Aspergillus fumigatus activate human corneal epithelial cells via Toll-like receptor 2 and 4

Abstract

To determine Toll-like receptors (TLR) 2 and TLR4 expression in human corneal epithelial tissue and cell line (THCE), and its activation by aspergillus fumigatus (AF) in inflammatory response. The expression of TLR2 and TLR4 protein in human corneal epithelial tissue and THCE was detected by Western blot and immunocytochemistry. THCE was challenged with AF mycelium fragment (5 x 10(6)/ml) and supernatant extract agent (equivalent to bovine serum albumin 10 microg/ml). IL-8 and TNF-alpha in THCE supernatant were detected by ELISA at 1, 2, 4 and 8 h post stimulation. The protein of IkappaBalpha in THCE cells was assayed by Western blot at 30 min, 1 h and 2 h after treatment. Antibody blocking test was utilized to evaluate the effect on IL-8 and TNF-alpha expression of THCE by blocking TLR2 and (or) TLR4 before challenge with AF agent. TLR2 and TLR4 protein were expressed in human corneal epithelial tissue and THCE. The IL-8 and TNF-alpha level in THCE supernatant was elevated at 1 h, increased to (64.71 +/- 5.15) pg/ml and (32.46 +/- 3.28) pg/ml (AF mycelium challenge group), (94.94 +/- 11.92) pg/ml and (48.70 +/- 3.32) pg/ml (AF supernatant challenge group) 8 h post-challenged, which was 3.0 times and 2.5 times, 4.5 times and 3.5 times to that of control group respectively (P < 0.01). The activity of IkappaBalpha in THCE cells was decreased to 10.31 +/- 1.30 (gray scale value) and 8.15 +/- 2.37 at 30 min after challenged with AF mycelium or supernatant extract agent compared to 51.57 +/- 5.58 and 49.23 +/- 3.49 of control group (P < 0.01), and was reverted at 2 h. The secretion of IL-8 and TNF-alpha was partly inhibited by blocking TLR2 or TLR4 (P < 0.05), obviously inhibited by blocking TLR2 and TLR4 (50% and 40% compared to that of control group) (P < 0.01) when challenged with AF mycelium. And that was markedly inhibited by blocking TLR4 or blocking TLR2 and TLR4 when challenged with AF supernatant (P < 0.01). The secretion of IL-8 and TNF-alpha was not inhibited by blocking TLR2 when challenged with AF supernatant (P > 0.05). AF agent may induce human corneal epithelial cells express inflammatory cytokines via TLR-NF-kappaB pathway. TLR2 and TLR4 possibly mediate the recognition to AF mycelium, and TLR4 may dominate the recognition to AF supernatant agent.