Extracellular vesicles from A23187-treated neutrophils cause cGAS-STING-dependent IL-6 production by macrophages

Abstract

In response to several types of bacteria, as well as pharmacological agents, neutrophils produce extracellular vesicles (EVs) and release DNA in the form of neutrophil extracellular traps (NETs). However, it is unknown whether these two neutrophil products cooperate to modulate inflammation. Consistent with vital NETosis, neutrophils challenged with S. aureus, as well as those treated with A23187, released significantly more DNA relative to untreated or fMLF-treated neutrophils, with no lysis occurring for any condition. To test the hypothesis that EVs generated during NETosis caused macrophage inflammation, we isolated and characterized EVs from A23187-treated neutrophils (A23187-EVs). A23187-EVs associated with neutrophil granule proteins, histone H3, transcription factor A, mitochondrial (TFAM), and nuclear and mitochondrial DNA (mtDNA). We showed that DNA from A23187-EVs, when transfected into macrophages, led to production of IL-6 and IFN-α2, and this response was blunted by pre-treatment with the STING inhibitor H151. Next, we confirmed that A23187-EVs were engulfed by macrophages, and showed that they induced cGAS-STING-dependent IL-6 production. In contrast, neither EVs from untreated or fMLF-treated cells exhibited pro-inflammatory activity. Although detergent-mediated lysis of A23187-EVs diminished IL-6 production, removal of surface-associated DNA with DNase I treatment had no effect, and A23187-EVs did not induce IFN-α2 production. Given these unexpected results, we investigated whether macrophage mtDNA activated the cGAS-STING signaling axis. Consistent with mitochondrial outer membrane permeabilization (MOMP), a defined mechanism of mtDNA release, we observed macrophage mitochondrial membrane depolarization, a decrease in cytosolic Bax, and a decrease in mitochondrial cytochrome c, suggesting that macrophage mtDNA may initiate this EV-dependent signaling cascade. All together, these data demonstrate that A23187-EVs behave differently than transfected NET- or EV-DNA, and that neutrophil-derived EVs could be used as a model to study NF-κB-dependent STING activation.

Keywords: EVs; STING; cGAS; ectosomes; exosomes; inflammation; microvesicles.

Figure 1

Neutrophils treated with NET agonists release EVs. Following 20 minutes incubation with buffer or indicated agonists, conditioned media was collected and assayed for cytotoxicity (A, n≥3) and extracellular DNA (B, n≥3). Values are expressed as fold change relative to neutrophils left alone in buffer (control, CTL). Data are presented as the mean of three or more experiments ± SEM. P-values were determined using a one-way ANOVA with Dunnett’s post-test (* p < 0.05, ** p < 0.01 vs. CTL). EV subtypes (spontaneous-EVs (far left, scale bar 200 nm), fMLF-EVs (middle left, scale bar 200 nm), A23187-EVs (middle right, scale bar 200 nm), and S. aureus-EVs (far right, scale bar 200 nm)) were processed and then imaged on a Hitachi 7700 TEM at 80kV HR. Shown are representative electron micrographs at 10,000X magnification (C, n=3). A23187-EVs were conjugated to beads, stained with anti-CD63-PE, and analyzed by flow cytometry. Shown is a representative histogram gated on bead-conjugated EVs (D, n=3). Equivalent protein concentrations (1µg top and 9µg bottom) of cell lysates (CL) or EVs were separated by SDS-PAGE and blotted for flotillin-1 and GRP94. Shown are representative images of four or more experiments (E).

Figure 2

A23187-EVs associated with NET proteins and DNA, and transfected DNA activated cGAS-STING. Neutrophil elastase (NE), myeloperoxidase (MPO), citrullinated histone H3 (cit-H3), histone H3 (His-H3), and TFAM were detected by immunoblotting. Equivalent protein concentrations of cell lysates (CL) and EVs were separated by SDS-PAGE prior to immunoblotting (1µg for MPO; 3µg for NE, cit-H3, and His-H3; 9µg for TFAM). Shown are representative images of four experiments (A). EV-DNA was isolated from EV subsets and resolved with gel electrophoresis. Shown is a representative image of five experiments (B). EVs were treated or without with DNase I for 1 hour prior to isolating DNA. Primers specific for 18S rRNA and tRNA^Leu were used to amplify nuclear (C) and mitochondrial (D) DNA, respectively, and DNA copy numbers were quantified by ddPCR. Values are expressed as DNA copies per EV-cell equivalents. Bars represent the average of three or more experiments ± SEM. P-values were determined using a two-way ANOVA with Dunnett’s post-test (**** p < 0.0001 vs. spontaneous-EVs). A23187-EV-DNA was transfected for 18 hours at the indicated concentrations, and cytokines in supernatants were measured by ELISA (E, n=3). Bars represent the average of experimental data ± SEM. P values were determined using a two-way ANOVA with Sidak’s post-test (* p <0.05 vs. CTL, ^# p <0.05 vs. CTL). EV-DNA was isolated from A23187-EVs and then encapsulated in DOTAP. Macrophages were pre-treated with and without H151 (1 µM), then transfected for 18 hours. Supernatants were collected and analyzed by ELISA for IL-6 (F, n=4) and IFN-α2 (G, n=3). Bars represent the average of experimental data ± SEM. P values were determined using a two-way ANOVA with Sidak’s post-test (*** p < 0.001, **** p < 0.0001).

