The Role Of Complement C3 Mediated CD4~+T Cell Proliferation And Differentiation In The Pathogenesis Of SCADRs

Serious cutaneous adverse drug reactions(SCADRs,also known as severe drug eruption)has a variety of clinical manifestations,dangerous condition,complex pathogenesis and high mortality.SJS and TEN are common SCADRs which can be considered to be different stages of the same disease.SJS and TEN are rapidly changing,and the case-fatality rate of SJS is 5~10%and TEN is 30%.To elucidate the pathogenesis of SCADR,especially SJS/TEN,has become an significant topic in clinical practice.The present study suggests that SCADRs is a heterogeneous immune response mediated by T cells and is the result of interaction of host genes,microorganisms and drugs.Currently,known HLA risk alleles,T cells and complement systems participate in the pathogenesis of SCADRs.How can drug specific CD4~+T cells be activated,selectively proliferate and differentiate?whether there is a correlation between the complement system and the specific mechanism of T cell activation,at the same time,the mechanisms by which the complement system mediates cell-mediated immune response remain unclear.The study found that the complement fragment C3a can bind to the C3a receptor(C3aR)on the surface of T cells,and affect the proliferation and differentiation of CD4~+T cells.On the basis of the previous work of our research group,the relationship between complement active fragment C3a and CD4~+T lymphocyte subsets proliferation and differentiation and the possible mechanism are discussed.We intend to elucidate the regulatory mechanism of C3a-C3aR as a activating or enhancing co-stimulating signal and then activating the PI3K/Akt/FoxO1signaling pathway to mediate the proliferation and differentiation of drug specific CD4~+T cells.The study will further reveal the pathological development of severe drug eruption and provide new ideas and clues for the prevention and treatment of severe drug eruption.Objective Research for the present SCADRs from hospital is to understand the incidence situation of drug eruption,through regression analysis to investigate the factors affecting prognosis of severe drug eruption;The clinical study is conducted to investigate the correlation between complement activation and the number of CD4~+T lymphocyte subsets by detecting the complement related indices and CD4~+T lymphocyte subsets by flow cytometry at different stages in patients with severe drug eruptions;Through animal experiments with TCE sensitized female BALB/c mice,specific proteins and transcription factors by adopting molecular biology method to detect PI3K/Akt pathway cascading reactions,in order to investigate the role of C3a-C3aR as or enhancing co-stimulatory signal in the activation of PI3K/Akt/FoxO1signaling pathways,and then mediate the regulation mechanism of drug specific CD4~+T cell proliferation and differentiation in severe drug eruption.The study will further reveal the pathogenesis and development of severe drug eruption,and provide basic data for the study of the pathogenesis of severe drug eruption.Methods The present patients study:The patients suffering from SCADRs victims were investigated under unified standard.Descriptive statistical analysis was performed on the medical records of patients and Logistic regression analysis was used to explore the factors influencing the outcome of severe drug eruption treatment;The clinical study:the blood routine,parameters of liver and kidney function,blood glucose and C3,C3a,CD55 and CD59 of complement related indexes were detected in patients with severe drug eruption.CD4~+T cell subsets(Th1,Th2,Th17 and Treg)were detected by Flow cytometry,as well as ELISA method was used to detect the effects of IFN-gamma,IL-4,IL-17 and IL-10 cytokines;Animal experiments:Spleen lymphocytes of sensitized BALB/c female mice treated with TCE and C3a in vitro,the expression of cytokines(IFN-gamma,IL-4,IL-17,IL-10)was detected by the ELISA method and T cell-expressed receptors for C3a(C3aR)were detected by Western bolt.CD4~+T lymphocyte subsets(Th1,Th2,Th17 and Treg)were detected by flow cytometry.Real-time PCR detected the mRNA expression of T cell subsets specific transcription factors T-bet,GATA-3,ROR-?t and Foxp3,and the protein expression levels of PI3K,p-PI3K,Akt,p-Akt,FoxO1 and p-FoxO1 were detected by Western bolt.Meanwhile the C3a receptor antagonist and PI3K specific inhibitor were pretreated to observe the changes of the above indexes.Results(1)The present patients study:(a).The allergenic drugs:the main drugs that cause SJS/TEN are antiepileptic and antigals.Common drugs are carbamazepine,allopurinol and phenytoin sodium.(b).The hospitalization days and the incubation period:the average hospitalization days for patients with severe drug eruption is(13.20±10.80)days,and(15.52±20.85)days as the average incubation period days.Hospitalization days and the incubation period between different types of drug eruption is no statistical difference(P>0.05).(c).Involving organs:in 42 cases of severe drug eruption,36 cases with abnormal blood(85.71%),30 cases of liver function damage(71.43%),29 cases of fever(69.05%),24 cases of mucosal injury(57.14%),22 cases of respiratory infection(52.38%),and 18 cases of renal impairment(42.86%).(d).Treatment outcome:the total effective rate of severe drug eruption was 78.57%,in which the total effective rate of ED was 83.33%,SJS was 72.73%,AGEP was 85.71%,and TEN was 60%.(e).Logistic regression analysis:the dosage of glucocorticoid(OR=2.822,P<0.01),ALT(OR=1.391,P<0.01),albumin propagated(OR=1.336,P<0.01),fasting glucose(OR=1.075,P<0.05)is the influence factor of severe drug eruption treatment outcome.