Figure 1.
ATP, cholesterol crystal or 7-ketocholesterol increased inflammasome formation in Asc+/+ BMMs.
(A) Representative confocal fluorescence images and summarized data depicting the effect of ATP (0–7.5 mM, 16 h) on the colocalization between Nlrp3 and Asc or caspase-1 in Asc+/+ BMMs. (B) Representative confocal fluorescence images and summarized data depicting the effect of cholesterol crystal (0–1 mg/ml, 16 h) on the colocalization between Nlrp3 and Asc or caspase-1 in Asc+/+ BMMs. (C) Representative confocal fluorescence images and summarized data depicting the effect of 7-ketocholesterol (0–20 µg/ml, 16 h) on the colocalization between Nlrp3 and Asc or caspase-1 in Asc+/+ BMMs. * P<0.05 vs. untreated control group (n = 6).
Figure 2.
Effects of ATP, cholesterol crystals and 7-Ketocholesterol on Nlrp3 inflammasome formation in Asc+/+ and Asc−/− BMMs.
(A) Representative confocal fluorescence images depicting the effects of cholesterol crystals (CHC, 0.5 mg/ml, 16 h), 7-ketocholesterol (7-Ket, 10 µg/ml, 16 h), or ATP (2.5 mM, 16 h) on the colocalization between Nlrp3 and Asc from Asc+/+ and Asc−/− BMMs. (B) Summarized data showing colocalization efficiency between Nlrp3 and Asc. (C) Representative confocal fluorescence images depicting the effects of cholesterol crystals (CHC, 0.5 mg/ml, 16 h), 7-ketocholesterol (7-Ket, 10 µg/ml, 16 h), or ATP (2.5 mM, 16 h) on the colocalization between Nlrp3 and caspase-1 from Asc+/+ and Asc−/− BMMs. (D) Summarized data showing colocalization efficiency between Nlrp3 and caspase-1. * P<0.05 vs. untreated Asc+/+ control group; # P<0.05 Asc−/− vs. Asc+/+ group (n = 6).
Figure 3.
Effects of ATP, cholesterol crystals and 7-Ketocholesterol on IL-1β production in Asc+/+ and Asc−/− BMMs.
BMMs were stimulated with ATP (0–7.5 mM) (A), cholesterol crystals (CHC, 0–1 mg/ml) (B) or 7-ketocholesterol (7-Ket, 0–20 µg/ml) (C) for 16 h and IL-1β concentrations in the supernatants were determined by ELISA. * P<0.05 vs. untreated Asc+/+ control group; # P<0.05 Asc−/− vs. Asc+/+ group (n = 6).
Figure 4.
Asc gene knockout inhibited ATP-induced caspase-1 activation in BMMs.
(A) Western blot analysis showing the effect of ATP (2.5 mM, 16 h) on active caspase-1 expression and the quantitative analysis (lower blot). (B) Summarized data of FLICA assays quantifying the relative caspase-1 activity compared to control. * P<0.05 vs. untreated Asc+/+ control group; # P<0.05 vs. Asc+/+ ATP group (n = 6).
Figure 5.
Increased lipid accumulation in BMMs with activation of Nlrp3 inflammasomes by ATP.
BMMs were primed with LPS (1 ng/ml) for 3 h and treated with ATP (2.5 mM, 16 h), IL1β (0.5 ng/ml), MSU (100 µg/ml), or IL18 (25 nM). Some group of BMMs were treated with ATP or MSU in the presence of caspase-1 inhibitor WEHD (0.15 µg/ml) or IL1R antagonist (IL1Ra, 40 ng/ml). Then, BMMs were loaded with oxLDL (10 µg/mL) for 16 hours. (A) Light microscopic images show oil red O stained BMMs. Cells were counterstatined with hematoxylin. (B) Summarized data showing area percentage of each cell positive for Oil red O staining in BMMs. * P<0.05 vs. untreated Asc+/+ control group; # P<0.05 vs. Asc+/+ ATP group; $ P<0.05 vs. Asc+/+ MSU group (n = 6).
Figure 6.
