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. 2020 Nov 20;9:e59704. doi: 10.7554/eLife.59704

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© 2020, Green et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 5. — (A) IL-1β release was determined by ELISA on supernatants from wild-type (WT) or Lrrc8a knockout (KO) bone-marrow-derived macrophages (BMDMs). Naïve or LPS-primed (1 µg mL⁻¹, 4 hr) BMDMs were stimulated with either vehicle, ATP (5 mM), nigericin (Nig, 10 µM), silica (300 µg mL⁻¹) or imiquimod (IQ, 75 µM) for 2 hr (n = 5). (B) Cell death determined by an LDH assay of cells treated in (A) (n = 3). (C) Western blot of Triton x-100 insoluble crosslinked ASC oligomers and soluble total BMDM cell lysates (cell lysate + supernatant) probed for ASC and caspase-1. LPS-primed (1 µg mL⁻¹, 4 hr) WT or Lrrc8a KO BMDMs were stimulated with either nigericin (Nig, 10 µM), ATP (5 mM) or imiquimod (IQ, 75 µM) for 2 hr (n = 3). (D) IL-1β release from LPS-primed (1 µg mL⁻¹, 4 hr) WT or Lrrc8a KO BMDMs pre-treated with a vehicle control (DMSO), flufenamic acid (FFA, 100 µM) or NS3728 (NS3, 10 µM) and then stimulated with ATP (5 mM) or nigericin (10 µM) for 2 hr (n = 3). (E) Western blot of Triton x-100 insoluble crosslinked ASC oligomers and soluble total BMDM cell lysates (cell lysate + supernatant) probed for ASC, caspase-1 and IL-1β. LPS-primed (1 µg mL⁻¹, 4 hr) WT or Lrrc8a KO BMDMs were pre-treated with a vehicle control, flufenamic acid (FFA, 100 µM) or NS3728 (NS3, 10 µM) and stimulated with nigericin (10 µM, 2 hr) (n = 5). (F–G) IL-1β detected by ELISA in the peritoneal lavage (F) or plasma (G) from WT mice. Mice were pre-treated intraperitoneally (i.p.) with a vehicle control, NS3728 (NS3, 50 mg kg⁻¹) or MCC950 (MCC, 50 mg kg⁻¹) and LPS (1 µg). 4 hr after injection with LPS, mice were anaesthetised and injected with additional vehicle control, NS3728 (NS3, 50 mg kg⁻¹) or MCC950 (MCC, 50 mg kg⁻¹) before i.p. injection of ATP (100 mM, 500 µL, 15 min) (n = 5). (H–I) IL-1β detected by ELISA in the peritoneal lavage (H) or plasma (I) from Lrrc8a KO and WT littermates as treated in (F) (n = 3–5). **p<0.01, ***p<0.001, ****p<0.0001 determined by a one-way ANOVA with Dunnett’s (vs vehicle control) post hoc analysis (F,G) or a two-way ANOVA with Tukey’s post hoc analysis (A,B,D,H,I). Values shown are mean plus the SEM.

Figure 5—figure supplement 1. — (A) IL-1β release was determined by ELISA on supernatants from wild-type (WT) or Lrrc8a knockout (KO) bone-marrow-derived macrophages (BMDMs). Naïve or LPS-primed (1 µg mL⁻¹, 4 hr) BMDMs were stimulated with either vehicle, ATP (5 mM), nigericin (Nig, 10 µM), silica (300 µg mL⁻¹) or imiquimod (IQ, 75 µM) for 2 hr (n = 5). (B) Cell death determined by an LDH assay of cells treated in (A) (n = 3). (C) Western blot of Triton x-100 insoluble crosslinked ASC oligomers and soluble total BMDM cell lysates (cell lysate + supernatant) probed for ASC and caspase-1. LPS-primed (1 µg mL⁻¹, 4 hr) WT or Lrrc8a KO BMDMs were stimulated with either nigericin (Nig, 10 µM), ATP (5 mM) or imiquimod (IQ, 75 µM) for 2 hr (n = 3). (D) IL-1β release from LPS-primed (1 µg mL⁻¹, 4 hr) WT or Lrrc8a KO BMDMs pre-treated with a vehicle control (DMSO), flufenamic acid (FFA, 100 µM) or NS3728 (NS3, 10 µM) and then stimulated with ATP (5 mM) or nigericin (10 µM) for 2 hr (n = 3). (E) Western blot of Triton x-100 insoluble crosslinked ASC oligomers and soluble total BMDM cell lysates (cell lysate + supernatant) probed for ASC, caspase-1 and IL-1β. LPS-primed (1 µg mL⁻¹, 4 hr) WT or Lrrc8a KO BMDMs were pre-treated with a vehicle control, flufenamic acid (FFA, 100 µM) or NS3728 (NS3, 10 µM) and stimulated with nigericin (10 µM, 2 hr) (n = 5). (F–G) IL-1β detected by ELISA in the peritoneal lavage (F) or plasma (G) from WT mice. Mice were pre-treated intraperitoneally (i.p.) with a vehicle control, NS3728 (NS3, 50 mg kg⁻¹) or MCC950 (MCC, 50 mg kg⁻¹) and LPS (1 µg). 4 hr after injection with LPS, mice were anaesthetised and injected with additional vehicle control, NS3728 (NS3, 50 mg kg⁻¹) or MCC950 (MCC, 50 mg kg⁻¹) before i.p. injection of ATP (100 mM, 500 µL, 15 min) (n = 5). (H–I) IL-1β detected by ELISA in the peritoneal lavage (H) or plasma (I) from Lrrc8a KO and WT littermates as treated in (F) (n = 3–5). **p<0.01, ***p<0.001, ****p<0.0001 determined by a one-way ANOVA with Dunnett’s (vs vehicle control) post hoc analysis (F,G) or a two-way ANOVA with Tukey’s post hoc analysis (A,B,D,H,I). Values shown are mean plus the SEM.