Mitochondrial ROS induces NLRP3-dependent lysosomal damage and inflammasome activation¹

BMDM preparation

As previously described (19), bone marrow was harvested from the tibiae and femora of WT or NLRP3^−/− B6 mice (gifts from Dr. Lisa Borghesi, Dr. Olivera Finn, and Dr. Timothy Billiar). Cells were grown in the presence of 20% FBS (Gemcell), 2 mM L-glutamine (Cellgro), 500 U penicillin/500 µg streptomycin (Lonza), 1 mM sodium pyruvate (MP Biomedicals), and 30% L cell supernatant made from culturing L cell fibroblasts (CCL-1 from American Type Culture Collection) for about six days. Media of monocytes was replaced after four days. Cells were then harvested and grown in IMDM (HyClone), called cIMDM when supplemented with 10% FBS, 2 mM L-glutamine, 500 U penicillin/500 µg streptomycin and were harvested with Cellstripper (Cellgro) for experiments from day 8–25.

TLO Preparation

Tetanolysin O (Enzo) was reduced as previously described (19). Serum containing media was washed out prior to the addition of TLO to cells. 100 ng/ml was added in serum free media to appropriate dishes and incubated at 37°C for the duration of imaging as indicated.

MTT assay

WT or NLRP3^−/− BMDM were plated the day before the experiment at 500,000 cells/well in a 12 well tissue culture plate (Costar) in cIMDM, as described above. Cells were incubated for 4 hours with 1 µg/ml LPS (Sigma). 50 mM KCl, 100 µM or 500 µM MitoTEMPO (Enzo), or 100 µM Ac-YVAD-CMK (Alexis) were added during the last 30 minutes of LPS treatment and then re-added when media was removed and 1ml fresh phenol red-free supplemented IMDM (Gibco) was added to the wells. 20 µM of nigericin sodium salt (Sigma) or 30 mM ATP (Sigma) was added to the wells and incubated at 37°C for 5–30 minutes. After supernatants were removed, cells were washed once with 1× DPBS (Cellgro) and the Vybrant® MTT cell proliferation assay kit (Invitrogen/Molecular Probes) was used according to the manufacturer’s instructions. Samples were then assayed in duplicate for OD using a PowerWave XS microplate spectrophotometer. Percent MTT converted to formazan in the sample was calculated by (mean OD of sample-mean OD of blank)/(mean OD of untreated control- mean OD of blank) X 100. Percent unconverted MTT was graphed.

LDH Release Assay

WT and NLRP3−/− BMDM were treated as described above for the MTT assay. 1% Triton X-100 (Fisher Scientific) was incubated with cells for five minutes as a positive control for complete cell lysis. Supernatants were removed and assayed in duplicate using an LDH cytotoxicity kit (Cayman Chemical) for OD using a PowerWave XS microplate spectrophotometer (BioTek). Percent LDH release was calculated as (mean OD value of sample-mean OD value of blank)/(mean OD value of TritonX-100 control sample – mean OD of blank) X100.

Quantification of ATP remaining in cell

WT or NLRP3^−/−BMDM were treated as described for the LDH assay. Supernatants were removed. Using CellTiter-Glo Luminescent Cell Viability Assay (Promega), ATP levels remaining in the cell lysates were determined using a Berthold Orion microplate luminometer according to the kit instructions and Simplicity 2.1 software. Results were recorded as a percentage of ATP remaining in cells based on untreated control cells.

Seahorse Assay

WT or NLRP3^−/− BMDM were plated the night before at 500,000 cells/well in a Seahorse 24 well V7 microplate. Cells were LPS primed at 1 µg/ml for four hours. Media was removed from wells and cells were incubated in buffered growth media for an hour after pre-treatment with nitrite. 20 µM nigericin was added into wells in unbuffered media prior to inserting plate into Seahorse XF24 Extracellular Flux analyzer (Seahorse Bioscience). After 21.5 minutes of equilibration, media was injected as a control, oligomycin (20 µM) was injected into each well at 48 minutes, followed by FCCP (75 µM) at 73 minutes, and rotenone (20 µM) at 99 minutes. OCR was recorded as pMoles/min. Averages of three wells were taken per data point.

