Cutting edge: TNF-alpha mediates sensitization to ATP and silica via the NLRP3 inflammasome in the absence of microbial stimulation

Abstract

The Nlrp3 inflammasome is critical for the activation of caspase-1 in response to danger signals and particulate matter. However, its role in sterile inflammation remains unclear because prestimulation of phagocytic cells with microbial molecules is required for caspase-1 activation. We show here that exposure of macrophages and dendritic cells to TNF-alpha promotes ATP- or silica-mediated caspase-1 activation and IL-1beta secretion in the absence of microbial stimulation. The effect of TNF-alpha was abolished in macrophages deficient in TNF receptor I and II, Nlrp3, or ASC, whereas that induced by TLR ligands required MyD88/Trif. In addition to TNF-alpha, IL-1alpha and IL-1beta promoted caspase-1 activation via Nlrp3 in response to ATP. Remarkably, macrophages tolerized to TNF-alpha, but not to LPS, retained full sensitivity to ATP stimulation via Nlrp3. These results provide a mechanism by which danger signals and particulate matter mediate inflammation via the Nlrp3 inflammasome in the absence of microbial infection.

Figure 1

TNF-α induces caspase-1 activation in response to ATP and silica in the absence of microbial stimulation. A, BMDM were stimulated for 6 hrs with TNF (100 ng/ml), CD40L (10μg/ml), agonistic anti-Fas antibody (10μg/ml), IFN-γ (100 U/ml) or LPS (10 ng/ml). When indicated ATP (5 mM) was added for the last 30 minutes B, BMDM were stimulated for 6 hrs with PMA (100 ng/ml), RANKL (1 μg/ml), IL-1α (10 ng/ml), IL-1β (10 ng/ml). When indicated ATP (5 mM) was added for the last 30 minutes. C, BMDM were stimulated with TNF-α and then stimulated with ATP, silica or infected with S. Typhimurium (Salm). (A–C) Extracts were prepared from cell and culture supernatants and immunoblotted with caspase-1 antibody. Arrows denote procaspase-1 (p45) and its processed p20 subunit. Results are representative of three separate experiments.

Figure 2

TNF-α promotes IL-1β secretion and caspase-1 activation via TNFR-I/II and the Nlrp3 inflammasome. A–B, BMDC from wild-type (WT), TnfrI/II deficient (TNFRI/II-DKO) and Nlrp3 deficient (Nlrp3-KO) mice were stimulated with TNF-α for 6 hrs and then stimulated, or not, with ATP for 30 minutes. IL-1β was measured 4 hrs after stimulation in cell-free supernatants by ELISA. Values represent mean ± SD of triplicate cultures. * p < 0.01 between WT and mutant macrophages. C, BMDM were stimulated as in panels A and B. Extracts were immunoblotted with caspase-1 antibody. Arrows denote procaspase-1 (p45) and its processed p20 subunit. (A–B) Results are representative of three separate experiments.

Figure 3

TNF-mediated activation of caspase-1 requires gene transcription. A, BMDM were pretreated with 50 μM of cyclohexemide (CHX) or the NF-κB inhibitor BAY 11-7082 (20μM) for 1 hour, and then stimulated with TNF-α for 6 hrs. When indicated ATP was added for the last 30 minutes. B, BMDM from WT or MyD88^−/−/Trif^−/− mice (DKO) were stimulated for 6 hrs with LPS (1ug/ml), BLP (1ug/ml), poly IC (1ug/ml) or CpG (1ug/ml). When indicated ATP (5 mM) was added for the last 30 minutes. A and B, extracts were immunoblotted with caspase-1 antibody. Arrows denote procaspase-1 (p45) and its processed p20 subunit Results are representative of three separate experiments.

Figure 4

TNF-α, but not LPS, induces ATP-mediated IL-1β secretion and caspase-1 activation via Nlrp3 in tolerized macrophages. A–D, BMDM were left unstimulated or stimulated with TNF-α (100 ng/ml) (A–B) or LPS (C and D) for 6 or 24 hrs and then stimulated with ATP (5 mM) for 30 min. A–C, IL-1β was measured after stimulation in cell-free supernatants by ELISA. Values represent mean ± SD of triplicate cultures. n. d. refers to undetectable. * p < 0.01 between WT and mutant macrophages. B–D, Extracts were immunoblotted with caspase-1 antibody. Arrows denote procaspase-1 (p45) and its processed p20 subunit. (A–D) Results are representative of three separate experiments.