Carbon monoxide negatively regulates NLRP3 inflammasome activation in macrophages

Abstract

Inflammasomes are cytosolic protein complexes that promote the cleavage of caspase-1, which leads to the maturation and secretion of proinflammatory cytokines, including interleukin-1β (IL-1β) and IL-18. Among the known inflammasomes, the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3)-dependent inflammasome is critically involved in the pathogenesis of various acute or chronic inflammatory diseases. Carbon monoxide (CO), a gaseous molecule physiologically produced in cells and tissues during heme catabolism, can act as an anti-inflammatory molecule and a potent negative regulator of Toll-like receptor signaling pathways. To date, the role of CO in inflammasome-mediated immune responses has not been fully investigated. Here, we demonstrated that CO inhibited caspase-1 activation and the secretion of IL-1β and IL-18 in response to lipopolysaccharide (LPS) and ATP treatment in bone marrow-derived macrophages. CO also inhibited IL-18 secretion in response to LPS and nigericin treatment, another NLRP3 inflammasome activation model. In contrast, CO did not suppress IL-18 secretion in response to LPS and poly(dA:dT), an absent in melanoma 2 (AIM2)-mediated inflammasome model. LPS and ATP stimulation induced the formation of complexes between NLRP3 and apoptosis-associated speck-like protein, or NLRP3 and caspase-1. CO treatment inhibited these molecular interactions that were induced by LPS and ATP. Furthermore, CO inhibited mitochondrial ROS generation and the decrease of mitochondrial membrane potential induced by LPS and ATP in macrophages. We also observed that the inhibitory effect of CO on the translocation of mitochondrial DNA into the cytosol was associated with suppression of cytokine secretion. Our results suggest that CO negatively regulates NLRP3 inflammasome activation by preventing mitochondrial dysfunction.

Keywords: interleukin-18; mitochondria; nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3.

Fig. 1.

Carbon monoxide (CO) suppressed interleukin (IL)-1β and IL-18 secretion in vitro and in vivo. A: bone marrow-derived macrophages (BMDMs) were stimulated with lipopolysaccharide (LPS, 10 ng/ml) for 6 h and then stimulated with ATP (2 mM) for an additional 1 h in the presence or absence of CO [250 parts/million (ppm)]. IL-1β and IL-18 in the supernatants were measured by ELISA. Data are representative of 3 experiments. B: CO exposure was initiated at different time points (−2 h, 2 h before LPS treatment; 0 h, cotreatment with LPS; +3, 3 h after LPS treatment; +5, 5 h after LPS treatment). IL-1β secretion was analyzed by ELISA. Data are representative of 3 experiments. C: J774A.1 macrophages were stimulated with LPS (200 ng/ml) for 4 h, followed by ATP (3 mM) for 1 h. IL-1β and IL-18 secretion was analyzed by ELISA. Data are representative of 3 experiments. D: LPS-primed BMDMs were stimulated with nigericin (3.3 μM). IL-18 in the supernatant was analyzed by ELISA. E: LPS-primed BMDMs were transfected with poly(dA:dT) (1 μg/ml). IL-18 in the supernatant was analyzed by ELISA. Data are representative of 3 experiments. All data represent means ± SE. **P < 0.01 by unpaired, 2-tailed Student's t-test (A–D). F: mice were exposed to 250 ppm CO or ambient room air (RA) for 1 h before receiving ip injections of LPS (2.5 mg/kg) or PBS; n = 6/group. Blood and lungs were collected 24 h after LPS treatment. The levels of IL-1β and IL-18 in serum and lung homogenates were analyzed by ELISA. Data are representative of 2 experiments. All data represent means ± SE. *P < 0.05 vs. RA + PBS, ^#P < 0.05 vs. CO + PBS, and ^†P < 0.05 vs. RA + LPS by 2-way ANOVA with Bonferroni's posttests.

Fig. 2.

CO inhibited caspase-1 activation in macrophages. BMDMs were incubated with LPS for 6 h, followed by stimulation with ATP for the indicated time in the presence or absence of CO. Cell lysates were analyzed by immunoblotting for caspase-1, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3), IL-1β, P2X₇ receptor, apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and β-actin. Data are representative of 3 experiments.

