23 accelerated the production of IL-17A by Vγ4 T cells to aggravate graft rejection.4.IL-17A produced by Vγ4 T cells promoted the accumulation of DC in DLN to activateαβT cells After deleption of Vγ4 T cells,MHCⅡhighCD86+CD11c+DC and IL-17A+CD4+αβT cells in DLN were prominently reduced.WT or Il-17a-/-Vγ4 T cells were transferred to the skin transplantation area of Tcrδ-/-mice,the number of MHCⅡhighCD86+CD11c+DC and IL-17A+CD4+αβT cells in DLN was notably declined in Il-17a-/-group compared with WT control.These results suggested that IL-17A produced by Vγ4 T cells in the early stage of acute rejection could enhance the expression of IL-17A in CD4+αβT cells which would play a role in the subsequent rejection.In conclusion,we found that Vγ4 T cells migrated from dermis into epidermis through CCR6-CCL20 pathway,and secreted a large amount of IL-17A upon the stimulation of IL-1βand IL-23 in the early stage of acute rejection.Vγ4 T cell-derived IL-17A promoted DC maturation and migration to the draining lymph nodes,and activatedαβT cells to express IL-17A.This study extended the understanding ofγδT cells in the rejection of skin transplantation,which may provide new ideas and clues for the mechanism research and prevention of human skin graft rejection in the future.Part Two: The The effect of γδ T cells on wound healing and the underlying mechanismsBackgroud: γδ T cells are an important part of skin immune defense,participate in psoriasis,tumors,skin transplantation rejection,microbial infection and wound healing.DETC specificly express Vγ5Vδ1 TCR,only distributed in murine epidermal layer.Vγ1 and Vγ4 T cells are main subsets of γδ T cells in the peripheral circulation of mice,and are distributed in the skin tissue but only in the dermis.After skin injury,DETC can be rapidly activated and secrete IGF-1,which are the major source of IGF-1 in epidermal tissue to promote wound repair.Vγ1 and Vγ4 T cells are involved in allergic reactions,autoimmune diseases,anti-tumor immunity and infection immunity.However,whether Vγ1 and Vγ4 T cells affect wound healing is not clear.IL-17 A is an inflammatory cytokine that plays an important role in the initiation and development of inflammatory responses.γδ T cells are the early source of IL-17 A in the initial stage of inflammation,while Th17 cells are the main source of IL-17 A in the later stage of inflammation.It has been reported that IL-17 A is necessary for effective wound healing and can induce epidermal keratinocytes to express antimicrobial peptide beta defensin 3（β-defensin 3）and Reg IIIγ.However,Rodero MP et al.reported the opposite result,that IL-17 A may inhibit wound healing.In the skin transplantation model,we found that Vγ4 T cells migrated from dermis to epidermis through the CCL20-CCR6 pathway,and they were the main source of IL-17 A in the early stage of transplantation.However,the effect of Vγ 4 T cells and IL-17 A on skin wound healing needs to be further elaborated.Keratinocytes and langerhans cells are the major sources of IL-1β and IL-23 in the skin.In γδ T cell-mediated skin diseases,IL-1β and IL-23 strongly promote the secretion of IL-17 A by γδ T cells.We observed that IL-1β and IL-23 could induce Vγ4 T cells to aggravate the rejection of skin transplantation.Interestingly,IL-17 A produced by Vγ4 T cells in the transplanted area can in turn enhance the expression of IL-1β and IL-23 in the epidermal tissue.However,it is not clear whether IL-1β/IL-23 affects the expression of IL-17 A in γδ T cells during wound healing.DETC-derived IGF-1 can significantly promote the proliferation and migration of keratinocytes,which is critical for timely and effective wound healing.Whether IL-17 A secreted by Vγ4 T cells could affect the expression of IGF-1 in DETC and wound healing is worth exploring.On this basis,we will study the effect of Vγ4 T cells on wound healing and pro-healing function of DETC,mainly including the following aspects: 1.Do Vγ4 T cells