Fig. 4. NTPDase1 modulates P2X₇-dependent IL-1β release from macrophages.

Macrophages (peritoneal or BMMΦ) were primed with LPS (or other TLR ligands as indicated), washed, and the medium was replaced with fresh medium containing ATP (0.1 to 5 mM) or nigericin (5 µM). Supernatants were analyzed for IL-1β concentration by ELISA.

A) IL-1β released by LPS-primed peritoneal macrophages 60 min after treatment with 2 mM ATP in the presence or absence of apyrase (Apy; 2 U) or P2X₇ antagonists (100 µM RB-2, 25 µM A438079 or 3 µM KN-62). Data show mean + SD of one representative experiment performed in duplicate and are representative of least three independent experiments. Data show mean + SD of one representative experiment performed out of at least three independent experiments. In the presence of 2 mM ATP IL-1β levels ranged from 1.7 to 2.3 ng/10⁶ cells and 0.8 to 1.1 ng/10⁶ cells for Entpd1^−/− (gray bars) and Entpd1^+/+ (filled bars) macrophages, respectively.

B) IL-1β released by LPS-primed peritoneal macrophages 30 min after ATP treatment (0.1 to 4.0 mM). Data show mean + SEM of n ≥ 3 independent experiments. **p<0.01, ***p<0.001, two-way ANOVA with Bonferroni post hoc test.

C) IL-1β mRNA in Entpd1^−/− and Entpd1^+/+ peritoneal macrophages primed for 3 hours with LPS (10 ng/mL) or not (CTRL) was measured by qPCR. Data show mean + SEM of RNA purified from macrophages obtained from 9 (Entpd1^−/−) and 8 (Entpd1^+/+) mice, respectively. NS (no significant differences), p>0.05.

D) IL-1β released by LPS-primed peritoneal macrophages 30 min after 1 mM ATP or nigericin treatment was measured. The inhibitors of transcription (8 µM Actinomycin D), protein synthesis (10 µg/mL cycloheximide [CHX]), caspases (20 µM z-VAD), or caspase-1 (20 µM YVAD), were added either 15 min before LPS pre-stimulation/priming (pre) and removed before ATP stimulation, or 15 min prior to the addition of ATP when LPS was not yet removed from the media. Data show mean + SEM of n ≥ 3 experiments. After the indicated treatment, significantly less IL-1β was measured when compared to ATP 1mM (***p<0.001) for both Entpd1^−/− (gray bars) and Entpd1^+/+ (filled bars). There were no significant differences between Entpd1^−/− and Entpd1^+/+ macrophages for both nigericin (Nig) and Nig + Apy (NS, p>0.05), two-way ANOVA with Bonferroni post hoc test.

E) ATP-induced IL-1β release by peritoneal macrophages primed with various TLR ligands. Cells were pre-stimulated with an agonist to either TLR4 (10 ng/mL LPS, or ultra pure LPS (uLPS); positive controls), TLR2 (100 ng/mL Pam₃CSK₄ [Pam₃]), TLR5 (1 µg/mL flagellin, “Flag”) or TLR3 (1 µg/mL poly I:C) and then further stimulated for 30 min with 1 mM ATP. IL-1β released by these cells was quantified by ELISA. Data show mean + SEM of 3 to 8 independent experiments. **p<0.001, two-way ANOVA with Bonferroni post hoc test.

F) IL-1β released by BMMΦ primed for 24 hours with LPS (+) or DMEM (−) and then stimulated 30 min with or without ATP (0, 0.5 or 1.0 mM), was measured. Data show mean + SEM of one experiment performed in triplicate.