Superoxide production and enhanced NOX2 induction through JAK/STAT signalling mediate IL-6-induced inflammatory responses of colon epithelial cells

Abstract

Background

IL-6 plays an important role in the pathogenesis of inflammatory bowel disease (IBD), which is supported by close correlation between IL-6 production and severity of the disease in IBD patients. IL-6 enhances expression of adhesion molecules that are required for the recruitment process of neutrophils and monocytes to lesion sites. The regulation of those molecule expressions by NF-κB, a redox-sensitive transcription factor, implies that IL-6-induced adhesion molecule expression may also be associated with reactive oxygen species (ROS) producing activity of IL-6. Present study focused on whether NADPH oxidase is involved in IL-6-induced ROS production and changes in the protein expressions.

Methods

The IL-6-induced adhesion of monocytes (U937 cell line) to colon epithelial cells (HT-29 cell line) was examined by co-culture of HT-29 cells with U937 cells that were already labelled with BCECF-AM (10 μg/ml). After 3 h treatment with IL-6, BCECF fluorescence from adhered cells was measured. To identify signalling pathway, siRNA transfection, RT-PCR and Western blot analyses were performed. ROS was measured by lucigenin chemiluminescence assay.

Results

IL-6 significantly increased U937 monocytic cell adhesion to HT-29 colonic epithelial cells, which was accompanied by upregulation of adhesion molecules (ICAM-1 and VCAM-1) and repression of junction proteins (E-cadherin and claudin). Concurrently, IL-6 significantly increased superoxide production in a time-dependent manner, which matched significant induction of NOX2 and its regulatory subunits. The IL-6-induced ROS production and protein expression changes were attenuated by pretreatment with NADPH oxidase inhibitors (VAS-2840, and DPI) and STAT3 inhibitor (stattic), but not by inhibitors against other enzymes, such as cytosolic COX-2 (celecoxib), mitochondria (antimycin A), xanthine oxidase (allopurinol), and iNOS (NAME). Similarly, tofacitinib, a JAK inhibitor, and sttattic blocked IL-6-induced superoxide production and NOX2 induction.

Conclusions

Taken together, our results suggest that IL-6-activated JAK/STAT signalling is linked to superoxide production and NOX2 induction, resulting in IL-6-induced inflammatory responses of colon epithelial cells.