The Role Of IDO In The Fungal Immune Tolerance Of Fungal Keratitis And Its Regulatory Mechanisms

Objective: To explore the expression and role of Indoleamine 2,3-dioxygenase in mediating immune tolerance and determine if pattern recognition receptor regulate Indoleamine 2,3-dioxygenase in fungal keratitis.Methods: Three models were used for this study.Based on this,this study was divided into three parts: 1.31 cases of patients with fungal keratitis at the Department of Ophthalmology(The Affiliated Hospital of Qingdao University)from January 2012 to December 2014 were included.A total of 31 patients were divided into three groups according to keratomycosis severity,which were scored visually with aid of a slit lamp.Corneal epithelial scrapings were collected,and the m RNA was analyzed using q RT-PCR.Controls were 6 normal corneal tissues remaining after corneal transplantation.By using immunofluorescence staining,the localization and expression of IDO were examined in corneal tissues of patients with fungal keratitis.Besides,we further investigated IDO expression in 31 cases of fungal keratitis versus 6 normal human corneas.We used immunofluorescence staining,q RT-PCR and Western blot to detect the localization and expression of IDO in the murine models with fungal keratitis.2.To explore the effect of A.fumigatus infection on IDO expressions in THCECs,we treated the cells with inactivated A.fumigatus spores and detected expressions of IDO by q RT-PCR.In order to select the optimal concentration,we stimulated THCECs with three different concentrations(5 × 106 /ml,5 × 107 /ml and 5 × 108 /ml)in 12-well plates.Moreover,q RT-PCR and ELISA were used to detect IDO and inflammatory cytokine after pretreatment of 1-MT.Furthermore,healthy C57BL/6 mice were randomly divided into control group(The mice were pretreated with PBS,central corneal epithelium were scraped about 2 mm in diameter,and the corneas were covered by contact lens),infection group(Central corneal epithelium were scraped about 2 mm in diameter,and the corneas were innoculated with fungi and covered by the cornea contact lens)and 1-MT +infection group(1-MT,as IDO inhibitors,was pretreated and the central corneal epithelium were scraped.The corneas were inoculated by fungi and covered by the contact lens).Disease score was evaluated with the scoring system.The corneas collected were used for q RT-PCR,immunofluorescence and flow cytometry in order to compare the disease score,detect expression and location of IDO,the effect on inflammatory cytokine,and the balance between Th17 and Treg in the cornea,lymph gland,spleen before and after IDO inhibition.3.THCECs were incubated by a variety of extracted or synthetic microbial components,representing the ligands to Dectin-1,TLR2,TLR4 respectively,and the m RNA expression of IDO was detected by q RT-PCR.The q RT-PCR ? ELISA and Western blot were used to test the effect of Dectin-1 on the expression of IDO and inflammatory cytokines in the THCECs incubated with A.fumigates which were pretreated with 1-MT,agonist and inhibitor of Dectin-1.Results: 1.(1)In the patients with fungal keratitis,IDO-positive cells in infected corneal tissues were detected with strong green fluorescence.No immunoreactivity of IDO was detected in epithelial cells of healthy corneal tissue.(2)The q RT-PCR results showed that,compared with normal cornea,IDO m RNA expression was significantly increased in human corneal epithelium with fungal infection.Moreover,the expressions of IDO correlated with the severity of keratomycosis.Transcript level measured by q RT-PCR was consistent with IDO protein expression determined by immunofluorescent staining.(3)A.fumigatus infection increased IDO m RNA expression in the corneal epithelium at 1day and began to decline at 3 days.Besides,the results of Western blot illustrated that IDO protein began to elevate in the corneas with A.fumigatus keratitis at 1 day after infection,continued to increase at 3 days and declined at 5 days.2.(1)In THCECs incubated with A.fumigatus,inactivated A.fumigatus spores could up-regulate the expressions of IDO,which presented the change trend of concentration dependence.The q RT-PCR results indicated that the expressions of IL-1 ? and IL-6 increased in comparision with the control group.Blockage of IDO by 1-MT further increased the production of these cytokines in comparison with the production in the A.fumigatus infection group.Meanwhile,the concentration of IL-1?and IL-6 in the supernatant was also up-regulated,which was consistent with the change of IDO m RNA levels.(2)In the murine models of fungal keratitis,the disease scores indicated that infection aggravated in the murine models pretreated with 1-MT in comparision with AF group.The q RT-PCR and ELISA results indicated IDO and inflammatory cytokine IL-1?,IL-6 increased in the cornea in comparison with the AF group.After pretreatment with 1-MT,corneal IL-17 and IL-23 m RNA was significantly increased while IL-10 and TGF-?m RNA was significantly decreased in comparison with the AF group.The results of flow cytometry displayed 1-MT induced the Th17 cells and inhibited Treg cells in comparison with the AF group.Differences between the two groups were statistically significant.3.(1)The m RNA expression of IDO in untreated THCECs was at a relatively low level,but was largely induced after exposure to A.fumigatus and curdlan which was the ligands to Dectin-1.IDO expression was not significantly induced by the TLR2 ligand Pam3csk4 and TLR4 ligands LPS.(2)Curdlan,the Dectin-1 ligand,significantly enhanced A.fumigatus-induced up-regulation of IDO.Further studies by Western blot also showed that IDO expression was up-regulated by A.fumigatus stimulation.This effect was enhanced by pre-treatment with curdlan while partly inhibited by laminarin.Conclusion: 1.IDO and downstream inflammatory cytokines play an important role in the immune responses of fungal keratitis.2.The activation of Dectin-1 may contribute to A.fumigatus spores-induced up-regulation of IDO,which can active Treg cells and inhibit Th17 resulting in immune tolerance and chronic inflammation.3.Regulation ofIDO can affect the inflammation response induced by A.fumigates.These novel findings shed light on the understanding of immune tolerance involved in fungal keratitis and might create new therapeutic targets to cure fungal keratitis.