Live cell microscopy

WT or NLRP3^−/− BMDM were plated at 250,000 cells/dish in 35 mm collagen coated glass bottom culture dishes (MatTek) the night before the experiment in cIMDM. Media was removed and cells were LPS primed (1 µg/ml) for three to four hours in fresh media. Inhibitors were added during the last 30 minutes of LPS priming at 37°C and were added back to the dish in fresh media before imaging, following washing out LPS containing media. YVAD was added at 100 µM, CA-O74 Me (Calbiochem) was added at 100 µM. 2µM MitoTracker Red CM-H2XRos (Invitrogen), 10 µM TMRM, or 2 µM LysoTracker Green DND-26 (Invitrogen) were added during the last 30 minutes of LPS priming. 10 µl/ml pSIVA (Imgenex) was added in 1 ml of complete IMDM two frames after the initiation of imaging after 30 minutes pre-incubation. Dishes were imaged on Nikon A1 inverted microscope with a Plan Fluor 40× objective with an 1.30 NA or Plan Apo 60× objective with an 1.40 NA, Photometrics Coolsnap HQ2 or Andor™ Technology iXon^EM + camera, NikonPiezo driven XYZ stage, and Tokai Hit Environmental chamber. For MitoTracker, LysoTracker, TMRM, and pSIVA, images were captured every 30 seconds for one hour. 20 µM nigericin or 3 mM ATP was added 2 minutes into imaging in 1ml of media. Videos were analyzed using Elements (Nikon) and Microsoft Excel.

IL-1β ELISA

WT or NLRP3^−/− BMDM were plated at 500,000 cells/well in a 12 well tissue culture plate the night before. Cells were LPS primed for four hours and treated as indicated. An IL-1β ELISA was performed as previously described (19). OD values were read at 450 nm with 570 nm background subtraction using BioTek® Powerwave XS Microplate Spectrophotometer and Gen5^TM Data Analysis Software.

IL-1β Western Blot

Lysates and supernantants were collected from 2.0 × 10⁶ BMDM after treating cells as indicated in serum-free IMDM. Supernatants were TCA precipitated and both lysates and supernatants were analyzed for IL-β via Western blot as previously described (19). Western blotting luminal reagent (Santa Cruz) was added before imaging on a Kodak Image Station 4000MM (Molecular Imaging Systems).

Statistical analysis

All data sets were analyzed via a student’s two-tailed t-test unless otherwise noted using Graphpad software. P values were as indicated and were considered significant if P<0.05.

NLRP3 inflammasome activation increases sensitivity of bone marrow derived macrophages to membrane damage

The NLRP3 inflammasome is present in a variety of immune cells, including macrophages. Due to the robustness of NLRP3 activation in murine bone marrow derived macrophages (BMDM), this system has frequently been employed to examine NLRP3 inflammasome activation and function. BMDM were primed with LPS for three to four hours to up-regulate inflammasome components as well as induce the translation of pro-IL-1β, a cytokine that is processed into its active form during NLRP3 inflammasome dependent caspase-1 activation. Following priming, BMDM from wild type C57/BL6 mice were treated with the potassium ionophore nigericin, a prototypical activator of the NLRP3 inflammasome.

To test membrane permeability, lactate dehydrogenase (LDH) was measured in the culture supernatants. Following nigericin treatment, LDH levels were increased in the supernatants of LPS primed wild type BMDM but not LPS primed NLRP3^−/− cells, indicating that the cell membrane integrity had been compromised in the former (Figure 1A). Pre-treatment of WT BMDM with 50 mM KCl to block potassium efflux significantly inhibited release of LDH. Non-LPS primed or BMDM treated with LPS alone did not release LDH above background levels.

An additional assay of cell viability, the conversion of MTT to formazan, which is carried out by mitochondrial reductases, showed similar results. LPS-primed WT and NLRP3 ^−/− BMDM differed significantly in MTT reduction after nigericin exposure (Figure 1B). We next tested whether ATP, a second well characterized stimulator of NLRP3 inflammasome, promoted similar responses. As with nigericin, 3mM ATP treatment resulted in an inflammasome dependent reduction of MTT conversion to formazan. Together, these results demonstrate that the NLRP3 inflammasome mediates membrane damage.

