Toll-like receptors (TLRs) expression and function in response to inactivate hyphae of Fusarium solani in immortalized human corneal epithelial cells - PubMed (original) (raw)

. 2007 Oct 17:13:1953-61.

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Toll-like receptors (TLRs) expression and function in response to inactivate hyphae of Fusarium solani in immortalized human corneal epithelial cells

Xiuming Jin et al. Mol Vis. 2007.

Abstract

Purpose: To evaluate the role of toll-like receptors (TLRs) in host responses to Fusarium solani by the use of cultured immortalized human corneal epithelial cells (HCEC) and to determine whether inactive hyphal fragments can induce an antifungal response in these cells.

Methods: Cultured HCEC cells were stimulated with inactive hyphal fragments from Fusarium solani, and the effect on expression of TLRs was determined by real-time polymerase chain reaction (PCR), immunofluorescence, and western blot analysis. Cells were also cocultured with hyphal fragment and hydrocortisone to determine whether hydrocortisone modulates the transcription of TLRs. The release of interleukin-6 (IL-6) and IL-8 was also measured using enzyme-linked immunosorbent assays (ELISA) in the presence and absence of specific blocking antibodies to TLR2 and TLR4.

Results: Incubation of HCECs with inactive hyphal fragments upregulated the expression of TLR2, 3, 4, and 6 mRNAs and increased the release of IL-6 and IL-8. Immunofluorescence staining and western blot analysis confirmed that expression of TLR2 and TLR4 was upregulated in response to hyphal fragments. This upregulation was further enhanced by cotreatment with hydrocortisone. Results from ELISA assays showed that the concentration of IL-6 was increased, and the concentration of IL-8 was decreased in supernatants of HCECs after treatment with both hydrocortisone and hyphal fragments. The release of IL-6 and IL-8 was also inhibited by incubation with anti-TLR2 and anti-TLR4 monoclonal antibodies.

Conclusions: HCECs are involved in the cornea immune response to fungal infections. TLR2 and TLR4 may play a crucial signaling role in response to Fusarium hyphae in HCECs. Glucocorticoids such as hydrocortisone can enhance the expression of the TLRs on the epithelium and thus may enhance the resistance to fungal infections in the cornea. These findings may provide crucial information for understanding the immune mechanisms of fungal keratitis and promote the design of new immune therapeutical approaches to fungal keratitis.

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Figures

Figure 1

Figure 1

Morphology and size of the killed hyphal fragments

Figure 2

Figure 2

Real time PCR shows the relative expression of TLR1–10 mRNA

Figure 3

Figure 3

Toll-like receptors 1–10 mRNA expression in hyphal fragment-treated human corneal epithelial cells compared with untreated human corneal epithelial cells

Figure 4

Figure 4

Toll-like receptors 1–10 mRNA expression in hydrocortisone treated human corneal epithelial cells compared with untreated human corneal epithelial cells

Figure 5

Figure 5

Toll-like receptors 1–10 mRNA expression in hydrocortisone and hyphal fragments-treated human corneal epithelial cells compared with levels in human corneal epithelial cells treated with hyphal fragments alone

Figure 6

Figure 6

The hyphae fragments-stimulated cells treated with anti-TLR2 and anti-TLR4 antibodies and stained by Cy3 and 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride

Figure 7

Figure 7

Western blot analyses detect the expression of TLR2 and TLR4 proteins in human corneal epithelial cells following different stimulation

Figure 8

Figure 8

The results of ELISA showed the release of IL-6 and IL-8 from HCEC under different stimulation

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