Autophagy Protects against Eosinophil Cytolysis and Release of DNA

Abstract

The presence of eosinophils in the airway is associated with asthma severity and risk of exacerbations. Eosinophils deposit their damaging products in airway tissue, likely by degranulation and cytolysis. We previously showed that priming blood eosinophils with IL3 strongly increased their cytolysis on aggregated IgG. Conversely, IL5 priming did not result in significant eosinophil cytolysis in the same condition. Therefore, to identify critical events protecting eosinophils from cell cytolysis, we examined the differential intracellular events between IL5- and IL3-primed eosinophils interacting with IgG. We showed that both IL3 and IL5 priming increased the eosinophil adhesion to IgG, phosphorylation of p38, and production of reactive oxygen species (ROS), and decreased the phosphorylation of cofilin. However, autophagic flux as measured by the quantification of SQSTM1-p62 and lipidated-MAP1L3CB over time on IgG, with or without bafilomycin-A1, was higher in IL5-primed compared to IL3-primed eosinophils. In addition, treatment with bafilomycin-A1, an inhibitor of granule acidification and autophagolysosome formation, enhanced eosinophil cytolysis and DNA trap formation in IL5-primed eosinophils. Therefore, this study suggests that increased autophagy in eosinophils protects from cytolysis and the release of DNA, and thus limits the discharge of damaging intracellular eosinophilic contents.

Keywords: DNA traps; IL3; IL5; IgG; MAP1LC3B; SQSTM1; autophagy; cytolysis; eosinophils.

Figure 1

IL5-primed cells produce the same amount of ROS and phospho(p)-cofilin on IgG as IL3-primed eosinophils but display reduced adhesion and p-p38. Blood eosinophils were primed with either IL3 (2 ng/mL) or IL5 (2 ng/mL) for 20 h and were seeded on coated heat-aggregated (HA)-IgG (IL3IgG or IL5IgG) for the indicated timepoints. IL3-primed eosinophils were also seeded in wells without coated HA-IgG (IL3). (A) Adhesion using Cell Tag 700 was performed in 96-well plates after 2 h on IgG. Representative wells are shown in the top panel. The graph shows average ± SEM for 4 experiments using 4 different donors, and paired Student’s t test between IL5IgG and IL3IgG is shown. (B,C) Western blots were performed to evaluate the level of phosphorylation of p38 MAPK (B) and cofilin (C). Primed eosinophils remained 1 h (B) and 2 h (C) on IgG. The intensity of the signals is relative to β-actin, and the average ratio was fixed at 100 for IL3IgG in (B), and IL3 in (C). Average ± SD of 3 experiments with 3 different donors is shown. One-way ANOVA: IL5IgG is significantly reduced compared to IL3IgG in (B) (#); both IL5IgG and IL3IgG are significantly reduced compared to IL3 in (C) (*), with no statistical difference between IL5IgG and IL3IgG (ns). (D) ROS production was measured using DRH-123 at the indicated timepoints on IgG. For positive control, eosinophils were treated with PMA (100 ng/mL). No difference was observed between IL5IgG, IL3IgG, and PMA for all timepoints (average ± SD of n = 4 experiments with 4 different donors).

Figure 2

Primed eosinophils produce SQSTM1-p62, LC3 and lipidated-LC3 (PE-LC3). Blood eosinophils were cultured without cytokine (rest) or were primed with either IL3 (2 ng/mL) or IL5 (2 ng/mL) for 20 h. A representative Western blot is shown and graphs show the average ± SEM of the ratio with β-actin from 3 experiments using 3 different donors. The ratio for rest was fixed at 1. SQSTM1-p62 is increased by cytokine priming while LC3 and PE-LC3 were not changed. One-way ANOVA was performed and p values compared to rest for SQSTM1-p62 are shown.

