TLRs Signaling In The Pathogenesis Of Aspergillus Fumigatus Keratitis

Part one: TLR2 and TLR4 signaling in immune responses against Aspergillusfumigatus in human corneal epithelial cellsPurpose.Cornea epithelial cells play an early and crucial role in the initiation of ocular surface responses to pathogens.Participation of Toll-like receptor(TLR)2 and TLR4, which are major forms of fungi receptors,may be involved in the Aspergillus fumigatus induced immune responses.The objective of the present study was to examine whether Aspergillus fumigatus antigens induce NF-κB activation and production of proinflammation cytokines,and whether the expression of Toll-like receptor(TLR)2 and TLR4 was amplified by Aspergillus fumigatus antigens in cultured immortalized human corneal epithelial cells(THCEs),which may contribute to our knowledge of the mechanism by which the host comea can successfully defend against invasive fungi.Methods.THCEs were stimulated with inactive antigens from Aspergillus fumigatus.The expressions of TLR2 and TLR4 were determined by Real-Time Quantitative PCR, immunofluorescence,and western blot.The activation of pIκB was determined by western blot in the presence or absence of TLR2 or TLR4 antibody.The concentration of interleukin(IL)-1β,IL-6,TNF-αand IL-8 in the cell supernatant were also assessed by enzyme-linked immunosorbent assays(ELISA)in the presence or absence of TLRs antibodies or NF-κB inhibitor.Results.TLR2 and TLR4 were expressed in THCEs.Aspergillus fumigatus antigens induced the activation of corneal epithelial cells as characterized by TLR2 and TLR4 expression,activation of pIκB,and upregulation of IL-1β,IL-6,TNF-αand IL-8.The Aspergillus fumigatus antigens -induced NF-κB activation was blocked by TLR2 and TLR4 antibodies.In addition,the Aspergillus fumigatus antigens-induced production of IL-1β, IL-6,TNF-αand IL-8 in supernatants of corneal epithelial cells was also attenuated by NF-κB inhibitor.Conclusions.Aspergillus fumigatus antigens contribute to the inflammatory responses of corneal epithelium in a TLR2 and TLR4-NF-κB signaling pathway-dependent manner.Part two: Activation of Toll-Like Receptor 2 and 4 in a rat Model of Aspergillusfumigatus KeratitisPurpose.To establish,in the Wistar rat eye,a new model of Aspergillus fumigatus keratitis suitable for studies of pathogenesis and TLRs signaling pathways. Methods.Corneas of 280 rats were infected with spores of Aspergillus fumigatus. Aspergillus fumigatus spores were intrastromally injected into rat corneas(1×10~8 CFU per cornea).Corneas injected with sterile physiological saline served as controls.Rats underwent slit lamp examination(SLE)at 1,3,5,7,and 11 days post infection and were killed.Histopathologic analyses,quantitative fungal cultures,and myeloperoxidase(MPO) activity assays were performed at each time point.Corneal TLR2 and TLR4 levels were tested by real time quantitative PCR and western blot,IL-1βand IL-10 levels were measured by ELISA at each time point.Results.Aspergillus fumigatus keratitis developed in Wistar rat,as evidenced by high SLE scores and viable fungi per infected eye than in control rats at 1,3,5,7,and 11 days after infection,and the severity of which peaked on day 5 after infection.Histopathologic analysis and MPO assays of infected rat both revealed an influx of polymorphonuclear leukocytes(PMNs).Production of IL-1βand IL-10,which mediate neutrophil recruitment to the corneal stroma,was elevated in the corneal epithelium and stroma of infected corneas. Rats injected with Aspergillus fumigatus exhibited an increase in corneal mRNA and protein for TLR2 and TLR4 over controls.Conclusions.These studies demonstrate the establishment of Aspergillus fumigatus keratitis in the rat eye.These findings indicate that the cornea has functional TLR2 and TLR4,and activation of TLR2 and TLR4 through NF-κB may contribute to pathogenesis of keratomycosis.Part three: Expression of TLR2 and TLR4 in the Healthy and Fungi-infected CorneaPurpose.To investigate the toll-like receptor(TLR)2,TLR4 and cytokines levels in healthy corneas and corneas with fungal keratitis(FK).Methods.37 corneas with FK and 11 healthy corneas were examined for histopathology. The expression of TLR2 and TLR4 were evaluated by quantitative reverse transcription-polymerase chain reaction(QRT-PCR),western blot and immunofluorescence staining.The secretion of IL-1βand IL-10 in the corneas was determined by ELISA.Results.Of 37 human corneal buttons with FK obtained by keratoplasty,23(62.2%) showed positive culture identification and in which 17 showed Fusarium infection,5 showed Aspergillus infection,1 showed combined fungi infection.Corneal sections from FK group revealed a large proportion of polymorphonuclear leukocytes(PMNs).TLR2 and TLR4 mRNA was expressed in both healthy and FK corneas.The mRNA expression of TLR2 and TLR4 was downregulated,whereas the expression of IL-1βand IL-10 was upregulated in the FK corneas compared with the healthy corneas.The immunofluorescence staining showed that the expression of TLR2 and TLR4 was slightly stronger in the healthy corneas than that of FK corneas.The western blot also showed that the expression of TLR2 and TLR4 was slightly stronger in the healthy cornea than that of FK corneas.Conclusions.Fusarium species and Aspergillus species were the most commonly isolated pathogens of fungal keratitis.TLR2 and TLR4,as well as Th1/Th2 type cytokines may be implicated in the pathogenesis of fungi infection in the cornea,and may play key roles in fungal keratitis.