Fig. 2.

Caspase-1–dependent and –independent inflammasomes are activated during infection of human macrophages. (A) Primary human MDMs were infected with WT Lp, infected with T4SS⁻ Lp, or mock-infected for 20 h. Immunoblot analysis was performed on supernatants (sup) for cleaved caspase-1 (casp1 p10) and on lysates for full-length caspase-1 (pro-casp1). Lysates were reprobed for β-actin. Primary human MDMs (B) or PMA-differentiated THP-1 cells (C) were pretreated with 40 μM caspase-1 inhibitor [Ac-YVAD-cmk (YVAD)] or vehicle control (DMSO) and infected with WT Lp or mock-infected for 20 h. (D and E) PMA-differentiated THP-1 cells were transfected with control siRNA (CNTRL) or siRNA against caspase-1 and infected with WT Lp or mock-infected for 20 h. Immunoblot analysis was performed on lysates for pro-casp1, pro-casp4, and pro–IL-1β, and blots were reprobed for β-actin. Cell death was measured using a lactate dehydrogenase release assay and normalized to mock-infected cells. IL-1α, IL-1β, and TNF levels in the supernatants were measured by ELISA. (A and D) Western blots are representative of three independent experiments. Shown are the pooled results of four independent infections of cells from different donors (B) or the pooled results of four (C) or three (E) independent experiments in THP-1 cells. Each data point shows the mean of triplicate infected wells. *P < 0.05 by paired t test (B and E) or unpaired t test (C). NS, not significant. The dashed line is the limit of detection.