Figure 3

Autophagy is higher in IL5- versus IL3-primed eosinophils. Blood eosinophils were primed with IL3 (2 ng/mL) or IL5 (2 ng/mL) for 20 h and were seeded on coated HA-IgG for the indicated timepoints. Western blots for SQSTM1 and LC3 were performed and β-actin was used as a loading control. Representative blots and graphs with average ± SEM are shown. (A) The amount of PE-LC3 and SQSTM1-p62 was lower in IL5-primed versus IL3-primed eosinophils when interacting with IgG (n = 3 experiments using 3 different donors). For each timepoint, paired Student’s t tests to compare IL3 and IL5 were performed and p values < 0.05 are shown (ns = not significant). (B) Western blots were performed to measure PE-LC3 and SQSTM1-62 in cytokine-primed (IL3 or IL5) eosinophils on IgG for 1.5 h after treatment with bafilomycin-A1 (Baf.; 1 μM) or DMSO (vehicle only; (-)). The difference in amount of PE-LC3 with Baf. and vehicle only represent the autophagic flux (AF). Values for (-) were fixed at 1. Paired Student’s t tests were performed to evaluate the difference between Baf. and vehicle only (-). P values are shown; n = 6 experiments (6 subjects) for PE-LC3B and n = 7 (7 subjects) for SQSTM1-62.

Figure 4

Inhibition of the formation of autophagolysosomes leads to increased eosinophil cytolysis on IgG. Eosinophils were primed with either IL3 (2 ng/mL) or IL5 (2 ng/mL) for 20 h, treated with bafilomycin-A1 (Baf.; 1μM) or vehicle only [(-); DMSO] for 20 min, and seeded on HA-IgG for 4.5 h before addition of R110 for 30 min. Cytolysis was measured by fluorescence (485 nm_Ex/520 nm_Em). Values obtained with the controls (medium only and IL3-primed eosinophils seeded without IgG) were subtracted from the values obtained for cytokine-primed eosinophils on IgG (IL3IgG and IL5IgG). For each of the 4 experiments (4 different blood donors), the value for IL3IgG was fixed at 100. After Log10 transformation, one-way ANOVA followed by Holm-Sidak was performed and p values comparing Baf. versus no Baf. are shown. # indicates p < 0.001 between (-)IL3IgG and (-)IL5IgG, and * indicates p < 0.001 between Baf.IL3IgG and Baf.IL5IgG. A representative image of the 4 conditions is shown (bright-field; 20X objective of a Nikon Eclipse Ti inverted microscope).

Figure 5

IL3-primed eosinophils on IgG release DNA in the extracellular compartment. Blood eosinophils were primed with IL3 (2 ng/mL) for 20 h and were seeded on coated HA-IgG for 5 h. Eosinophils were stained for an actin regulatory protein, gelsolin (anti-gelsolin; green), filamentous actin (phalloidin, grey), and DNA (DAPI; blue). DNA clouds and projections are stained by DAPI.

Figure 6

Inhibition of the formation of autophagolysosomes in IL5-primed eosinophils on IgG leads to increased DNA projections and spreading. Eosinophils were primed with IL3 (2 ng/mL) or IL5 (2 ng/mL) for 20 h and were seeded on coated HA-IgG for 4.5 h. For IL5 priming, cells were treated with bafilomycin-A1 (Baf.; 1 μM) or DMSO ((-); vehicle only) for 20 min before seeding on IgG. Cells were fixed and stained with DAPI to visualize DNA. Three experiments using three different blood donors were performed. For each experiment, three to four fields per well were imaged for a total of about 450 cells using a 20× objective. (A) The number of DNA projections was counted and reported per 100 cells. One-way ANOVA followed by Holm-Sidak method: * p < 0.001 between IL5IgG and Baf.IL5IgG; # p < 0.001 between (-)IL3IgG and (-)IL5IgG. (B) The surface covered by DNA was measured by ImageJ and reported as area of DNA per cell. IL3IgG was fixed at 100. Student’s t test between IL5IgG and Baf.IL5IgG: ^ p < 0.0014. (C) A representative image of the three conditions is shown.