The lysosomal Ragulator complex activates NLRP3 inflammasome in vivo via HDAC6

Abstract

The cellular activation of the NLRP3 inflammasome is spatiotemporally orchestrated by various organelles, but whether lysosomes contribute to this process remains unclear. Here, we show the vital role of the lysosomal membrane-tethered Ragulator complex in NLRP3 inflammasome activation. Deficiency of Lamtor1, an essential component of the Ragulator complex, abrogated NLRP3 inflammasome activation in murine macrophages and human monocytic cells. Myeloid-specific Lamtor1-deficient mice showed marked attenuation of NLRP3-associated inflammatory disease severity, including LPS-induced sepsis, alum-induced peritonitis, and monosodium urate (MSU)-induced arthritis. Mechanistically, Lamtor1 interacted with both NLRP3 and histone deacetylase 6 (HDAC6). HDAC6 enhances the interaction between Lamtor1 and NLRP3, resulting in NLRP3 inflammasome activation. DL-all-rac-α-tocopherol, a synthetic form of vitamin E, inhibited the Lamtor1-HDAC6 interaction, resulting in diminished NLRP3 inflammasome activation. Further, DL-all-rac-α-tocopherol alleviated acute gouty arthritis and MSU-induced peritonitis. These results provide novel insights into the role of lysosomes in the activation of NLRP3 inflammasomes by the Ragulator complex.

Keywords: HDAC6; NLRP3 inflammasome; Ragulator complex; α-tocopherol.

Figure 1. NLRP3 inflammasome activation is impaired in myeloid‐specific Lamtor1 deficiency

1. A

   Effects of myeloid‐specific Lamtor1 deficiency on peritoneal IL‐1β production, CD11b⁺ Ly6C⁺ monocyte recruitment, and CD11b⁺ Ly6G⁺ neutrophil recruitment after an intraperitoneal injection of 600 μl alum solution (20 mg/ml, 6 h), Data are shown as means ± SEM. *P < 0.05 and ***P < 0.001 by the Student's t‐test, n = 6 mice (peritoneal IL‐1β production) and 4 mice (FACS analysis).

2. B

   Effects of myeloid‐specific Lamtor1 deficiency in an acute gouty arthritis model. MSU crystals (0.5 mg) or the PBS control were injected intra‐articularly into the tibia‐tarsal joint of Lamtor1 ^flox/flox and Lamtor1 ^flox/flox LysM‐Cre mice. Mice were assessed for joint swelling using electronic calipers. Images show photomicrographs (upper panel) and hematoxylin and eosin–stained sections (middle and lower panels) of ankle joints obtained at 24 h. Data are shown as the means ± SEM. ***P < 0.001 by the Student's t‐test, n = 7 mice each. Scale bars = 50 μm.

3. C

   Effects of Lamtor1 deficiency on IL‐1β secretion after NLRP3 inflammasome activation or TLR ligand stimulation. ELISA assay shows IL‐1β secretion in supernatants from Lamtor1 ^flox/flox and Lamtor1 ^flox/flox LysM‐Cre BMDMs treated with LPS (200 ng/ml, 4 h), followed by nigericin (15 μM, 1 h), ATP (10 mM, 1 h), or GPN (100 nM, 6 h). Data are shown as the means ± SEM. *P < 0.05 and **P < 0.01 by the Student's t‐test, n = 3 biological replicates.

4. D

   Effects of Lamtor1 deficiency on IL‐1β secretion after NLRP3 inflammasome activation in human THP‐1 monocytes. ELISA assay shows IL‐1β secretion in supernatants from WT cells, Lamtor1 KO different single clones, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight). Data are shown as the means ± SEM. ***P < 0.001 by one‐way ANOVA, n = 3 biological replicates.

5. E, F

   Effects of Lamtor1 deficiency on IL‐1β secretion after the induction of endogenous ROS by mitochondrial poisons or the administration of an exogenous ROS (hydrogen peroxide). ELISA assay shows IL‐1β secretion in supernatants after treatment with LPS (200 ng/ml), Rotenone (10 μM), or Antimycin (10 μg/ml) for 6 h or followed by hydrogen peroxide (3.86 mM). Data are shown as the means ± SEM. *P < 0.05 and ***P < 0.001 by the Student's t‐test, n = 3 biological replicates.

