Caspase-8 as an Effector and Regulator of NLRP3 Inflammasome Signaling

Abstract

We recently described the induction of noncanonical IL-1β processing via caspase-8 recruited to ripoptosome signaling platforms in myeloid leukocytes. Here, we demonstrate that activated NLRP3·ASC inflammasomes recruit caspase-8 to drive IL-1β processing in murine bone marrow-derived dendritic cells (BMDC) independent of caspase-1 and -11. Sustained stimulation (>2 h) of LPS-primed caspase-1-deficient (Casp1/11(-/-)) BMDC with the canonical NLRP3 inflammasome agonist nigericin results in release of bioactive IL-1β in conjunction with robust caspase-8 activation. This IL-1β processing and caspase-8 activation do not proceed in Nlrp3(-/-) or Asc(-/-) BMDC and are suppressed by pharmacological inhibition of caspase-8, indicating that caspase-8 can act as a direct IL-1β-converting enzyme during NLRP3 inflammasome activation. In contrast to the rapid caspase-1-mediated death of wild type (WT) BMDC via NLRP3-dependent pyroptosis, nigericin-stimulated Casp1/11(-/-) BMDC exhibit markedly delayed cell death via NLRP3-dependent apoptosis. Biochemical analyses of WT and Casp1/11(-/-) BMDC indicated that caspase-8 is proteolytically processed within detergent-insoluble ASC-enriched protein complexes prior to extracellular export during nigericin treatment. Although nigericin-stimulated caspase-1 activation and activity are only modestly attenuated in caspase-8-deficient (Casp8(-/-)Rip3(-/-)) BMDC, these cells do not exhibit the rapid loss of viability of WT cells. These results support a contribution of caspase-8 to both IL-1β production and regulated death signaling via NLRP3 inflammasomes. In the absence of caspase-1, NLRP3 inflammasomes directly utilize caspase-8 as both a pro-apoptotic initiator and major IL-1β-converting protease. In the presence of caspase-1, caspase-8 acts as a positive modulator of the NLRP3-dependent caspase-1 signaling cascades that drive both IL-1β production and pyroptotic death.

Keywords: apoptosis; caspase 1 (CASP1); caspase 8; dendritic cell; inflammasome; inflammation; interleukin 1 (IL-1); pyroptosis.

FIGURE 1.

Sustained nigericin stimulation induces delayed processing and release of mature, bioactive IL-1β in LPS-primed Casp1/11^−/− murine BMDC. A, WT and Casp1/11^−/− BMDC were primed with LPS (100 ng/ml) for 4 h prior to stimulation with nigericin (10 μm) for 30 min and 1, 2, 4, or 6 h, and the extracellular medium was collected and assayed for IL-1β by ELISA. BMDC, which were stimulated for 30 min with nigericin (Nig), were primed with LPS for 5.5 h. Results are the mean ± S.E. of 16 experiments. The differences in IL-1β release between WT and Casp1/11^−/− BMDC treated with LPS and nigericin at all time points were significant (p < 0.001) by one-way ANOVA analysis and Bonferroni post-test. B, BMDC were stimulated as in A, and the extracellular media and cell lysates were collected and processed for Western blot analysis of IL-1β. The data are representative of results from three experiments. C, HEK^TM-Blue-IL-1R reporter cells were incubated for 18 h with either the indicated concentrations of recombinant murine IL-1β standards (upper panel) or with diluted aliquots of conditioned medium from WT or Casp1/11^−/− BMDC treated as indicated (lower panel). SEAP activity released by the reporter cells was measured, and the data points represent the mean ± S.E. of 2–5 replicates from two experiments (upper panel) or triplicates from a single experiment (lower panel) representative of two identical experiments. Parallel wells of the reporter cells were incubated with the IL-1β standards or conditioned medium sample in the presence of 10 μg/ml IL-1Ra (anakinra). D, WT and Casp1/11^−/− BMDC were primed with LPS (100 ng/ml) and stimulated with nigericin (10 μm) as in A; the extracellular medium was collected and assayed for bioactive IL-1 by the HEK^TM-Blue-IL-1R reporter assay. Data points mean ± S.E. of triplicates from a single experiment representative of two identical experiments. E, detergent-insoluble lysates from WT and Casp1/11^−/− BMDC treated with LPS and nigericin as described in A were cross-linked with DSS and run on a 12% polyacrylamide gel. Western blot analysis was used for detection of monomeric (mono.), dimeric (dimer), and oligomeric (oligo.) ASC was performed. F, BMDC were primed and stimulated as in A and B, and the extracellular media and cell lysates were collected and processed for Western blot analysis of IL-1β, caspase-1, caspase-8, and NLRP3. The data are representative of results from three experiments.

