Expression And Role Of IL-36? In Fungal Keratitis

Purpose:To determine the expression of interleukin-36 alpha(IL-36?)and its role in fungal keratitis(FK).Methods:1.The corneal epithelial tissues of patients with fungal keratitis and the peripheral corneal epithelial tissues of healthy donors after corneal transplantation were scraped to extract mRNA,and reverse transcription polymerase chain reaction(RT-PCR)was applied to evaluate IL-36?mRNA expression.Corneal specimens from patients with fungal keratitis and healthy donors were collected,and HE staining(Hematoxylin-Eosin Staining)was used to observe the pathological changes in cornea,while immunohistochemistry(IHC)was performed to detect the expression of IL-36?in human cornea.C57BL/6 mouse cornea was infected by Aspergillus fumigatus(AF)to establish in vivo fungal keratitis model.Corneal inflammation of mice was observed on the 1st,3rd,and 5~thh day post infection,and clinical scores were recorded.RT-PCR and Western Blot were used respectively to assess the mRNA and protein level of IL-36?in mice cornea.Besides,immunofluorescence(IF)was performed to further determine the expression of IL-36?in mice corneal tissues.2.Mice were subconjunctivally injected with recombinant mouse IL-36 alpha(rm IL-36?)or recombinant control IgG before infected by Aspergillus fumigatus.Corneal inflammation were observed with slit-lamp biomicroscope and inflammation clinical scores were recorded on the 3~rdd day after infection.MPO detection was applied to assess the quantity of neutrophils in mouse corneal tissues.3.Mice were divided into IgG control group,IgG+AF group and rm IL-36?+AF group.Using RT-PCR or Western Blot to detect IL-1?,IL-6,TNF-?mRNA or protein level in mice cornea on the 3~rdd day post infection.4.mouse macrophage RAW264.7 cells was stimulated by inactivated hyphae of Aspergillus fumigatus,and IL-36?mRNA and protein expression in cells were evaluated.RAW264.7 cells were treated with exogenous rmIL-36?and cells were collected to detect the level of IL-1?and IL-6 mRNA and protein.RAW264.7 cells were pretreated with rmIL-36?,then cells were stimulated with Aspergillus fumigatus inactivated hyphae,and the mRNA or protein level of IL-1?,IL-6 in cells was detected.5.RAW264.7 cells were pretreated with recombinant mouse IL-36Ra(rmIL-36Ra)to block IL-36 receptor(IL-36R),after that cells were stimulated with rmIL-36?or inactivated hyphae.mRNA and protein expression of IL-1?,IL-6 in cells was determined.Results:1.The expression of IL-36?m RNA in corneal epithelium of patients with fungal keratitis was significantly higher than that of the healthy donors.Immunohistochemistry indicated that the expression of IL-36?protein in fungal infected cornea was significantly higher than in normal cornea,and mainly located in corneal epithelial.Compared with the control group,the ulcer area and the degree of turbidity in cornea of C57BL/6 mice infected by Aspergillus fumigatus were increased,and the clinical score elevated,which reached peak at 3 days after infection.Meanwhile,the IL-36?mRNA and protein level were significantly increased in infected mice corneal tissues,and the elevation trend was consistent with the severity of corneal inflammation.Immunofluorescence images showed that the expression of IL-36?protein was significantly enhanced in infected mice cornea in contrast to the control group,mainly localized in the corneal epithelial.2.Compared with the IgG+AF group,the corneal ulcer area and turbidity in cornea of rmIL-36?+AF group were increased,same as the clinical score.Meanwhile,the MPO level in cornea were up-regulated.3.The mRNA and protein levels of IL-1?,IL-6 and TNF-?in cornea of rm IL-36?+AF group were higher than those in IgG+AF group.4.Compared with the control group,the mRNA and protein expression of IL-36?in cells stimulated by inactivated hyphae was increased.The IL-1?,IL-6 mRNA and protein level in rmIL-36?group cells was upregulated compared with that of the IgG control group,but lower than that of the IgG+AF group.In contrast to the IgG+AF group,cells of rmIL-36?+AF group presented with significantly higher IL-1?,IL-6 expression.5.Compared with the IgG+AF group,the expression of IL-1?,IL-6 was decreased in IL-36Ra+AF group;while the expression of IL-1?,IL-6 m RNA and protein was decreased in IL-36Ra+rm IL-36?group comparing to the rmIL-36?group.Conclusion:1.IL-36?might participate in the inflammatory response of fungal keratitis.2.IL-36?could promote the infiltration of neutrophils in the corneal tissue,and up-regulate the expression of IL-1?,IL-6 and TNF-?.3.IL-36?could regulate Aspergillus fumigatus induced IL-1?and IL-6 expression through IL-36R.