15-Epi-LXA4 and 17-epi-RvD1 restore TLR9-mediated impaired neutrophil phagocytosis and accelerate resolution of lung inflammation

Abstract

Timely resolution of bacterial infections critically depends on phagocytosis of invading pathogens by polymorphonuclear neutrophil granulocytes (PMNs), followed by PMN apoptosis and efferocytosis. Here we report that bacterial DNA (CpG DNA) and mitochondrial DNA impair phagocytosis and attenuate phagocytosis-induced apoptosis in human PMNs through Toll-like receptor 9 (TLR9)-mediated release of neutrophil elastase and proteinase 3 and subsequent down-regulation of the complement receptor C5aR. Consistently, CpG DNA delays pulmonary clearance of Escherichia coli in mice and suppresses PMN apoptosis, efferocytosis, and generation of proresolving lipid mediators, thereby prolonging lung inflammation evoked by E. coli Genetic deletion of TLR9 renders mice unresponsive to CpG DNA. We also show that aspirin-triggered 15-epi-lipoxin A₄ (15-epi-LXA₄) and 17-epi-resolvin D1 (17-epi-RvD1) through the receptor ALX/FPR2 antagonize cues from CpG DNA, preserve C5aR expression, restore impaired phagocytosis, and redirect human PMNs to apoptosis. Treatment of mice with 15-epi-LXA₄ or 17-epi-RvD1 at the peak of inflammation accelerates clearance of bacteria, blunts PMN accumulation, and promotes PMN apoptosis and efferocytosis, thereby facilitating resolution of E. coli-evoked lung injury. Collectively, these results uncover a TLR9-mediated endogenous mechanism that impairs PMN phagocytosis and prolongs inflammation, and demonstrate both endogenous and therapeutic potential for 15-epi-LXA₄ and 17-epi-RvD1 to restore impaired bacterial clearance and facilitate resolution of acute lung inflammation.

Keywords: TLR9; aspirin-triggered LXA4 and RvD1; neutrophils; phagocytosis; resolution of inflammation.

Fig. 1.

CpG DNA impairs phagocytosis-induced neutrophil apoptosis. (A and B) Human PMNs (5 × 10⁶ cells per milliliter) (A) or whole blood (B) was cultured with CpG DNA for 60 min and then mixed with opsonized FITC-labeled E. coli at a ratio of 1:7 for 30 min. Extracellular fluorescence was quenched with 0.2% trypan blue and intracellular fluorescence was analyzed with flow cytometry. Results are means ± SEM (n = 6 different blood donors). *P < 0.05, **P < 0.01. (C and D) PMNs were cultured for 4 h (C) or 24 h (D) with yeast at a ratio of 1:5 and stained with acridine orange (10 µg/mL), and apoptosis was assessed by nuclear morphology (condensed or fragmented chromatin) under a fluorescence microscope. Results are means ± SEM (n = 6 different blood donors). *P < 0.05, **P < 0.01. (E and F) Caspase-8 (E) and caspase-3 (F) activity was assessed at 4 h of culture with flow cytometry using FITC-labeled Z-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethyl ketone and Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone, respectively. Results are means ± SEM (n = 4 different blood donors). *P < 0.05 (Dunn’s multiple contrast hypothesis test). RFU, relative fluorescence unit.

Fig. 2.

CpG DNA inactivates C5aR through inducing NE and PR3 release in human neutrophils. (A and B) PMNs (5 × 10⁶ cells per milliliter) were cultured with CpG DNA for 60 min. Cell-surface expression of CD11b and C5aR (CD88) was analyzed by flow cytometry using anti-CD11b mAb (clone D12) and anti-CD88 mAb (clone S5/1), respectively. Representative histograms (A) and concentration-dependent actions of CpG DNA after correction with staining with appropriate isotype-matched irrelevant antibodies (IgG) (B). Results are means ± SEM (n = 4 or 5 different blood donors). ^#P < 0.05, ^##P < 0.01 vs. vehicle (0 µg/mL) (Wilcoxon–Wilcox test). (C) Proteome profiler array on human PMNs challenged with CpG DNA (1.6 μg/mL) or vehicle (control) for 30 min and densitometry analysis of Src kinases. Blots and densitometry data are representative of three independent arrays with different blood donors. (D and E) NE (D) and PR3 levels (E) in the culture medium were measured at 1 h post CpG DNA by colorimetric assays using specific substrates. Results are means ± SEM (n = 5 independent experiments). ^##P < 0.01 vs. vehicle (0 µg/mL) (Wilcoxon–Wilcox test). (F) Surface C5aR expression. Human PMNs were incubated with neutrophil elastase inhibitor IV (20 µM), cathepsin G inhibitor (CGI; 20 µM), 1,10-phenantroline (Phen; 4 mM), PMSF (2 mM), or neutralizing anti-PR3 Ab (5 µg/mL) for 30 min and then challenged with CpG DNA (1.6 μg/mL) for 60 min. Surface expression of C5aR was analyzed by flow cytometry. Results are means ± SEM (n = 4 or 5 different blood donors). *P < 0.05, **P < 0.01 (Dunn’s multiple contrast hypothesis test). (G) NEI restores phagocytosis. PMNs were preincubated with NEI or CGI for 30 min and challenged with CpG DNA (1.6 μg/mL) in the presence of opsonized FITC-labeled E. coli at a ratio of 1:7 for 30 min. Extracellular fluorescence was quenched with 0.2% trypan blue and intracellular fluorescence was analyzed with flow cytometry. Results are means ± SEM (n = 5 or 6 different blood donors). *P < 0.05, **P < 0.01 (Dunn’s multiple contrast hypothesis test). (H) NE and PR3 down-regulate C5aR expression. PMNs were challenged with purified human NE or PR3 for 60 min. Results are means ± SEM (n = 5 different blood donors). ^#P < 0.05, ^##P < 0.01 vs. control (0 μg/mL) (Wilcoxon–Wilcox test).

