Pretreatment With TLR2and TLR4Ligand Modulates Innate Immunity In Corneal Fibroblasts Challenged With Aspergillus Fumigatus

Background:Fungal keratitis is a kind of infective corneal disease caused high-rate of blindness. China is an area with a high incidence of the disease. Relevant clinical epidemiology investigation showed that the incidence of fungal keratitis was rising year by year, therefore, prevention and treatment of fungal keratitis has become an important clinical subject of eye disease caused blindness in China.One of the important reasons of blindness caused by fungal keratitis is excessive inflammatory reaction in innate immunity to fungal infection. TLRs play an important role in immune response of cornea to fungi. TLR2recognizes its ligand as zymosan and TLR4recognizes lipopolysaccharide. Studies showed that pretreatment with low concentration of TLRs ligand induced low-reaction to the "deadly" stimulation and low-secretion of inflammatory cytokines of host, the phenomenon was called "TLRs tolerance". Reports showed that TLRs pretreatment inhibited excessive inflammatory reaction of PMNs. Studies about tolerance induced with with TLRs ligand pretreatment in corneal fibroblasts challenged with Aspergillus fumigatus were few, even less about synergy of different TLRs in tolerance.Purpose:To study the innate immunity in telomerase-immortalized human stroma fibroblasts (THSFs) challenged with Aspergillus fumigatus hyphae after co-pretreatment with Zymosan(TLR2special ligand) and LPS(TLR4special ligand). Methods:THSFs were cultured in6-well plates at a density of4×105cells per well, and pretreated with different concentration of zymosan(104ng/mh103ng/m、100ng/ml、10ng/ml) for4hours, then challenged with high dose of Aspergillus fumigatus hyphae4hours. The expression of TNF-a and IL-6was detected by RT-PCR. THSFs were pretreated with the best concentration of zymosan and/or lipopolysaccharide (LPS) at different time periods (6h,12h,18h,24h or30h) and then re-stimulated with A. fumigatus hyphae. The expression of inflammatory cytokines (TNF-a, IL-6and IL-8) detected by RT-PCR and ELISA. Co-pretreated THSFs with the differrnt concentration of zymosan and LPS (103ng/ml Zymosan+10ng/ml LPS、100ng/ml Zymosan+10ng/ml LPS) for various periods (6h,12h,18h,24h and30h), then stimulated with A. Fumigatus hyphae for4h. The culture media and cells were harvested at the indicated time-points for measurement of cytokines (TNF-a, IL-6and IL-8) mRNA and protein levels. Statistical analysis was determined by one-way analysis of variance (ANOVA) and Student’s t test using SPSS (version13.0). Differences were considered statistically significant at P<0.05.Results:1. Pretreatment with10000ng/ml Zymosan led to maximum suppression. In the certain concentration range, pretreatment of THSFs with Zym suppressed gene expression of inflammatory cytokines (TNF-a and IL-6).2. Co-pretreatment with10000ng/ml Zym and10ng/ml LPS suppressed gene expression and protein secretion more strongly compared with pretreatment with zymosan or LPS alone.Conclusions:Pretreatment of THSFs with TLR2specific ligand, zymosan resulted in a state of A. fumigatus hyphae tolerance. Co-pretreatment with TLR2and4ligands (zymosan and LPS) led to a more strongly state of A. fumigatus hyphae tolerance.