Abstract: Ni Chunya, Gao Lujuan, Chen Lianjun, Dou Xia. Department of Dermatology, Huashan Hospital, Fudan University, Shanghai 200040, China Corresponding author: Dou Xia, Email: douxia@medmail.com.cn 【Abstract】 Objective To evaluate the in vitro effect of the house dust mite allergen rDer p1 on the production of thymic stromal lymphopoietin （TSLP） by human keratinocytes. Methods Human HaCaT keratinocytes were cultured in the presence of different concentrations （0.1, 1 and 10 mg/L） of rDer p1, SLGIKV （a specific agonist of protease-activated receptor-2, 500 μmol/L） and VKGILS （a specific antagonist of protease-activated receptor-2, 500 μmol/L） alone or in combination for different durations. Subsequently, enzyme-linked immunosorbent assay （ELISA） and fluorescence-based quantitative PCR were performed to detect the expression levels of TSLP protein in the culture supernatant of and TSLP mRNA in these cells respectively, confocal laser scanning microscopy was used to visualize intracellular calcium influx which reflected the activation of protease-activated receptor-2 （PAR2）. Those HaCaT cells cultured in serum-free medium served as the blank control group. Statistical analysis was done by t test and one-way analysis of variance. Results The level of TSLP protein was （155.5 ± 5.9） ng/L, （228.8 ± 28.7） ng/L, and （166.2 ± 8.8） ng/L in the culture supernatant of keratinocytes after 12-hour treatment with rDer p1 of 1 mg/L and 10 mg/L as well as SLGIKV respectively, significantly higher than that in the blank control group （（54.3 ± 13.9） ng/L, all P < 0.01）. The TSLP mRNA expression in keratinocytes peaked at 8 hours after the stimulation with rDer p1 of 10 mg/L and SLGIKV separately, with the relative expression levels being （3.28 ± 0.27） folds and （2.15 ± 0.26） folds, respectively, which were significantly different from those at 4 and 24 hours （all P < 0.01）. Both SLGIKV and rDer p1 of 10 mg/L promoted intracellular calcium influx in keratinocytes, while the pretreatment with VKGILS obviously inhibited the rDer p1-induced increase in intracellular calcium influx. Conclusions The house dust mite allergen rDer p1 may induce the production of the pro-inflammatory cytokine TSLP by human keratinocytes partly via activating PAR2 receptors.

Key words: Dermatitis, atopic, Dermatophagoides pteronyssinus, Keratinocytes, Thymic stromal lymphopoietin