The roles of surfactant protein D during Aspergillus fumigatus infection in human corneal epithelial cells

AIM: To investigate roles of surfactant protein D (SP-D) and relative cytokines in human corneal epithelial(HCE) cells exposed to aspergillus fumigatus (AF) antigens.METHODS: HCE cells cultured in vitro with AF antigens and sampled at 0, 0.5, 1 hour, 2, 4, 6 and 8 hours. The expression of SP-D mRNA was evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).The expression of SP-D protein was shown by ELISA and immunocytochemistry SP methods. The expression of NF-κB and relative downstream cytokines such as TNF-α, IL-1β, IL-8 and IL-10 in supernatant fluid were measured by ELISA.RESULTS: SP-D mRNA and protein were detected in untreated HCE cells. The expression of SP-D and the relative downstream cytokines rose after being stimulated with AF antigens. SP-D mRNA began to rise at 0.5 hour and the most significantly peak was in 2 hours. The protein of SP-D in supernatant fluid had the same trend with mRNA. Immunocytochemistry of SP-D showed positive expression and gradually increased to 6 hours, and then the expression began to decline. NF-κB was activated after treated by AF antigens and the changes had correlation with SP-D. TNF-α, IL-1β, IL-8 and IL-10 began to rise after given AF antigens 1 hour and were 1.82, 1.43, 1.12 and 1.28 times higher than the untreated HCE cells separately. The expression of TNF-α and IL-1β reached the peak at 2 hours, separately 2.80 and 2.86 times than the untreated. The expression of IL-8 and IL-10 gradually increased with a time-dependent manner.CONCLUSION: HCE cells exists SP-D and it may play a significant role in pathogenesis of keratomycosis. AF may induce human corneal epithelial cells to express inflammatory cytokines via SP-D and NF-κB pathway. SP-D possibly mediates the recognition to AF mycelium.

Effects of COX-2 inhibitor NS-398 on IL-10 expression in rat fungal keratitis

·AIM:To investigate the expression of interleukin-10 (IL-10) and the effect of NS-398(COX-2 inhibitor) on the expression of IL-10 in fungal keratitis in rats,and analyze its effects on anti-fungus immunity.·METHODS:Ninety Wister rats were randomly divided into 3 groups.Group A was blank control group (10 eyes).Group B was fungal keratitis group (40 eyes).Group C was fungal keratitis group treated with NS-398 (40 eyes).PAS staining,100g/L potassium hydroxide (KOH) smear and fungal culture confirmed the successful establishment of fungal keratitis model.After the central epithelium was scraped,Fusarium solani colonies were applied and contact lens was put on the right cornea of group B and C,and plane contact lens was put on the left cornea of control eyes.Phosphate buffered saline (PBS) eyedrops were given for group B and NS-398 eyedrops for group C.The expression of IL-10 on corneas of group B and C on the 1st day,3rd days,7th days,and 14th days were detected by immunohistochemistry and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).·RESULTS:Histopathologic examination showed neutrophil infiltration and severe tissue necrosis in ulcer cornea.PAS staining confirmed the existence of hyphae and spores in the superficial layer of stroma.In the blank and control groups almost no expression of IL-10 was detected at any observing points.In group B the expression of IL-10 increased at first and decreased thereafter.Its expression also showed significant difference at any observing points (P￡?0.01).Compared with group B,the expression of IL-10 in group C showed no difference on the 1stday,decrease on the 3rd day,but a significant increase on the 7th day and 14th day.·CONCLUSION:IL-10 takes part in the occurrence and development of fungal keratitis.NS-398 can upgrade the expression of IL-10 in fungal keratitis in the later period of the ulcer.Meanwhile,pathologic observation showed a slightly corneal opacity.IL-10 may play an important role in the process of cornea anti-damage repair.·