(2)The clinical study:(a).Serum complement component assay:compared with the control group,the serum complement protein C3 decreased(P<0.05)in the patients with drug eruption and the activity of C3 increased after treatment,and there was statistical significance between the two groups before and after treatment(P<0.05);The level of serum C3a in drug eruption patients was higher than that in control group(P<0.01).After treatment,the level of C3a began to decrease(P<0.01);The mean fluorescence intensity of complement activation regulated protein CD55 in lymphocytes and monocytes from drug eruption group decreased compared with control group(P<0.05),and the mean fluorescence intensity of CD59 in lymphocyte and granulocyte decreased(P<0.05).(b).CD4~+T cell subsets:the number of Th1 and Th17 cells increased compared with control group(P<0.01),while the number of Th2 and Treg cells decreased(P<0.05);Th1 and Th17 subsets decreased after treatment,while the number of Th2 and Treg cells increased,and there was statistically significant between the two groups before and after the treatment(P<0.05).In patients with drug eruption,the ratio of Th1/Th2 and Th17/Treg increased before treatmentand decreased after treatment(P<0.01),and there was statistical significance between the two groups before and after treatment(P<0.05).(c).Cytokines:Compared with the control group,IFN-?and IL-17 xpression increased in the patients with drug eruption(P<0.05),and the expression of IFN-?and IL-17 decreased after treatment(P<0.05).IL-4 and IL-10 showed high expression in the control group,and the serum content of IL-4 and IL-10 were significantly lower in patients with drug rash(P<0.05)and increased after treatment(P<0.05).(d).The correlation analysis:C3a was positively correlated with Th1 and Th17 cell subsets.The correlation coefficients were r=0.651,r=0.545,respectively,which was statistically significant(P<0.05).However,C3a was weak negatively correlated with Th2 and Treg cells,and the correlation coefficients were r=-0.056,r=-0.005,respectively,which was no statistical significance(P>0.05).(3)Animal experiments:(a).Cytokines:Spleen lymphocytes of sensitized mice treated with TCE and C3a,IFN-?and IL-17 expression quantity increased(P<0.05),and IL-4 and IL-10 expression decreased(P<0.05).The expression levels of IFN-?and IL-17 were continuously decreased with the use of C3aR antagonist and PI3K inhibitor,while expression of IL-4 and IL-10 increased(P<0.05).(b).CD4~+T cell subsets:After TCE and C3a treatment,the number of Th1 and Th17 cells was higher than that in the control group(P<0.05),while the number of Th2 and Treg cells decreased(P<0.05).Th1 and Th17 cells decreased with pretreatment of C3aR antagonist and PI3K inhibitor,while the number of Th2 and Treg cells increased(P<0.05).(c).The expression of C3aR on the surface of CD4~+T cells:After the spleen lymphocytes of sensitized mice were treated with TCE,the expression of C3aR on the surface of CD4~+T cells increased(P<0.05).On this basis,C3a was also added to the medium to co-incubate with cells,and the results showed that the expression of C3aR was further increased(P<0.05),and the expression of C3aR decreased after pretreatment with C3aR antagonist(P<0.05).(d).The mRNA expression of T cell subsets specific transcription factors:The spleen lymphocytes of sensitized mice were treated with TCE and C3a,and the mRNA expression of T-bet and ROR-?t increased(P<0.05),while the mRNA expression of GATA-3 and Foxp3 decreased(P<0.05).After pretreatment with C3aR antagonists and PI3K inhibitors,the mRNA expression of T-bet and ROR-?t decreased with the increase of GATA-3 and Foxp3 mRNA expression level(P<0.05).(e).The expression levels of PI3K,p-PI3K,Akt,p-Akt,FoxO1 and p-FoxO1 protein:The protein expression of PI3K,Akt and FoxO1decreased in spleen lymphocytes of sensitized mice by treatment with TCE and C3a,while the phosphorylated products p-PI3K,p-Akt and p-FoxO1 showed high expression(P<0.05).After pretreatment with C3aR antagonists and PI3K inhibitors,the expression of PI3K,Akt and FoxO1 was elevated,but the expression of p-PI3K,p-Akt and p-FoxO1 was inhibited and the protein expression was decreased significantly(P<0.05).Conclusion The main conclusions of this paper:(1)The main drugs that cause SJS/TEN are antiepileptic and anti-gout drugs including carbamazepine,allopurinol and phenytoin.Major complications of severe drug eruption include abnormal blood system,liver function damage,fever,mucosal damage and respiratory infection.The total effective rate of treatment was 78.57%,of which SJS was 72.73%and TEN was 60.00%.(2)The major factors influencing prognosis of severe drug eruption are glucocorticoid dosage,alanine aminotransferase,serum albumin and fasting blood glucose.(3)Activation of complement system and differentiation of CD4~+T lymphocytes may be an important link in the pathogenesis of severe drug eruption.(4)TCE can activate T lymphocytes of sensitized mice in vitro,which may be related to up-regulating the expression of C3aR on the surface of CD4~+T cells,and then regulate the proliferation and differentiation of CD4~+T cells by PI3K/Akt/FoxO1 signaling pathway cascade through the C3a-C3aR axis.(5)Complement fragment C3a could bind their respective G protein-coupled receptors C3aR expressed on T cells to provide co-stimulatory signals that enhance proliferation and differentiation of Th1 and Th17 cell and limit generation and stability of Th2 and Treg cells through the PI3K/Akt/FoxO1 signaling pathways,which amplify the cellular immune response may mediate the pathological process of severe drug eruption.