Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury

Abstract

Neutrophils dominate the early immune response in pathogen-induced acute lung injury, but efforts to harness their responses have not led to therapeutic advancements. Neutrophil extracellular traps (NETs) have been proposed as an innate defense mechanism responsible for pathogen clearance, but there are concerns that NETs may induce collateral damage to host tissues. Here, we detected NETs in abundance in mouse models of severe bacterial pneumonia/acute lung injury and in human subjects with acute respiratory distress syndrome (ARDS) from pneumonia or sepsis. Decreasing NETs reduced lung injury and improved survival after DNase I treatment or with partial protein arginine deiminase 4 deficiency (PAD4+/-). Complete PAD4 deficiency (PAD4-/-) reduced NETs and lung injury but was counterbalanced by increased bacterial load and inflammation. Importantly, we discovered that the lipoxin pathway could be a potent modulator of NET formation, and that mice deficient in the lipoxin receptor (Fpr2-/-) produced excess NETs leading to increased lung injury and mortality. Lastly, we observed in humans that increased plasma NETs were associated with ARDS severity and mortality, and lower plasma DNase I levels were associated with the development of sepsis-induced ARDS. We conclude that a critical balance of NETs is necessary to prevent lung injury and to maintain microbial control, which has important therapeutic implications.

Keywords: Immunology; Innate immunity; Neutrophils; Pulmonology.

Figure 1. Neutrophil extracellular traps (NETs) are elaborated during MRSA- and PAO1-induced lung injury models.

(A) Mice were instilled intratracheally (i.t.) with methicillin-resistant Staphylococcus aureus (MRSA) at 5 × 10⁷ CFU/mouse and sacrificed at indicated time points (n = 3–12). (A) Bronchoalveolar lavage (BAL) total protein concentration, (B) lung vascular permeability to radioactive albumin, (C) excess lung water, and (D) BAL WBC were all increased after infection. (E) BAL cytospin and (F) differential counts are composed mainly of neutrophils. Scale bar: 20 μm. (G) NETs (neutrophil elastase–DNA complexes) in BAL and (H) plasma. (I) BAL NETs and protein concentration are positively correlated (r = Pearson’s correlation coefficient). (J–M) Mice were instilled i.t. with Pseudomonas aeruginosa strain PAO1 at 1 × 10⁷ or 5 × 10⁷ CFU/mouse and sacrificed at 24 hours (n = 3–4). (J) BAL WBC, (K) BAL total protein concentration, (L) BAL NETs, and (M) plasma NETs were all increased after infection. Data were analyzed using 1-way ANOVA and Dunnett’s post test. *P ≤ 0.05,**P ≤ 0.01, ***P ≤ 0.001. ns, not significant.

Figure 2. NETs are visualized ex vivo and in vivo in infected lungs.

(A and B) Bronchoalveolar lavage (BAL) from mice infected with (A) methicillin-resistant Staphylococcus aureus (MRSA) or (B) Pseudomonas aeruginosa strain PAO1 was settled on a slide and stained ex vivo with SYTOX Green DNA dye (green), neutrophil elastase (NE) antibody (red), and histone H2B antibody (blue). (C–H) Lung 2-photon intravital microscopy. (C) LysM-GFP mice (green neutrophils) were challenged with MRSA (2 × 10⁷ CFU, i.t.), injected with Texas Red–dextran i.v. to stain the vasculature, and observed from 3 to 5 hours after infection. (D) GFP and Texas Red signals were quantified, showing neutrophils accumulating in the lungs. (E) MRP8-mTmG mice (red vasculature, green neutrophils) were challenged i.t. with 5 × 10⁶ CFU mCherry-PAO1 (pink, arrows) and observed from 3 to 5 hours after the infection. (F and G) MRP8-mTmG mice (red vasculature, blue neutrophils) were challenged with PAO1 (5 × 10⁶ CFU, i.t.) and observed from 3 to 5 hours after the infection. Extracellular DNA was stained with SYTOX Green. (H) WT mice injected with FITC-dextran (green vasculature) were challenged i.t. with 5 × 10⁶ CFU mCherry-PAO1 (pink, arrows) and observed 17 hours after the infection. Extracellular DNA was stained with SYTOX Blue. Scale bars: 20 μm (A and H), 10 μm (B and E), and 50 μm (C, F, and G).

