Study On Immune Mechanism Of CCL17/CCR4 Axis Mediated Drug Eruption

Objectives:Drug eruption,a common adverse drug reaction,is an important public health issue due to the potential to cause life-threatening anaphylaxis and severe drug eruption.However,the mechanism involved in drug eruption remains unclear.Although a number of studies have shown that CCL17/CCR4 axis plays a pivotal role in regulating allergic diseases,its role in drug eruption remains unclear.Here,we aim to explore the role and the underlying mechanism of CCL17/CCR4 axis in drug eruption.Methods:In order to find out the different immune mechanism of severe drug eruption between HIV-infected patients with severe drug eruption and non-HIV infected patients with severe drug eruption.The peripheral blood samples of HIV-infected patients with severe drug eruption(HIV+SDE+),non-HIV infected patients with severe drug eruption(HIV-SDE+)and HIV-infected patients without severe drug eruption(HIV+SDE-)were collected.The Human Chemokine Antibody Array were employed to measure the chemokine protein expression in sera,and different data was carried out through bioinformatics analysis.At the time,we found the common immune mechanism of severe drug eruption in different population,therefore,the peripheral blood samples of HIV+SDE+,HIV-SDE+,HIV+SDE-and healthy donors were collected.Then ELISA of serum were performed in four groups and identified up-regulation of chemokines in all SDE patients.Furthermore,we sought to uncover the role of CCL17/CCR4 axis.1.In clinical samples,CD4+T,Th1,and Th2 cell subgroups in PBMC of four groups were detected by flow cytometry.Then IL-4 and IL-13 levels were further detected by ELISA of serum in four groups.Finally,the amount of CCL17-CD4+T cells were detected by flow cytometry.2.In vitro,D10.G4.1 mouse Th2 cell line was employeded and the CCR4 gene was knocked out by CRISPR/Cas9 technology.Then it was divided into four groups:CCL17 stimulated CCR4+Th2 cells,non-CCL17 stimulated CCR4+Th2 cells,CCL17 stimulated CCR4-Th2 cells and non-CCL17 stimulated CCR4-Th2 cells.At first,CCR4 internalization in Th2 cells elicited by CCL17,chemotaxis of CCR4+Th2 cells evoked by CCL17 and Th2 cell viability stimulated by CCL17 were evaluated among four groups by confocal microscopy,flow cytometry and CCK8,respectively.On the other hand,PCR-Array were used to screen Th2 cells mRNA expression with/without CCL17 stimulation.Further,ELISA was carried out using supernatant of four groups to calculate secreted IL-4,IL-5 and IL-13 levels,and western blotting was used to analyze the expression of p-ERK,ERK,p-STAT3 and STAT3.After ERK inhibitors treated CCL17 stimulated CCR4+Th2 cells and without CCL17 stimulated CCR4+Th2 cells,ELISA and western blotting were used to detect the level of downstream secreted proteins and analysis of protein expression,respectively.At last,the apoptosis rate of keratinocytes was evaluated by annexin V-FITC/PI double-staining and flow cytometry,when Th2 cells of four groups were co-cultured with keratinocytes for 24h,respectively.3.The efficacy of CCR4 antagonist(Compound47)and dexamethasone(DXM)were respectively performed in a Nevirapine-induced drug eruption rat model.Then it was divided into four groups:control group,non-treatment group,Compound47group and DXM group.Cytokine levels and CD4+ T and Th2 cell subgroups in peripheral blood of four groups were detected by ELISA and flow cytometry.The pathological manifestations of rat skin,keratinocyte apoptosis and the number of mast cells among four groups were detected by HE staining,Caspase-3 staining and toluidine blue staining,respectively.The expression of p-ERK,ERK,p-STAT6,STAT6 and pro-Caspase 