P2X7 receptor-stimulated secretion of MHC class II-containing exosomes requires the ASC/NLRP3 inflammasome but is independent of caspase-1

Abstract

We recently reported that P2X7 receptor (P2X7R)-induced activation of caspase-1 inflammasomes is accompanied by release of MHC class II (MHC-II) protein into extracellular compartments during brief stimulation of murine macrophages with ATP. Here we demonstrate that MHC-II containing membranes released from macrophages or dendritic cells (DCs) in response to P2X7R stimulation comprise two pools of vesicles with distinct biogenesis: one pool comprises 100- to 600-nm microvesicles derived from direct budding of the plasma membrane, while the second pool is composed of 50- to 80-nm exosomes released from multivesicular bodies. ATP-stimulated release of MHC-II in these membrane fractions is observed within 15 min and results in the export of approximately 15% of the total MHC-II pool within 90 min. ATP did not stimulate MHC-II release in macrophages from P2X7R knockout mice. The inflammasome regulatory proteins, ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain) and NLRP3 (NLR family, pyrin domain containing 3), which are essential for caspase-1 activation, were also required for the P2X7R-regulated release of the exosome but not the microvesicle MHC-II pool. Treatment of bone marrow-derived macrophages with YVAD-cmk, a peptide inhibitor of caspase-1, also abrogated P2X7R-dependent MHC-II secretion. Surprisingly, however, MHC-II release in response to ATP was intact in caspase-1(-/-) macrophages. The inhibitory actions of YVAD-cmk were mimicked by the pan-caspase inhibitor zVAD-fmk and the serine protease inhibitor TPCK, but not the caspase-3 inhibitor DEVD-cho. These data suggest that the ASC/NLRP3 inflammasome complexes assembled in response to P2X7R activation involve protease effector(s) in addition to caspase-1, and that these proteases may play important roles in regulating the membrane trafficking pathways that control biogenesis and release of MHC-II-containing exosomes.

Figure 1. P2X7R-dependent release of MHC-II from inflammatory murine BMDM

A, B. C57BL/6 BMDM were plated 2× 10⁶/well, and pretreated with IFN-γ (2 ng/ml) for 16–18 h before LPS (1 μg/ml) priming for 4 h. Cells were transferred to either BSS or DMEM and stimulated with 5 mM ATP for the indicated times. The extracellular media and cell lysates were separately collected and processed for western blot analysis. The blot membrane was first probed with anti-MHC-II antibody, then stripped and sequentially probed with antibodies against caspase-1. These data are representative of similar time course studies from 6 separate experiments with BSS and 4 separate experiments with DMEM. C. A standard curve was generated from a series of dilutions corresponding to the indicated number of IFN–γ and LPS-primed BMDM per lane. Densitometric analysis performed with Image J version 1.39u imaging software (59) was used to correlate MHC-II band intensities with corresponding cell number. D. IFN–γ and LPS-primed BMDM were stimulated without or with 5 mM ATP for the indicated times and the released MHC-II was quantified as the percentage of total cellular MHC-II content based on the panel C standard curve The data points in the bottom panel represent the means±SE from 8 experiments using different preparations of C57BL/6 BMDM; the western blot in the upper panel is from a representative experiment. E. IFN–γ and LPS-primed BMDM from wildtype C57BL/6 mice were transferred to BSS and incubated for the indicated times in the absence of ATP stimulation. The extracellular media samples collected at each time point were processed for analysis of MHC-II and caspase-1 content. Serial dilutions of the cell lysates were processed in parallel for analysis of total cellular MHC-II content. The western blot data are representative of observations from 3 separate experiments. F. IFN–γ and LPS-primed BMDM from P2X7R-knockout (P2X7R^−/−) mice were transferred to BSS and stimulated for the indicated times in the presence of 5 mM. The extracellular media samples collected at each time point were processed for analysis of MHC-II and caspase-1 content. Serial dilutions of the cell lysates were processed in parallel for analysis of total cellular MHC-II content. These data are representative of observations from 5 separate experiments.

