TIM4 Expression And Function In The Distinct Subsets Of Dendritic Cells Residing At Skin And Its Draining Lymph Nodes

Background: T cell immunoglobulin and mucin(TIM)family represents an evolutionarilyconserved group of type 1 membrane proteins,which consists of eight members(encoding TIM-1 to TIM-4 and putative TIM-5 to TIM-8)located on chromosome 11B1.1 in mice and three members(Tim-1,Tim-3 and Tim-4)located on chromosome 5q33.2 in human.Previous data reveal a profound immunoregulatory role of TIM members in infectious,allergic,autoimmune,and neoplastic disorders.Further research into the underpinning mechanisms indicate that TIM-1,TIM-2 and TIM-3,which are predominantly expressed on T lymphocytes,govern the proliferation and activation of T cell subsets,including T helper cells 1(Th1),Th2,Th17 as well as regulatory T cells(Treg).In contrast,TIM-4 is primarily expressed on antigen-presenting cells(APC),such as conventional dendritic cells(DC)and macrophages.As a costimulatory molecule on the surface of APCs,TIM-4 regulates the differentiation of T cell sublineages through binding to its natural ligand TIM-1 or other unknown ligands;as a phosphatidylserine(PS)receptor for capturing apoptotic bodies,TIM-4 modifies the types and intensities of immune responses by adjusting the apoptotic processes of immune cells.Skin DCs characterize a heterogeneous cell population residing at the interphase with the external environment,which process and present ingested antigens to T cells within skin-draining lymph nodes(LN)and regulate their immunofunctions.As the sole DC subset in the epidermis,Langerhans cells(LC)mediate immune surveillance as well as tolerance at the first line of body defense.Dermal DCs are comprised of dermal LCs in transit,dermal CD207+ DCs(CD207+ DDC),dermal CD207-CD11b-DCs(CD207-CD11bDDC)and dermal CD207-CD11b+ DCs(CD207-CD11b+ DDC).Diverse subtypes of DCs in the skin-draining LNs are essentially involved in T cell immunity,involving LN migrated LCs(m LC),LN migrated CD207+ DDCs(CD207+ m DDC),LN tissue-resident CD207+CD8+ DCs,LN conventional CD207-CD4-CD8+ DCs,LN CD207-CD4-CD8-DCs,LN CD207-CD4+CD8-DCs and LN CD207-CD4+CD8+ DCs.To the best of our knowledge,the significance of TIM-4 in the skin and skin-draining LN DCs remains unexplored.Objective:Our study here aimed at clarifying the expression level of TIM-4 in the distinct subsets of DCs within the skin and its draining LNs,exploring the impact of TIM-4 on the homeostatic maintenance of skin-relating DCs,investigating the significance of TIM-4 in the immune functions of epidermal LCs and evaluating the influence of TIM-4 upon cutaneous contact hypersensitivity(CHS)and autoimmune disease psoriasis.Materials and methods: We assessed the expression level of TIM-4 in epidermal LCs,dermal LCs in transit,CD207+ DDCs,CD207-CD11b-DDCs,CD207-CD11b+ DDCs,skin-draining LN m LCs,LN CD207+ m DDCs,LN tissue-resident CD207+CD8+ DCs,LN conventional CD207-CD4-CD8+ DCs,LN CD207-CD4-CD8-DCs,LN CD207-CD4+CD8-DCs and LN CD207-CD4+CD8+DCs from normal C57BL/6 wild-type(WT)mice(designated as TIM-4 WT mice);we compared the frequencies of distinct DC subsets residing at the epidermis,dermis and skin-draining LNs between TIM-4 WT mice and TIM-4 knock-out(KO)C57BL/6 mice(designated as TIM-4 KO mice);we evaluated the impact of TIM-4 deficiency on the in vitro phagocytosis and spontaneous maturation of LCs;we also compared the in vivo migration and the repopulation in response to ultra-violet(UV)-mediated skin inflammation between TIM-4-intact and TIM-4-deficient LCs;applying dinitrofluorobenzene(DNFB)-induced contact hypersensitivity(CHS)mouse model,we tested the influence of TIM-4 ablation on murine skin-related immune reaction;utilizing imiquimoid(IMQ)-induced psoriasis-like dermatitis mouse model,we explored the role of TIM-4 in the pathogenesis of psoriasis.Results: For the first time,we discovered that epidermal LCs expressed TIM-4 at a low level,and displayed an increase of TIM-4 expression upon migration to skin-draining LNs;CD207-CD11b-DDCs and CD207-CD11b+ DDCs barely expressed TIM-4;CD207+ DDCs had a relatively high level of TIM-4 expression,and lowered its expression after migration;LN tissue-resident CD207+CD8+ DCs expressed a moderate level of TIM-4;among LN conventional DCs,CD207-CD4+CD8-DCs and CD207-CD4+CD8+ DCs highly expressed TIM-4 while CD207-CD4-CD8-DCs and CD207-CD4-CD8+ DCs have limited expression of TIM-4.By means of comparing TIM-4 WT mice and TIM-4 KO mice,we demonstrated that genetic deletion of TIM-4 significantly upregulated the frequencies of epidermal LCs(1.10 ± 0.39% in WT mice vs.1.63 ± 0.48% in KO mice,P = 0.017),LN CD207-CD4+CD8-DCs(25.8 ± 7.1% in WT mice vs.41.44 ± 4.22% in KO mice,P = 0.029)and LN CD207-CD4+CD8+ DCs(6.00 ± 0.16% in WT mice vs.11.04 ± 1.16% in KO mice,P = 0.004)as well as downregulated LN CD207-CD4-CD8-DCs(63.25 ± 8.15% in WT mice vs.43.06 ± 3.11% in KO mice,P = 0.01),whereas the percentages of the remaining skin-relevant DCs remained unaltered.Moreover,the epidermal LCs of TIM-4-deficient mice displayed normal in vitro phagocytic and maturational abilities,unharmed migration upon the stimulation as well as local repopulation under UV-mediated inflamed state.Furthermore,lack of TIM-4 did not affect DNFB-induced CHS response.However,TIM-4 deficiency exacerbated the severity of IMQ-induced psoriasis-like skin inflammation.Conclusion: In conclusion,our results indicated that the expression patterns of TIM-4 differed in the distinct subsets of DCs within skin and skin-draining LNs that CD207+ DDCs and the CD4-positive compartments of LN conventional CD207-DCs highly expressed TIM-4.Notably,TIM-4 specifically regulated the homeostasis of epidermal LCs,LN CD207-CD4+CD8-DCs,LN CD207-CD4+CD8+ DCs and LN CD207-CD4-CD8-DCs.Additionally,TIM-4 might play a protective role in the pathogenesis of psoriasis.