Nigericin alters mitochondrial function independently of NLRP3

To explore how inflammasome activating stimuli impact mitochondrial function, we measured ATP levels in LPS primed BMDM following nigericin exposure. Both WT and NLRP3^−/− BMDM treated with nigericin contained reduced amounts of ATP compared to non-nigericin treated cells, suggesting either decreased synthesis, increased consumption, or release of ATP into the culture supernatant (Figure 2A). ATP levels in nigericin treated cells were not preserved by co-exposure to KCl, consistent with a NLRP3-independent event.

To further characterize nigericin-induced alterations in mitochondrial function, we used the Seahorse assay to measure oxygen consumption rate (OCR) in WT and NLRP3^−/− BMDM. Basal OCR levels in non-primed WT and NLRP3^−/− BMDM were comparable, while both showed reduced OCR levels four hours after LPS priming. Nigericin rapidly increased OCR levels in both LPS primed WT and LPS primed NLRP3^−/− BMDM, indicating a stress response in mitochondrial respiration (Figures 2B and 2C). Following nigericin exposure, cells from both cell types were unable to respond to oligomycin or FCCP, which respectively inhibit ATP synthesis and uncouple electron transport. In contrast, LPS primed WT or NLRP3^−/− BMDM decreased OCR in response to oligomycin, followed by increased OCR in response to FCCP, which could be blocked by rotenone. These data show that LPS priming and nigericin each disrupt mitochondrial respiration in BMDM independently of NLRP3, with more severe dysfunction occurring after nigericin treatment. Taken together, these data demonstrate that nigericin reduces OCR and diminishes cellular ATP levels independently of NLRP3 in BMDM.

Loss of mitochondrial membrane integrity and cell death are NLRP3 dependent

Since WT and NLRP3^−/− BMDM differ greatly in their ability to convert MTT through mitochondrial reductases (Fig 1B), we further probed potential effects of NLRP3 on mitochondria using MitoTracker Red to measure membrane integrity. Using live cell microscopy, we found that nigericin- or ATP- treated, LPS primed WT and NLRP3^−/− BMDM showed distinct differences in mitochondrial integrity (Figures 3A– C, and Supplementary Videos 1 and 2). Approximately 30% of WT BMDM exhibited complete loss of mitochondrial integrity, while no loss was detected in NLRP3^−/− BMDM in one hour (Figure 3D). In LPS primed untreated or non-primed nigericin treated BMDM, no loss of mitochondrial integrity was seen (Figures 3B,C). Furthermore, both nigericin and ATP induced MMP could be significantly reduced with exposure to KCl (Figure S1A). Loss of mitochondrial membrane integrity correlated with exposure of phosphatidylserine (PS) at the plasma membrane (Figures 3A and 3D). PS exposure was inhibited by the addition of KCl (Figure 3E). These data demonstrate that NLRP3 inflammasome increases sensitivity of BMDM to loss of mitochondrial membrane integrity and subsequent cell death.

We investigated mtDNA localization in cells using PicoGreen, a dsDNA specific dye which labels mitochondria and nuclei (20). We observed a decrease in mitochondrial PicoGreen staining in LPS-primed WT BMDM, but not in NLRP3^−/− BMDM after 1h exposure to nigericin (Supplementary Video 3 and 4). Loss of mtDNA in WT cells was prevented by KCl (data not shown), and coincided with loss of MitoTracker Red (Supplementary Video 3).

Lysosomal membrane permeabilization precedes mitochondrial integrity breakdown during NLRP3 activation

A previously proposed model for NLRP3 activation suggested that following phagocytic uptake of indigestible particles known to trigger IL-1β secretion, rupture of phagolysosomes would release proteases into the cytosol leading to assembly and activation of the NLRP3 inflammasome (7). We evaluated one aspect of this model using live cell imaging. Lysosomes labeled with Lysotracker Green lose fluorescence within 15 min after exposure to nigericin in LPS-primed WT cells (Figure 4A), consistent with either loss of lysosomal acidity or a loss of lysosomal membrane integrity in a process termed LMP. Labeled lysosomes in NLRP3^−/− BMDM however retained fluorescence after nigericin treatment, demonstrating that LMP is NLRP3 dependent. LMP was also induced in WT, but not NLRP3^−/− BMDM, by two other NLRP3 inflammasome activators, the pore forming toxin tetanolysin O (TLO) (Figure S1 B) and ATP (and data not shown). LMP always preceded MMP in both nigericin and ATP treated WT BMDM (Figure 4B). In nigericin treated cells, LMP occurred within 15 minutes of exposure to the stimulus, while MMP typically required between 30–60 min. In ATP treated cells, LMP occurred within 10 minutes of exposure to the stimulus and MMP typically required 15 to 30 min. In addition, all WT BMDM exposed to nigericin underwent LMP, while on average no more than 60–70% underwent MMP within 60 min (Figure 4C). In contrast, NLRP3^−/− BMDM did not undergo LMP or MMP with the 60 min experiment. LMP was blocked in WT BMDM by KCl, consistent with NLRP3 dependence of the process (Figure S1 C). Our results demonstrate that NLRP3 inflammasome activation results in loss of membrane integrity in lysosomes and mitochondria in kinetically distinct events.

