Abstract
Purpose:
To determine the Toll-like receptor (TLR) and pore forming signals used by toxigenic Staphylococcus aureus (S. aureus) bacteria in conjunctival goblet cells to activate the NLRP3 inflammasome to produce IL-1β.
Methods:
Cultured human goblet cells were incubated for 72 hrs with siRNA for NLRP3, TLR 1, 2, and 6; scrambled siRNA; or vehicle. Cells were then stimulated with no additions or S. aureus RN6390. NLRP3 activation was determined by measuring pro-IL1β expression by western blotting and mature IL-1β secretion by ELISA. The amount of TLR-1 and -2 expression in cultured cells was measured by Q-PCR. Cultured goblet cells were stimulated by no additions, α toxin, toxigenic S. aureus RN6390, and S. aureus ALC837 that only lacked α toxin. Caspase-1 activity was measured by FLICA along with pro-IL-1 β expression and mature IL-1β secretion.
Results:
NLRP3 siRNA completely decreased NLRP3 expression, pro-IL-1β expression, and mature IL-1β secretion stimulated by S. aureus RN6390. Use of non-target siRNA had no effect. When cells were stimulated with S. aureus RN6390, TLR2 siRNA completely blocked pro-IL-1β expression and mature IL-1β secretion. Non-target siRNA had no effect. Stimulation of pro-IL-1β expression and mature IL-1β secretion by S. aureus RN6390 in the presence of TLR1 siRNA was significantly, but not completely decreased. In contrast in the presence of TLR6 siRNA, S. aureus RN6390 did not significantly alter pro-IL-1β expression and mature IL-1β secretion. Values were the same as in the presence of non-target siRNA. TLR1 was expressed at much lower levels than TLR2 in cultured goblet cells. S. aureus RN6390, but not α toxin nor S. aureus ALC837, stimulated pro-IL-1β expression and mature IL-1β secretion. The α toxin was active as it stimulated caspase-1 activity to the same level as S. aureus RN6390. Simultaneous addition of α toxin and S. aureus ALC837 restored pro-IL-1β expression and mature IL-1β secretion to the same level as produced by S. aureus RN6390.
Conclusions:
We conclude that in conjunctival goblet cells toxigenic S. aureus stimulates the NLRP3 inflammasome to secrete mature IL-1β by activating TLR2, to a lesser extent TLR1, but not TLR 6 and by the pore forming action of its α toxin.