Ligation Of FasL Regulates Function Of Dendritic Cells And Its Effect In Anti-fungal Immunity

In 1973,Steinman and Cohn found a unique morphological cell with dendritic protrusions in the spleen from mice and named it dendritic cell（DC）.Dendritic cell,known as the most potent antigen-presenting cell（APC）,is an initiator of immune response,because of its acknowledged characteristic--stimulating the activation and proliferation of na?ve T cells,while M?,B cells are only to activate memory T cells or activated T cells.What’s more,the activated DC can produce a large amount of inflammatory chemokines or cytokines such as TNF-αand IL-6.And these inflammatory chemokines or cytokines promote the progress of SIRS,even cause MODS by septicemia.Therefore,it is of great significance to take research on the functional regulation of DC.Nowadays,researches on apoptosis and innate immunity is still the hotspot,and the increasing number of people pay more attention to the apoptosis-related molecules mediated effect in innate immune response.It is well known that Fas/FasL is the classic pathway to induce apoptosis and recent evidences demonstrate that Fas and FasL also exert non-apoptosis effect.When Fas interact with FasL,the cell expressing FasL is regulated via FasL-mediated reverse signaling.Related experiments in vivo and in vitro manifest that FasL participates in the process of thymocytes maturation and T cells activation and proliferation as a costimutory molecule.What’s more,thymocytes-mediated positive selection was facilitated owing to FasL regulating the affinity between TCR and MHC-I/II but its mechanism remains to be uncovered.Since FasL is expressed on DC,we wonder whether FasL mediates reverse signaling in DC.Our previous work shows that activating anti-Fas antibody Jo-2 contributes to the maturation of DC and secretion of cytokines（such as IL-1β）and chemokines instead of apoptosis.To find out the effect of FasL on DC,we applied Fas-Fc fusion protein to stimulate BMDC,after which the secretion of inflammatory cytokines,DC phenotypic characters,DC phagocytosis and its capability of antigen-presentation were detected.The results showed that stimulation of FasL induced the improved secretion of proinflammatory mediators such as IL-6,and TNF-α,increased expression of CD40 and MHC-II,enhanced antigen presentation capability and decreased phagocytosis capability in WT BMDC.In order to confirm the results,we used FasL^gld mice and FasL△Intra mice（mutation in the intracellular segment of FasL and the extracellular segment still works）.The results showed that BMDC from FasL^gldld or FasL△Intra mice secreted less proinflammatory mediators than WT BMDC,further verifying the effect of FasL in DC.Furthermore,utilization of various inhibitors of signaling pathways demonstrated that the FasL triggered signaling is relevant to the mitogen-activated protein kinases including extracellular signal-regulated kinase,p38,and c-Jun N-terminal kinase.These data suggested that activated transmembrane FasL could transmit reverse signals which may contribute to the activation and maturation of DC.Therefore,deep investigation on the reverse signals of FasL will shed new light on the functional regulation of DC.Furthermore,our results showed that the activation of FasL produced a large amount of inflammatory cytokines,which plays a key role in the development and progress of SIRS.We wonder what is the effect of FasL in the progress of SIRS.Therefore,we established systemic infection model by injecting a certain amount of Candida albicans into WT,FasL^gld or FasL△Intra mice through caudal vein.The results demonstrated that FasL^gldld and FasL△Intra mice were less sensitive to fugal infection and had longer life expectancy and there was less production of inflammatory factors such as IL-6 and TNF-αafter infection,which led to a conclusion that FasL-mediated reverse signal is involved in inflammatory damage in fungal systemic infection.It is well known that TLR（Toll-like receptors）and CLR（C-type lectin receptors）detect bacteria or fungal infection and CLR recognizes and binds the exposed ligand in fungal such asβ-glucan and mannose.CLR including dectin-1,dectin-2,Mannose receptor,DC-SIGN,Mincle and so on,ligation of CLR enables DC to secret inflammatory factors and chemokines.Combining with previous findings,we wonder if there is crosstalk between FasL and CLR signals.So we cultivated BMDC from WT,FasL△Intra mice in vitro,respectively,and then BMDC was treated with 2μg/ml zymd（activator of dectin-1）,2μg/ml mannan（activator of dectin-2）for 24h.The results showed that BMDC from FasL△Intra mice secreted less inflammatory factors such as IL-6 and TNF-α,indicating that FasL promoted CLR-mediated effects;Reversely,we used Fas-Fc to stimulate the BMDC from the WT and CLR dysfunction mice(dectin-1^-/-and dectin-2^-/-mice).The results showed that the secretion of IL-6 and TNF-αdecreased in CLR dysfunction mice.These results verified the crosstalk between FasL and CLR signals,although its molecular mechanism needs to be further studied.To sum up,ligation of FasL induced production of proinflammatory mediators relevant to the activation of mitogen-activated protein kinases in dendritic cells.In the progress of acute systemic fungal infection,FasL facilitated the secretion of inflammatory factors and its cross talk with CLR might play a role in anti-fungal immunity.The study on the effect of FasL-mediated reverse signal on DC functional regulation and the underlying mechanism will help to understand the non-classical effect of FasL and shed new light in the DC treatment of diseases.