Lysosomal membrane permeabilization causes secretion of IL-1β in human vascular smooth muscle cells

Abstract

Objective: IL-1β secretion by the inflammasome is strictly controlled and requires two sequential signals: a priming signal and an activating signal. Lysosomal membrane permeabilization (LMP) plays a critical role in the regulation of NLRP3 inflammasome, and generally acts as an activating signal. However, the role of LMP controlling NLRP3 inflammasome activation in human vascular smooth muscle cells (hVSMCs) is not well defined.

Methods: LMP was induced in hVSMCs by Leu-Leu-O-methyl ester. Cathepsin B was inhibited by CA-074 Me. Cytokine release, mRNA, and protein were quantified by enzyme-linked immunosorbent assay, quantitative PCR, and Western blot, respectively. NF-κB activity was analyzed by immunostaining of the NF-κB p65 nuclear translocation and using the dual-luciferase reporter assay system.

Results: LMP had both priming and activating roles, causing an upregulation of proIL-1β and NLRP3 and the secretion of mature IL-1β from unprimed hVSMCs. LMP activated the canonical NF-κB pathway. The priming effect of LMP was inhibited by CA-074 Me, indicating an upstream role of cathepsin B.

Conclusions: These data support a novel role of LMP as a single stimulus for the secretion of IL-1β from hVSMCs, implying the possibility that hVSMCs are an important initiator of the sterile inflammatory response caused by lysosomal disintegration.

Keywords: Cathepsin B; Human vascular smooth muscle cells (hVSMCs); Inflammasome; Lysosomal membrane permeabilization (LMP); NF-κB.

Fig. 1

Single LMP stimulation causes the secretion of mature IL-1β from unprimed human VSMCs. a HASMCs were treated with LLME (0-12.5 mM), and the IL-1β concentrations were measured in the supernatant by LEGENDplex Multi-Analyte Flow Assay Kit after 24 h incubation. **p < 0.005 versus unstimulated cells. b Western blot of IL-1β, pro-IL-1β, caspase-1, and procaspase-1 in supernatant derived from HASMCs stimulated with LLME (2.5 mM) for 24 h. c IL-1β concentration in supernatant. HASMCs were cultured with increasing concentrations of YVAD or MCC950 and exposed to LLME (2.5 mM) for 24 h. *p < 0.05, **p < 0.005 versus LLME-stimulated cells without YVAD (5–500 µM), or MCC950 (5–500 µM). d IL-1β concentration in supernatant. Unprimed or TNFα-primed HASMCs were cultured with 2.5 mM LLME, 5 µM ionomycin, or 1 µM nigericin. *p < 0.05, **p < 0.005 versus unstimulated cells in unprimed, or TNFα-primed HASMCs. e Time course of the level of IL-1β in supernatant from LLME (2.5 mM)- or silica (500 µg/mL)-stimulated HASMCs. f Time course of the level of IL-1β in supernatant from HCASMCs and HPASMCs stimulated with LLME (2.5 mM). *p < 0.05, **p < 0.005 versus unstimulated cells. g LMP detection by FITC dextran in cell culture. HASMCs were stained for 1 h with FITC dextran (0.5 mg/mL) and then stimulated with LLME (2.5 mM) for 10 or 60 min. Note the swelling and bursting of the punctate after LLME stimulation (FITC dextran staining). Scale bar: 10 µm. h Time course of the number of live HASMCs stimulated with LLME (2.5 mM) or silica (500 µg/mL). i Time course of the number of live MLs stimulated with LLME (0–12.5 mM). j IL-1β concentration in supernatant. Unprimed MLs were stimulated with LLME (0–12.5 mM) or ATP (2.5 mM) for 24 h. LPS (1 µg/mL)-primed MLs were stimulated with LLME (2.5 mM) or ATP (2.5 mM) for 4 h. a, c–f, h–j Results are given as mean ± SEM (n = 3). Statistical significance was evaluated by Student’s t test