6. G

   Effects of Lamtor1 reconstitution in Lamtor1‐deficient macrophages on IL‐1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL‐1β secretion in BMDM supernatants from Lamtor1 ^flox/flox cells, Lamtor1 ^flox/flox LysM‐Cre cells, and Lamtor1 ^flox/flox LysM‐Cre cells reconstituted with full‐length Lamtor1, all of which were treated with nigericin (15 μM, 1 h) after priming with LPS (200 ng/ml, 4 h). Data are shown as the means ± SEM. *P < 0.05 and **P < 0.01 by one‐way ANOVA, n = 3 biological replicates.

7. H

   Effects of Lamtor1 deficiency on IL‐1β secretion after NLRP3 inflammasome activation in human THP‐1 monocytes. ELISA assay shows IL‐1β secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with full‐length Lamtor1, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/ml, 2 h). Data are shown as the means ± SEM. ***P < 0.001 by one‐way ANOVA, n = 3 biological replicates.

8. I

   Effects of Lamtor1 deficiency on IL‐18 secretion after NLRP3 inflammasome activation in human THP‐1 monocytes. ELISA assay shows IL‐18 secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with full‐length Lamtor1, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/ml, 2 h). Data are shown as the means ± SEM. ***P < 0.001 by one‐way ANOVA, n = 3 biological replicates.

9. J

   Effects of Lamtor1 deficiency on IL‐1β secretion after Pyrin inflammasome activation. ELISA assay showing IL‐1β secretion in supernatants from Lamtor1 ^flox/flox and Lamtor1 ^flox/flox LysM‐Cre BMDMs treated with TcdB (0.5 μg/ml, 3 h) after LPS (200 ng/ml, 4 h) stimulation. Data are shown as the means ± SEM. **P < 0.01 no significant difference by one‐way ANOVA, n = 3 biological replicates.

10. K

    Effects of Lamtor1 deficiency on IL‐1β secretion after AIM2 inflammasome activation. ELISA assay showing IL‐1β secretion in supernatants from Lamtor1 ^flox/flox and Lamtor1 ^flox/flox LysM‐Cre BMDMs treated with Poly(dA:dT; 5 μg/ml, overnight) and Lipofectamine 3000 after LPS (200 ng/ml, 4 h) stimulation. Data information: Data are shown as the means ± SEM. n.s no significant difference by one‐way ANOVA, n = 3 biological replicates.

Source data are available online for this figure.

Figure EV1. Additional data related to Fig 1

1. A

   Effects of myeloid‐specific Lamtor1 deficiency (Lamtor1 ^flox/flox LysM‐Cre) on serum IL‐1β production following intraperitoneal injection of LPS. ELISA assay shows IL‐1β secretion in serum from Lamtor1 ^flox/flox and Lamtor1 ^flox/flox LysM‐Cre mice after intraperitoneal injection of LPS (10 mg/kg, 6 h). Data are pooled from three independent experiments. Data are shown as the mean ± SEM. *P < 0.05 by the Student's t‐test, n = 12 mice (WT) and 10 mice (KO).

2. B–D

   Western blotting showing the confirmation of Lamtor1 knockout (B), exogenous expression of Lamtor1‐Flag in BMDM and THP‐1 cells (D). (C) Effects of Lamtor1 deficiency on IL‐1β secretion after TLR ligand stimulation. ELISA assay shows IL‐6 secretion in supernatants from Lamtor1 ^flox/flox and Lamtor1 ^flox/flox LysM‐Cre BMDMs treated with LPS (1 μg/ml, overnight) or R848 (10 μg/ml, overnight). Data are shown as means ± SEM. *P < 0.05 and **P < 0.01 by the Student's t‐test, n = 3 biological replicates.

Source data are available online for this figure.

Figure 2. NLRP3 inflammasome activation is impaired in Lamtor1‐deficient macrophages

1. A, B

   Effects of Lamtor1 deficiency on Caspase‐1 processing after NLRP3 inflammasome activation. Western blot shows caspase‐1/p10, pro‐caspase‐1, Lamtor1, and β‐actin. Lamtor1 ^flox/flox and Lamtor1 ^flox/flox LysM‐Cre BMDMs (A) or WT and Lamtor1 KO THP‐1 cells (B) were treated with nigericin (15 μM, 1 h) after LPS [200 ng/ml, for 4 h (BMDM) or 2 h (THP‐1)] stimulation. Data are shown as the means ± SEM. ***P < 0.001 by the Student's t‐test, n = 3 biological replicates.

