ATP Release from Dying Autophagic Cells and Their Phagocytosis Are Crucial for Inflammasome Activation in Macrophages

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, P2X7 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

Inflammasome Activation in Human and Mouse Macrophages Engulfing Autophagic Dying Cells

Phagocytosis of PAMPs, DAMPs and certain dying cells can activate the inflammasome pathway in macrophages. In our study, we show that both human and mouse macrophages display a pro-inflammatory response to autophagic dying MCF-7 and Ba/F3 cells, but not to living, apoptotic, necrotic or necrostatin-1 treated ones. When we investigated this phenomenon, further it was found that caspase-1 was activated and IL-1β was processed and then secreted in a MyD88-independent manner. Neither caspase-1 inhibited nor caspase-1 deficient macrophages could trigger IL-1β release due to the lack of key component for pro-IL-1β cleavage and maturation before its secretion. Next we clarified which inflammasome is activated by autophagic dying cells and found that NALP-3 deficient macrophages displayed reduced IL-1β secretion, which was also observed in macrophages in which the NALP-3 gene was knocked down. Next, we investigated the upstream mechanism of NALP-3 inflammasome activation triggered by autophagic dying cells. Our results show that during phagocytosis of autophagic dying MCF-7 and Ba/F3 cells exogenous ATP is acting through P2X7 receptor, initiates K+ efflux, inflammasome activation and secretion of IL-1β from human and mouse macrophages. Calreticulin exposure on autophagic dying MCF-7 cells do not play role in inflammasome activation. ATP was secreted from human macrophages during co-incubation with autophagic dying MCF-7 cells which did not release ATP. However, autophagic dying Ba/F3 cells were the source the ATP which activated the P2X7 receptor and lead to inflammasome activation in mouse macrophages. We further showed that pannexin-1 channel is responsible for ATP secretion from autophagic dying Ba/F3 cells. MCF-7 and Ba/F3 cells dying with involvement of autophagy were capable of preventing crude LPS-induced pro-inflammatory cytokine release but pro-inflammatory cytokines were produced and secreted from human macrophages triggered by autophagic dying cells as a result of the secreted IL-1β. Finally, it was observed that injection of autophagic dying cells intraperitoneally induced an acute inflammatory reaction by recruiting neutrophils and monocytes/macrophages.d

EPO mediates neurotrophic, neuroprotective, anti-oxidant and anti-apoptotic effects via downregulation of miR-451 and miR-885-5p in SH-SY5Y neuron-like cells

Erythropoietin (EPO) is a neuroprotective cytokine, which has been applied in several animal models presenting neurological disorders. One of the proposed modes of action resulting in neuroprotection is post-transcriptional gene expression regulation. This directly brings to mind microRNAs (miRNAs), which are small non-coding RNAs that regulate gene expression at the post-transcriptional level. It has not yet been evaluated whether miRNAs participate in the biological effects of EPO or whether it, inversely, modulates specific miRNAs in neuronal cells. In this study, we employed miRNA and mRNA arrays to identify how EPO exerts its biological function. Notably, miR-451 and miR-885-5p are downregulated in EPO-treated SH-SY5Y neuronal-like cells. Accordingly, target prediction and transcriptome analysis of cells treated with EPO revealed an alteration of the expression of genes involved in apoptosis, cell survival, proliferation, and migration. Low expression of miRNAs in SHSY5Y was correlated with high expression of their target genes, vascular endothelial growth factor A, matrix metallo peptidase 9 (MMP9), cyclin-dependent kinase 2 (CDK2), erythropoietin receptor, Mini chromosome maintenance complex 5 (MCM5), B-cell lymphoma 2 (BCL2), and Galanin (GAL). Cell viability, apoptosis, proliferation, and migration assays were carried out for functional analysis after transfection with miRNA mimics, which inhibited some biological actions of EPO such as neuroprotection, anti-oxidation, anti-apoptosis, and migratory effects. In this study, we report for the first time that EPO downregulates the expression of miRNAs (miR-451 and miR-885-5p) in SH-SY5Y neuronal-like cells. The correlation between the over-expression of miRNAs and the decrease in EPO-mediated biological effects suggests that miR-451 and miR-885-5p may play a key role in the mediation of biological function

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

<p>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).</p

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

<p>(<b>A</b>) Primed resident macrophages were co-incubated with Ba/F3 cells that were IL-3-depleted, or IL-3-depleted and also pretreated with 3<b>-</b>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>B</b>, <b>C</b>) 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).</p

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

<p>(<b>A</b>) 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>B</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.</p

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

<p>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).</p

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

<p>(<b>A</b>) Primed resident macrophages were co-incubated with dying autophagic (AU) cells in the presence of KCl. Macrophages were treated with adenosine diphosphatase (apyrase) (<b>B</b>) and purinergic receptor inhibitor (KN-62) (<b>C</b>), or pannexin-1 channel inhibitor (CBX) (D) and co-incubated with IL-3-depleted dying autophagic cells. (<b>B</b>, <b>D</b>) 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).</p