3 in rat tissues among four groups were detected by Western blotting.Results:1.In clinical samples,CCL17 levels were much more in peripheral blood serum of HIV+SDE+patients(n=15)than in HIV-SDE+patients(n=15),and CCL17 levels were much more in HIV+SDE-patients(n=10)than in healthy donors(n=20)(P=0.037,and P=0.034,respectively).Furthermore,Th2 cell subgroup in HIV+SDE+patients were up-regulated when compared to HIV+SDE-patients and HIV-SDE+patients(P=0.002,and P=0.010,respectively).IL-4 levels were much more in peripheral blood serum of HIV+SDE+patients than in HIV-SDE+patients and HIV+SDE-patients(P=0.034,and P=0.023,respectively),and IL-3 levels were much more in peripheral blood serum of HIV-SDE+ patients than in healthy donors(P<0.001).The enhancement by CCL17 was more pronounced in human CD4+T cells,when compared to healthy donors(P=0.016).2.In vitro,CCL17 promotes CCR4 internalization in Th2 cells.The chemottraction of CCL17 stimulated CCR4+Th2 cells was much greater than in non-CCL17 stimulated CCR4+Th2 cells and CCL17 stimulated CCR4-Th2 cells(P=0.003,and P=0.011,respectively).Otherwise,when with/without CCL17 stimulated Th2 cells for 72 hours,the proliferation of CCL17 stimulated CCR4+Th2 cells was significantly higher than those in CCR4+Th2 cells without CCL17 stimulation and CCR4-Th2 cells with CCL17 stimulation(P=0.026,and P<0.001,respctively).Further,IL-4/IL-13 production by CCL17 stimulated CCR4+Th2 cells were up-regulated when compared to other two groups(P=0.002,and P=0.032,respctively)(P=0.012,and P=0.019,respctively).Western blotting confirmed that CCL17 stimulated CCR4+Th2 cells had higher expression of p-ERK/ERK and p-STAT3/STAT3 than other two groups(P<0.001,and P<0.0001,respectively)(P=0.004,and P=0.011,respectively).After the use of ERK inhibitors,CCR17 stimulated CCR4+Th2 cells expressed protein p-ERK/ERK and p-STAT3/STAT3 ratio decreased(P<0.0001,and P=0.011,respectively),IL-4 and IL-13 secretion decreased(P<0.0001,and P=0.006,respectively),compared with CCR17 stimulated CCR4+Th2 cells without ERK inhibitors.Finally,co-cultured of keratinocytes with CCL17 stimulated CCR4+Th2 cells result in enhanced apoptosis rate of keratinocytes,which were higher than that of non-CCL17 stimulated CCR4+Th2 cells and CCL17 stimulated CCR4-Th2 cells(P=0.003,and P<0.001,respectively).3.In the rat model of nevirapine-induced drug eruption(DE),rashes improved,ear swelling subsided,and body temperature decreased when CCR4 antagonist(Compound47)and DXM were administrated,respectively.Compared with non-treatment group,CCR4 antagonist and DXM treatment abolished the Th2 cells population(P<0.001,and P<0.001,respectively),and mast cells(P=0.001,and P=0.001,respectively).And CCR4 antagonist treatment reduced the levels of IL-4,IL-5 and IL-13(P=0.039,P=0.003 and P=0.014,respectively),Furthermore,CCR4 antagonist and DXM treatment reduced the levels of p-STAT6/STAT6(P<0.001,and P<0.0001,respectively).Conclusion(s):Taken together,our data provide proof of concept that CD4+T cells represent an important source of CCL17,which potentially plays a role via a mechanism in the development of drug eruption.Furthermore,CCL17 evokes CCR4 internalization and mediates chemotaxis,proliferation and IL-4/IL-13 secretion of Th2 cells through activation of phosphorylation ERK/STAT3 pathway.IL-4/IL-13 further induces keratinocytes apoptosis.CCR4 antagonist Compound47 is effective for suppression of Th2-mediated allergic inflammation by reduce Th2 cell subsets,IL-4/IL-13 levels,and mast cell counts in nevirapine-induced DE rats.It may inhibit the activation of phosphorylation STAT6 pathway to reduce apoptosis of keratinocytes,thus relieving rash symptoms of DE rats.