IL-6 induces NLRP3 inflammasome activation through JAK/STAT3-dependent NOX2 induction in colon epithelial cells

Abstract

Background: IL-6, in addition to TNFα, plays an important role in the pathogenesis of inflammatory bowel disease (IBD), which is supported by clinical observations of close correlation between IL-6 production and severity of the disease in IBD patients. IL-6 plays a role in the recruitment process of neutrophils and monocytes to lesion sites, resulting in aggravated and chronic inflammation. Recently, TNFα is shown to prime TLR-independent NLRP3 inflammasome activation. IL-6, however, has not been shown such activity. The present study aims to investigate whether IL-6 induces NLRP3 inflammasome formation, and NADPH oxidase is involved in that process.

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 labeled with BCECF-AM (10 μg/mL). After 3 h treatment with IL-6, BCECF fluorescence from adhered cells was measured. To identify signaling pathway, siRNA transfection, RT-PCR and Western blot analyses were performed. Reactive oxygen species (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), NLRP3, caspase-1, and IL-1β. Concurrently, IL-6 significantly increased ROS production in a time-dependent manner, which matched significant induction of NOX2 and its regulatory subunits. The IL-6-induced ROS production and increased expression of NLRP3, caspase-1, and IL-1β were attenuated by pretreatment with NADPH oxidase inhibitors (VAS-2840, DPI and apocyanin), but not by inhibitors against other enzymes, such as cytosolic COX-2 (celecoxib), mitochondria (antimycin A), xanthine oxidase (allopurinol), and iNOS (NAME) in HT-29 cells. Similarly, inhibitors of JAK (tofacitinib) and STAT3 (stattic) suppressed IL-6-induced ROS production, NOX2 induction, and the changes in inflammasome proteins in HT-29 cells.

Conclusions: Taken together, our results suggest that IL-6 induces NLRP3 inflammasome activation through JAK/STAT-dependent NOX2 induction in HT-29 colonic epithelial cells.