Mitochondrial reactive oxygen species induces NLRP3-dependent lysosomal damage and inflammasome activation

Abstract

The nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome drives many inflammatory processes and mediates IL-1 family cytokine release. Inflammasome activators typically damage cells and may release lysosomal and mitochondrial products into the cytosol. Macrophages triggered by the NLRP3 inflammasome activator nigericin show reduced mitochondrial function and decreased cellular ATP. Release of mitochondrial reactive oxygen species (ROS) leads to subsequent lysosomal membrane permeabilization (LMP). NLRP3-deficient macrophages show comparable reduced mitochondrial function and ATP loss, but maintain lysosomal acidity, demonstrating that LMP is NLRP3 dependent. A subset of wild-type macrophages undergo subsequent mitochondrial membrane permeabilization and die. Both LMP and mitochondrial membrane permeabilization are inhibited by potassium, scavenging mitochondrial ROS, or NLRP3 deficiency, but are unaffected by cathepsin B or caspase-1 inhibitors. In contrast, IL-1β secretion is ablated by potassium, scavenging mitochondrial ROS, and both cathepsin B and caspase-1 inhibition. These results demonstrate interplay between lysosomes and mitochondria that sustain NLRP3 activation and distinguish cell death from IL-1β release.

Figure 1. NLRP3 dependent cell membrane damage

(A)LPS primed or unprimed WT and NLRP3^−/− BMDM were treated with 20 µM nigericin or 3 mM ATP for thirty minutes with or without KCl. LDH release was recorded as the percent released into the supernatant by five minutes of Triton treatment. LDH assay was allowed to develop for two hours and readings were taken as an average of two wells. Error bars represent mean ± s.d. (n=3, *, P<0.01; LPS Nig WT vs KO: P=0.009; WT LPS Nig vs WT LPS Nig KCl: P=0.004). (B) Conversion of MTT reagent added to cell lysates was measured as absorbance of the colored product formazan. Data were acquired as the percent of absorbance of each well/absorbance of Triton-X treated control cells and graphed as MTT reagent remaining unconverted by mitochondrial reductases. Readings were taken as an average of two wells. Error bars represent mean ± s.d. (n=4, ***, P<0.0001; n=3, **, P=0.0155).

Figure 2. Loss of cellular ATP and decrease in OCR are NLRP3 independent

WT or NLRP3 ^−/− BMDM were LPS primed and (A) treated with nigericin for thirty minutes with or without KCl. Supernatants were collected and ATP levels measured. Error bars represent ± s.d. (n≥6, *, P=0.0103; ns, p=0.7941) WT BMDM (B) or NLRP3^−/− BMDM (C) were treated with nigericin immediately before inserting plate in Seahorse machine. OCR was recorded as an average of 3–4 wells. Media (line a), oligomycin (b), FCCP (c), and rotenone (d) were added as indicated. Error bars represent ± s.d.(n=4, at 45 min, ns, P=0.3099).

Figure 3. Loss of mitochondrial membrane permeability and cell death are NLRP3 dependent

(A) WT and NLRP3 ^−/− BMDM were LPS primed and treated with 20 µM nigericin. Cells were stained with MitoTracker red (red) and pSIVA (green), imaged for one hour with a confocal live cell microscope. Images from one representative experiment shown. Scale bar= 50 µm (B) Loss of MitoTracker red signal over time was recorded via live cell microscopy. Cells were left untreated or treated with 3mM ATP. Data was normalized to initial signal with background subtraction. (n≥9, at 45 min *, P=0.0382 for WT LPS ATP vs KO LPS ATP; **, P=0.0021 for WT LPS vs WT LPS ATP). (C) Cells were imaged as in (B) with 20 µM nigericin (n=6; at 40 min. ***, P= 0.0001; **, P=0.0015) (D) Percentage of cells/field that lost MitoTracker signal or gained pSIVA signal over 1 hour of confocal live cell imaging. Error bars represent ± s.d. (MitoTracker n=4; **, P=0.002; pSIVA, n=3; **, P=0.002) (E) Cells were treated with or without 50 mM KCl and imaged for one hour on a confocal live cell microscope. Percent of cells/field that were pSIVA positive at 60 min recorded. Error bars represent ± s.d. (n=3; ***, P=0.0001).