Figure 3

Macrophages engulf A23187-EVs in an actin- and PI3K-dependent manner. PKH67-labelled A23187-EVs were co-cultured with macrophages for the indicated times before analysis by flow cytometry (A). Bars represent the average of three experiments ± SEM. P values were determined using a one-way ANOVA with Dunnett’s post-test (** p < 0.01, *** p < 0.001 vs. time 0). Macrophages were pre-treated for 2 hours with the inhibitors cytochalasin D (B) and wortmannin (C) at the indicated concentrations. PKH67-stained A23187-EVs were co-cultured with macrophages 1.5 hours, and analyzed by flow cytometry. Bars represent the average of three experiments ± SEM. P values were determined using a one-way ANOVA with Dunnett’s post-test (* p < 0.05, ** p < 0.01, **** p < 0.0001).

Figure 4

A23187-EVs cause macrophage IL-6 production. Macrophages were treated for 18 hours with 1 µg/mL of transfected S. aureus DNA (SA-DNA) or 1 µg/mL EV subtypes and supernatants were analyzed by ELISA (A). A portion of A23187-EVs were lysed prior to ultracentrifugation with 0.05% TX-100, and paired whole and lysed A23187-EVs were used to treat macrophages at equivalent protein concentration as above (B). Bars represent the averages of four or more experiments ± SEM. P values were determined using one-way ANOVA with Tukey’s post-test (** p < 0.01, *** p < 0.001). A23187-EVs treated without (top panel) and with (bottom panel) TX-100 were processed and imaged on a Hitachi 7700 TEM at 80kV HR. Shown are representative electron micrographs at 10,000X magnification (C, scale bar 500 nm, n=3).

Figure 5

A23187-EVs cause cGAS-STING-dependent IL-6 production but do not cause IFN-α2 production. Macrophages were left in buffer or pre-treated with the inhibitors g140 (A, cGAS inhibitor, 5µM) or H151 (B, STING inhibitor, 1µM). Macrophages were treated with A23187-EVs at the indicated concentrations, supernatants were collected after 18 hours, and IL-6 analysis was performed by ELISA. Bars represent the average of seven or more experiments ± SEM. P values were determined using a two-way ANOVA with Sidak’s post-test (* p < 0.05, *** p < 0.001 vs. CTL). Macrophages were treated with the indicated concentrations of A23187-EVs and cytokines were measured (C, n=11). Bars represent the average of experimental data ± SEM. P values were determined using a two-way ANOVA with Sidak’s post-test (* p <0.05, **** p <0.0001; ^# p < 0.05 vs. CTL).

Figure 6

A23187-EVs cause macrophage mitochondrial dysfunction. S. aureus DNA (SA-DNA, left panel) and A23187-EVs (right panel) were treated with and without DNase I, then co-cultured with macrophages for 18 hours. Supernatants were analyzed by ELISA for IL-6 (A). Bars represent the average of 8 experiments ± SEM. P values were determined using a paired t-test (left panel, ** p < 0.01) and a two-way ANOVA with Sidak’s post-test (right panel, ns, not significant,). Macrophages were left untreated, or were treated at the indicated times with A23187-EVs or CCCP, stained using JC-1, then analyzed by flow cytometry for loss of mitochondrial membrane polarization (B). Data was normalized to CCCP treatment and bars represent the average of 5 experiments ± SEM. P values were determined using a one-way repeated measures ANOVA with Dunnett’s post-test (* p < 0.05, *** p < 0.001 vs. time 0). Following treatment of macrophages with A23187-EVs for the indicated time points, supernatants were collected and assayed LDH as a measure for cytotoxicity (C, n=3). Values are expressed as fold change relative to untreated macrophages. Data are presented as the mean of 3 experiments ± SEM. Macrophages were treated at the indicated times with spontaneous-EVs or CCCP, then analyzed by flow cytometry as above (D). Bars represent the average of three experiments ± SEM. P values were determined using a one-way repeated measures ANOVA with Dunnett’s post-test (* p < 0.05, *** p < 0.001 vs. time 0). Macrophages were treated with A23187-EVs at the indicated time points, subcellular fractionation performed, then 10 µg lysates were resolved by SDS-PAGE gel and blotted for Bax, cytochrome c, and loading controls GAPDH and TOM20, as indicated (E). A representative result from four experiments is shown.