(a) Peritoneal macrophages from Map1lc3b^−/− mice and Becn1^+/− mice and the corresponding wild-type littermate mice were stimulated with LPS, followed by ATP treatment (L A) for 30 min. Cell lysates (Cells) and culture supernatants (Sup.) were analyzed by immunoblotting for caspase-1 and IL-1β. (b) Peritoneal macrophages were stimulated with LPS, followed by ATP treatment for 1 h. Secretion of cytokines into the media was analyzed by enzyme-linked immunosorbent assay (ELISA). (c) BMDM from Map1lc3b^+/+ or Map1lc3b^−/− mice were incubated with LPS, followed by ATP treatment for 30 min (immunoblotting) or 1 h (ELISA). Cell lysates were analyzed by immunoblotting for caspase-1 and IL-1β. Cell culture supernatants were analyzed by ELISA for cytokine secretion. (d) LPS-primed BMDM from Map1lc3b^−/− mice and Becn1^+/− mice were co-incubated with Z-YVAD-FMK (10 μM) or DMSO, followed by ATP stimulation for 1 h. IL-1β secretion into the media was analyzed by ELISA. Statistical significance was determined by the Student's t-test. *P < 0.05. Data are representative of three or four experiments (mean and s.d. in b,c,d).

(a,b) BMDM from Map1lc3b^−/− (a) or Becn1^+/− (b) mice were incubated with LPS (10 ng/ml) and then stimulated with ATP (1 mM) for 30 min. Morphological changes of mitochondria were examined by TEM. Arrows indicate mitochondria. Scale bars represent 500 nm. (c) Macrophages from Map1lc3b^−/− mice and Becn^+/− mice were labeled with MitoSOX and analyzed by flow cytometric analyses. Representative histograms are shown. (d) Macrophages stimulated with LPS and ATP were stained with MitoTracker Deep Red and MitoTracker Green for 15 min. Representative flow cytometry plots are represented. Data are representative of three experiments.

(a) J774A.1 macrophages exposed to EtBr (0, 10, 100 and 500 ng/ml) were stimulated with LPS and ATP. Cytokine secretion was analyzed by ELISA. Lysates and supernatants were immunoblotted for caspase-1 and IL-1β. (b) J774A.1 or ρ⁰ macrophages (EtBr; 100 ng/ml) were incubated with LPS, followed by ATP (15 min and 30 min). Lysates were immunoblotted for caspase-1 and ASC. (c) J774A.1 l or ρ⁰ macrophages were incubated with LPS, and ATP. Cytokine secretion was analyzed. (d) LPS-primed macrophages were incubated with rotenone (5 μM) or DMSO 1 h before ATP. Cytokine secretion was analyzed. Lysates were immunoblotted for caspase-1 and IL-1β. (e) J774A.1 or ρ⁰ macrophages were incubated with LPS and rotenone, followed by ATP. IL-1β secretion was analyzed by ELISA (f) Peritoneal macrophages pre-incubated with Mito-TEMPO for 1 h were stimulated with LPS and ATP. Cytokine secretion was analyzed. (g,h) Peritoneal macrophages isolated from Map1lc3b^−/− (g) or Becn1^+/− (h) mice were incubated with Mito-TEMPO (500 μM) for 1 h, followed by LPS and ATP. IL-1β secretion was analyzed by ELISA. Lysates were immunoblotted for caspase-1. *P < 0.05 (Student's t-test). Data represent three experiments (mean and s.d. in a,c,d,e,f,g,h).

(a) LPS-primed BMDM from Map1lc3b^+/+ or Map1lc3b^−/− mice were co-incubated with cyclosporine A or DMSO, followed by ATP stimulation for 1 h. Cytokine secretion was analyzed by ELISA. (b) LPS-primed BMDM from Becn1^+/+ or Becn1^+/− mice were co-incubated with cyclosporine A (10 μM) or DMSO, followed by ATP stimulation. Cytokine secretion was analyzed by ELISA. (c) LPS-primed BMDM were incubated with rotenone (5 μM) or vehicle (DMSO) 1 h before ATP stimulation. Cytosolic mtDNA copy number was measured by quantitative PCR. (d) Macrophages pre-incubated with Mito-TEMPO (500 μM) for 1 h were incubated with LPS, followed by ATP stimulation. Cytosolic mtDNA copy number was measured by quantitative PCR. (e) LPS-primed BMDM were incubated with cyclosporine A (10 μM), rotenone (5 μM) or DMSO, followed by ATP stimulation. Cytosolic mtDNA copy number was measured by quantitative PCR. (f) LPS-primed BMDM from Map1lc3b^−/− mice and Becn1^+/− mice were stimulated with ATP for 30 min. Cytosolic mtDNA copy number was measured by quantitative PCR. Statistical significance was determined by the Student's t-test. *P < 0.05. Data are representative of three experiments (mean and s.d.).

