Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis

Abstract

We report that in the presence of signal 1 (NF-κB), the NLRP3 inflammasome was activated by mitochondrial apoptotic signaling that licensed production of interleukin-1β (IL-1β). NLRP3 secondary signal activators such as ATP induced mitochondrial dysfunction and apoptosis, resulting in release of oxidized mitochondrial DNA (mtDNA) into the cytosol, where it bound to and activated the NLRP3 inflammasome. The antiapoptotic protein Bcl-2 inversely regulated mitochondrial dysfunction and NLRP3 inflammasome activation. Mitochondrial DNA directly induced NLRP3 inflammasome activation, because macrophages lacking mtDNA had severely attenuated IL-1β production, yet still underwent apoptosis. Both binding of oxidized mtDNA to the NLRP3 inflammasome and IL-1β secretion could be competitively inhibited by the oxidized nucleoside 8-OH-dG. Thus, our data reveal that oxidized mtDNA released during programmed cell death causes activation of the NLRP3 inflammasome. These results provide a missing link between apoptosis and inflammasome activation, via binding of cytosolic oxidized mtDNA to the NLRP3 inflammasome.

Figure 1. Mitochondrial dysfunction is linked to NLRP3 inflammasome activation

(A-C) IL-1β and TNF-α concentrations (ELISA) in BMDM culture supernatants treated with (A) NLRP3 activating stimuli or (B) LPS (1 μg/ml) plus ATP (5 mM, final 2 hr culture) in the presence of increasing doses of chloramphenicol. Also shown is an LDH release assay of the same BMDM. (C) The effect of Cyclosporin A (CsA) on LPS+ATP-induced IL-1β in WT BMDM. (D) Mitochondrial membrane potential (ΔΨ_m) measurements during NLRP3 inflammasome activation. BMDM were treated with UV-killed CP (UVCP, MOI=10) or live CP (MOI=10), treated with ATP (5 mM) or staurosporine (STS, 5 μM) for 24 hr and examined for TMRM incorporation. (E) Changes in ΔΨ_m were monitored over time by TMRM incorporation. BMDM were treated with live CP (MOI=10), ATP (5 mM), STS (5 μM), nigericin (NIG, 10μM), or alum (130 μg/ml). (F and G) Changes in ΔΨ_m due to ATP treatment as measured by by TMRM microscopy. A representative image of mitochondrial TMRM fluorescence and associated 3D intensity map are shown in (F). The kinetic profile shown in (G) depicts the mean value +/- the SEM at each time point. (H and I) Extracellular K⁺ affects (H) IL-1β secretion and (I) ΔΨ_m as measured by TMRM incorporation. Four hours after LPS priming (1 μg/ml) or after CP infection (MOI=10), BMDM were transferred to culture media containing either 5 mM or 100 mM K⁺. Six h after LPS priming, BMDM were then treated with ATP (5 mM), STS (5 μM) or NIG (10 μM) and cultured for an additional 2 hr before culture supernatants were collected and IL-1β concentration was measured. CP infected BMDM were cultured for an additional 20 hr before supernatants were collected for IL-1β measurement. (J and K) Oxygen consumption rate (OCR) was measured in LPS primed macrophages. (J) Base line OCR measurements 2 hr after ATP (5 mM), STS (5 μM) or NIG (10 μM) treatment. (K) The change of OCR was monitored after 1 μM oligomycin, 1 μM FCCP, and 1 μM Rotenone sequential additions. (I) Kinetics of OCR were monitored in macrophages in response to NLRP3 activation. Data shown are representative of two or more independent experiments (means ± SD). * p< 0.05, ** p<0.01, ***p<0.001.

