The lysosomal Ragulator complex activates NLRP3 inflammasome in vivo via HDAC6 (2022)
Figure 1

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

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. * indicates P < 0.05 and *** indicates P < 0.001 by Student's t-test, n = 6 mice (peritoneal IL-1β production) and 4 mice (FACS analysis).

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 Lamtor1flox/flox and Lamtor1flox/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. *** indicates P < 0.001 by Student's t-test, n = 7 mice each. Scale bars = 50 μm.

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 Lamtor1flox/flox and Lamtor1flox/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. * indicates P < 0.05 and ** indicates P < 0.01 by Student's t-test, n = 3 biological replicates.

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. *** indicates P < 0.001 by one-way ANOVA, n = 3 biological replicates.

E, F Effects of Lamtor1 deficiency on IL-1β secretion after the induction of endogenous ROS by mitochondrial poisons or the administration of an exogeneous 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. * indicates P < 0.05 and *** indicates P < 0.001 by Student's t-test, n = 3 biological replicates.

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 Lamtor1flox/flox cells, Lamtor1flox/flox LysM-Cre cells, and Lamtor1flox/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. * indicates P < 0.05 and ** indicates P < 0.01 by one-way ANOVA, n = 3 biological replicates.

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. *** indicates P < 0.001 by one-way ANOVA, n = 3 biological replicates.

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. *** indicates P < 0.001 by one-way ANOVA, n = 3 biological replicates.

J Effects of Lamtor1 deficiency on IL-1β secretion after Pyrin inflammasome activation. ELISA assay showing IL-1β secretion in supernatants from Lamtor1flox/flox and Lamtor1flox/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. ** indicates P < 0.01 indicates no significant difference by one-way ANOVA, n = 3 biological replicates.

K Effects of Lamtor1 deficiency on IL-1β secretion after AIM2 inflammasome activation. ELISA assay showing IL-1β secretion in supernatants from Lamtor1flox/flox and Lamtor1flox/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 are shown as the means ± SEM. n.s indicates no significant difference by one-way ANOVA, n = 3 biological replicates.