Fig. 3.

Effect of CO on NLRP3 inflammasome complex formation. A: BMDMs were incubated with LPS for 6 h, followed by stimulation with ATP for 15 min in the presence or absence of CO. Interaction between ASC and NLRP3 was analyzed by immunoprecipitation and immunoblotting. Expression of ASC and β-actin was analyzed by immunoblotting using the identical total cell lysates. Data are representative of 3 experiments. B: BMDMs were incubated with LPS for 6 h, followed by stimulation with ATP for 15 min in the presence or absence of CO. Interaction between NLRP3 and caspase-1 or NLRP3 and ASC was analyzed by immunoprecipitation and immunoblotting. Expression of NLRP3 and β-actin was analyzed by immunoblotting using the identical total cell lysates. Data are representative of 3 experiments.

Fig. 4.

Effect of CO on mitochondrial reactive oxygen species (ROS) production in response to LPS and ATP. A: LPS-primed BMDMs were stained with MitoSOX for 15 min before stimulation with ATP for 15 min in the absence or presence of CO, and then analyzed by flow cytometry. The x-axis shows the fluorescent signal intensity, and the y-axis represents the cell number normalized as a percentage of the maximum (%max). Data are representative of 3 experiments. B: LPS-primed macrophages were treated with Mito-TEMPO (100 μM) for 30 min before incubation with ATP for 1 h in the presence or absence of CO. IL-18 secretion in the supernatants was analyzed by ELISA. Data are representative of 3 experiments. All data represent means ± SE. **P < 0.01 by unpaired, 2-tailed Student's t-test (A and B).

Fig. 5.

CO inhibited decrease of mitochondrial membrane potential in response to LPS and ATP. A: LPS-primed BMDMs were stained with tetramethylrhodamine, ethyl ester (TMRE) for 15 min, followed by stimulation with ATP for 15 min. BMDMs treated with carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP, 20 μM) for 15 min were used as a positive control. MFI, mean fluorescence intensity. The x-axis shows the fluorescent signal intensity, and the y-axis represents the cell number normalized as a percentage of the maximum. Data are representative of 3 experiments. B: LPS-primed BMDMs were stained with TMRE for 15 min, followed by stimulation with ATP for 15 min in the presence or absence of Mito-TEMPO (100 μM). The x-axis shows the fluorescent signal intensity, and the y-axis represents the cell number normalized as a percentage of the maximum. Data are representative of 3 experiments. C: LPS-primed BMDMs were stained with TMRE for 15 min, followed by stimulation with ATP for 15 min in the presence or absence of CO. The x-axis shows the fluorescent signal intensity, and the y-axis represents the cell number normalized as a percentage of the maximum. Data are representative of 3 experiments. D: LPS-primed BMDMs were stained with MitoTracker Deep Red and MitoTracker Green for 15 min, followed by stimulation with ATP for 15 min in the presence or absence of CO. Data are representative of 3 experiments. E: LPS-primed macrophages were incubated with rotenone (5 μM) or DMSO for 45 min before stimulation with ATP for 1 h in the presence or absence of CO. Cytokine secretion was analyzed by ELISA. Data are representative of 3 experiments. F: LPS-primed macrophages were incubated with 5 or 10 μM of cyclosporine A or DMSO for 1 h before stimulation with ATP for 1 h. IL-18 secretion was measured by ELISA. Data are representative of 3 experiments. All data represent means ± SE. **P < 0.01 or *P < 0.05 by unpaired, 2-tailed Student's t-test (A–C, E, and F).

Fig. 6.

CO decreased release of mitochondrial DNA (mtDNA) into cytosol. A: LPS-primed macrophages were incubated with ATP for 15 min in the presence or absence of CO. Cytosolic mtDNA copy number was measured by quantitative PCR. Data are representative of 3 experiments. B: BMDMs were transfected with 3 μg of DNase I for 4 h and then stimulated with LPS, followed by ATP stimulation for 1 h in the absence or presence of CO. Cytokine secretion into the supernatants was analyzed by ELISA. Data are representative of 3 experiments. All data represent means ± SE. **P < 0.01 by unpaired, 2-tailed Student's t-test (A and B).