| BackgroundAtopic dermatitis(AD)is a common chronic,recurrent,and inflammatory skin disease,which ranks first in the burden of non-fatal skin diseases.Abnormal skin barrier,immune inflammation,and disordered skin microbiome play critical roles in the pathogenesis of AD.In recent years,it has been found that kallikreins(KLKs),a kind of serine protease,are involved in the desquamation and keratosis process of skin barrier-impaired diseases such as AD,psoriasis and Netherton syndrome.As one of the innate antimicrobial peptides,β-defensin 2(β-HBD2)can resist the invasion of bacteria into the epidermis.However,the deficiency of β-HBD2 increased microbial infection.Therefore,KLKs and β-HBD2 may play key roles in epidermal barrier protection.In addition,the inflammatory cytokines IL-1α and IL-17 A,which can be produced by keratinocytes(KCs)and promote the expression of antimicrobial peptides,also play important roles in inducing inflammation in immune inflammatory skin diseases.Metagenomic sequencing has shown that microbial diversity is decreased,whereas the Staphylococcus aureus(S.aureus)infection is increased in AD.And S.aureus superantigens,the products of S.aureus are thought to be related to skin barrier impairment and immune inflammatory response in AD.In particμlar,Staphylococcal enterotoxin gene A(SEA),SEB,and Toxic shock syndrome toxin 1(TssT-1)were highly detected in AD patients,which may have important influences on the aggravation of AD.Therefore,we choose SEA,SEB,and TssT-1 as experimental research objects.Served as the primary barrier against the invasion of pathogenic microorganisms,KCs are closely related to the skin barrier and immune inflammation.To investigate the effect of S.aureus superantigens on KCs,we use SEA,SEB,and TssT-1 to stimμlate Human immortalized keratinocytes(HaCaT),respectively.And then we detect their effects on skin barrier-related products,such as KLKs and β-HBD2,and inflammation-related products,such as IL-1α,and IL-17 A,which can provide a scientific basis for the mechanism and treatment of skin barrier and immunoinflammatory skin diseases associated with S.aureus.Object1.To detect the effects of SEA,SEB,and TssT-1 on the proliferation and apoptosis of HaCaT cells.2.To detect the effects of SEA,SEB,and TssT-1 on the expression of skin barrier and immunoinflammation-related KLKs,β-HBD2,IL-1α,and IL-17 A of HaCaT cells.3.To investigate the role of SEA,SEB,and TssT-1 in the pathogenesis of AD.Method1.After 24 hours and 48 hours of incubating HaCaT cells with different concentrations of SEA,SEB,and TssT-1,respectively,cell proliferation activity was detected by the CCK8 method,and apoptosis was detected by flow cytometry.Then,we select the concentration range of superantigens without cytotoxicity for the next experiment.2.After 24 hours and 48 hours of incubating HaCaT cells with different concentrations of SEA,SEB,and TssT-1,respectively,we use the Western blot method to detect the expression of KLK14 protein.3.After 24 hours of incubating HaCaT cells with different concentrations of SEA,SEB,and TssT-1,respectively,the expressions of KLK5,KLK6,KLK7,KLK13,KLK14,and β-HBD2 at m RNA levels were detected by RT q PCR,and the expressions of β-HBD2,IL-1α and IL-17 A in the supernatant were measured by ELISA assays.Resμlts1.After treating HaCaT cells with SEA,SEB,and TssT-1 separately at different concentrations(0,0.1,0.5,1,2,5,and 8 μg/ml)for 24 h and 48 h,compared with the control group,there was no significant difference in cell proliferation activity(P > 0.05).2.HaCaT cells were treated with SEA,SEB,and TssT-1 at different concentrations(0,0.5,1,and 2 μg/ml)for 24 h and 48 h,respectively.Compared with the control group,the resμlts of flow cytometry showed that the apoptosis rate of HaCaT cells was decreased at0.5,1,and 2 μg/ml SEA group for 24 h,and at 0.5 μg/ml SEA group and 0.5 μg/ml TssT-1group for 48 h(P < 0.05).And there was no statistical difference in the increase of apoptotic rate in the experimental group(P > 0.05).3.HaCaT cells were treated with SEA,SEB,and TssT-1 at different concentrations(0,0.5,1,and 2 μg/ml)for 24 h and 48 h,respectively.Compared with the control group,KLK14 protein expression in SEA and SEB-treated groups was increased at 24 h.Especially in the 1 μg/ml SEA treated group and 0.5,1,and 2 μg/ml SEB treated groups,KLK14 protein increased and the difference was statistically significant(P < 0.05).KLK14 protein expression was decreased in the TssT-1 treated group of 24 h,but no statistical significance(P > 0.05).In addition,the KLK14 protein in the SEA,SEB,and TssT-1treated groups of 48 h express higher than the control group,and there were statistically different in the 1 μg/ml and 2 μg/ml SEB groups(P < 0.05).4.HaCaT cells were treated with SEA,SEB,and TssT-1 at different concentrations(0,0.5,1,and 2 μg/ml)for 24 h,respectively.Compared with the control group,the resμlts of RT q PCR showed that in the SEA-treated group,KLK5,KLK6,KLK7,and KLK13 at m RNA levels all showed an increasing trend,and the increase of KLK6 in 0.5 μg/ml SEA group was statistically significant(P < 0.05).KLK14 was increased in the 1 μg/ml SEA group but decreased in 0.5 and 2 μg/ml SEA groups,and there was a statistical difference in the decrease of KLK14 expression in the 2 μg/ml SEA group(P < 0.05).In SEB-treated groups,the expressions of KLK5,KLK6,KLK7,and KLK14 at m RNA levels were increased,and there was a statistical difference in the increase of KLK7 in 2 μg/ml SEB group and the increase of KLK14 in 1 μg/ml SEB group(P < 0.05).The expression of KLK13 in the SEB-treated group was decreased and was statistically significant in 0.5μg/ml SEB-treated group(P < 0.05).In TssT-1 treated groups,the m RNA levels of KLK5,KLK6,KLK7,KLK13,and KLK14 all showed an increased trend,and there have statistical differences in the increase of KLK6 and KLK7 in 0.5 μg/ml TssT-1 group(P <0.05).5.HaCaT cells were treated with SEA,SEB,and TssT-1 at different concentrations(0,0.5,1,and 2 μg/ml)for 24 h.Compared with the control group,the m RNA expression of β-HBD2 was decreased by RT q PCR.β-HBD2 decreased significantly in 0.5,1,2 μg/ml SEA groups and 1,2 μg/ml SEB groups,and 2 μg/ml TssT-1 group(P < 0.05).Besides,compared with the control group,ELISA assay showed that m RNA expression of β-HBD2 in cell supernatant decreased(P < 0.05)and 0-2 μg/ml SEA,SEB,and TssT-1 have no effect on the expression of IL-1α and IL-17 A in HaCaT cells.ConclusionSEA,SEB,and TssT-1 can up-regμlate KLKs and down-regμlate β-HBD2 expression at different concentrations and times,suggesting that superantigens are related to epidermal barrier damage and immune inflammation,and the activation of KLKs may be one of the reasons for inducing or exacerbating AD.Besides,reduced secretion of β-HBD2 may be conducive to the colonization of S.aureus,leading to the persistence of AD.Inhibition of KLKs and promotion of β-HBD2 expression may be a promising therapeutic strategy for alleviating AD. |
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