affect wound healing and the IGF-1 expression in DETC? 2.How do Vγ4 T cells influence the IGF-1 production in DETC? 3.What signal pathways are involved in the effect of Vγ4 T cells on the IGF-1 secretion by DETC?Results: 1.Vγ4 T cells delayed wound healing by inhibiting DETC secretion of IGF-1.1.1 Vγ4 T cells delayed wound healing by acting on DETC.We cleared Vγ4 T cells and Vγ1 T cells of WT mice respectively,and observed that the wound healing was markedly accelerated after the depletion of Vγ4 T cells,while the wound healing was not significantly changed after the removal of Vγ1 T cells,suggesting that Vγ4 T cells could delay the wound healing.However,the recontitution of Vγ4 T cells to the back of Tcrδ-/-mice did not promote wound healing,suggesting that Vγ4 T may indirectly affect wound healing.It has been reported that DETC accelerate wound healing.The freshly separated DETC alone or the mixture of DETC and Vγ4 T cells was transferred back to the wound beds in Tcrδ-/-mice,it was shown that the wound healing of latter group was delayed compared with that of the former.These results suggested that Vγ4 T cells may delay wound healing through DETC.1.2 Vγ4 T cells inhibited DETC to secrete IGF-1 and thus affected wound healing.WB and immunohistochemistry were used to detect the expression level of IGF-1 in the epidermis around wound of the mice with Vγ4 T cell depletion and the control group.It was found that the expression level of IGF-1 was prominently increased after the depletion of Vγ4 T cells.The production of IGF-1 in the epidermis around wound was significantly increased when DETC was transferred back to the Tcrδ-/-mice,but the expression of IGF-1 was not increased observably when DETC was transferred back with Vγ4 T cells.r IGF-1 injected into the wound margin of Vγ4 T cell-depletion mice could promote wound healing,while IGF-1 neutralization antibody could delay wound healing.These results suggested that Vγ4 T cells could inhibit the expression of IGF-1 in DETC and thus affect wound repair.1.3 Vγ4 T cells did not affect the number and morphology of DETC but only inhibited the expression of IGF-1 in DETC.The proportion,number and morphology of DETC in the epidermis of wound margin were detected in Vγ4 T cell-depletion mice and control mice.It was found that the proportion,number and morphology of DETC did not change significantly after the removal of Vγ4 T cells.DETC was isolated from the epidermis around wound in Vγ4 T cell-depletion mice and control mice.Surface activation markers such as CD25,CD44,CD62 L and CD69 were analyzed.The results showed that there was no significant difference in the expression of activation markers between the two groups,suggesting that the expression of DETC activation markers could not be affected by Vγ4 T cells.When the activation receptors such as Vγ5 TCR,NKG2 D and JAML were detected,it was demonstrated that although the expression of JAML on DETC in the epidermis was not significantly changed after the clearance of Vγ4 T cells,the expression level of Vγ5 TCR was slightly increased and the expression level of NKG2 D was prominently increased.These results indicated that Vγ4 T cells did not affect the expression of activation markers but inhibited the expression of activation receptors on the surface of DETC.Flow cytometry was conducted to measure the expression level of IGF-1 in DETC at the wound margin of Vγ4 T cell-depletion mice and control mice.The results showed that the expression level of IGF-1 in DETC was increased after the removal of Vγ4 T cells.These results suggested that the effect of Vγ4 T cells on DETC was not the number and activation markers but the expression of IGF-1.2.Vγ4 T cells secreted IL-17 A and migrated to the marginal epidermis to inhibit wound healing.2.1 Vγ4 T cells depended on IL-17 A to delay wound healing.We detected the expression levels of IFN-γ and IL-17 A in the wound margin of Vγ4 T cell-depletion mice and control mice,and