*P 0.05 and **P 0.01. genome-wide complementary DNA (cDNA) microarray to assess the differentially expressed genes in corneal epithelial cells after challenge, with or without flagellin pretreatment (15). Among the genes differentially regulated by flagellin pretreatment, the expression of IL-24 and its downstream unfavorable regulator, SOCS3, was notably upregulated in response to contamination and significantly suppressed by flagellin pretreatment. IL-24, along with IL-19, IL-20, IL-22, and IL-26, belongs to the IL-20 subfamily of the IL-10 superfamily of cytokines (16). IL-19, IL-20, and IL-24 (named as IL-20R cytokines here) share the IL-20R1 and IL-20R2 heterodimer receptor. IL-20 and IL-24 can also bind to the IL-22R1 and IL-20R2 receptor complex (17). After binding to their receptors, IL-20R cytokines transmission through the Janus kinase-signal transducer and activator of transcription 3 (JAK/STAT3) pathway to regulate downstream gene expression (18, 19). The SOCS family of proteins, including eight users, play an important role in the unfavorable regulation of cytokine-JAK-STAT signaling pathway (20). While SOCS1 inhibits STAT1 activation in the IFN- signaling cascade (21), SOCS3 is usually a major unfavorable regulator of the IL-6-STAT3 signaling pathway (22). SOCS3 binds to both JAK and cytokine receptors and targets them for ubiquitination and proteasomal degradation (23). SOCS3 can also directly inhibit JAK activity (24). Increased expression of IL-24 and SOCS3 has been found within inflamed tissues in various diseases, such as skin inflammation (e.g. psoriasis), inflammatory bowel disease (IBD), and rheumatoid arthritis (25C27). IL-24 exerts protective effects against intracellular pathogens, such as and (28, 29). Studies in different mouse models have proven the crucial importance of SOCS3 in restraining inflammation and promoting optimal levels of protective immunity against the infection (30). On the other hand, some pathogens have Squalamine lactate evolved to modify host SOCS3 expression to evade the immune response. For example, virus and computer virus can stimulate SOCS3 expression to suppress type I interferon (IFN) production, thereby subverting the host immune response (31, 32). To date, the functions and mechanism of IL-24 in bacterial keratitis have not been Squalamine lactate explored. Given the reported importance of IL-24 in inflammation and host protection against contamination, we hypothesized that IL-24 would enhance the host defense against keratitis. Surprisingly, we found that IL-24 increased the susceptibility of mouse cornea to contamination. Our results indicate that induces the early expression of IL-24, resulting in a suppression of early protective mucosal immune responses. Materials and methods Animals Wild-type C57BL/6 mice (8 weeks; female) were purchased from your Jackson Laboratory (Bar Harbor, ME, USA). All animal procedures were performed in compliance with the ARVO Statement for the use of animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee of Wayne State University. Mouse model of Keratitis Mice were anesthetized with an intraperitoneal injection of Ketamine (90 mg/kg) and Xylazine (10 mg/kg) before surgical procedures. Mouse corneas were scratched gently with a sterile 26-gauge needle to produce three 1-mm incisions to break the epithelial barrier. Purified flagellin (500ng in 5l PBS) was applied topically to the hurt cornea as an vision drop. PBS was used as control. Twenty-four hours later, the corneas were scratched Squalamine lactate again and inoculated with 1.0104 CFUs of in 5l PBS. Administration of siRNA, recombinant protein, or neutralizing antibody All the siRNAs used in this study were SMARTpool (a mixture of 4 siRNAs) ON-TARGETplus siRNAs designed Squalamine lactate by GE Dharmacon Organization (Lafayette, CO, USA). Mice were subconjunctivally injected twice with siRNA targeting to a specific gene (50 picomoles in 5l RNase-free water) over 2 days. Six hours after the second siRNA injection, mouse corneas were inoculated with around the corneas. Isolation of mouse corneal epithelial cells Razor knife was tailored to approximately 5 mm wide in the edge and placed in a Castroviejo razor Squalamine lactate knife breaker and holder. Mice were euthanized by cervical dislocation. Under the microscope, corneal epithelial cells were surgically scraped off from the basement membrane. Cells were collected to Rabbit polyclonal to AIP the razor knife from the basement membrane. Liquid nitrogen was used to snap-frozen the cells and cool off the tip of a sharp surgical scalper at the same time. Cells were immediately transferred into a pre-cool, 1.5 ml Eppendorf tubes placed on dry ice by scraping the razor blade with the scalper. Cells were processed for RNA isolation, protein extraction or stored at ?80C freezer for later use. Clinical examination,.