Experimental Studies On The Relationship Between The Toll-like Receptors And The Innate Immune Of The Cornea

PART 1Modulation of Expression and Function of Toll-like Receptors in Human Corneal Epithelial Cells and Corneal Fibroblast by GlucocorticoidsPurpose: To evaluate the effect of glucocorticoids on the expression and function of Toll-like receptors (TLRs) in human corneal epithelial cells and human corneal fibroblasts (HCF).Methods: Three different concentrate of hydrocortisone were used to stimulate the cultured immortalized human corneal epithelial cell line (HCEC) and HCF. The cellular abundance of the mRNAs for TLR1-10 was determined by reverse transcription and real-time polymerase chain reaction analysis. The releases of IL-6 and IL-8 from cultured HCEC and HCF were measured with enzyme-linked immunosorbent assays (EIA). The proteins of TLR2, 3, 4 and 9 were also comparedby immunofluorescence staining and western blot.Results: TLR1-10 mRNA was expressed in both HCEC and HCF, but the expression existed diversity. In HCEC the hydrocortisone enhanced most of the TLR mRNA expression, especially in TLR2, 3, 4 and 9. The protein expression of TLR2, 3, 4 and 9 in hydrocortisone treated HCEC was also higher than untreated cells. EIA showed the concentration of IL-6 was increased in hydrocortisone treated HCEC in a dose-dependent manner. But in HCF the expression of TLRs were markedly inhibited by hydrocortisone in both mRNA and protein level. And the concentration of IL-6 and IL-8 was decreased in hydrocortisone treated HCF in a dose-dependent manner.Conclusions: TLRs expressed diversely in HCEC and HCF, and these expressions can be modulated differently by hydrocortisone suggest the two cells may take different role in the cornea innate immune and cornea inflammation. Hydrocortisone enhance the expression and function of TLRs in HCEC provides evidence for a novel function of glucocorticoids in innate immune and indicate that use of topical glucocorticoids may enhance the innate immune of corneal epithelium and benefit certain cornea infections.PART 2Toll-like Receptor (TLR) Expression and Function in Response to Inactivate Hyphae of Fusarium Solani in Human Corneal Epithelial Cell LinePurpose: To evaluate the role of Toll-like receptors (TLRs) in the host responses to Fusarium solani in immortalized human corneal epithelial cells and determine whether exposure to the inactive hyphal fragments induces an antifungal response in these cells.Methods: Inactive hyphal fragments from Fusarium solani was used to stimulate the cultured Immortalized Human Corneal Epithelial Cells (HCEC). The cellular abundance of the mRNAs for TLR1-10 was determined by real-time PCR. Then hydrocortisone was used to co-culture with hyphal fragment in HCEC to investigate whether hydrocortisone can modulate TLR mRNA expression in HCEC that is induced by hyphal fragments. The release of IL-6 and IL-8 from cultured HCEC with or without stimulation by hyphal fragments was measured with enzyme-linked immunosorbent assays (EIA). We also block the TLR2 and TLR4 with specific monoclonal antibodies to observe the release of IL-6 and IL-8 in HCEC under hyphal fragments treatment. The proteins expression of TLR2 and TLR4 were also evaluated by immunofluorescence staining and western blot.Results: Incubation of HCEC with hyphal fragments from Fusarium solani for 48h increased the expression of TLR2, 3, 4 and 6 mRNAs and the release of IL-6 and IL-8. Immunofluorescence staining and western blotting indicated that hyphal fragments also enhanced the expression of TLR2 and TLR4 in protein level. The expression of TLR2 and TLR4 in protein level which induced by hyphal fragment can be further enhanced by hydrocortisone. EIA showed the concentration of IL-6 was increased after hydrocortisone was used to co-culture with hyphal fragment in HCEC, however the release of IL-8 was inhibited. The release of IL-6 and IL-8 can also be inhibited by anti-TLR2 and anti-TLR4 monoclonal antibody.Conclusions: HCEC is involved in the cornea immune response to fungal infections. TLR2 and TLR4 may play the crucial role in signaling of Fusarium hyphae in HCEC. The glucocorticoids enhance the expression of the TLRs on the epithelium may benefit for the anti-fungal infection of the epithelium. These findings may provide crucial information for understanding the immune mechanisms of fungal keratitis, and provide hope for designing new immune therapeutical approaches to fungal keratitis.PART 3 Toll-like Receptor (TLR) Expression and Function in Response to Inactivate Hyphae of Fusarium Solani in Human Corneal FibroblastPurpose: To evaluate the role of TLRs in the host response to Fusarium solani in human corneal fibroblast (HCF).Methods: The deactive hyphal fragments from Fusarium solani were used to stimulate the cultured HCF. The cellular abundance of the mRNAs for TLR1-10 was determined by Real-time polymerase chain reaction analysis. Next Three different concentration of hydrocortisone were used to co culture with hyphal fragment in HCF to investigate whether hydrocortisone can modulation of TLR mRNA expression in HCF by hyphal fragments. The releases of IL-6 and 8 from cultured HCF with or without stimulation were measured with enzyme-linked immunosorbent assays (ELISA). We also block the TLR2 and TLR4 with specific monoclonal antibodies (mAbs) to observe the release of IL-6 and IL-8 in HCF under hyphal fragments treatment. The proteins of TLR2 and 4 were also evaluated by immunofluorescence staining.Results: Incubation of HCF with hyphal fragments from Fusarium solani for 48h increased the expression of TLRs mRNAs (especially in TLR2 and TLR4) and the release of IL-6 and IL-8. And this increase can be downregulated by hydrocortisone. The release of IL-6 and IL-8 can also be inhibited by anti-TLR2 and anti-TLR4 monoclonal antibody. Immunofluorescence staining indicated that hyphal fragments may enhance the expression of TLR2 and TLR4 in protein level.Conclusions: HCF is involved in the cornea response to invasive fungal infections. TLR2 and TLR4 may play crucial role in signaling of Fusarium hyphae in HCF. Hydrocortisone may enhance the invasive abilities of fungi through decrease the expression of TLRs. These findings may provide crucial information for understanding the immune mechanisms of fungal keratitis, and provide hope for designing new immune therapeutical approaches to fungal keratitis.PART 4 Expression of Toll-like Receptors in the Healthy and HSV Infected CorneaPurpose: To investigate the expression of toll-like receptors (TLRs) in healthy cornea and the cornea with active or non-active herpetic stromal keratitis (HSK).Methods: Eight corneas with HSK (included 5 active and 3 non-active cases) and 8 healthy corneas were evaluated for TLR1-10 mRNA by reverse transcribed polymerase chain reaction (RT-PCR) and relative real-time PCR. The proteins of TLR2, 9 in three different groups were also compared by immunofluorescence staining. The data were analyzed with SPSS11.5.Results: TLR1-10 mRNA was expressed in both healthy and HSK corneas. In the healthy cornea, the mRNA expression of TLR1, 2, 3, 4 and 6 was higher; while the expression of TLR7, 8 and 9 was lower relative to TLR 5 and 10. All the TLRs mRNA expression in the active HSK corneas were upregulated, especially TLR4, 7, 8 and 9 compared with the healthy corneas. But in non-active HSK corneas, only TLR7 mRNA expression upregulated while the others downregulated. The immunofluorescence staining showed the expression of TLR 9 was slight stronger in the cornea with active HSK than that of non-active and healthy cornea.Conclusions: TLRs are expressed diversely in the healthy cornea and could have an important role in the cornea innate immunity. TLR4, 8 and 9 may be implicated in the pathogenesis of active HSV infection in the cornea, while TLR7 may play a key role on HSK whether it is active or not.