Study On The Mechanism Of Qingreliangxue Recipe In Regulating Peripheral Helper T Cell Immunotherapy For Blood Heat Type Psoriasis

Psoriasis is a common chronic inflammatory skin disease with various clinical manifestations,mainly characterized by erythema and scale.The pathogenesis of psoriasis is complex and unclear,and it is generally believed to be caused by the interaction of multiple factors such as genetics and environment.Various CD4+T lymphocytes are involved in the pathogenesis,including Th1,Th2,Th9,Th17,Th22,Treg cells and so on.Tph cells are a new subset of CD4+T cells,defined as PD-1+CXCR5-CD4+T cells.These cells express PD-1 and ICOS,and mainly secrete cytokines CXCL13 and IL-21,but lack the high expression of CXCR5.Many studies have shown that Tph cells are involved in the pathogenesis of a variety of immune-related diseases.However,the characteristics and roles of Tph cells in psoriasis remain unclear.Psoriasis is known as "Baibi" in Traditional Chinese Medicine."Traditional Chinese hemo-modulating therapy" has already been the main theoretical foundation of treating psoriasis by syndrome differentiation and "Blood-heat syndrome" is the most syndrome of psoriasis.On the basis of these theories and clinical practice,Professor Yanping Bai developed the Heat-clearing and blood-cooling recipe.The efficacy and safety of this recipe have been confirmed by clinical trials and experimental studies.However,it is not clear whether the Heat-clearing and blood-cooling recipe may treat psoriasis with blood-heat syndrome through Tph cells.Objectives:1.The aim is to study the characteristics and significance of Tph cells in psoriasis with blood-heat syndrome.2.The aim is to study the characteristics and significance of Tph cell subsets in psoriasis with blood-heat syndrome.3.We want to explore the relationships between Tph cells and Th17 or Tfh cells in psoriasis with blood-heat syndrome.4.We want to observe whether the the Heat-clearing and blood-cooling recipe may treat psoriasis with blood-heat syndrome through Tph cells.Methods:27 psoriasis patients with blood-heat syndrome and 13 healthy controls were enrolled.1.Flow cytometry was used to detect the Tph cell frequency,the expression of functional molecules(CD38,HLA-DR and ICOS)on the surface of Tph cells,and the levels of CXCL13 and IL-21 secreted by Tph cells in peripheral blood of the two groups.The infiltration of Tph cells in the skin lesions of patients and healthy controls was detected by immunofluorescence technique,and the correlations between the above indicators and Psoriasis Area and Severity Index(PASI)were analyzed.2.Flow cytometry was used to detect the frequency of Tph cell subsets(Tphl,Tph2 and Tph17)and the expression levels of functional molecules(CD38,HLA-DR and ICOS)on the surface of each subset in peripheral blood.The correlations between the above indexes and the severity of psoriasis were analyzed.ROC curve was used to analyze the sensitivity and specificity of Tph 17 cells in the diagnosis of psoriasis with blood-heat syndrome.3.Flow cytometry was used to examine frequencies of Th17 and Tfh cells in two groups.The correlations between these two cell frequencies and PASI,the relationships between Tph cells and Th17 or Tfh cells,and the relationships between CXCL13+Tph cells and CXCR5+Th17 or Tfh cells in patients were analyzed.4.The changes of PASI score and cell frequency of Tph,CXCL13+Tph,Tfh,Th17 and CXCR5+Th17 in patients before and after the treatment of Heat-clearing and blood cooling recipe were compared.Results:1.Compared with healthy controls,the Tph cell frequency,HLA-DR+Tph cell ratio,the ability of Tph cells to secrete CXCL13 in psoriasis patients with blood-heat syndrome were all significantly increased(all P values<0.0001),and the above indicators were all positively correlated with the PASI score.However,the ratio of CD38+and ICOS+Tph cells and the ability of Tph cells to secrete IL-21 did not significantly change,and there was no correlation with the PASI score.In addition,the number of Tph cells infiltrated in the lesions of patients was significantly increased(P<0.0001),but there was no significant correlation with the PASI score.2.Compared with healthy controls,the frequency of Tph17 cells in peripheral blood of psoriasis patients with blood-heat syndrome was higher than that of healthy controls(P=0.004),and it was positively correlated with the PASI score.The percentages of CD38+,HLA-DR+,ICOS+and CD38+HLA-DR+ICOS+Tph17 were increased(P=0.048,P=0.02,P=0.0013,P=0.015),but they were not correlated with the PASI score.There were no significant differences in the frequency of Tph1,Tph2 and CXCR3+CCR6+PD-1+CXCR5-CD4+T cells and the expression of CD38,HLA-DR and ICOS on Tphl or Tph2 cells between the two groups,and they were not correlated with the PASI score.In addition,the area under the ROC(AUC)of Tph17 cell frequency was 0.788(P=0.004),the cut-off value was 20.30%,the sensitivity was 0.815,and the specificity was 0.692.3.Compared with healthy controls,Th17 and Tfh cell frequencies in peripheral blood of psoriasis patients with blood-heat syndrome were increased(P=0.029,P=0.004),and were both positively correlated with PASI score.In patients,the frequency of Tph cells was positively correlated with the frequency of Th17 and Tfh cells.The frequency of CXCL13+Tph cells was also positively correlated with that of CXCR5+Th17 and Tfh cells.4.The PASI score,the frequency of Tph cells,the level of CXCL13 secreted by Tph cells,and frequencies of Th17,Tfh and CXCR5+Th17 cells in psoriasis patients with blood-heat syndrome were all significantly decreased after treatment with Heat-clearing and blood cooling recipe(P<0.01,P<0.01,P<0.01,P<0.05,P<0.05,P<0.05).Conclusions:1.Tph cells and Tph17 cells may be involved in the immunological pathogenesis of psoriasis with blood-heat syndrome.2.Tph cells may be related to Th17 and Tfh cells in psoriasis with blood-heat syndrome.3.Heat-clearing and blood-cooling recipe may be used to treat psoriasis with blood-heat syndrome by regulating Tph cell immunity.