2. C

   Effects of Lamtor1 deficiency on GSDMD processing after NLRP3 inflammasome activation. Western blot shows GSDMD, Lamtor1, and β‐actin. Lamtor1 ^flox/flox and Lamtor1 ^flox/flox LysM‐Cre BMDMs were treated with nigericin (15 μM, 1 h) after LPS (200 ng/ml, 4 h) stimulation. Data are shown as the means ± SEM. *P < 0.05 by the Student's t‐test, n = 3 biological replicates.

3. D

   Effects of Lamtor1 deficiency on pyroptosis after NLRP3 inflammasome activation. Lamtor1 ^flox/flox and Lamtor1 ^flox/flox LysM‐Cre BMDMs were treated with nigericin (15 μM, 1 h) after LPS (200 ng/ml, 4 h) stimulation, and the percentage of propidium iodide positive cells was counted by flow cytometry. Data are shown as the means ± SEM. ***P < 0.001 by the Student's t‐test, n = 3 biological replicates.

4. E

   Effects of Lamtor1 deficiency on IL‐1β dynamics. Western blot shows IL‐1β and β‐actin. WT cells and Lamtor1 KO cells were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) stimulation.

5. F

   Effects of the active form of GSDMD reconstitution on Lamtor1 KO THP‐1 cells. ELISA assay shows IL‐1β secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with the active form of GSDMD, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/ml, 2 h). Data are shown as means ± SEM. n.s. indicates P > 0.05 by one‐way ANOVA, n = 3 biological replicates.

6. G

   Confirmation of the reconstitution of the active form Caspase‐1 (p10 and p20) or GSDMD (GSDMD‐NT).

7. H

   Effects of Lamtor1 deficiency on the priming phase. Lamtor1 ^flox/flox and Lamtor1 ^flox/flox LysM‐Cre BMDMs were treated with LPS (200 ng/ml, 4 h). Data are shown as the means ± SEM. ***P < 0.001 by the Student's t‐test, n = 3 biological replicates.

8. I

   Effects of IL‐1β and active form of Caspase‐1 reconstitution on Lamtor1 KO THP‐1 cells. ELISA assay shows IL‐1β secretion in supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with IL‐1β, the active form of Caspase‐1, all of which were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight) and LPS (200 ng/ml, 2 h). Data are shown as the means ± SEM. ***P < 0.001 by the Student's t‐test and n.s. indicates P > 0.05 by one‐way ANOVA, n = 3 biological replicates.

Source data are available online for this figure.

Figure 3. mTORC1 activity is not involved in the inflammasome activation phase

1. Representative images of mutant Lamtor1. PMA‐primed Lamtor1 KO THP‐1 macrophages reconstituted with full‐length or truncated Lamtor1. Scale bars = 5 μm.

2. Effects of the Lamtor1 point mutation on IL‐1β secretion after NLRP3 inflammasome activation. ELISA assays show IL‐1β secretion in THP‐1 cell supernatants from WT cells, Lamtor1 KO cells, and Lamtor1 KO cells reconstituted with full‐length Lamtor1 or variants of Lamtor1 in which G2 was replaced with alanine (G2A); all cell types were then treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight). Data are shown as the means ± SEM. *P < 0.05 and ***P < 0.001 by one‐way ANOVA, n = 3 biological replicates.

3. Effects of Lamtor1 deficiency on mTORC1 activity. Immunoblot analysis of Lamtor1 ^flox/flox and Lamtor1 ^flox/flox LysM‐Cre BMDMs treated with LPS (200 ng/ml, 4 h).

4. Effect of the rapamycin treatment on IL‐1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL‐1β secretion in supernatants from PMA (50 nM, overnight) primed WT THP‐1 cells treated with rapamycin at the indicated concentration for 2 h, then treated with LPS (200 ng/ml, 2 h) and nigericin (15 μM, 1 h). Data are shown as the means ± SEM. **P < 0.01, and n.s indicates no significant difference by one‐way ANOVA, n = 3 biological replicates.