FIGURE 2.

NLRP3 inflammasomes mediate the delayed IL-1β release and caspase-8 activation responses to sustained nigericin stimulation of Casp1/11^−/− BMDC. A, WT, Asc^−/−, and Nlrp3^−/− BMDC were primed with LPS (100 ng/ml) for 4 h before stimulation with nigericin (Nig) for 30 min and 2, 4, or 6 h. The extracellular medium was collected and assayed for IL-1β by ELISA. BMDC were primed with LPS for 5.5 h prior to nigericin stimulation for 30 min. Results are the mean ± S.E. of three experiments. B, detergent-insoluble lysate fractions were cross-linked with DSS, and ASC oligomerization was assayed by Western blot analysis in WT and Nlrp3^−/− BMDC stimulated with LPS alone or LPS plus a 30-min and 2-, 4-, or 6-h nigericin stimulus. C, WT and Nlrp3^−/− BMDC were primed and stimulated as in A, and the extracellular media and cell lysates were collected and processed for Western blot analysis of IL-1β, caspase-1, and caspase-8. The data are representative of results from three separate experiments. D, BMDC were primed with LPS in low serum-containing medium (0.1% calf serum) followed by stimulation with nigericin either in a NaCl-based buffered saline solution or in a high K⁺-containing (130 mm KCl) buffered saline solution, and the amount of IL-1β released was quantified by ELISA. Results are from a single experiment with each condition performed in triplicate.

FIGURE 3.

Nigericin-induced assembly of NLRP3 inflammasomes mediates rapid pyroptosis in WT BMDC but delayed apoptosis in Casp1/11^−/− BMDC. A, WT, Casp1/11^−/−, and Nlrp3^−/− BMDC were LPS-primed (or left unprimed “w/o LPS” as indicated) for 4 h and then stimulated with nigericin (Nig) (10 μm). At the indicated times, extracellular medium was collected and assayed for lactate dehydrogenase (LDH) activity. Parallel samples of unstimulated cells were permeabilized with Triton X-100 to induce maximal LDH release for normalization of the LDH released from nigericin-stimulated cells (% of Max). Data points represent the mean ± S.E. of 6–12 replicates from four (WT) or two (Casp1/11^−/−, Nlrp3^−/−, and nonprimed WT) identical experiments. B, WT and Casp1/11^−/− BMDC were primed and stimulated as in Fig. 1F, and the extracellular media and cell lysates were collected and processed for Western blot analysis of caspase-1, caspase-8, and caspase-3; this same membrane was used for the Western blot analysis shown in Fig. 1F. C, WT, Casp1/11^−/−, and Nlrp3^−/− BMDC were LPS primed for 4 h and then stimulated with or without nigericin (10 μm) for 240 min (arrow indicates the addition of nigericin), and the accumulation of fluorescent propidium²⁺·DNA complexes was quantified every 5 min. Propidium fluorescence is expressed as a percentage of maximal dye accumulation after Triton X-100 permeabilization. Data points represent the mean ± S.E. of 2–8 replicates from four (WT) or two (Casp1/11^−/− and Nlrp3^−/−) identical experiments. D, viability of WT, Asc^−/−, and Nlrp3^−/− BMDC stimulated with LPS (4 h) or LPS (4 h) followed by a 3–4-h nigericin stimulus was assayed using the redox potential indicator dye, alamarBlue®. Viability is expressed as a percentage relative to untreated BMDC. Results are the mean ± S.E. of seven independent wells per condition from two experiments. ***, p < 0.001 by ANOVA. E and F, viability of WT and Casp1/11^−/− BMDC stimulated as described in B was measured using the redox potential indicator dye, alamarBlue®. Results are the mean ± S.E. of 2–3 experiments.

FIGURE 4.

Sustained nigericin stimulation induces caspase-1-independent release of mature IL-1β in DC primed by TRIF-independent TLR2 signaling. A, WT and Casp1/11^−/− BMDC were primed with Pam₃CSK₄ (2 μg/ml) or LPS (100 ng/ml) for 4 h prior to stimulation with nigericin (Nig) for 0, 5, or 6 h. Results are the mean ± S.E. from three independent wells for each condition. B, BMDC were primed with LPS or Pam3CysK4 for 4 h prior to stimulation with nigericin for 0.5, 1, 2, or 4 h. Western blotted samples of cell lysates and extracellular media were probed for IL-1β. Results are representative of two experiments. ns, not significant.