Fig. 3.

CpG DNA impairs bacterial clearance and delays resolution of E. coli pneumonia in mice. Female C57BL/6 mice were injected intratracheally with 5 × 10⁶ live E. coli with or without CpG DNA (1 μg/g b.w., i.p.). (A and C) At 6, 24, or 48 h, the lungs were removed without lavage and analyzed for E. coli content (A) and tissue myeloperoxidase (MPO) activity (C). (B) Lung tissue sections from naïve mice (control) and mice challenged with E. coli and CpG DNA were stained with hematoxylin and eosin. (Scale bars, 100 µm.) (D–J) In separate groups of mice, bronchoalveolar lavage fluid protein concentration (D), neutrophil (E) and monocyte/macrophage numbers (F), percentage of annexin V-positive (apoptotic) neutrophils (G), percentage of macrophages containing apoptotic bodies (H), and lavage fluid levels of 15-epi-LXA₄ (I) and RvD1 (J) were determined. Results are means ± SEM (n = 5 to 7 mice per group). *P < 0.05, **P < 0.01 (Dunn’s multiple contrast hypothesis test).

Fig. 4.

15-Epi-LXA₄ restores impaired phagocytosis-induced neutrophil apoptosis. (A–C) Human PMNs (5 × 10⁶ cells per milliliter) were cultured for 10 min with 15-epi-LXA₄ and then with CpG DNA (1.6 μg/mL) for 60 min. Surface expression of CD11b (A) and C5aR (CD88) (B) was assessed by flow cytometry; neutrophil elastase release was measured with a colorimetric assay using N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide as a substrate (C). Results are means ± SEM (n = 4 or 5 different blood donors). *P < 0.05, **P < 0.01. (D) Purified human neutrophil elastase was incubated with 15-epi-LXA₄ for 10 min and then elastase activity was monitored. Results are means ± SEM (n = 3 independent experiments). (E) PMNs were cultured with 15-epi-LXA₄ for 10 min and CpG DNA for 60 min and then with opsonized FITC-labeled E. coli (7 bacteria per neutrophil) for 30 min. Extracellular fluorescence was quenched with 0.2% trypan blue and intracellular fluorescence was analyzed with flow cytometry. Results are means ± SEM (n = 6 different blood donors). **P < 0.01, ***P < 0.001. (F–H) Neutrophils were cultured with 15-epi-LXA₄ or DPI (20 μM) for 10 min, followed by CpG DNA for 60 min and then with yeast (5 yeast particles per cell). (F) Apoptosis was assessed by nuclear morphology at 24 h of culture following staining with acridine orange (10 µg/mL). Results are means ± SEM (n = 4 to 6 different blood donors). (G and H) Caspase-8 (G) and caspase-3 (H) activity was assessed at 4 h of culture with flow cytometry using FITC-labeled Z-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethyl ketone and Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone, respectively. Results are means ± SEM (n = 4 different blood donors). *P < 0.05, **P < 0.01 (Dunn’s multiple contrast hypothesis test).

Fig. 5.

15-Epi-LXA₄ and 17-epi-RvD1 facilitate bacterial clearance and enhance resolution of lung inflammation. (A) Female C57BL/6 mice were treated with 15-epi-LXA₄ (125 ng/g b.w., i.p.), 17-epi-RvD1 (25 ng/g b.w., i.p.), or vehicle 6 h after intratracheal instillation of 5 × 10⁶ live E. coli plus CpG DNA (1 μg/g b.w., i.p.). Mice were killed at the indicated times, and the lungs were processed for analysis without lavage or bronchoalveolar lavage was performed. (B) E. coli content. (C) Lung tissue sections from naïve mice (control) and mice challenged with E. coli + CpG DNA and treated with 15-epi-LXA₄, 17-epi-RvD1, or vehicle. Hematoxylin and eosin stain. (Scale bars, 100 µm.) (D) Lung tissue MPO content. (E) Lung dry-to-wet weight ratio. (F–K) Bronchoalveolar lavage fluid protein concentration (F), total leukocyte (G), neutrophil (H), and monocyte/macrophage counts (I), percentage of annexin V-positive (apoptotic) neutrophils (gated as Ly6G⁺ cells) (J), and percentage of macrophages containing apoptotic bodies (K) were determined. Results are means ± SEM (n = 6 or 7 mice per group). *P < 0.05, **P < 0.01 (Dunn’s multiple contrast hypothesis test).