Fig. 8: Defective ETT promotes NLRP3 inflammasome activation in mice.

a, Immunoblotting of supernatants and lysates from primary BMDMs isolated from Arfrp1^fl/fl or Arfrp1^fl/fl;LysM-Cre (Arfrp1^ΔMye) mice. Isolated cells were primed with 1 μg ml⁻¹ LPS for 4 h, followed by treatment with 2 mM ATP, 5 μM nigericin, 50 μM CL097 or 50 μM R837 for 40 min as indicated. Antibodies recognizing both p45 and p20 fragments of caspase-1 (CASP1), p31 and p17 fragments of IL-1β, NLRP3 and ARFRP1 were used. An antibody against tubulin was used as a loading control. Short exposures (s.e.) and long exposures (l.e.) of IL-1β and CASP1 fragments are shown. b, ELISA assay of IL-1β and TNF-α in supernatants of isolated BMDMs from mice treated as described for a. Mean ± s.d., N = 3. Data were analyzed with an unpaired two-sided t-test. c, Survival curves of Arfrp1^fl/fl or Arfrp1^fl/fl;LysM-Cre (Arfrp1^ΔMye) mice injected with LPS (15 mg kg⁻¹) in the presence of vehicle or 50 mg kg⁻¹ MCC950. The survival of mice after LPS injection was monitored every 6 h. n = 8 for vehicle-treated Arfrp1^fl/fl mice group; n = 8 for MCC950-treated Arfrp1^fl/fl mice group; n = 10 for vehicle-treated Arfrp1^ΔMye mice group and n = 9 for MCC950-treated Arfrp1^ΔMye mice group. Data were analyzed with a log-rank (Mantel–Cox) test. d, ELISA assay of IL-1β (left panel) and TNF-α (right panel) levels in serum collected from Arfrp1^fl/fl or Arfrp1^fl/fl;LysM-Cre (Arfrp1^ΔMye) mice at 3 h after peritoneal injections of LPS (15 mg kg⁻¹) in the presence of vehicle or 50 mg kg⁻¹ MCC950. Mean ± s.d., n = 8 for vehicle-treated Arfrp1^fl/fl mice group; n = 8 for MCC950-treated Arfrp1^fl/fl mice group; n = 10 for vehicle-treated Arfrp1^ΔMye mice group and n = 9 for MCC950-treated Arfrp1^ΔMye mice group. Data were analyzed with a two-sided Mann–Whitney U-test. e, Proposed model of NLRP3 inflammasome activation as described in Discussion. Data shown in a are representative of three independent experiments.

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