Inhibition of Efferocytosis by Extracellular CIRP-Induced Neutrophil Extracellular Traps

Abstract

Phagocytic clearance of apoptotic cells by the macrophages (efferocytosis) is impaired in sepsis, but its mechanism is poorly understood. Extracellular cold-inducible RNA-binding protein (eCIRP) is a novel damage-associated molecular pattern that fuels inflammation. We identify that eCIRP-induced neutrophil extracellular traps (NETs) impair efferocytosis through a novel mechanism. Coculture of macrophages and apoptotic thymocytes in the presence of recombinant murine CIRP (rmCIRP)-induced NETs significantly inhibited efferocytosis. Efferocytosis was significantly inhibited in the presence of rmCIRP-treated wild-type (WT), but not PAD4^-/- neutrophils. Efferocytosis in the peritoneal cavity of rmCIRP-injected PAD4^-/- mice was higher than WT mice. Milk fat globule-EGF-factor VIII (MFG-E8), an opsonin, increased macrophage efferocytosis, whereas the inhibition of efferocytosis by NETs was not rescued upon addition of MFG-E8, indicating disruption of MFG-E8's receptor(s) α_vβ₃ or α_vβ₅ integrin by the NETs. We identified neutrophil elastase in the NETs significantly inhibited efferocytosis by cleaving macrophage surface integrins α_vβ₃ and α_vβ₅ Using a preclinical model of sepsis, we found that CIRP^-/- mice exhibited significantly increased rate of efferocytosis in the peritoneal cavity compared with WT mice. We discovered a novel role of eCIRP-induced NETs to inhibit efferocytosis by the neutrophil elastase-dependent decrease of α_vβ₃/α_vβ₅ integrins in macrophages. Targeting eCIRP ameliorates sepsis by enhancing efferocytosis.

Figure 1:. eCIRP-induced NETs inhibit efferocytosis.

(A, B) A total of 5 × 10⁵ peritoneal macrophages were cultured with 1.5 × 10⁶ CFSE-labeled apoptotic cells in presence with PBS or various concentrations of NETs. After 1 h of incubation, cells were washed, fixed with 2% PFA, and efferocytosis was assessed by flow cytometry. Efferocytosis was determined as the percentage of CFSE-positive cells present in F4/80⁺ macrophages. Data were obtained from 3 independent experiments and expressed as means ± SE (n=7 samples/group). The groups were compared by one-way ANOVA and SNK method (*p<0.05; ***p<0.001 vs. PBS-treated group). (C) Confirmation of phagocytosis of apoptotic cells in F4/80 and CFSE double-positive population by image stream analysis. Representative images showing co-localization of F4/80 and CFSE double positive cells indicate efferocytosis. Scale bar: 7 μm. BMDN, bone marrow-derived neutrophil(s); MPO, myeloperoxidase; CFSE, carboxyfluorescein succinimidyl ester; PFA, paraformaldehyde; NETs, neutrophil extracellular traps; rmCIRP, recombinant mouse cold-inducible RNA-binding protein.

Figure 2:. eCIRP-induced wild-type, but not PAD4^−/− neutrophils inhibit efferocytosis.

(A, B) BMDN (1× 10⁶) isolated from WT or PAD4^−/− mice were stimulated with PBS or rmCIRP (1 μg/mL) for 4 h to allow them to form NETs. Peritoneal macrophages (5 × 10⁵) and CFSE-labeled apoptotic cells (Ao) (1.5 × 10⁶) were separately added to PBS- or rmCIRP-treated WT or PAD4^−/− PMN. Cells were continued to culture for 1 h. Cells were then stained with PE-F4/80 Abs and assessed efferocytosis by flow cytometry. Data were obtained from 3 independent experiments and expressed as means ± SE (n=7 samples/group). The groups were compared by one-way ANOVA and SNK method. *p<0.05 vs. rmCIRP(−) PMN(−); #p<0.05 vs. rmCIRP(+) WT PMN(+). (C, D) WT and PAD4^−/− mice were injected with rmCIRP (5 mg/kg; i.p.). After 4 h of injection with rmCIRP, a total of 1 × 10⁷ CFSE-labeled apoptotic cells were injected i.p. into the mice. After 1 h, peritoneal washout cells were collected, stained with PE-F4/80 Ab, and assessed efferocytosis by flow cytometry. Data were obtained from 3 independent experiments and expressed as means ± SE (n=6 mice/group). The groups were compared by one-way ANOVA and SNK method. *p<0.05 vs. WT mice. (E-J) WT and PAD4^−/− mice were injected with rmCIRP (5 mg/kg; i.p.). After 20 h of injecting the mice with rmCIRP, peritoneal washout cells were harvested and stained with macrophage marker PE-F4/80 and neutrophil marker APC-Ly6G Abs. The contents of (E-G) macrophages and (H-J) neutrophils in the peritoneal cavity were assessed my flow cytometry. Data were obtained from 3 independent experiments and expressed as means ± SE (n=6 mice/group). The groups were compared by one-way ANOVA and SNK method. *p<0.05 vs. PBS-treated WT/PAD4^−/− mice. PAD4, peptidylarginine deiminase 4; PMN, polymorphonuclear leukocytes; Ao, apoptotic cells.

Figure 3:. NETs decrease integrin-mediated efferocytosis.

