Abstract
Purpose: :
To evaluate the function of separate or combined pretreatment with Toll-Like Receptor (TLR)2 and TLR4 agonists in regulating corneal immune responses to Aspergillus fumigatus (Af) infection and the underlying mechanisms in vitro.
Methods: :
Telomerase-immortalized human stroma fibroblasts (THSFs) and telomerase-immortalized human corneal epithelial cells (THCEs) were pretreated with low-dose TLR4 agonist lipopolysaccharide (LPS) or TLR2 agonist Zymosan (Zym) respectively for various times and then challenged with a high dose of Af. At various time points postinfection, cell-free supernatants were assayed for IL-6 and TNF-α by ELISA. Cytokines (IL-6, TNF-α), antimicrobial peptides (CCL20, Tβ4), TLR4, TLR2, MyD88, IΚBα, MAPK3, AP-1 and TRIF gene-expression levels were assayed by real time RT-PCR. Protein expressions of p-Erk and IΚB-α were determined by Western blot. Polymorphonuclear leukocyte (PMN) migration was assayed using 24-well Transwell filters. In the combined pretreatment, THSFs were pretreated with Zym and LPS and were then treated with Af. Cytokines production was examined by ELISA and RT-PCR.
Results: :
Pretreatment of THCEs with low-dose Zym and pretreatment of THSFs with low-dose LPS resulted in diminished production of TNFα, IL6, TLR, MyD88, MAPK3, AP-1 and p-Erk, elevated expression of CCL20, TRIF and IΚBα, and suppression of PMN migration upon subsequent Af challenge. Pretreatment of THSFs with low-dose Zym combined with LPS significantly inhibited the release of inflammatory cytokines in response to Af challenge vs control or LPS/Zym alone group.
Conclusions: :
TLR4 and TLR2 agonists pretreatment could induce corneal cellular reprogramming and reduce fungal inflammation. Combined pretreatment of THSFs with TLR4 and TLR2 agonists enhances inhibitory effects of reprogramming on inflammation. The downregulation of TLR and MyD88, impaired NF-ΚB and MAPK3 pathway activity and upregulation of TRIF may play significant roles in mediating corneal antifungal reprogramming.
Keywords: immune tolerance/privilege • inflammation • cornea: basic science