observed that the proportion of IL-17A+Vγ4 T cells was significantly promoted after skin injury,and the expression level of IL-17 A was remarkably decreased after the depletion of Vγ4 T cells.No similar change was observed in IFN-γ.Wounds were conducted on the back of Ifn-γ-/-mice,Il-17a-/-mice and Tcrδ-/-mice,and cell transfusion experiments were performed to further explore which cytokines secreted by Vγ4 T cells were involved in wound healing.The results indicated that IL-17 A,rather than IFN-γ,produced by Vγ4 T cells was the major cytokine that delayed wound healing.In addition,subcutaneous injection of r IL-17 A at the edge of the wound after the clearance of Vγ4 T cells inhibited wound healing,while the administration of IL-17 A neutralizing antibody（20 μg/ wound）significantly facilitated wound healing.These results indicated that Vγ4 T cells relayed on IL-17 A to suppress wound healing.2.2 Vγ4 T cells were the major early source of IL-17 A in the epidermis after skin injury and migrated from dermis to epidermis through CCR6-CCL20.By analyzing the proportion of Vγ4 T cells in the epidermis,dermis and DLN after skin injury,we observed that the ratio of Vγ4 T cells significantly increased in the epidermis but decreased in the dermis after skin injury,while there was no significant change in DLN.The subsets of cells expressing IL-17 A in the marginal epidermis was detected,and it was found that more than 50% of IL-17A+T cells were Vγ4 T cells,and only less than 5% of IL-17A+T cells were DETC.After the clearance of Vγ4 T cells,the expression of IL-17 A decreased prominently in the epidermis,while the expression of IL-17 A did not change markedly in the dermis.The production of IL-17 A in the epidermis around wound was significantly increased when Vγ4 T cells rather than DETC were reconstituted to the wound beds of Tcrδ-/-mice,suggesting that Vγ4 T cells were the primary source of IL-17 A in the epidermis on the early stage of wound healing.Flow cytometry revealed that almost all the Vγ4 T cells infiltrating the epidermis were positive for CCR6,while blocking CCL20 significantly reduced the proportion of infiltrated Vγ4 T cells in epidermis,indicating that the migration of Vγ4 T cells into the epidermis after skin injury was dependent on the CCR6-CCL20 pathway.2.3 IL-17 A inhibited IGF-1 expression of DETC in epidermal tissues after skin injury.Low,medium and high doses of r IL-17 A were injected into the subcutaneous around wound of WT mice respectively,and it was demonstrated that high dose of r IL-17A（200ng/ wound）prominently blocked wound repair,while low dose（2ng/ wound）or medium dose（20ng/ wound）could not show the dampening effect on healing.The injection of IL-17 A neutralizing antibody into the subcutaneous around wound significantly facilitated the expression of IGF-1 in the epidermis and the proportion of IGF-1 positive DETC,while the injection of r IL-17 A into the subcutaneous around wound showed the opposite results.Beside the degree of change was positively correlated with the dose size of neutralizing antibody and cytokine.3.IL-1β and IL-23 were the mediators of Vγ4 T cells to inhibit the IGF production in DETC.3.1.IL-1β and IL-23 directly prevented DETC from producing IGF-1.In vitro,DETC was co-cultured with primary keratinocytes isolated from neonatal mouse skin,and it was demonstrated that r IL-17 A significantly inhibited the expression of IGF-1 in DETC,indicating that IL-17 A prevented DETC from producing IGF-1 through epidermal cells.DETC stimulated by r IL-1β and r IL-23 can significantly inhibit the expression of IGF-1 in vitro.WT mice treated with r IL-1β and r IL-23 showed significantly reduced IGF-1 expression and declined proportion of IGF-1 positive DETC in the epidermis around wound,as well as delayed wound healing.While treatment with IL-1β/IL-23 neutralizing antibodies demonstrated the opposite effects.These results sufficiently indicated that IL-1β and IL-23 can directly