Toxigenic Staphylococcus aureus-induced mucin secretion by conjunctival goblet cells is dependent upon IL-1β via Ca²⁺ and ERK1/2 signaling

Abstract

Purpose : Mucins are considered the first line of defense for the conjunctiva and protect the underlying epithelium by entrapping bacteria and preventing bacterial invasion. We previously demonstrated that pathogenic S. aureus activates the NLRP3 inflammasome in conjunctival goblet cells, resulting in secretion of the pro-inflammatory cytokine IL-1β and mucins. The following studies were performed to determine if NLRP3 inflammasome activation is required for S. aureus-induced mucin secretion and if this secretion is dependent upon the release of mature IL-1β.

Methods : Cultured human goblet cells were incubated for 72 h with siRNA for NLRP3 or TLR 2; scrambled siRNA; or vehicle. Cells were then stimulated with toxigenic S. aureus RN6390 or vehicle control. Additional goblet cell cultures were stimulated with IL-1β or S. aureus RN6390 in the presence of (i) an IL-1 receptor antagonist (IL-1Ra, Anakinra), (ii) an intracellular Ca²⁺ chelator (BAPTA/AM), (iii) a extracellular regulated kinase (ERK) 1/2 inhibitor (U0126), or (iv) a vehicle control. High molecular weight glycoconjugate (HMGC) secretion including MUC5AC was measured by enzyme linked lectin assay (ELLA).

Results : NLRP3 and TLR2 siRNA significantly inhibited goblet cell HMGC secretion induced by toxigenic S. aureus RN6390, while scrambled siRNA had no effect. Mature IL-1β alone stimulated goblet cell secretion in a dose-dependent manner. The IL-1Ra significantly blocked goblet cell HMGC secretion stimulated by S. aureus RN6390 or IL-1β alone. In addition, IL-β-induced mucin secretion was blocked by chelating intracellular Ca²⁺ or inhibiting ERK1/2 activity.

Conclusions : Goblet cell-derived mature IL-1β mediates S.aureus-induced mucin secretion by increasing intracellular [Ca²⁺] and activating ERK1/2. Moreover, the S. aureus-induced production of mature IL-1β is dependent upon the triggering of both NLRP3 and TLR2 expressed on goblet cells. These data reveal the pluripotent activities of goblet-cell-derived IL-1β induced by pathogenic S. aureus, an inducer of immune cell recruitment that clears pathogens and inducer of mucin secretion that enhances the ocular surface barrier to infection.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.