title: Toll-like receptors (TLRs) expression and function in response to inactivate hyphae of Fusarium solani in immortalized human corneal epithelial cells.
abstract: OBJECTIVE
  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.
authors.affiliation: Eye Center, Affiliated Second Hospital, School of Medecine, Zhejiang University, Hangzhou, China.
authors.forename: Xiuming
authors.initials: X
authors.lastname: Jin
authors.forename: Qin
authors.initials: Q
authors.lastname: Qin
authors.forename: Lili
authors.initials: L
authors.lastname: Tu
authors.forename: Xiangtian
authors.initials: X
authors.lastname: Zhou
authors.forename: Yi
authors.initials: Y
authors.lastname: Lin
authors.forename: Jia
authors.initials: J
authors.lastname: Qu
mesh_terms: D015153:Blotting, Western; D002461:Cell Line, Transformed; D003199:Computer Systems; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005670:Fusarium / Q000276:immunology*; D054884:Host-Pathogen Interactions / Q000276:immunology*; D006801:Humans; D006854:Hydrocortisone / Q000494:pharmacology; D025301:Hyphae / Q000276:immunology*; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D051195:Toll-Like Receptor 2 / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D051193:Toll-Like Receptors / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D015850:Interleukin-6; D016209:Interleukin-8; D012333:RNA, Messenger; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D051193:Toll-Like Receptors; D006854:Hydrocortisone