affect the production of IGF-1 in DETC.3.2.IL-1β and IL-23 were the mediators of IL-17 A to inhibit the expression of IGF-1 in the epidermis of wound margin.r IL-1β and r IL-23 directly inhibited DETC from producing IGF-1 when DETC were co-cultured with keratinocytes,but r IL-17 A could not directly display the inhibitory effect.It has been shown that blockage of IL-17 A alone at the edge of the wound can accelerate wound repair,while simultaneous injection of r IL-1β and r IL-23 with blockage of IL-17 A can inhibit wound repair.Injection of r IL-17 A alone into the margin of the wound delayed wound healing,but r IL-17 A injection together with IL-1β/IL-23 blockage promoted healing.These results suggested that IL-1β and IL-23 may act as mediators betweem IL-17 A and IGF-1.3.3.Positive feedback loops were formed in the epidermis of the wound margin between IL-17 A and IL-1β/IL-23.It has been demonstrated that r IL-17 A can enhance the m RNA and protein expression levels of IL-1β and IL-23 in keratinocytes in vitro.The expression levels of IL-1β and IL-23 in the epidermis around wound of Il-17a-/-mice were significantly lower than those of WT mice in vivo.The neutralizing antibody of IL-17 A injected into the margin of wound can reduce the expression levels of IL-1β and IL-23 in the epidermis,and the higher the neutralizing dose of IL-17 A,the lower the expression levels of IL-1β and IL-23.In addition,injection of r IL-17 A at the margin significantly increased the expression of IL-1β and IL-23 in the epidermis at the margin,and the higher the injection dose of r IL-17 A,the higher the expression levels of IL-1β and IL-23.These results suggested that IL-17 A was an important factor in promoting the production of IL-1β and IL-23 in the marginal epidermis after skin injury.The clearance of Vγ4 T cells and the cell transfusion experiment confirmed that IL-17 A can promote the expression of IL-1β and IL-23.According to the literature that IL-1β and IL-23 promoted the expression of IL-17 A,we believed that IL-17 A may form a positive feedback loop with IL-1β and IL-23 to regulate wound healing after skin injury.3.4.IL-1β and IL-23 regulated the expression of IGF-1 in DETC depending on NF-κb and STAT3 signaling pathways.Subcutaneously injection of cytokines or neutralizing antibodies at the margin of the wound in vivo and DETC stimulated by cytokines in vitro revealed that IL-1β inhibited the expression of IGF-1 by DETC,while IL-23 may only enhance IGF-1 expression by cooperating with IL-1β.The phosphorylation levels of downstream signaling pathways such as NF-κb and STAT3 was detected.IL-1β and IL-23 were shown to increase the phosphorylation levels of NF-κb and STAT3,and accelerate the transfer of NF-κb and STAT3 from the cytoplasm to the nucleus.Moreover,blocking NF-κb signaling pathway rather than STAT3 signaling pathway can significantly promote the expression level of IGF-1 in DETC.Therefore,we considered that NF-κb signaling pathway played a more important role than STAT3 signaling pathway in regulating IGF-1 expression of DETC by IL-1β.In conclusions,Vγ4 T cells were the main source of IL-17 A in epidermis around wound and migrated from dermis to epidermis through CCR6-CCL20 pathway at the early stage after skin trauma.In addition,IL-17 A derived from Vγ4 T cells indirectly prevented DETC from producing IGF-1 by enhancing the expression of IL-1β/IL-23 in epidermis.IL-1β/IL-23 blocked IGF-1 expression in DETC by activating the NF-κb and STAT3 signaling pathways,and the more important mechanism by which IGF-1 was regulated was the NF-κb signaling pathway activated by IL-1β.This study aimed at exploring the effect of Vγ4 T cells on wound healing and underlying mechanisms in the regulation between Vγ4 T cells and DETC.To some extent it complements the theory of wound healing and may provides new ideas and clues for human wound repair in the future. |
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