Pretreatment With Lipopolysaccharide Modulates Innate Immunity In Cornea

[Background] Fungal keratitis is a potentially vision-threatening disease that requires prompt diagnosis and treatment to prevent vision loss. Recently, there has been a gradual increase in the number of severely infected corneal ulcers. It is estimated that nearly half of corneal ulcers may be secondary to fungal infection, and Fusarium and Aspergillus are the most common pathogens of fungal keratitis in certain parts of the world. Fungal keratitis is an opportunistic fungal infection of the cornea, which always occurs after the epithelial integrity of the cornea has been breached, exposing underlying fibroblasts. The corneal epithelium and corneal fibroblast serve as cellular barrier of the cornea and participates in the host innate immune response to invading organisms.The ability of corneal fibroblasts to recognize and respond to invading pathogens is attributed largely to the Toll-like receptor (TLR) family. Among these receptors, TLR4 is capable of sensing lipopolysaccharide (LPS), Previous studies in our and other laboratories have shown that TLR2 and TLR4 play crucial roles in the recognition of pathogen-associated molecular patterns (PAMPs) presented by Aspergillus fumigatus (A. fumigatus). Activation of TLRs induces the production of proinflammatory cytokines and chemokines that recruit polymorphonuclear leukocytes (PMN) to the corneal stroma as well as antimicrobial molecules that kill the invading pathogens.Recent studies showed that pretreatment with a TLR ligand could induce cell reprogramming with decreased production of proinflammatory cytokines in response to subsequent immune challenges.[Objective] We tested the hypothesis that LPS can induce cell reprogramming of telomerase-immortalized human stroma fibroblasts (THSF).[Methods] THSF or THCE were challenged with a TLR4 ligand, LPS, for various periods. At the end of the culture period, the culture media were collected for the measurement of cytokines IL-6, IL-8 and TNF-a by ELISA. In separate experiments, THSF were pretreated with low-dose LPS for various times and then challenged with a high dose of LPS or A. fumigatus hypha for 4h. The culture media were harvested for the measurement of cytokines and antimicrobial peptides by ELISA. PMN migration was assayed using 24-well Transwell filters. Following the second stimulation for 1h, the cells were harvested for measurement of cytokines mRNA and antimicrobial peptides mRNA.[Results]1. Effect of LPS on cytokine release in corneaLPS did not enhance the release of TNF-a in THSF, but induced IL-8 secretion in a dose-dependent manner. A time-course study showed that LPS had no apparent effect on the release of TNF-a in THSF. However, LPS induced the release of IL-8 in a time-dependent manner. LPS did not enhance the release of IL-8 in THCE, but A. fumigatus did.2. Effect of LPS pretreatment on the production of proinflammatory cytokine and chemokine induced by LPS or A. fumigatusPretreatment of THSF with low-dose LPS resulted in diminished production of cytokines upon subsequent LPS or A. fumigatus challenge.3. Effect of LPS pretreatment on the expression of antimicrobial genes induced by LPS or A. fumigatus in THSFPretreatment of THSF with low-dose LPS resulted in elevated expression of antimicrobial peptides (CCL20 and Tβ4) upon subsequent A. fumigatus challenge. Pretreatment of THSF with low-dose LPS resulted in elevated expression of antimicrobial peptides CCL20 but not LL-37, upon subsequent LPS challenge.4. Effect of LPS pretreatment on PMN migrationFurthermore, we used 24-well Transwell inserts to test whether LPS pretreatment had an effect on the migration of PMN. Besides, the results showed that the cell supernatant from THSF pretreated with LPS elicited less migrated PMN than did the non-pretreated controls did.[Conclusion] We observed that pretreatment of THSF with LPS resulted in diminished production of cytokines, elevated expression of antimicrobial peptides and suppression of PMN migration upon subsequent LPS or A. fumigatus challenge, indicating that LPS induces cell reprogramming. Therefore, LPS pretreatment may induce protective mechanisms that modulate the host inflammatory response and provide an innate defense against fungal infection in the cornea.