Figure 4. Loss of lysosomal membrane permeability is NLRP3 dependent

(A) WT and NLRP3^−/− BMDM were LPS primed, stained with LysoTracker Green and imaged via live cell confocal microscope with 20 µM nigericin treatment for 30 min. Data from one representative experiment shown. Scale bar= 50 µm (B) WT BMDM were LPS primed, treated with 20 µM nigericin or 3 mM ATP, and stained with MitoTracker Red and LysoTracker Green. Cells were imaged for one hour after nigericin or ATP addition via live cell microscopy. Data were recorded as time to loss of signal, each dot represents one cell with larger dots representing multiple cells. Cells that did not lose MitoTracker Red were recorded as data points beyond 60 min imaging period. Data from five fields are displayed. (C) WT BMDM or NLRP3 KO BMDM were LPS primed and nigericin treated, stained with TMRM and LysoTracker Green, and imaged with live cell microscopy for 1 hr. Percent of cells/field staining positive for each of the two dyes was recorded. n≥5 fields.

Figure 5. IL-1 beta processing and secretion is kinetically distinct from MMP

WT BMDM were LPS primed or unprimed and (A) treated with 20 µM nigericin for 5–30 minutes. Supernatants were collected and analyzed for IL-1β using ELISA. Error bars represent ± s.d. n=3 (B) WT BMDM were treated with nigericin for thirty minutes. 50 mM KCl was added after 0–25 minutes following nigericin treatment. Supernatants were collected and analyzed via IL-1β ELISA. Error bars represent ± s.d. n≥3 (*, P=0.0294 for LPS Nig KCl 25 vs LPS Nig). (C) WT BMDM were LPS primed and treated with nigericin for 5–30 minutes or (D) were LPS primed and then had 50 mM KCl added 0–25 minutes following nigericin treatment. Supernatants were collected for LDH release analysis. Data shown as percentage of LDH release by Triton-X 100 treated control cells. Error bars represent ± s.d. n=4 (*, P=0.0475 for LPS Nig KCl 25 vs LPS Nig).

Figure 6. Mitochondrial ROS is required for NLRP3 inflammasome dependent events

(A) WT BMDM were LPS primed, treated with nigericin following YVAD exposure, and labeled with MitoTracker Red and LysoTracker Green. Cells imaged via live cell microscopy for 1 hr. Data presented as positive cells per field at 60 min. Error bars represent ± s.d. n=5. WT BMDM were LPS primed and treated with nigericin following 100 µM YVAD, or 100 µM or 500 µM MitoTEMPO (Mito T). Supernatants were analyzed for (B) IL-1β via ELISA or (C) LDH release as percentage of LDH release by Triton-X 100 treated control cells. Error bars represent ± s.d. n=3 (D) WT BMDM were LPS primed, nigericin and 500 µM MitoTEMPO treated. LysoTracker and MitoTracker (not shown) MFI recorded via live cell microscopy. The LysoTracker MFI was normalized to initial signal and background corrected. LysoTracker data are presented as one line/cell from five representative fields and split into two groups: cells that lost MitoTracker signal after 60 minutes of imaging (top panel) and cells that retained MitoTracker signal throughout the experiment (lower panel). (E) WT BMDM were LPS primed and nigericin or ATP treated with or without 500 µM MitoTEMPO. Cells were labeled with MitoTracker Red and imaged via live cell microscopy. Data are presented as percent of cells/field retaining MitoTracker signal. Error bars represent ±s.d. n≥ 10 fields. (**, P=0.0061; ***, P< 0.0001).