(A, B) Peritoneal macrophages (5 × 10⁵) and CFSE-labeled apoptotic cells (1.5 × 10⁶) in the presence of NETs (1000 ng/mL) with or without rmMFG-E8 (2 μg/mL). After 1 h of incubation, the cells were collected, washed, and stained with PE-F4/80 Ab and assessed efferocytosis in macrophages by flow cytometry. Data were obtained from 3 independent experiments and expressed as means ± SE (n=6 mice/group). The groups were compared by one-way ANOVA and SNK method. *p<0.05 vs. NETs(−) rmMFG-E8(−); #p<0.05 vs. NETs(−) rmMFG-E8(+). (C-F) Impaired surface expression of integrins in NETs-treated macrophages. Peritoneal macrophages (5 × 10⁵) were treated with NETs at various doses. After 1 h of stimulation, the macrophages were washed, stained with (C, D) anti- α_vβ₃ and (E, F) α_vβ₅ integrin Abs and detected integrins’ expression by flow cytometry. Data were obtained from 3 independent experiments and expressed as means ± SE (n=9 samples/group). The groups were compared by one-way ANOVA and SNK method. *p<0.05 vs. PBS-treated macrophages. rmMFG-E8, recombinant mouse milk fat globule-EGF-factor VIII.

Figure 4:. NE cleaves integrins causing impaired efferocytosis.

(A, B) Murine peritoneal macrophages (5 × 10⁵) were cultured with CFSE-stained apoptotic cells (1.5 × 10⁶) in presence of NETs (1000 ng/mL) and DNase I (500U/mL). After 1 h of cell culture, the cells were washed and stained with PE-F4/80 Ab and assessed efferocytosis using flow cytometry. (C-F) Assessment of surface expression of integrins following treatment of the macrophages with various doses of rmNE. After treatment of the peritoneal macrophages (5 × 10⁵) with rmNE for 4 h the cells were washed and stained with (C, D) anti-α_vβ₃ and (E, F) α_vβ₅ integrin Abs and assessed their expression by flow cytometry. (G, I) Peritoneal macrophages (5 × 10⁵) and CFSE-labeled apoptotic cells (1.5 × 10⁶) were co-cultured in the presence of various doses of rmNE. After 1 h, the cells were washed and stained with PE-F4/80 Ab and the efferocytosis was assessed by flow cytometry. Data were obtained from 3 independent experiments and expressed as means ± SE (n=7–9 samples/group). The groups were compared by one-way ANOVA and SNK method. *p<0.05 vs. PBS-treated macrophages. (H, J) Peritoneal macrophages (5 × 10⁵) and CFSE-labeled apoptotic cells (1.5 × 10⁶) were co-cultured in the presence of rmNE (1000 ng/mL) with or without NE-I (100 μM). After 1 h, the cells were washed and stained with PE-F4/80 Ab and assessed efferocytosis by flow cytometry. Data were obtained from 3 independent experiments and expressed as means ± SE (n=6 samples/group). The groups were compared by one-way ANOVA and SNK method. *p<0.05 vs. NE(−) NE-I(−); #p<0.05 vs. NE(+) NE-I(−). NE, neutrophil elastase; NE-I, NE-inhibitor.

Figure 5:. NE inhibitor reverses NET-dependent impairment of efferocytosis.

(A, B) Murine peritoneal macrophages (5 × 10⁵) and CFSE-labeled apoptotic cells (1.5 × 10⁶) in presence of NETs (1000 ng/mL) and various doses of NE-I. After 1 h of cell culture, the cells were washed and stained with PE-F4/80 Ab and assessed efferocytosis using flow cytometry. Data were obtained from 3 independent experiments and expressed as means ± SE (n=8 samples/group). The groups were compared by one-way ANOVA and SNK method. *p<0.05 vs. NETs(−) NE-I(−); #p<0.05 vs. NETs(+) NE-I(–). (C) Murine peritoneal macrophages (5 × 10⁵) were plated in 24-well plate. CFSE-stained apoptotic cells (1.5 × 10⁶) were added to the macrophages. The cells were treated with NETs (1000 ng/mL) and various doses of NE-I. After 1 h of incubation, media was carefully removed, washed the cells with PBS, followed by fixing and stained the cells with PE-F4/80 Ab. Efferocytosis was detected by using fluorescent microscope at ×400 original magnification. Representative images in each group were chosen from 10 randomly captured images. Scale bar: 40 μm.

Figure 6:. CIRP^−/− mice have increased efferocytosis in sepsis.

(A, B) WT and CIRP^−/− mice were underwent sham and CLP operation. After 5 h of surgery all mice were injected with CFSE-labeled apoptotic cells (1 × 10⁷) via i.p. injection. After 1 h of injection of apoptotic cells, peritoneal washout cells were collected and stained with PE-F4/80 Ab and assessed efferocytosis by flow cytometry. Data were obtained from 3 independent experiments are expressed as means ± SE (n=6 mice/group). The groups were compared by one-way ANOVA and SNK method. *p<0.05 vs. relevant (WT or CIRP^−/−) sham mice; #p<0.05 vs. WT CLP mice.

Figure 7:. Schematic Summary.

eCIRP is released during sepsis, which induces neutrophils to produce NETs. NE contained in the NETs cleaves α_vβ₃ and α_vβ₅ integrins expressed on the surface of macrophages. This leads to decreased binding of apoptotic cell’s opsonin MFG-E8 to the macrophage, thereby impairing the bridge formation between apoptotic cell and macrophage and inhibiting efferocytosis.