Regulation Of Staphylococcus Epidermidis On Malassezia Furfur-mediated Inflammatory Response In Keratinocytes

Objective:Malassezia is a lipophilic yeast,a resident flora in human skin and mucous membrane.It not only exists as an opportunistic pathogen,but also participates in a variety of skin diseases,such as pityriasis versicolor,Malassezia folliculitis,dandruff,seborrheic dermatitis,atopic dermatitis and psoriasis.In recent years,some research results indicated that Staphylococcus epidermidis may play a role in Malassezia-related inflammatory skin diseases.Research results have shown that Staphylococcus epidermidis was the dominant bacterial flora in the skin lesions of patients with scalp dandruff.More staphylococci were colonized in the skin lesions of patients with facial seborrheic dermatitis.Staphylococcus epidermidis was dominant in children with mild atopic dermatitis.As a resident opportunistic pathogen of the skin,Staphylococcus epidermidis was mainly discussed in the research of catheter-related infections.Studies on the interaction with other skin flora have shown that Staphylococcus epidermidis can inhibit the growth and local colonization of harmful microorganisms.It can also down-regulate the skin inflammation induced by Staphylococcus aureus or Cutibacterium acnes.However,the role of Staphylococcus epidermidis in Malassezia-related inflammatory skin diseases and mechanisms are still unclear.This study aims to explore the role of Staphylococcus epidermidis in the skin inflammation induced by Malassezia furfur.Through co-culture with keratinocytes,we found that Staphylococcus epidermidis selectively inhibited the expression of IL-6 induced by Malassezia furfur,and NF-?B pathway participated in the regulation of this process.In mouse experiments,we found that Staphylococcus epidermidis reduced the epidermis thickening and the infiltration of dermal cells induced by Malassezia furfur.Methods:1.Firstly,the MTS method was used to detect the cytotoxicity of different concentrations of Malassezia furfur or Staphylococcus epidermidis on keratinocytes,and the concentration that had no effect on cell proliferation and survival was chosen for subsequent experiments to avoid the influence of cytotoxicity on the expression of inflammatory cytokines.Then,real-time quantitative PCR technology and ELISA method were used to detect the mRNA level and protein level of keratinocyte inflammatory cytokines stimulated by Malassezia furfur or(and)Staphylococcus epidermidis at different time points.2.In order to explore the mechanism of the effect of Staphylococcus epidermidis,we applied western blot experiment to detect the translation level of IL-6 upstream pathway proteins.Samples of Malassezia furfur or(and)Staphylococcus epidermidis stimulation at different time points were collected to detect the expression of phosphorylated protein and receptor proteins.Keratinocytes were pretreated with pathway inhibitors,and the secretion of IL-6 was detected by ELISA.By comparing the changes before and after inhibitor pretreatment,the role of the pathway proteins in inhibiting the expression of IL-6 was further confirmed.3.Experiment in vivo was carried out to further investigate the role of Staphylococcus epidermidis on Malassezia furfur-treated keratinocytes and other possible effects in the dermis.Under the mouse anesthesia,the hair on the back skin was shaved,and the back skin was taped to destroy the skin barrier.Re-suspended Malassezia furfur or/and Staphylococcus epidermidis in olive oil were applied with sterile cotton swabs to the back skin,and olive oil alone was used for the negative control group.On the 4^thday,the skin changes were observed with naked eyes and the skin lesions were biopsied,fixed in formalin and embedded in paraffin,and then HE staining and immunohistochemical staining were performed.Results:1.In the cell viability experiment,compared with the control group,the cell viability of the Malassezia furfur or Staphylococcus epidermidis:keratinocytes=50:1group was significantly reduced.Compared with the control group,the cell viability of the Staphylococcus epidermidis:keratinocytes=20:1 group co-cultured for 24h was significantly reduced.Real-time quantitative PCR experiment results showed that 2h co-culture of keratinocytes with Malassezia furfur significantly increased,the mRNA expression of pro-inflammatory factors IL-1?,IL-1?,IL-6 and IL-8,while the Staphylococcus epidermidis had no effect on the mRNA expression of the above inflammatory cytokines.Compared with the Malassezia furfur group,the mRNA expression of IL-6 in keratinocytes co-treated by Malassezia furfur and Staphylococcus epidermidis was reduced,and ELISA experiments showed the same trend of the secretion of IL-6 in the cell supernatant,while the expression of other inflammatory cytokines was not inhibited.2.Western-blot results showed that there was difference in the expression of phosphorylated protein of NF-?B and JNK pathways between the Malassezia furfur group and the co-treated group,but the difference varied among different time points.Ah R downstream transcription factor CYP1A1 was detected.After pathway inhibitors pretreatment,NF-?B pathway inhibitors reversed the inhibitory effect of Staphylococcus epidermidis on Malassezia furfur-induced IL-6 expression,suggesting that NF-?B pathway participated in the regulatory process,while JNK pathway inhibitors didn't change the difference between groups suggesting that it was not involved in the regulatory process.3.In the mouse experiment,it was observed by naked eye that Malassezia furfur induced desquamation and thickening of the skin,and HE staining showed increased infiltration of dermal cells.Compared with the control group,Staphylococcus epidermidis reduced the infiltration of dermal cells(651 cells/mm²).Staphylococcus epidermidis reduced the epidermal thickness and dermal cell infiltration induced by Malassezia furfur.Immunohistochemical staining showed that the expression of IL-6and IL-17 in the epidermis and dermis was very low,and the difference was not statistically significant.Conclusion:1.Staphylococcus epidermidis selectively inhibited the expression of IL-6 mRNA and the secretion of IL-6 in keratinocytes induced by Malassezia furfur,and did not inhibit IL-1?,IL-1?,TNF-?and IL-8 mRNA expression.Staphylococcus epidermidis alone has no obvious inhibitory effect on the expression of IL-1?,IL-1?,TNF-?,IL-6 and IL-8.2.The NF-?B pathway was involved in the biological process of Staphylococcus epidermidis inhibiting the IL-6 secretion of keratinocytes induced by Malassezia furfur,while JNK pathway and Ah R-CYP1A1 pathway were not involved in the regulatory process.3.Epicutaneous application of Malassezia furfur caused desquamation and thickening of the back skin of mice,accompanied by increased dermal cell infiltration.Exposure to the combination of Staphylococcus epidermidis and Malassezia furfur reduced the epidermal thickness and the number of dermis cells compared to Malassezia furfur-alone treatment.In summary,the results of this study indicated that the resident skin bacterium Staphylococcus epidermidis had a potiential inhibitory effect on skin inflammation caused by Malassezia furfur.