Figure 6. N. gonorrhoeae LOS induces NLRP3-dependent IL-1β secretion and pyronecrosis in THP-1 cells.

A) and B) N. gonorrhoeae -conditioned media (GC-CM), N. gonorrhoeae-conditioned media 100kD retentate (CM100R) and filtrate (CM100F) were prepared as described in the materials and methods. THP-1 cells were incubated with live N. gonorrhoeae (GC) at an MOI of 1 or conditioned media preparations from and equivalent amount of bacteria for 4 hours and secreted IL-1β and cell death were assessed as previousl described. C) and D) THP-1-derived cell lines stably transduced with shRNA expressing retrovirus (described in Figure 2) were incubated with the indicated concentration of E. coli derived LPS or purified gonococcal LOS from strains PID2 or DOV (15, 35). After 4 hours, cell death was determined by measurement of LDH release into the media and secreted IL-1β was determined by ELISA. The shRNA’s are directed to knock down expression as follows: shCON - negative control (scrambled sequence with base content equal to shASC); shASC – shRNA directed against Apoptotic Speck Containing-protein; shNLRP3 – shRNA directed against NLRP3. LDH release and IL-1β secretion into the media that was not detectable is indicated by an asterisk (*). Representative experiments (of three) are shown, the bars indicate mean values of triplicate measurements with error bars representing the SEM.