Expression of interleukin-9 in murine cornea with herpetic stromal keratitis

Abstract

Background Herpetic stromal keratitis (HSK) is an immunopathologic eye disorder mediated by CD4+ T lymphocytes.Interleukin-9 (IL-9) is a cytokine linked to the process of many immune inflammatory diseases,but whether IL-9 is involved in the immunopathology of HSK remains unclear.Objective This study was to investigate the expression of IL-9 in murine cornea during the development of HSK and its relationship with the degree of HSK.Methods One hundred and eighty clean BALB/c mice were divided into the normal control group (20 mice) and experimental group (160 mice).HSK models were established by scratching on the surface of the cornea followed by inoculation of 1 × 106 plague forming unit(PFU) herpes simplex virus type 1 (HSV-1) KOS strain.Change of ocular surface was examined under the slit lamp biomicroscopy before and I day,3,5,7,8,10,14,21 days after inoculation of HSV-1.The corneas of mice were collected on the time points mentioned above.The relative expression level of IL-9 mRNA in the cornea of mice was detected by real-time PCR.Immunocytochemical localization of IL-9 protein in murine cornea was viewed by a confocal microscope after preparation of the corneal cryostat sections.All experimental manipulations were undertaken in accordance with the institutional guidelines for the care and use of laboratory animals.Results Punctiform,dendriform or geographic defect in corneal epithelium were seen 3 days and healing 6 days after inoculation of HSV-1.However,edema and opacity of the corneal stroma appeared 7 days and peaked 14 days following the inoculation.Real-time PCR assay showed that very little of IL-9 mRNA (A260/A280) was expressed in the cornea in the mice of the control group.But in 1 day,3,5,7,8,10 and 14 days,the relative level of IL-9 mRNA was 6.37±0.45,5.66±0.53,3.93±0.35,3.62±0.34,3.23±0.18,2.57±0.14,2.19±0.20,with a significant difference among the various time points (F=92.764,P=0.000),and IL-9 mRNA in 1 day,3,5,7,8,10 and 14 days after inoculation was significantly higher than before inoculation (P＜0.05);while no markedly difference was found in IL-9 mRNA expression between the 21-day group after inoculation and the normal control group (P =0.340).IL-9 protein was expressed in the epithelial layer,stromal layer and endothelial cell layer of the corneas,with a stronger green fluorescence in the HSK mice,but no expression of IL-9 protein was seen in the control mice.Conclusions HSK model can be successfully created by corneal scratching and inoculation of HSV-1 KOS strain in BALB/c mouse.IL-9 is involved in the pathogenesis and development of HSK mediated by immunoresponse. Key words: Herpes simplex virus type 1 ; Cornea; Interleukin-9 ; Stromal keratitis