ATP release from dying autophagic cells and their phagocytosis are crucial for inflammasome activation in macrophages

Abstract

Pathogen-activated and damage-associated molecular patterns activate the inflammasome in macrophages. We report that mouse macrophages release IL-1β while co-incubated with pro-B (Ba/F3) cells dying, as a result of IL-3 withdrawal, by apoptosis with autophagy, but not when they are co-incubated with living, apoptotic, necrotic or necrostatin-1 treated cells. NALP3-deficient macrophages display reduced IL-1β secretion, which is also inhibited in macrophages deficient in caspase-1 or pre-treated with its inhibitor. This finding demonstrates that the inflammasome is activated during phagocytosis of dying autophagic cells. We show that activation of NALP3 depends on phagocytosis of dying cells, ATP release through pannexin-1 channels of dying autophagic cells, P(2)X(7) purinergic receptor activation, and on consequent potassium efflux. Dying autophagic Ba/F3 cells injected intraperitoneally in mice recruit neutrophils and thereby induce acute inflammation. These findings demonstrate that NALP3 performs key upstream functions in inflammasome activation in mouse macrophages engulfing dying autophagic cells, and that these functions lead to pro-inflammatory responses.

Figure 1. Autophagy is induced in apoptotic Ba/F3 cells by IL-3 depletion.

Ba/F3 cells were kept without IL-3 for 6 h (dying AU). (A) Both living and dying autophagic Ba/F3 cells were stained with anti-LC3 antibody or acridine orange stain to demonstrate increased autophagosome formation. Arrows represent the increased autophagy with IL-3 depletion. Scale bars are 10 µm. (B) Proteins in western blots of samples from dying autophagic cells were detected with anti-LC3 antibody. Chloroquine (CQ) was used as lysosomal inhibitor. The right panel presents the quantification of the western blot. Data represent the mean ± SEM of 13, 19, 5 and 6 independent experiments for IL-3+, IL-3-, IL-3+CQ+ and IL-3-CQ+, respectively. (C) Cell death was quantified by flow cytometric analysis of dying autophagic cells by using Annexin-V-FITC/PI staining. Data represent the mean ± SEM of 7, 10, 3 and 9 independent experiments for IL-3+, IL-3−, IL-3+CQ+, IL-3−CQ+, respectively. PS: Phosphatidylserine, PI: Propidium iodide (D) Proteins obtained from dying autophagic cells were detected with caspase-3 antibody. Anti-actin poyclonal antibody was used to show that equal amounts of proteins were loaded in western blots. For simplicity, parts from the same western blots are shown separately in parts B and D. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Figure 2. Apoptotic and necrotic Ba/F3 cells do not show autophagic activity.

(A) Ba/F3 cells were treated with different doses of doxorubicin in the presence of IL-3 for 16 h. Immunoblotting with anti-LC3 antibody was done to detect LC3 protein in cells. Ba/F3 cells which were not treated with doxorubicin in IL-3 containing medium were used as control for the experiment. (B) Necrosis in Ba/F3 cells was induced by freeze-thaw. Anti-actin polyclonal antibody was used to show that equal amount of proteins were loaded. For simplicity, parts from the same western blots are shown separately. Cell death was checked by flow cytometric analysis of dying cells by using Annexin-V-FITC/PI staining. Relevant controls (autoflourescent and Annexin-V-FITC positive cells) are also included in the figure.

Figure 3. Resident peritoneal macrophages release IL-1β while engulfing dying autophagic Ba/F3 cells.

(A) IL-3-depleted cells, live cells, apoptotic cells (treated with 10 µM doxorubicin), and necrotic Ba/F3 cells were co-incubated with primed resident macrophages. As a control condition, IL-1β was quantified from supernatant of only dying autophagic Ba/F3 cells in order to analyze if they secrete IL-1β by themselves. ATP, which is a stimulus for the inflammasome activation, was used as a positive control. (B) Primed thioglycollate-elicited macrophages were co-incubated with IL-3-depleted dying autophagic cells. Primed macrophages (control) but not co-incubated with any type of Ba/F3 cells. Unpaired non-bias two-tailed student t-test was used for statistical analysis in part B. Data represent the mean ± SEM of three and four independent experiments in parts A and B, respectively; all experiments were performed in triplicates. (*p<0.05, **p<0.01, ****p<0.0001).

Figure 4. Uptake of dying autophagic cells and autophagic features of dying cells are required for macrophages to release IL-1β.