5. Effect of rapamycin treatment on Caspase‐1 processing. Western blot shows caspase‐1/p20. LPS (200 ng/ml, 4 h) primed BMDMs were treated with rapamycin at indicated concentrations followed by nigericin (15 μM, 1 h). Data are shown as the means ± SEM. n.s indicates no significant difference by one‐way ANOVA, n = 3 biological replicates.

6. Effect of Torin‐1 treatment on IL‐1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL‐1β secretion in supernatants from WT BMDMs pretreated with Torin‐1 (0, 1, 10, 100, or 250 nM) for 2 h before LPS (200 ng/ml, 4 h) and nigericin (15 μM, 1 h) treatment (left panel). Western blot shows phospho‐p70‐S6K from BMDMs pretreated with Torin‐1 at the indicated concentrations for 2 h before LPS and nigericin treatment (right panel). Data are shown as the means ± SEM. n.s indicates no significant difference by one‐way ANOVA, n = 3 biological replicates.

Source data are available online for this figure.

Figure EV2. Effect of combined TFEB and Lamtor1 deficiency on IL‐1β secretion

1. Effects of Lamtor1 deficiency on TFEB nuclear localization in THP‐1 cells. Representative images of WT cells, Lamtor1 KO cells, or Lamtor1 KO cells reconstituted with Lamtor1; all cell types were stained with endogenous TFEB. Scale bar = 30 μm. Data are shown as means ± SEM. ***P < 0.001 by one‐way ANOVA. TFEB nuclear translocation was quantified over 10 images in each experiment, and each dot indicates the mean of a single experiment.

2. Western blot showing TFEB knockout in addition to Lamtor1.

3. Effects of combined TFEB and Lamtor1 deficiency on IL‐1β secretion after NLRP3 inflammasome activation. ELISA assay shows IL‐1β secretion in supernatants from Lamtor1 ^flox/flox, Lamtor1 ^flox/flox LysM‐Cre, and Lamtor1 ^flox/flox TFEB ^flox/flox LysM‐Cre BMDMs treated with ATP (10 mM, 1 h) after LPS (200 ng/ml, 4 h) stimulation. Data are shown as means ± SEM. ***P < 0.001, and n.s indicates no significant difference by one‐way ANOVA, n = 3 biological replicates.

Source data are available online for this figure.

Figure 4. Interaction between Lamtor1 and HDAC6 is necessary for inflammasome activation

1. A

   Effects of the HDAC6 inhibitor treatment on IL‐1β secretion after NLRP3 inflammasome activation. ELISA assays show IL‐1β secretion and caspase‐1 processing (p10) in BMDMs upon NLRP3 inflammasome activation following pretreatment with tubacin (left to right: 10 and 20 mM) or rocilinostat (left to right: 30 and 60 mM). Data are shown as the means ± SEM. ***P < 0.001 by one‐way ANOVA, n = 3 biological replicates.

2. B–F

   Co‐immunoprecipitation assay showing the interaction between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1–Flag co‐immunoprecipitated with HDAC6–myc from lysates of HEK293T cells transfected with the indicated plasmids (B, C), Lamtor1–Flag co‐immunoprecipitated with endogenous HDAC6 from the lysate of Lamtor1‐Flag knocked‐in THP‐1 cells. (D) Endogenous Lamtor1 co‐immunoprecipitated with endogenous HDAC6 from lysates of WT THP‐1 cells (E, F). Effects of NLRP3 inflammasome activation on the interactions between Lamtor1 and HDAC6. Co‐immunoprecipitation assay shows interactions between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1–Flag co‐immunoprecipitated with HDAC6–myc from lysates of HEK293T cells transfected with the indicated plasmids. Data are shown as means ± SEM. *P < 0.05 and **P < 0.01 by one‐way ANOVA, n = 3 biological replicates.

3. G

   PLA to confirm endogenous binding between Lamtor1 and HDAC6. Lamtor1‐HDAC6 complexes are shown in red, and nuclei are depicted in blue. In situ PLA signals were quantified over nine images in each experiment. Data are shown as the means ± SEM. ***P < 0.001 by the Student's t‐test. The data are representative of three experiments that showed consistent results. Scale bars, 10 μm.

