The Mechanism Of Qingre Liangxue Recipe In Intervening TIGIT Signaling Pathway To Regulate T Cell Immunotherapy For Psoriasis

Introdution:Psoriasis is an immune-mediated chronic and recurrent skin disorder which can be called"Baibi" in Traditional Chinese Medicine.A diverse clinical features of psoriasis can be found in clinic,but the main characters are erythema and scale.The pathogenesis of psoriasis is extremely complicated,which associates with immune-related genes,environmental factors,multifunctional cells and inflammatory cytokines.Since the pathogenesis of psoriasis is still unclear,plenty of researches showed that the dysfunction of immunocytes especially CD4+ T cells have crucial roles in the development of psoriasis.Activation of CD4+ T cells need stimulate by T cell receptor and a co-stimulatory molecule on the T cell by the major histocompatibility complex(MHC)peptide and co-stimulatory molecules on the antigen presenting cell(APC).The co-stimulatory molecules involves in the development of diseases such as autoimmune disease and tumor by affecting the function of T cells.The co-stimulatory molecules is composed by two types of functional molecules which are co-stimulatory receptors and co-inhibitory receptors.The co-inhibitory receptors,also called immune checkpoint,play an important role in the maintenance of immune homeostasis,also can suppress the over-activation and over-proliferation of T cell by binding with the ligands.T cell immunoglobulin and ITIM domain(TIGIT)is a novel identified co-inhibitory receptors which has lower autimmune-like toxicity than CTLA-4,PD-1.Several sesearches showed that the function of CD4+ T cells can be suppressed by activating the TIGIT signaling.All of these make us hypothesize that TIGIT may be involved in the pathogenesis of psoriasis.This may provide some new method and thought target in psoriasis.After the Ming and Qing dynasties,"Traditional Chinese hemo-modulating therapy" has already been the main theoretical foundation of treating psoriasis by syndrome differentiation.Several large samples epidemiological studies showed that "Blood-heat syndrome" is the main syndrome of psoriasis.On the basis of these theories and clinical practice,Professor Yanping Bai developed the Heat-clearing and blood cooling recipe.The clinical efficacy has been proved by clinical trials,cell and animal experiments.Because of the Chinese herbs can treat diseases by acting on several targets.So selecting the best target become the key point in researching Chinese herbal compound.Objective:To explore the effect of TIGIT in the immune pathogenesis and the clinical significance of Heat-clearing and blood cooling recipe in psoriasis.We observe the TIGIT+CD4+T cells and cytokines expression and the mechanism of TIGIG signaling in psoriasis,and analyze their associations with disease severity.In addition,we investigated whether the Heat-clearing and blood cooling recipe could treat psoriasis by affecting the TIGIT expression on CD4+T cells.Methods:The expression of TIGIT on CD4+T cells was evaluated by flow cytometric analysis and qRT-PCR.IFN-y,IL-17A and IL-10 production were measured by CBA.All of the results were compared between 28 patients with blood-heat type psoriasis and 14 healthy controls.The infiltration of TIGIT+CD4+T cells in lesion of psoriasis patients were detected by imunohistochemistry and immunofluorescence.The relationships between Psoriasis Area and Severity Index(PASI)score of patients and the frequency of TIGIT+CD4+T cells and the levels of IFN-γ,IL-17A,IL-10 were analyzed.To turther explore the function of TIGIT in the pathogenesis of psoriasis,the proliferation and function of CD4+T cells were measured by using functional anti-human TIGIT antibody and recombinant human CD155/Fc proteinin to interfere the TIGIT signaling.In addition,after 2 months of Heat-clearing and blood cooling recipe treatment,changes of the frequency of TIGIT+CD4+T cells and the levels of IFN-γ,IL-17A,IL-10 were detected.Results:(1)the frequency of TIGIT+CD4+T cells significantly decreased in patients with blood-heat type psoriasis compared with HC(P<0.05),but the result of immunohistochemistry and immunofluorescence was negative.The qRT-PCR assay also showed that the mRNA expression level of TIGIT was significantly lower in CD4+T cells from PV(P<0.01).Further analysis indicated a negative correlation between PASI score and TIGIT expression(P<0.01).The plasma levels of INF-y,IL-17A were significantly higher in PV compared with HC(P<0.01,P<0.01),but IL-10 was on the opposite(P<0.05).To investigate whether those cytokines had relationships with TIGIT,we analysis the correlation of the levels of INF-γ,IL-17A,IL-10 with the frequency of TIGIT+CD4+T cells.The dates showed INF-γ,IL-17A negatively correlated with the frequency of TIGIT+CD4+T cells(P=0.0149,-0.49;P=0.0069;r=-0.72,P<0.01).But IL-10 had positively correlation with the frequency of TIGIT+CD4+T cell(r=0.62,P=0.0004)(2)48h after anti-CD3 and anti-CD28 antibodies stimulation on CD4+ T cells,the division and proliferation of T cells in 5ug/ml and 2ug/ml concentration is the strongest.Blockade of the TIGIT signaling pathway by functional anti-human TIGIT antibody significantly increased supernatant productions of IFN-γ and IL-17A(P<0.05,P<0.05),but no statistical difference was founded in the IL-10 level and CD4+T cells proliferation.Furthermore,addition of recombinant human CD 155/Fc protein efficiently increased the supernatant production of IL-10,and decreased the production of IFN-γ and IL-17A,and inhibited the proliferation of CD4+T cells(P<0.05,P<0.05,P<0.05,P<0.01).(3)The frequency of TIGIT+CD4+T cells and the level of IL-10 were significantly increased in patients after treatment with Heat-clearing and blood cooling recipe(P<0.01,P<0.01).In addition,there was also a significant change in the plasma levels of INF-γ,IL-17A and PASI score after treatment(P<0.05,P<0.01,P<0.01).Conclusion:The expression of TIGIT was decreased in patients with blood-heat type psoriasis and associated with the PASI score.And down-regulate of TIGIT on CD4+T cells might contribute to the pathogenesis of psoriasis and activation of TIGIT pathway may be a potential therapeutic target in psoriasis.