(A) Primed resident macrophages were co-incubated with Ba/F3 cells that were IL-3-depleted, or IL-3-depleted and also pretreated with 3-methyladenine (3-MA) or necrostatin-1. Cell death was checked by flow cytometric analysis of dying autophagic cells by using Annexin-V-FITC/PI staining. For simplicity, parts of the same western blot are shown separately. (B, C) CMTMR-stained macrophages were treated with cytochalasin D (cytD) to inhibit phagocytosis, and cytD treated/non-treated macrophages were co-incubated with CFDA-stained dying autophagic Ba/F3 cells. R2 is the region which shows both the upper left quadrant (macrophages which do not engulf dying cells) and upper right quadrant (macrophages which engulf dying cells). The cells out of R2 region is also shown in the figure. The macrophages were also co-incubated with the conditioned medium (CM) from dying autophagic cells. In parts A and C, control cells are macrophages, which were primed but not co-incubated with any type of Ba/F3 cells. Data represent the mean ± SEM of two independent experiments; all experiments were performed in triplicates, (***p<0.001).

Figure 5. Uptake of dying autophagic cells leads to NALP3 and caspase-1 mediated IL-1β release in macrophages.

Primed thioglycollate-elicited macrophages from wild type and from CASPASE-1 (A) or NALP3 (C) knockout mice were co-incubated with IL-3-depleted cells. ATP was used as a positive control. (B) Resident macrophages treated with Z-YVAD-fmk (specific caspase-1 inhibitor) were co-incubated with IL-3-depleted dying cells. (D) Wild type and NALP3 or CASPASE-1 deficient macrophages were co-incubated with IL-3 depleted dying autophagic (AU) and living Ba/F3 cells and phagocytosis was measured by flow cytometry. In parts A, B and C, control cells are primed macrophages but not incubated with any type of Ba/F3 cells. Data represent the mean ± SEM of two independent experiments for part A, three independent experiments for part B, one experiment for part C, and two independent experiments for part D; all experiments were performed in triplicates, (***p<0.001).

Figure 6. Analysis of upstream mechanisms of NALP3 inflammasome activation in peritoneal macrophages engulfing dying autophagic cells.

(A) Primed resident macrophages were co-incubated with dying autophagic (AU) cells in the presence of KCl. Macrophages were treated with adenosine diphosphatase (apyrase) (B) and purinergic receptor inhibitor (KN-62) (C), or pannexin-1 channel inhibitor (CBX) (D) and co-incubated with IL-3-depleted dying autophagic cells. (B, D) ATP concentrations in culture media, (E) ATP concentrations in conditioned medium (CM) collected from Ba/F3 cells after 6 h of IL-3 depletion and CBX treated/non-treated dying AU cells (without macrophages). In parts A, B, C and D, control cells are primed macrophages but not co-incubated with any type of Ba/F3 cells. Data represent the mean ± SEM of pooled data from three experiments for A, two experiments for B, four experiments for C and D, and three experiments for E; experiments were performed in triplicates; (**p<0.01,**** p<0.0001).

Figure 7. Intraperitoneal injection of IL-3-depleted dying autophagic Ba/F3 cells induces a sterile inflammatory response (neutrophil influx).

Dying autophagic, apoptotic or necrotic cells and living cells were intraperitoneally injected into wild type BALB/c mice. Equal volumes of D-PBS were injected in mice as negative controls. Peritoneal exudate cells (PECs) were collected 16 h later and monocytes, macrophages, eosinophils and neutrophils were stained with anti-mouse antibodies F4/80-APC with CD11b-APC-Cy7 and Ly-6G-APC with CD11b-APC-Cy7 and analyzed on BD LSR-II. Graphs represent the number of macrophages (F4/80^high CD11b^high), monocytes (F4/80^medium CD11b^high), eosinophils (F4/80^medium CD11b^medium) and neutrophils (CD11b⁺ Ly6G^high) in PECs after injection of AU, necrotic, living or apoptotic Ba/F3 cells. (***p<0.001, ****p<0.0001).

Figure 8. Crude LPS-induced IL-6 release from macrophages is inhibited by dying autophagic cells

Thioglycollate-elicited peritoneal macrophages pre-treated with crude LPS were co-incubated with IL-3-depleted dying autophagic (AU) cells or with doxorubicin treated apoptotic cells. Data represent the mean ± SEM of pooled data from one experiment performed in five replicates; (****p<0.0001).