TSLPR/STAT5 signaling modulates innate immunity in corneal fibroblasts triggered by Aspergillus fumigatus

Abstract

To study the primary role of TSLPR/STAT5 signaling in inflammatory responses triggered by Aspergillus fumigatus(AF) in telomerase-immortalized human stromal fibroblasts (THSF). Experimental study. Baseline expression of TSLPR in THSF was assessed by immunofluorescence analysis. Human recombinant TSLP was added. At 5, 15, 30 and 60 min after incubation, cells were harvested for Western blot to assess the protein levels of p-STAT5 and STAT5. After stimulated with AF hyphae for 1, 3, 6, 12, 24 and 48 h, cells were collected for measurement of mRNA of TSLPR and IL-7Rα. After incubated with AF hyphae for 12, 24 and 48 h, cells were harvested for Western blot to assess the protein levels of TSLPR, p-STAT5 and STAT5. Incubation with anti-TSLPR antibody was performed for 4 h, and at 24 h after AF hyphae were added, cells were harvested to assess the protein levels of p-STAT5 and STAT5. Immunofluorescence staining evidenced that expression of TSLPR was visualized in THSF. Western blot assay showed that p-STAT5 protein was increased and peaked at 30 min after stimulation with hTSLP (AF group: 8.87±0.75; control group: 1.00±0.14; P<0.01). RT-PCR revealed that the expression levels of TSLPR mRNA were increased after incubation with AF hyphae for 3, 6, 12 and 24 h (AF group: 0.000 50±0.000 07, 0.001 20±0.000 11, 0.002 30±0.000 25 and 0.001 70±0.000 17; control group: 0.000 20±0.000 03, 0.000 20±0.000 05, 0.000 20±0.000 03 and 0.000 20±0.000 04; t=-9.955, -17.329, -16.735 and -18.214, P<0.01), but the expression levels of IL-7Rα mRNA were not increased significantly (t=-0.684,-0.029,-0.319,-1.034, P>0.05). In comparison with the control group, after being challenged with AF hyphae for 24 h, both TSLPR and p-STAT5 protein were increased significantly (p-STAT5: 9.46±2.08 vs. 1.00±0.06; TSLPR: 1.80±0.27 vs. 1.00±0.34; t=-7.055, -3.170, P<0.01). Western blot showed that the elevated p-STAT5 expression levels observed after AF hyphae stimulation can be inhibited by TSLPR antibody (anti-TSLPR-AF: 0.55 ± 0.20; CTR Ab-AF: 1.00 ± 0.08; t=3.506, P<0.05). TSLP/TSLPR/STAT5 signaling pathway plays an important role in inflammatory responses triggered by AF in THSF.