4. H

   NanoBRET assay to confirm endogenous binding between Lamtor1 and HDAC6 in living cells. HEK293T cells were transiently transfected with NanoLuc‐fused Lamtor1 and HaloTag‐fused HDAC6 using Lipofectamine 2000. Luminescence was measured 48 h after transfection following the addition of Nano‐Glo Luciferase Assay Substrate (Promega). Data are shown as the means ± SEM. ***P < 0.001 by the Student's t‐test, n = 3 biological replicates.

5. I

   Immunoblot analysis of the truncated form of Flag‐tagged Lamtor1 mutant (∆145–161, Met1–144; ∆95–161, Met1–Val94) co‐immunoprecipitated with HDAC6–myc from lysates of HEK293T cells transfected with the indicated plasmids.

6. J

   Effect of truncated Lamtor1 mutant on NLRP3 inflammasome activation in THP‐1 cells. ELISA assay showing IL‐1β secretion in supernatants from WT cells, Lamtor1 KO cells, Lamtor1 KO cells reconstituted with full‐length Lamtor1, and Met1‐Val94 Lamtor1 cells; all cell types were treated with nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight). Data are shown as the means ± SEM. ***P < 0.001 and n.s. indicates no significant difference by the Student's t‐test, n = 3 biological replicates.

Source data are available online for this figure.

Figure 5. Lamtor1 interacts with NLRP3 in the presence of HDAC6

1. A

   Yeast two‐hybrid (Y2H) assays for Lamtor1 interactions with NLRP3. Positive Y2H interaction between Lamtor1 as prey, and NLRP3 as bait. Lamtor2 as prey was used as a positive control and the empty bait vector as a negative control.

2. B

   PLA to confirm endogenous binding between Lamtor1 and NLRP3. Lamtor1–NLRP3 complexes are shown in red, and nuclei are depicted in blue. In situ PLA signals were quantified over 10 images in each experiment. Data are shown as the means ± SEM. ***P < 0.001 by the Student's t‐test. The data are representative of three experiments that showed consistent results. Scale bars = 5 μm.

3. C, D

   Immunoblot analysis of Lamtor1‐V5 co‐immunoprecipitated with NLRP3–Flag from lysates of HEK293T cells transfected with the indicated plasmids.

4. E, F

   Immunoblot analysis of the truncated form of V5‐tagged Lamtor1 mutant (∆145–161, Met1‐Ser144) co‐immunoprecipitated with NLRP3–flag from lysates of HEK293T cells transfected with the indicated plasmids.

5. G

   Effect of the HDAC6 knockdown on NLRP3–Lamtor1 interactions. Immunoblot analysis of Lamtor1–V5 co‐immunoprecipitated with NLRP3–Flag from lysates of HEK293T cells transfected with the indicated plasmids. Data are shown as the means ± SEM. **P < 0.01 by the Student's t‐test, n = 3 biological replicates.

Source data are available online for this figure.

Figure 6. ASC speck originates near the lysosomes

1. A, B

   Representative images of Lamtor1 and ASC. PMA‐treated THP‐1 cells stably expressing GFP‐tagged ASC and tdTomato‐tagged Lamtor1 were primed with LPS (200 ng/ml, 2 h) and then stimulated with nigericin for the indicated duration. Time shows after nigericin stimulation (min:s). Scale bars = 1 μm.

2. C

   Effects of Lamtor1 deficiency on ASC speck formation after NLRP3 inflammasome activation. Representative images of PMA‐primed THP‐1 macrophages stably expressing GFP‐tagged ASC treated with or without LPS (200 ng/ml, 2 h) and nigericin (15 μM, 1 h). Scale bars = 10 μm. Data are shown as the means ± SEM. ***P < 0.001, and n.s indicates no significant difference by one‐way ANOVA, n = 4 independent experiments.

Source data are available online for this figure.

Figure 7. DL‐all‐rac‐α‐tocopherol inhibits Lamtor1 and HDAC6 interactions and suppresses inflammasome activation

1. A

   Schematic showing the use of a NanoBRET assay to search for inhibitors of Lamtor1–HDAC6 interactions in a library of natural compounds.