(a) BMDM were transfected with 3 μg of DNase, LDH or heat-inactivated DNase for 4 h and then stimulated with LPS, followed by ATP stimulation for 1 h. Cytokine secretion into supernatants was analyzed by ELISA. (b) BMDM were transfected with DNase, LDH or heat-inactivated DNase for 4 h and then stimulated with LPS, followed by ATP stimulation for 15 min. Cytosolic mtDNA copy number was measured by quantitative PCR. (c) BMDM from aim2^+/+ or aim2^−/− mice were primed with LPS (200 ng/ml) for 4 h, and then were transfected with 1 μg of mtDNA for 6 h, followed by stimulation with ATP for 1 h. IL-1β secretion was analyzed by ELISA. . *P < 0.05, versus aim2^−/− cells treated with LPS and ATP (Student's t-test). (d) LPS-primed BMDM from aim2^−/− mice were transfected with mtDNA, poly(dA:dT) or mtDNA pre-digested by DNase for 6 h, followed by stimulation with ATP for 1 h. Cytokine secretion was analyzed by ELISA. *P < 0.05 (Student's t-test). Data are representative of three experiments (mean and s.d.).

(a) LPS-primed peritoneal macrophages from Map1lc3b^−/− (a) or Becn1^+/− (b) mice were incubated with glybenclamide (50–100 μM), Bay 11-7082 (12 μM) or DMSO (15 min), followed by ATP. Cytokine secretion was analyzed. (c) LPS-primed nlrp3^−/− macrophages were incubated with rotenone, followed by ATP (1 h). Cytokine secretion was analyzed. (d) LPS-primed nlrp3^−/− BMDM were stimulated with ATP (15 or 30 min). Cytosolic mtDNA copy number was measured by qPCR. Cytosolic cytochrome c and caspase-1 activation was analyzed by immunoblotting. (e) LPS-primed nlrp3^−/− or asc^−/− BMDM were stimulated with ATP (15 min). Cytosolic mtDNA copy number was measured by qPCR. *P < 0.05, nlrp3^−/− or asc^−/− versus WT cells treated with LPS and ATP (Student's t-test). (f) LPS-primed macrophages were stained with MitoSOX prior to ATP (15 min), and analyzed by flow cytometry. Representative histograms are shown. (g) Macrophages stimulated with LPS and ATP were stained with MitoTracker Deep Red. Representative cytometry plots are shown. (h) LPS-primed peritoneal macrophages from asc^−/− and nlrp3^−/− mice were stimulated with ATP (30 min), and analyzed by Annexin V flow cytometry. Data represent three experiments (mean and s.d. in a,b,c,d,e).

(a,b) Male map1lc3b^+/+ and map1lc3b^−/− mice were intraperitoneally injected with LPS (12 mg/kg). (a) Serum IL-1β and IL-18 at 24 h after the injection was analyzed by ELISA. (b) Survival of map1lc3b^+/+ (n =11) and map1lc3b^−/− mice (n =10) after LPS challenge. Mice were monitored over a period of 2 weeks. (c,d) Male map1lc3b^−/− mice (c) and becln1^+/− (d) mice were treated with cecal ligation and puncture (CLP) or with sham-laparotomy (Sham). Serum IL-1β and IL-18 at 24 h after CLP or sham-operation was analyzed by ELISA. In a,c,d, each symbol represents an individual mouse; small horizontal lines indicate the mean. P values, unpaired two-tailed Student's t-test (a,c,d) or Log-rank test; (b) Data are representative of two experiments.