Fig. 2

LMP primes the NLRP3 inflammasome of HASMCs. a, b NLRP3 and IL1B mRNA expression in HASMCs stimulated with LLME (2.5 mM) or silica (500 µg/mL) for 1–24 h. Values are relative to unstimulated cells at 24 h. c Western blot of NLRP3, pro-IL-1β, ASC, and β-actin in cell lysate derived from HASMCs stimulated with LLME (2.5 mM) for the indicated times. β-Actin was re-blotted from the stripped membrane for NLRP3 and proIL-β. The blot of ASC was derived from the same samples. d, e NLRP3 and IL-1β mRNA expression in MLs stimulated with LLME (2.5 mM) for 1–24 h. All MLs stimulated with LLME were dead at 24 h. Results are given as means ± SEM (n = 3). *p < 0.05, **p < 0.005 versus control cells at each time period, statistical significance was evaluated by Student’s t test

Fig. 3

LLME treatment activates the canonical NF-κB pathway in HASMCs. a NF-κB activation was evaluated using an NF-κB luciferase reporter system. Cells were stimulated with or without 2.5 mM LLME for the indicated times. The relative fold change from the unstimulated condition is shown. Results are given as mean ± SEM (n = 6). *p < 0.05, **p < 0.005 versus unstimulated cells, Student’s t test. b Representative confocal images of immunofluorescence staining of NF-κB p65 (green). Cells were stimulated with LLME (2.5 mM) for 1 or 2 h. Cells were stained with DAPI for visualization of the nucleus (white). Scale bars: 40 µm. c Quantitation of the nuclear translocation of NF-κB p65 by immunofluorescence staining. The mean fluorescence intensity of NF-κB p65 merged with the nucleus in each cell is plotted. Bars indicate mean ± SD (n = 143–231). Mann–Whitney U test was used for the statistical analysis. d IκBα protein was analyzed after 0–60 min LLME stimulation. e IL-1β concentration in supernatant from LLME-stimulated HASMCs with MG132 (0–10 µM). Results are given as mean ± SEM (n = 3). *p < 0.05, **p < 0.005 versus LLME-stimulated cells without MG132, Student t test. (Color figure online)

Fig. 4

Cathepsin B is involved in LMP-induced priming of HASMCs. a ROS detection by CellROX in cell culture. HASMCs were stained with CellROX and stimulated with LLME (2.5 mM) and DPI (0–40 µM) for 1 h. Results are given as means ± SEM (n = 3). **p < 0.005 versus LLME-stimulated cells without DPI, Student’s t test. b, c IL-1β concentration in supernatant. HASMCs were cultured with increasing concentrations of DPI (B) or CA-074 Me and exposed to LLME (2.5 mM) for 24 h. Results are given as means ± SEM (n = 3). *p < 0.05, **p < 0.005 versus LLME-stimulated cells without DPI or CA-074 Me, Student t test. d, e NLRP3 and IL1B mRNA expression in HASMCs stimulated with or without LLME (2.5 mM) in the presence or absence of CA-074-Me (50 µM), DPI (20 µM) or MG132 (10 µM). The relative fold change from unstimulated cells in each condition is shown. Results are given as mean ± SEM (n = 3). *p < 0.05, **p < 0.005 versus control cells, Student’s t test. f NF-κB activation was evaluated using an NF-κB luciferase reporter system. HASMCs were stimulated with LLME (2.5 mM) in the presence or absence of CA-074-Me (50 µM), DPI (20 µM) and MG132 (10 µM) for 2 h. The relative fold change from the control cells is shown. Results are given as mean ± SEM (n = 9). *p < 0.05, **p < 0.005 versus control cells, Student’s t test. g Evaluation of the nuclear translocation of NF-κB p65. Representative confocal images of the immunofluorescence staining of NF-κB p65 (green). Cells were treated with LLME (2.5 mM) for 1 h in the presence or absence of CA-074-Me (50 µM), DPI (20 µM) and MG132 (10 µM). Cells were stained with DAPI for visualization of the nucleus (white). Scale bar, 40 µm. h Quantitation of the nuclear translocation of NF-κB p65 by immunofluorescence staining. The mean fluorescence intensity of NF-κB p65 merged with the nucleus in each condition cell is plotted. Bars indicate means ± SD (n = 93–145). A Mann–Whitney U test was used for the statistical analysis. i IκBα protein was analyzed after 0–60 min LLME (2.5 mM) stimulation. HASMCs were pretreated with CA-074Me (50 µM) for 1 h. (Color figure online)