Figure 3.
Knocking out Atg5 in the eosinophil lineage results in increased bacterial clearance in C rodentium–infected mice. (A) Flow cytometry. Eosinophils isolated from the bone marrow and peripheral blood of CtrlII5tg and Atg5eoΔII5tg mice were primed with recombinant mouse (rm) GM-CSF (25 ng/mL), followed by stimulation with rmC5a (10−8 M). Unprimed eosinophils were incubated with PMA (25 nM), and eosinophil degranulation was determined by measuring the increase in plasma membrane expression of surrogate marker CD63 (n = 4). (B) Flow cytometry. Freshly purified bone marrow and peripheral blood eosinophils of CtrlII5tg and Atg5eoΔII5tg mice were primed with rmGM-CSF, subsequently activated with rmC5a, and incubated with opsonized E coli–GFP in the presence or absence of DNase I. After the incubation, supernatants were collected, and the reduction of live bacteria was determined (n = 3). (C) Quantification of double-stranded DNA (dsDNA). Released dsDNA in the supernatants of isolated eosinophils from the bone marrow and peripheral blood of CtrlII5tg and Atg5eoΔII5tg mice after combined GM-CSF/C5a or PMA treatment was quantified by using PicoGreen fluorescent dye (Thermo Fisher Scientific, Waltham, MA) (n = 4). (D) Flow cytometry. Control and Atg5eoΔ mice were infected with C rodentium for 12 days, and relative numbers of infiltrated colonic eosinophils (CD45+/Siglec-F+) and neutrophils (CD45+/Ly6G+) were determined. Colonic lamina propria eosinophils from infected mice were analyzed for their activation and degranulation status by using markers Siglec-F and CD63, respectively (n = 15-16). (E) Bacterial clearance in vivo. Control and Atg5eoΔmice were infected with C rodentium, and the numbers of bacterial CFUs were assessed by plating homogenized cecum, colon, mesenteric lymph nodes (MLNs), and spleen on agar plates (n = 15-16). (F) Confocal microscopy. Colon tissues of control and Atg5eoΔ mice were obtained 12 days after infection with C rodentium. Hematoxylin and eosin staining images show the areas that were analyzed. Scale bars, 100 µm. Tissues were stained with monoclonal anti-EPX antibody (green) and propidium iodide (PI) (red). Representative confocal microscopy images are shown. White circles indicate colocalization of extracellular DNA and EPX. Scale bars, 10 µm. Right: quantification of EPX+ infiltrating eosinophils was performed by counting cells in 10 randomly selected high-power fields, each covering the area of 22.5 × 10−3 mm2 using the automatic digital slide scanner Pannoramic MIDI II. Quantification of the DNA-releasing eosinophils was determined manually (n = 5). (G) Immunoblotting. Left: bone marrow eosinophils of CtrlII5tg and Atg5eoΔII5tg mice were untreated or primed with rmGM-CSF for 5 minutes. Protein lysates were analyzed for phosphorylated Stat3 (Tyr705), phosphorylated p38 (Thr180/Tyr182), and phosphorylated p44/42 (Thr202/Tyr204) protein expression. Stat3, p38, p44/42, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels were analyzed as loading controls. Right: bone marrow eosinophils of CtrlII5tg and Atg5eoΔII5tg mice were untreated or primed with rmIL-5, rmGM-CSF, and rmIL-33 for 5 minutes. Protein lysates were analyzed for protein expression of phosphorylated Stat3 (Tyr705). Stat3 and GAPDH served as loading controls. Representative immunoblots of 2 independent experiments are shown. Values are means ± standard error of the mean, or single data are presented in scatter dot plots in which the medians are indicated as red lines. *P < .05; **P < .01; ***P < .001. n.s., not significant.

Knocking out Atg5 in the eosinophil lineage results in increased bacterial clearance in C rodentium–infected mice. (A) Flow cytometry. Eosinophils isolated from the bone marrow and peripheral blood of CtrlII5tg and Atg5eoΔII5tg mice were primed with recombinant mouse (rm) GM-CSF (25 ng/mL), followed by stimulation with rmC5a (10−8 M). Unprimed eosinophils were incubated with PMA (25 nM), and eosinophil degranulation was determined by measuring the increase in plasma membrane expression of surrogate marker CD63 (n = 4). (B) Flow cytometry. Freshly purified bone marrow and peripheral blood eosinophils of CtrlII5tg and Atg5eoΔII5tg mice were primed with rmGM-CSF, subsequently activated with rmC5a, and incubated with opsonized E coli–GFP in the presence or absence of DNase I. After the incubation, supernatants were collected, and the reduction of live bacteria was determined (n = 3). (C) Quantification of double-stranded DNA (dsDNA). Released dsDNA in the supernatants of isolated eosinophils from the bone marrow and peripheral blood of CtrlII5tg and Atg5eoΔII5tg mice after combined GM-CSF/C5a or PMA treatment was quantified by using PicoGreen fluorescent dye (Thermo Fisher Scientific, Waltham, MA) (n = 4). (D) Flow cytometry. Control and Atg5eoΔ mice were infected with C rodentium for 12 days, and relative numbers of infiltrated colonic eosinophils (CD45+/Siglec-F+) and neutrophils (CD45+/Ly6G+) were determined. Colonic lamina propria eosinophils from infected mice were analyzed for their activation and degranulation status by using markers Siglec-F and CD63, respectively (n = 15-16). (E) Bacterial clearance in vivo. Control and Atg5eoΔmice were infected with C rodentium, and the numbers of bacterial CFUs were assessed by plating homogenized cecum, colon, mesenteric lymph nodes (MLNs), and spleen on agar plates (n = 15-16). (F) Confocal microscopy. Colon tissues of control and Atg5eoΔ mice were obtained 12 days after infection with C rodentium. Hematoxylin and eosin staining images show the areas that were analyzed. Scale bars, 100 µm. Tissues were stained with monoclonal anti-EPX antibody (green) and propidium iodide (PI) (red). Representative confocal microscopy images are shown. White circles indicate colocalization of extracellular DNA and EPX. Scale bars, 10 µm. Right: quantification of EPX+ infiltrating eosinophils was performed by counting cells in 10 randomly selected high-power fields, each covering the area of 22.5 × 10−3 mm2 using the automatic digital slide scanner Pannoramic MIDI II. Quantification of the DNA-releasing eosinophils was determined manually (n = 5). (G) Immunoblotting. Left: bone marrow eosinophils of CtrlII5tg and Atg5eoΔII5tg mice were untreated or primed with rmGM-CSF for 5 minutes. Protein lysates were analyzed for phosphorylated Stat3 (Tyr705), phosphorylated p38 (Thr180/Tyr182), and phosphorylated p44/42 (Thr202/Tyr204) protein expression. Stat3, p38, p44/42, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels were analyzed as loading controls. Right: bone marrow eosinophils of CtrlII5tg and Atg5eoΔII5tg mice were untreated or primed with rmIL-5, rmGM-CSF, and rmIL-33 for 5 minutes. Protein lysates were analyzed for protein expression of phosphorylated Stat3 (Tyr705). Stat3 and GAPDH served as loading controls. Representative immunoblots of 2 independent experiments are shown. Values are means ± standard error of the mean, or single data are presented in scatter dot plots in which the medians are indicated as red lines. *P < .05; **P < .01; ***P < .001. n.s., not significant.

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