Asc gene deletion and caspase-1 inhibition blocked cholesterol deposition in lysosomes.
BMMs were primed with LPS (1 ng/ml) for 3 h and treated with ATP (2.5 mM, 16 h) in the absence or presence of caspase-1 inhibitor WEHD (0.15 µg/ml), or IL1β (0.5 ng/ml) alone. Then BMMs were loaded with oxLDL (10 µg/mL) for 16 hours. (A) Representative confocal fluorescent images showing the colocalization between filipin (cholesterol) and Lamp1 (lysosome marker). An increase in the purple color in overlay images indicates increased cholesterol trapping in lysosomes. (B) Quantified and summarized data showing co-localization co-efficiency between filipin and Lamp1. (C) Effect of Asc gene deletion on cholesterol level in isolated lysosomes from BMMs. Lysosomes were isolated from Asc+/+ and Asc−/− BMMs and cholesterol concentration in these isolated lysosomes were determined using a standard fluorescence assay kit. Some groups of cells were pretreated with ATP in the presence of IL1R antagonist (IL1Ra, 40 ng/ml) or IL18 (25 nM) alone. * P<0.05 vs. untreated Asc+/+ control group; # P<0.05 vs. Asc+/+ ATP group (n = 6).
Figure 7.
ATP-induced lipid trafficking in lysosomes was blocked in Asc−/− BMMs.
Asc+/+ and Asc−/− BMMs were primed with low dose of LPS (1 ng/ml) for 3 h and treated with ATP (2.5 mM, 16 h) in the absence or presence of caspase-1 inhibitor WEHD (0.15 µg/ml). (A) BMMs were incubated with BSA-conjugated BODIPY FL-C5-lactosylceramide (LacCer) and lipid trafficking was tested by following LacCer trafficking. Representative confocal fluorescent images show the co-localization between LacCer (red color) and Lysotracker (green color). An increase in the yellow color in overlay images indicates increased LacCer trafficking to the lysosomes. (B) Quantified and summarized data showing the percent of lipid in lysosomes. (C) The ganglioside GM1 levels in isolated lysosomes from BMMs were determined by dot blot analysis. Represent dot blot image shows the ganglioside GM1 level in isolate lysosome homogenates as detected by cholera toxin-conjugated HRP. (D) Summarized analysis of ganglioside GM1 in lysosomes. * P<0.05 vs. untreated Asc+/+ control group; # P<0.05 vs. Asc+/+ ATP group (n = 6).
Figure 8.
Effect of inflammasome activation on macrophage migration in vitro.
Macrophage migration in vitro was assayed using Transwell inserts with a 5 µm porous membrane. The migratory cells on the lower side of insert membrane were quantified. BMMs in Transwell inserts were primed with LPS (1 ng/ml) for 3 h and treated with ATP (2.5 mM, 16 h), IL1β (0.5 ng/ml), MSU (100 µg/ml), or IL18 (25 nM) in the absence or presence of caspase-1 inhibitor WEHD (0.15 µg/ml) or IL1R antagonist (IL1Ra, 40 ng/ml). Quantification of the transwell assays reveals the ability of BMMs to migrate from inside Transwell inserts through the membrane upon stimulation. * P<0.05 vs. untreated Asc+/+ control group; # P<0.05 Asc−/− vs. Asc+/+ group (n = 6).
Figure 9.
Macrophage migration in vivo is attenuated by Asc-deficiency.
(A) GFP-expressing Asc+/+ and Asc−/− BMMs were obtained by Nucleofection of BMMs with GFP-encoding plasmids. Representative dot plots from flow cytometry analysis show the GFP expression and cell viability (PI) in Asc+/+ and Asc−/− BMMs post Nucleofection with GFP plasmids. (B) GFP-expressing Asc+/+ or Asc−/− BMMs were intravenously injected in Asc+/+ mice with Zymosan A-induced peritonitis. The numbers of GFP+ macrophages in peritoneal lavage fluids of Asc+/+ mice were quantified and compared. * P<0.05 vs. Asc+/+ BMMs (n = 4).