Figure 2. Apoptotic stimuli activate the NLRP3 inflammasome in macrophages

(A and B) BMDM were treated with UVCP (MOI=10), live CP (MOI=10), ATP (5 mM), STS (5 μM) or alum (130 μg/ml) and (A) LDH release was determined at the indicated time points or (B) cells were fixed, stained with DAPI, and condensed nuclei were enumerated. (C) Immunoblotting was used to analyze mouse caspase-1, pro-IL-1β, and IL-1β in culture supernatants and lysates of BMDM treated with (left to right): UVCP (MOI of 10, 8 hr), live CP (MOI of 10, 8 hr), LPS (1 μg/ml, 8 hr), ATP alone (5 mM), LPS+ATP, STS alone (5 μM), or LPS+STS (5 μM, final 2 hr of culture). (D and E) IL-1β or TNF-α secretion by (D) wild-type (WT) or Casp1^–/– or (E) WT, Asc^–/– or Nlrp3^–/– BMDM was measured by ELISA after UVCP, live CP (MOI=10 and 8 hr) or LPS (1 μg/ml, for 8 hr) treatment in presence of STS (2.5 μM or 5 μM) for the final 2 hr of culture. (F) IL-1β secretion was quantified by ELISA in BMDM primed with UVCP, live CP or LPS and then treated with ATP or STS at 0 h or 6 hr after priming. (G) BMDM were treated with live CP, LPS, LPS+ATP, or ATP alone as indicated. Cells were analyzed by immunofluorescence for IL-1β and DAPI. Data shown are representative of three or more independent experiments (means ± SD).

Figure 3. Bcl-2 inversely regulates mitochondrial dysfunction and NLRP3 inflammasome activation

(A) Bcl-2-neo^r or empty vector (EV, neo^r) was stably expressed in MCL macrophages, and Bcl2 expression was determined by immunoblot. (B and C) Bcl-2 overexpressing macrophages were treated with live CP (MOI=10, 24 hr), LPS (1 μg/ml, 5 hr), ATP (5 mM, final 2 hr of culture), LPS+ATP, STS (5 μM, final 2 hr of culture), LPS+STS, NIG (10 μM, final 2 hr), or LPS+NIG and then (B) IL-1β or TNF-α secretion was measured by ELISA or (C) immunoblotting was used to analyze mouse capsase-1, pro-IL-1β, or IL-1β in culture supernatants or cell lysates. (D and E) ΔΨ_m (D) and apoptotic cells (E) were measured after NLRP3 inflammasome stimulation at 8 hr for CP, or at 4 hr for ATP, STS and NIG. (F) BMDM from Bcl-2 transgenic mice were treated with live CP, LPS, LPS+ATP, LPS+STS, LPS+NIG, or LPS+alum (130 μg/ml, final 8 hr). IL-1β and TNF-α secretion were then measured using ELISA. (G) Validation of shRNA targeting Bcl-2 in stably-expressing MCL macrophages by immunoblot. (H and I) shBcl-2 macrophages were primed with LPS and treated with ATP (5 mM for 4 hr), and (H) LDH release, (I) IL-1β or TNF-α were then measured in supernatants. Data shown are representative of three or more independent experiments (means ± SD). * p<0.05, ** p< 0.01, *** p<0.001.

Figure 4. Salmonella type III secretion protein and invasion factor SipB induces mitochondrial depolarization and caspase-1 dependent IL-1β secretion

(A) Three hour-LPS-primed Nlrp3^–/– BMDM were exposed to wild-type (St wt) or nonflagellated mutant St (St ΔfljB/fliC) (MOI=5) and IL-1β was quantified in culture supernatants by ELISA. (B and C) Three hour-LPS-primed BMDM were exposed to various strains of St (MOI=5) and (B) cell viability was determined by LDH release assay or (C) ΔΨ_m was measured by TMRM incorporation. (D and E) IL-1β or TNF-α were quantified in culture supernatants by ELISA in (D) WT and Casp1^–/– macrophages or in (E) WT and Bcl-2 overexpressing macrophages that were exposed to St (MOI=5). Data shown are representative of three or more independent experiments (means ± SD).* p<0.05, ** p< 0.01, *** p<0.001.