IL-1β processing and secretion kinetically precedes loss of mitochondrial membrane integrity

A major function of the NLRP3 inflammasome in immunity is to regulate secretion of a family of non-conventionally secreted cytokines, including IL-1β. To probe the role of mitochondria and lysosomes in cytokine secretion, we performed kinetic analysis of processed IL-1β secretion. Processed IL-1β was detected in the supernatants of LPS primed WT BMDM 15 minutes after nigericin exposure, and levels continued to increase over thirty minutes of nigericin exposure (Figure 5A). Similar kinetics of IL-1β secretion were observed in LPS primed ATP treated WT BMDM (data not shown). Western blotting was used to confirm that released IL-1β was the cleaved biologically active form, not unprocessed precursor (Figure S2). IL-1β release was significantly decreased by addition of KCl five minutes after nigericin exposure, with progressively less inhibition occurring with KCl added at later times within the 30 min nigericin incubation period (Figure 5B). Since LPS primed, nigericin exposed BMDM lose mitochondrial integrity and expose plasma membrane PS by about 30 minutes (Figures 3 and 4), this suggests that IL-1β secretion precedes cell death and MMP, and correlates kinetically with LMP. LDH release occurred with nearly identical kinetics to IL-1β secretion and was also inhibited by KCl, consistent with compromised plasma membrane integrity before cell death (Figures 5C–D).

IL-1β secretion distinct from organelle damage and cell death

To characterize the mechanisms underlying MMP, LMP, and IL-1β processing and secretion, we examined the requirement for caspase-1 in each process. Ac-YVAD-cmk (YVAD), a caspase-1 inhibitor, did not prevent the loss of lysosomal membrane integrity or mitochondrial membrane integrity in WT BMDM treated with nigericin (Figure 6A). Furthermore, YVAD pre-treatment blocked processing and secretion of IL-1β but did not significantly inhibit LDH release from WT BMDM (Figures 6B and 6C). While caspase-1 activity is required for the processing and secretion of mature IL-1β, it does not appear to be required in its active form to trigger LMP, MMP, or cell death.

We and others have shown the cathepsin B inhibitor, CA-074 Me, inhibits secretion of processed IL-1β (7, 19). Cathepsin B is a lysosomal protease that is likely released during LMP. Since processing and secretion of IL-1β correlates kinetically with LMP (Figures 4 and 5), we tested whether cathepsin B participates in MMP. CA-074 Me did not inhibit LMP or MMP induced by nigericin in LPS primed BMDM (Figure S3 A). These results show that the role of NLRP3 inflammasome in IL-1β processing and secretion is distinct from effects on lysosomal and mitochondrial membrane integrity in its dependence on caspase-1 and cathepsin B.

NLRP3 dependent organelle damage and cytokine release is mediated by mitochondrial ROS

Next we examined the potential role for mitochondrial ROS in IL-1β processing and secretion, LDH release, LMP, and MMP. MitoTEMPO, a mitochondria specific ROS scavenger, reduced both IL-1β secretion after nigericin or ATP exposure (Figure 6B) and LDH release after nigericin exposure in LPS primed WT BMDM (Figure 6C) (17). In addition, 500 µM MitoTEMPO also delayed LMP and reduced subsequent MMP in nigericin treated WT BMDM (Figures 6D and 6E and Supplemental Table I). Furthermore, LMP was significantly delayed and MMP reduced when ATP exposed WT BMBM were treated MitoTEMPO (Figure 6E and Figure S3 B). Together, these data suggest that mitochondrial ROS production is an early event that may lead to NLRP3 activation, as well as inflammasome dependent LMP, IL-1β secretion, MMP, and cell death.