Glyburide

At 3 h prior to infection, the medium was changed to complete medium (RPMI) plus 1% fetal bovine serum with or without 100 ng/ml ultrapure lipopolysaccharide (LPS; Sigma, Shanghai, China). The PMs were stimulated with N. caninum for the indicated times (1–24 h) at various multiplicities of infection (MOI = 1:1, 3:1, and 5:1). PMs treated with medium alone or LPS were used as negative controls, and cells treated with ATP (5 mM, 30 min; Sigma, Shanghai, China) were used as positive controls, as extracellular ATP is a conventional NLRP3 inflammasome agonist [14 (link)].
To monitor the role of the inflammasome in response to N. caninum infection in PMs, PMs were pre-treated with 100 μM Ac-YVAD-CHO (an inhibitor of caspase-1 and -4; Enzo Life Science, Lausen, Switzerland), 100 μM zVAD-fmk (an inhibitor of pan-caspase; Selleck, Shanghai, China) or 100 μM glyburide (an inhibitor of NLRP3 by inhibiting K⁺ efflux; Selleck, Shanghai, China) for 45 min before stimulation, and the PMs were then stimulated with LPS priming plus N. caninum for 12 h at an MOI of 3:1 (parasite:cell), and PMs cultured with 0.2% DMSO were used as a negative control.

PMφ was pretreated with either 100 μM Ac-YVAD-CHO (an inhibitor of caspase-1 and -4; Enzo Life Science, Switzerland) or 10 μM zVAD-fmk (an inhibitor of pan-caspase; Selleck, Shanghai) for 1 h before A. sobria stimulation (MOI=10) to explore the roles of caspase-1 p20 in activated IL-1β secretion (Malyshev et al., 2004 (link); Cao et al., 2005 (link)). PMφs were pretreated with either 25 μM CA-074 methyl ester (CA-074 Me, an inhibitor of cathepsin B, MedChemExpress, USA) or 50 μM Glyburide (an inhibitor of NLRP3 by inhibiting K⁺ efflux; Selleck, Shanghai) for 1 h before stimulation to block NLRP3 receptor activation and explore its roles in caspase-1 p20 production and IL-1β p17 secretion (Lamkanfi and Dixit, 2009 (link); Liu et al., 2013 (link)). PMφs were pretreated with these NLRP3 inflammasome inhibitors through different ways to detect its roles in inflammatory response by measuring cytokines levels of IL-1β, IL-6, IL-12, and TNF-α.