Figure 5. Mitochondrial DNA colocalizes with NLRP3

A) Flag-NLRP3 and empty vector (EV) stably-expressing 293 cells were exposed to BrdU-labeled mitochondrial DNA (mtDNA) in presence of lipofectamine. Cell lysates were collected 3 hr later and immunoprecipitated. mtDNA was detected by BrdU dot-blot, and NLRP3 Ab was used for immunoblotting as a loading control. (B and C) YFP-tagged NLRP3 stably-expressing 293 cells were exposed to BrdU-labeled mitochondrial DNA plus lipofectamine. Three hr after exposure, cells were fixed, permeabilized, treated with DNase I (10 U/ml, 30 min), and stained with BrdU Ab. (B) Co-localization of YFP-NLRP3 and mtDNA was analyzed by structured illumination microscopy, and (C) shows an orthogonal view from a z-series.

Figure 6. Oxidized mitochondrial DNA binds to NLRP3 and activates the inflammasome

(A) BMDM were preloaded with BrdU (10 μM), and cells were treated with LPS (1 μg/ml, 3 hr), followed by 3-MA (2.5 mM, 1 hr), ATP (5 mM, 1 h) or NIG (10 μM, 1 h). Cell lysates were collected and immunoprecipitated with anti-NLRP3 Ab, then detected by dot-blots probed with anti-BrdU or anti-8OH-dG Abs. As a loading control, NLRP3 immunoblot was performed. (B) BrdU-preloaded BMDM were treated with LPS+ATP, cell lysates were immunoprecipitated with Ab against NLRP1, NRLP3, or AIM2. Immune dot-blots with BrdU Ab, and respective control immunoblots are shown. (C) Mitochondrial COX1 DNA associates with NLRP3 immunoprecipitates. COX1 DNA was amplified from the NLRP3 immunoprecipitates by PCR. (D) BMDM were stimulated with or without LPS for 3 hr, then loaded with 2.5 uM MitoSOX for 20 min. Mitochondrial ROS was measured every 30 s thereafter. ATP was added 240 s after monitoring was initiated. Relative mitochondrial ROS increase at 30 min after ATP was shown. (E and F) Mitochondrial DNA was extracted from BMDM after NLRP3 inflammasome stimulation, and amounts of 8-OH-dG were quantified by (E) ELISA and (F) LC-MS-MS (means ± SD). (G) 8-OH-dG immune dot-blots were performed in WT, Casp1^–/– and Nlrp3^–/– BMDM after LPS+ATP treatment. LPS-primed BMDM were treated with 3MA for 2 hr, followed by stimulation with ATP for 30 min. Cell lysates were collected and NLRP3 Immunoprecipitation and 8-OH-dG dot-blot were performed in WT, Casp1^–/– and Nlrp3^–/– BMDM. (H and I) LPS-primed (H) WT or (I) Nlrp3^–/– BMDM were treated with exogenous 8-OH-dG incorporated DNA (oxDNA, 2 μg/ml) or control DNA for 8 hr. IL-1β and TNFα amounts were quantified in supernatants by ELISA (means ± SD). (J) LPS-primed Aim2^–/– BMDM were treated with exogenous oxDNA (2 μg/ml) and IL-1β and TNFα concentrations were determined in culture supernatants by ELISA (means ± SD).

Figure 7. 8-OH-dG inhibits mtDNA from binding to and activating the NLRP3 inflammasome

(A and B) BMDM were stimulated with LPS (1 μg/ml) in the presence of deoxyguanosine (dG) and 8-OH-dG, then treated with (A) ATP ± NIG or (B) poly(dA:dT) (2 μg/ml), and IL-1β release was quantified by ELISA (means ± SD). (C) BMDM were primed with LPS (1 μg/ml, 3 hr) in the presence of dG or 8-OH-dG (200 μM), pretreated with 3-MA (2.5 mM for 1 hr), and then treated with ATP (5 mM, 1 hr). IP with anti-NLRP3 Ab was performed, and BrdU or 8-OH-dG dot-blotting was carried out by immunoblotting. NLRP3 is shown as a loading control. Data shown are representative of three or more independent experiments. *p<0.05.