Fig. 7: Unconventional autophagy during apoptosis reduces IL-1β release by co-cultured BMDMs.

a, b The indicated Bax-/- MEFs engineered for ATG16L1 expression were transduced with BH3-only proteins (a) or treated with 2 μM MTX (b) and, 7 h later, supplemented with the ecto-ATPase inhibitor ARL67156 (3 μM). Apoptotic cells were collected 20 h (a) or 22 h (b) later and added to BMDMs pre-activated with LPS (100 ng/ml, 4 h). After 6 h, the supernatant was tested for IL-1β (ELISA). Graphs show mean values of IL-1β concentration −/+ s.d. of triplicate experimental points (n = 3 biological replicas; n.s. P > 0.05, *P < 0.05, ***P < 0.001 two-tailed Student’s t-test). Numeric P-values are shown. c P2X7 inhibitors reduce IL-1β secretion by BMDMs. Engineered Bax-/- MEFs expressing ATG16L1-Nt were treated as in (a) and the apoptotic bodies added to BMDMs pre-incubated for 45 min with control medium (condition 1) or the P2X7 channel inhibitors KN-62 (condition 2) and JNJ (condition 3), as indicated. After 6 h, supernatants were tested for IL-1β. Graphs show data as in (a) (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 two-tailed Student’s t-test). Numeric P-values are shown. d Secretion of mature IL-1β from BMDMs cultured with dying MEFs expressing ATG16L1-Nt (top) or HCT116 cells harbouring split ATG16L1 (bottom). BMDMs were incubated as in (a) with apoptotic bodies of the indicated cells transduced with tBID. Supernatants were collected 6 h later for anti-IL1β immunoprecipitation plus anti-IL-1β Western blot (top, MEFs) or directly subjected to anti-IL-1β Western blot (bottom; HCT116 cells). ATP-treated BMDMs (5 mM, 30 min) provide a positive control. Crossreactive bands of the immunoprecipitating antibody (Ig) confirm equal loading (top). e Increased caspase-1 (Casp-1) cleavage in BMDMs cultured with dying MEFs expressing ATG16L1-Nt. BMDMs were treated as in (d) and lysed for Western blot. f Gasdermin D inhibitor DMF reduces IL-1β secretion by BMDMs. Bax-/- MEFs expressing ATG16L1-Nt were treated as in (c) and the apoptotic bodies tested for IL-1β secretion by BMDMs in the absence or presence of DMF. Data are displayed as in (c). Green bars represent ATP treatment. Source data are provided as a Source Data file.