Figure 2. Infection with N. gonorrhoeae induces caspase-1-dependent cytokine production by THP-1 cells and PBMC-derived monocytes.

A) THP-1 cells at 1×10⁶ cell/ml were incubated with gonococci (GC) at the indicated MOI as noted in the experimental methods. At 4 hours, extracellular bacteria were killed by addition of gentamicin. Supernatants were collected after 4 hours (black bars) and 20 hours (white bars) and secreted IL-1β was measured by ELISA. B) Primary monocytes were isolated from PBMC by adherence. The cells were seeded at 1×10⁶ cell/ml and incubated with GC at the indicated MOI. At 4 hours, extracellular bacteria were killed by addition of gentamicin and supernatants were collected. C) THP-1-derived cell lines stabily transduced with shRNA expressing retrovirus were infected with GC at MOI of 2.0 and IL-1β production determined at 4 hours as described in A. 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. D) Bone marrow derived macrophages were isolated from C57/B6 mice which were either wild type (WT) or bearing genetic knock out of the genes encoding NLRP3 (Nlrp3^−/−) or ASC (Asc ^−/−), cultured and infected with GC (MOI-0.2) as described in the materials and methods. At the indicated time points, culture supernatant was removed and assayed for the presence of IL-1β using ELISA. E) Immunoblot analysis for activated caspase-1 (P10 subunit) and control protein, actin, was performed on protein extracts from cellular infections described in (C) as described in the materials and methods. F) shRNA-expressing THP-1 cells were infected with GC (MOI-2.0) as described in (C) and secreted IL-18 was measured using ELISA. G) THP-1 cells were infected with pathogenic and commensal Neisseria species at the indicated MOI as described in A; N. gonorrhoeae (GC), Neisseria flavescens (N.f.), or Neisseria cinerea (N.c.). Secreted IL-1β was measured using ELISA. Experiments were performed in triplicate and results from representative experiments are shown. Error bars represent the standard error of the mean for duplicate or triplicate measurements of IL-1β.