Interleukin-32 Induced Thymic Stromal Lymphopoietin Plays A Critical Role In The Inflammatory Response Of Human Corneal Epithelium

Objective:To investigate the role of Interleukin-32(IL-32)and thymic storal lymphopoietin(TSLP)in the inflammatory response of human corneal epithelial cells and its regulatory mechanism.Methods:(1)The expression of TSLP,IL-6,TNF?m RNA and protein in HCECs was detected by RT-q PCR and ELISA with different concentrations of IL-32.The expression of TSLP protein in ex vivo model of human corneal tissues was detected by immunohistochemical staining(2)Western blot was used to detect the activity of caspase-1 in corneal epithelial cells of each group.The expression of caspase-1?TSLP m RNA and protein were detected by Western blot,RT-q PCR and ELISA in the presence of a caspase-1 inhibitor VX-765.(3)The HCECs exposed to IL-32(10ng/m L)were preincubated in the absence or presence of Isotype Ig G antibody(5?g/m L),TSLP antibodies(5?g/m L),NF-k B activation inhibitor quinazoline(NF-k B-I,10?M)or Caspase-1 inhibitor VX-765 for 1 h.The cultures treated with IL-32 for 4 h were subjected to RT and real-time PCR to measure m RNA,and those treated for 48 h were used to evaluate protein levels in cell lysates by ELISA.Results:(1)IL-32 stimulated HCECs could induce the expression of TSLP,IL-6,TNF?m RNA and protein levels in a dose-dependent and time manner(p <0.05).TSLP protein was normally produced by corneal epithelial cells,and mainly localized in the cytoplasm in the untreated corneal epithelial tissues.Stronger immunostaining throughout multiple layers of corneal epithelium was observed in the tissues exposed to IL-32(50ng/ml)for 48 hours;(2)IL-32 increased caspase-1 activation in a dose-dependent manner.IL-32 induced caspase-1 activity and the expression of TSLP m RNA and protein was significantly inhibited in the presence of a caspase-1 inhibitor(P<0.05);(3)Pre-treatment with TSLP antibodies?the NF-?B inhibitor quinazoline(NF-?B-I,10 ?M)and Caspase-1inhibitor VX-765 significantly suppressed the TNF? and IL-6 m RNA and protein expression in HCECs,but not with its isotype Ig G antibody(p <0.05).Conclusion:IL-32 and IL-32-induced TSLP are important cytokines involved in inflammatory responses,IL-32 induced TSLP m RNA and protein expression via caspase-1signaling;IL-32 and TSLP also increased the inflammatory response of human cornealepithelium through the caspase-1/TSLP/NF-?B signaling pathway.