FIGURE 5.

IL-1β release during sustained nigericin stimulation is similarly reduced by caspase-1 or caspase-8 ablation and pharmacological inhibition of either caspase. A and B, WT, Casp1/11^−/−, and Casp8^−/− Rip3^−/− BMDC were primed with LPS (100 ng/ml) for 4 h and then stimulated with nigericin (Nig) (10 μm) for 6 h in the presence or absence of varying doses of the caspase-1 inhibitor, YVAD, or the caspase-8 inhibitor, IETD, and the extracellular medium was collected and assayed for IL-1β by ELISA. Results are the mean ± S.E. from six independent wells for each condition and expressed as a percentage of WT BMDC treated with LPS + nigericin (6 h). ***, p < 0.001 or not significant (n.s.) by ANOVA. C and D, BMDC were treated exactly as described in Fig. 3, A and B, but IL-1β release is expressed as a percentage of each genotype treated with LPS + nigericin (6 h).

FIGURE 6.

Nigercin-stimulated IL-1β release from Casp1/11^−/− BMDC is mediated by caspase-8. A and B, kinetics of IL-1β release were assayed by ELISA from LPS-primed (100 ng/ml) and nigericin-stimulated WT and Casp1/11^−/− BMDC in the presence or absence of IETD (20 μm) or YVAD (10 μm). Results are the mean ± S.E. of three experiments. C, detergent-insoluble lysate fractions were cross-linked with DSS, and ASC oligomerization was assayed in WT and Casp1/11^−/− BMDC stimulated with LPS alone (5 h) plus 30 min nigericin (Nig) in the presence or absence of IETD (20 μm), YVAD (10 μm), and Z-VAD (50 μm). Results are representative of two similar experiments performed. D, LPS-primed WT or Casp1/11^−/− BMDC were stimulated with nigericin for 0.5 or 6 h in the presence or absence of YVAD (10 μm) or IETD (20 μm), and Western blot analysis of IL-1β, caspase-1, and caspase-8 from cell lysates and extracellular supernatants was performed. BMDC treated with 30 min of nigericin were LPS-primed for 5.5 h. Results are representative of three experiments performed.

FIGURE 7.

Caspase-8 is proteolytically processed within detergent-insoluble protein complexes prior to extracellular export from nigericin-stimulated dendritic cells. WT and Casp1/11^−/− BMDC were treated with LPS (4 h) and nigericin (Nig) (6 h) in the presence or absence of Z-VAD (50 μm) or IETD (20 μm). The extracellular media, detergent-soluble lysate fractions, and detergent-insoluble lysate fractions were collected and processed for Western blot analysis of caspase-8 (A) ASC (B), caspase-1 (C), and IL-1β (D). A and D, membrane with extracellular media samples was simultaneously probed with anti-IL-1β and anti-caspase-8 antibodies. Results are from one experiment representative of two similar studies.

FIGURE 8.

Comparative effects of nigericin stimulation on IL-1β processing, caspase-1 activation, caspase-8 activation, ASC oligomerization, propidium²⁺ influx, and cell viability in WT, Casp8^−/−Rip3^−/− and Rip3^−/− murine BMDC. A, WT, Casp8^−/− Rip3^−/− and Rip3^−/− BMDC were LPS-primed for 4 h and then stimulated with either nigericin (Nig) for 30 min or 6 h or stimulated with the pan-caspase inhibitor, Z-VAD, for 3 h. The extracellular media and detergent-soluble cell lysates were collected and processed for Western blot analysis for detection of IL-1β, caspase-1, caspase-8, and ASC. Results are representative of two experiments performed. B, cells were stimulated as in A and detergent-insoluble lysates (±DSS) were collected and processed for detection of ASC, caspase-8, or caspase-1. C, WT, Casp8^−/− Rip3^−/− and Rip3^−/− BMDC were LPS-primed and stimulated with nigericin (10 μm) in the absence or presence of Z-VAD for 40 min (arrow indicates the addition of nigericin), and the accumulation of fluorescent propidium²⁺·DNA complexes was quantified every 5 min. Data points represent the average ± range of values from two experiments. D, WT, Casp8^−/− Rip3^−/−, and Rip3^−/− BMDC were primed with LPS (100 ng/ml) for 4 h prior to stimulation with nigericin (10 μm) for 1, 2, or 6 h. Cell viability was assayed by the redox potential indicator dye, alamarBlue®. Results are representative of three similar experiments.