Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome - PubMed (original) (raw)

doi: 10.1038/ni.1980. Epub 2010 Dec 12.

Jeffrey Adam Haspel, Vijay A K Rathinam, Seon-Jin Lee, Tamas Dolinay, Hilaire C Lam, Joshua A Englert, Marlene Rabinovitch, Manuela Cernadas, Hong Pyo Kim, Katherine A Fitzgerald, Stefan W Ryter, Augustine M K Choi

Affiliations

• PMID: 21151103
• PMCID: PMC3079381
• DOI: 10.1038/ni.1980

Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome

Kiichi Nakahira et al. Nat Immunol. 2011 Mar.

Abstract

Autophagy, a cellular process for organelle and protein turnover, regulates innate immune responses. Here we demonstrate that depletion of the autophagic proteins LC3B and beclin 1 enhanced the activation of caspase-1 and secretion of interleukin 1β (IL-1β) and IL-18. Depletion of autophagic proteins promoted the accumulation of dysfunctional mitochondria and cytosolic translocation of mitochondrial DNA (mtDNA) in response to lipopolysaccharide (LPS) and ATP in macrophages. Release of mtDNA into the cytosol depended on the NALP3 inflammasome and mitochondrial reactive oxygen species (ROS). Cytosolic mtDNA contributed to the secretion of IL-1β and IL-18 in response to LPS and ATP. LC3B-deficient mice produced more caspase-1-dependent cytokines in two sepsis models and were susceptible to LPS-induced mortality. Our study suggests that autophagic proteins regulate NALP3-dependent inflammation by preserving mitochondrial integrity.

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Figures

Fig. 1. Absence of LC3B and heterozygous disruption of Beclin 1 enhance caspase-1 dependent cytokine secretion in macrophages

(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).

Fig. 1. Absence of LC3B and heterozygous disruption of Beclin 1 enhance caspase-1 dependent cytokine secretion in macrophages

(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).

Fig. 1. Absence of LC3B and heterozygous disruption of Beclin 1 enhance caspase-1 dependent cytokine secretion in macrophages

(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).

Fig. 1. Absence of LC3B and heterozygous disruption of Beclin 1 enhance caspase-1 dependent cytokine secretion in macrophages

(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).

Fig. 2. Depletion of autophagic proteins alters mitochondrial phenotype

(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.

Fig. 2. Depletion of autophagic proteins alters mitochondrial phenotype

(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.

Fig. 2. Depletion of autophagic proteins alters mitochondrial phenotype

(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.

Fig. 2. Depletion of autophagic proteins alters mitochondrial phenotype

(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.

Fig. 3. EtBr abolishes caspase-1 activation

(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).

Fig. 3. EtBr abolishes caspase-1 activation

(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).

Fig. 3. EtBr abolishes caspase-1 activation

(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).

Fig. 3. EtBr abolishes caspase-1 activation

(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).

Fig. 3. EtBr abolishes caspase-1 activation

(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).

Fig. 3. EtBr abolishes caspase-1 activation

(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).

Fig. 3. EtBr abolishes caspase-1 activation

(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).

Fig. 3. EtBr abolishes caspase-1 activation

(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).

Fig. 4. Autophagic protein deficiency promotes mtDNA release into cytosol through increased MPT

(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.).

Fig. 4. Autophagic protein deficiency promotes mtDNA release into cytosol through increased MPT

(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.).

Fig. 4. Autophagic protein deficiency promotes mtDNA release into cytosol through increased MPT

(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.).

Fig. 4. Autophagic protein deficiency promotes mtDNA release into cytosol through increased MPT

(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.).

Fig. 4. Autophagic protein deficiency promotes mtDNA release into cytosol through increased MPT

(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.).

Fig. 4. Autophagic protein deficiency promotes mtDNA release into cytosol through increased MPT

(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.).

Fig. 5. Cytosolic mitochondrial DNA (mtDNA) is involved in caspase-1 activation

(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.).

Fig. 5. Cytosolic mitochondrial DNA (mtDNA) is involved in caspase-1 activation

(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.).

Fig. 5. Cytosolic mitochondrial DNA (mtDNA) is involved in caspase-1 activation

(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.).

Fig. 5. Cytosolic mitochondrial DNA (mtDNA) is involved in caspase-1 activation

(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.).

Fig. 6. NALP3 mediates mitochondrial DNA release

(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).

Fig. 6. NALP3 mediates mitochondrial DNA release

(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).

Fig. 6. NALP3 mediates mitochondrial DNA release

(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).

Fig. 6. NALP3 mediates mitochondrial DNA release

(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).

Fig. 6. NALP3 mediates mitochondrial DNA release

(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).

Fig. 6. NALP3 mediates mitochondrial DNA release

(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).

Fig. 6. NALP3 mediates mitochondrial DNA release

(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).

Fig. 6. NALP3 mediates mitochondrial DNA release

(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).

Fig. 7. Autophagic protein deficiency augments caspase-1-mediated inflammatory responses in vivo

(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.

Fig. 7. Autophagic protein deficiency augments caspase-1-mediated inflammatory responses in vivo

(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.

Fig. 7. Autophagic protein deficiency augments caspase-1-mediated inflammatory responses in vivo

(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.

Fig. 7. Autophagic protein deficiency augments caspase-1-mediated inflammatory responses in vivo

(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.

Comment in

• Mitochondrial control of the NLRP3 inflammasome.
  Kepp O, Galluzzi L, Kroemer G. Kepp O, et al. Nat Immunol. 2011 Mar;12(3):199-200. doi: 10.1038/ni0311-199. Nat Immunol. 2011. PMID: 21321591 No abstract available.

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References

1. 1. He C, Klionsky DJ. Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet. 2009;43:67–93. - PMC - PubMed
2. 1. Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell. 2008;132:27–42. - PMC - PubMed
3. 1. Virgin HW, Levine B. Autophagy genes in immunity. Nat Immunol. 2009;10:461–70. - PMC - PubMed
4. 1. Saitoh T, et al. Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production. Nature. 2008;456:264–8. - PubMed
5. 1. Stutz A, Golenbock DT, Latz E. Inflammasomes: too big to miss. J Clin Invest. 2009;119:3502–11. - PMC - PubMed

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