2. B–F

   Effects of DL‐all‐rac‐α‐tocopherol on the interactions between Lamtor1 and HDAC6. Immunoblot analysis of Lamtor1–Flag co‐immunoprecipitated with HDAC6–myc from lysates of HEK293T cells transfected with the indicated plasmids after pretreatments with the DL‐all‐rac‐α‐tocopherol (B) or D ‐α‐tocopherol (E) (10 μM, overnight). Data are shown as means ± SEM. **P < 0.01 by the Student's t‐test, n = 3 biological replicates. IL‐1β secretion upon NLRP3 inflammasome activation after overnight pretreatment with DL‐all‐rac‐α‐tocopherol (C) or (D) ‐α‐tocopherol (F) at the indicated concentrations. ELISA assay showing IL‐1β secretion in supernatants from WT THP‐1 cells treated with LPS (200 ng/ml, 2 h) and nigericin (15 μM, 1 h) after priming with PMA (50 nM, overnight). Data are shown as means ± SEM. *P < 0.05 by one‐way ANOVA, n = 3 biological replicates. (D) IL‐6 secretion after overnight pretreatment with DL‐all‐rac‐α‐tocopherol. ELISA assay showing IL‐6 secretion in supernatants from WT BMDMs treated with LPS (200 ng/ml, overnight) and DL‐all‐rac‐α‐tocopherol at the indicated concentrations (overnight). Data are shown as means ± SEM. n.s indicates no significant difference by one‐way ANOVA, n = 3 biological replicates.

3. G

   Effects of DL‐all‐rac‐α‐tocopherol in an acute gouty arthritis model. MSU crystals (0.5 mg) suspended in 20 μl endotoxin‐free PBS or PBS control were injected intra‐articularly into the tibia‐tarsal joint (ankle) of C57BL/6 WT mice with or without DL‐all‐rac‐α‐tocopherol. Ankle joint swelling at 24 h was assessed with electronic calipers and with photomicrographs and hematoxylin and eosin–stained sections of ankle joints. Data are shown as means ± SEM. **P < 0.01 by the Student's t‐test, n = 6 mice. Scale bars = 30 μm.

4. H

   Effects of DL‐all‐rac‐α‐tocopherol in an MSU‐induced peritonitis model. Twelve hours after DL‐all‐rac‐α‐tocopherol pretreatment, 100 μl MSU solution (10 mg/ml) was intraperitoneally injected for 4 h. Peritoneal IL‐1β production was then measured by ELISA, and CD11b + Ly6G+ neutrophil recruitment was measured by FACS. Data are shown as means ± SEM. *P < 0.05 by the Student's t‐test, n = 4 mice (control) and n = 5 mice (DL‐all‐rac‐α‐tocopherol).

Source data are available online for this figure.

Figure EV3. Characteristics of DL‐all‐rac‐α‐tocopherol other than inflammasome activation

1. Effect of DL‐all‐rac‐α‐tocopherol on priming. Immunoblot analysis of NLRP3 inflammasome‐related proteins from lysates of BMDMs treated with DL‐all‐rac‐α‐tocopherol at an indicated concentration (overnight) followed by LPS treatment for 4 h (200 ng/ml).

2. Effect of DL‐all‐rac‐α‐tocopherol on the overall activity of HDAC6. Immunoblot analysis of acetylated α‐tubulin from lysates of BMDMs treated with DL‐all‐rac‐α‐tocopherol (10 μM) and rocilinostat (30 μM) for 2 h. Data are shown as means ± SEM. ***P < 0.001, and n.s indicates no significant difference by one‐way ANOVA, n = 3 biological replicates.

3. Chemotaxis of THP‐1 cells treated with DL‐all‐rac‐α‐tocopherol in response to CCL2. Chemotaxis of WT THP‐1 cells in response to CCL2 (25 ng/ml) in the presence of the indicated concentration of DL‐all‐rac‐α‐tocopherol as determined by Transwell assay (pore size, 5 μm; upper). Data are representative of three experiments. Data are shown as means ± SEM. n.s indicates no significant difference with one‐way ANOVA, n = 3 biological replicates.

Source data are available online for this figure.

Figure 8. The Ragulator complex regulates inflammasome activation through interactions with NLRP3 augmented by HDAC6

The Ragulator complex expressed on the lysosomal membrane interacts with HDAC6, which augments the interactions between Lamtor1 and NLRP3, resulting in the activation of the inflammasome.