Figure 2. P2X7R-induced release of MHC-II is potentiated by LPS priming and suppressed by peptide inhibitors of proteases but is independent of caspase-1

A, B. BMDM from wildtype C57BL/6 mice were pretreated with IFN-γ (2 ng/ml) for 16–18 h before priming with or without LPS (1 μg/ml) for 4 hr. The primed BMDM were then stimulated without or with 5 mM ATP for the indicated times in the absence or presence of 50 YVAD-cmk. The released MHC-II at each time point was quantified as the percentage of total cellular MHC-II content using the protocol in Figs. 1C and D. The data points in panel B represent the means±SE from 8 experiments that tested the effects of priming and 6 experiments that tested the effects of the YVAD-cmk inhibitor; the western blots in panel A are from representative experiments. C, D. BMDM from wildtype C57BL/6 mice (WT) or caspase^−/−mice were pretreated with IFN-γ (2 ng/ml) for 16–18 h before priming with LPS (1 μg/ml) for 4 hr. The primed BMDM were then stimulated without or with 5 mM ATP for the indicated times in the absence or presence of 50 μM YVAD-cmk. The released MHC-II at each time point was quantified as the percentage of total cellular MHC-II content using the protocol in Fig. 1C and D The data points in panel D represent the means±SE from 8 experiments with WT BMDM, 5 experiments with caspase-1^−/− BMDM, and 4 experiments with caspase-1^−/− BMDM treated with YVAD-cmk; the western blots in panel C are from representative experiments. E.. IFN–γ and LPS-primed BMDM from wildtype C57BL/6 mice were transferred to BSS and incubated with or without 50 μM YVAD-cmk, 50 μM DEVD-cho, 50 μM ZVAD-fmk, or 100 μM TPCK for 30 min prior to stimulation with 5 mM ATP for an additional 30 min. The extracellular media and cell lysates were separately collected and processed for western blot analysis of MHC-II, caspase-1, and cathepsin B. The data are representative of results from 3 separate experiments.

Figure 3. The inflammasome proteins, ASC and NLRP3, but not caspase-1, are required for maximal P2X7R-induced release of MHC-II

A., B. BMDM from WT, caspase-1^−/−, ASC^−/−, and NLRP3^−/− mice were pretreated with IFN-γ (2 ng/ml) for 16–18 h prior to LPS (1 μg/ml) priming for 4 h. The primed BMDM were then stimulated without or with 5 mM ATP for the indicated times in the absence or presence of 50 μM YVAD-cmk. The released MHC-II at each time point was quantified as the percentage of total cellular MHC-II content using the protocol in Figs. 1C and D. The data points in panel B represent the means±SE from 8 experiments with WT BMDM, 7 experiments each with ASC^−/−and NLRP3^−/− BMDM, and 5 experiments with caspase-1^−/− BMDM; the western blots in panel A are from representative experiments. The cells were transferred to BSS, and stimulated with 5 mM ATP for indicated times. The extracellular media and cell lysates were collected and processed for semi-quantitative western blot analysis. C. Lysate dilutions from WT, caspase-1^−/−, ASC^−/−, NLRP3^−/− BMDM were analyzed for their relative contents of MHC-II, procaspase-1 and ASC. D. BMDM from WT, ASC^−/−, and NLRP3^−/− mice were pretreated with IFN-γ (2 ng/ml) for 16–18 h prior to additional incubation without or with LPS (1 μg/ml) for 4 h. The cells were transferred to either Ca²⁺-containing or Ca²⁺-free BSS, and then stimulated with or without 5 mM ATP for 5 min. The extracellular media and cell lysates were collected and processed for western blot analysis of MHC-II, caspase-1, and cathepsin B. The data are representative of observations from 5 experiments.

Figure 4. P2X7R-induced release of MHC-II containing membranes includes both microvesicles and exosomes

A. 30 ~ 40 × 10⁶ BMDM were pretreated with IFN-γ (2 ng/ml) for 16–18 h prior to LPS (1 μg/ml) priming for 4 h. The cells were transferred to BSS, and then stimulated with 5 mM ATP for 15 min. The collected extracellular media were centrifuged successively at 300×g and 2000×g, concentrated by using CENTRIPREP, followed by layering on top of linear sucrose density gradients and overnight centrifugation. Each fraction from the sucrose gradient was weighed and then subjected to western blot analysis. The membrane was sequentially probed with anti-MHC-II, anti-caspase-1, anti-IL-1β, anti-LAMP-1, and anti-cathepsin B. Data are representative of results from 2 experiments. B. IFN-γ and LPS-primed BMDM were transferred to BSS and stimulated with 5 mM ATP for 15 min. The extracellular media was collected, sequentially centrifuged at 300×g, 2000×g, 10,000×g, and 100,000×g. The pellets from the 10,000×g, and 100,000×g spins processed by equilibrium sucrose density gradient centrifugation and the resulting fractions analyzed by western blot. The results are representative of 2 experiments. C. IFN–γ and LPS-primed BMDM were stimulated with 5 mM ATP for 15 min. The extracellular media was collected, sequentially centrifuged at 300×g, 2000×g, 10,000×g, and 100,000×g. The microvesicles obtained from the 10,000×g pellet and the exosomes obtained from the 100,000×g pellet were analyzed by electron microscopy. The scale bars are 100 nm and the the images are representative of observations from 2 experiments.

Figure 5. The ASC and NLRP3 inflammasome proteins are required for P2X7R-induced release of MHC-II exosomes but not MHC-II microvesicles

A. IFN-γ and LPS-primed BMDM were stimulated without ATP for 30 min or with ATP for 30 min. The extracellular media was collected, sequentially centrifuged at 300×g, 2000×g, 10,000×g, and 100,000×g. The microvesicles obtained from the 10,000×g pellet and the exosomes obtained from the 100,000×g pellet were analyzed by western blot analysis. The membrane was probed sequentially with antibodies against MHC-II, caspase-1, LAMP-1, and actin. The results are representative of observations from 3 experiments. B. IFN–γ and LPS-primed BMDM WT, ASC^−/−, or NLRP3^−/− mice were stimulated with 5 mM ATP for 30 min. The extracellular media fractions were collected and sequentially centrifuged at 300×g, 2000×g,, and 100,000×g. The microvesicles obtained from the 10,000×g pellet and the exosomes obtained from the 100,000×g pellet were analyzed by western blot analysis. The membrane was probed sequentially with antibodies against MHC-II, LAMP-1, and actin. The results are representative of data from 3 experiments with WT and 2 experiments each with ASC^−/− or NLRP3^−/− BMDM. C. The MHC-II bands from the western blot data in panel B were quantified by densitometry. The MHC-II band densities from the ASC^−/− and NLRP3^−/− sample lanes were normalized to the band densities measured in the WT sample lanes.

Figure 6. Comparison of MHC-II membranes released from immature and mature BMDC via constitutive or ATP-stimulated mechanisms

A. BMDC derived from C57BL/6 mice were primed with LPS (1 μg/ml) for 4 h, transferred to BSS, and stimulated with or without 5 mM ATP for 15 min. The extracellular media and the cell lysates were processed and analyzed by western blot sequentially probed with antibodies against MHC-II, caspase-1, IL-1β, and LAMP-1. The results are representative of observations from 3 experiments. B. C. D. BMDC were primed either without (for immature DC) or with 1 μg/ml LPS (for mature DC) for 24 h before further experimentation. B. Immature or mature DC was incubated in serum-free DMEM media for 24 hr before collection and processing of the extracellular fraction. C. D. Mature DC were transferred to BSS and stimulated either with or without 5 mM ATP for 20 min before collection and processing of the extracellular fraction. The collected extracellular media were sequentially centrifuged to eliminate dead cells and debris, followed by concentration. In panels B and C, the concentrated samples were directly loaded onto linear sucrose gradients. D. The concentrated medium samples were supplemented with or without Triton X-100 before loading onto linear sucrose gradients. The sucrose gradient samples were then centrifuged at 100,000×g for 16 h. 1 ml fractions were collected, weighed, precipitated by TCA, and analyzed by western blot.