Figure 3. Lung injury, bacterial counts, and survival in NETosis-impaired mice.

(A–C) Bone marrow neutrophils were isolated from WT or PAD4^–/– mice, pretreated with Cl-amidine (200 μM), and stimulated in vitro with ionomycin (4 μM) for 4 hours. (B) Neutrophil extracellular traps (NETs) in neutrophil supernatants were quantified by neutrophil elastase–DNA (NE-DNA) ELISA (n = 4) and (C) visualized by immunofluorescence (DNA, green; NE, red). Scale bar: 20 μm. (D–K) WT, PAD4^–/–, and PAD4^+/– littermates were challenged in vivo with methicillin-resistant Staphylococcus aureus (MRSA; 5 × 10⁷ CFU, i.t.). (E) Bronchoalveolar lavage (BAL) was fixed ex vivo and visualized by immunofluorescence (DNA, green; NE, red; citrullinated histone H3 [CitH3], blue). Scale bar: 20 μm. BAL, blood, and lung were collected at 24 hours. (F) NETs (NE-DNA ELISA), (G) CitH3-DNA complexes, and (H) protein content were quantified in BAL. (I) Bacterial counts in the lung. (J and K) IL-6 and IL-1β concentration in BAL. (B, F–K) Data were analyzed using 1-way ANOVA (n = 11–19). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. (L) Survival curves for WT, PAD4^–/–, and PAD4^+/– littermates challenged with MRSA (1 × 10⁸ CFU, i.t.). Survival curves were compared using Gehan-Breslow-Wilcoxon test (n = 10–20). ns, not significant.

Figure 4. Degradation of NETs with DNase I reduces lung injury and improves survival.

(A–H) WT mice were challenged with methicillin-resistant Staphylococcus aureus (MRSA; 5 × 10⁷ CFU, i.t.) and treated 2 hours later with DNase I (2,000 units, i.t.) to disrupt neutrophil extracellular traps (NETs). Bronchoalveolar lavage (BAL), blood, and lung tissue were collected 8 hours after infection. (B) BAL NETs (neutrophil elastase–DNA complexes), (C) BAL WBC, (D) BAL albumin concentration, (E) lung vascular permeability to albumin, (F) excess lung water, (G) bacterial counts in blood, and (H) BAL bacterial counts were quantified. (I and J) Survival experiment schema and curves for mice challenged with MRSA (5 × 10⁷ CFU, i.t.) and treated with DNase I or diluent control (i.t.) at 2, 10, 18, and 28 hours after infection (n = 24). (K–N) Mice were challenged with MRSA (7 × 10⁷ CFU, i.t.) and treated with vancomycin (150 mg/kg, i.p.) and DNase I (2,000–4,000 units, i.t.) or diluent control at 2, 10, 18, and 28 hours after infection (n = 10). (M) Body temperature loss and (N) blood lactate 24 hours after infection. (J and L) Survival curves were compared using Gehan-Breslow-Wilcoxon test. (B–H, M, N) Data were analyzed using Student’s t test (n = 3–10). *P ≤ 0.05,**P ≤ 0.01. ns, not significant.

Figure 5. Lipoxin pathway regulates NET production.

(A and B) Bone marrow neutrophils were isolated from WT or Fpr2^–/– mice, preincubated 1 hour with 300 nM lipoxin A₄ (LXA4), and stimulated in vitro with 100 nM PMA or 10⁸ CFU/ml methicillin-resistant Staphylococcus aureus (MRSA). (B) Neutrophil extracellular traps (NETs) in neutrophil supernatant were quantified by ELISA (neutrophil elastase–DNA [NE-DNA] complexes) (n = 4). PMA and MRSA increase NET production over nonstimulated neutrophils (^┼P ≤ 0.01) and LXA4 inhibits NET production compared with WT neutrophils treated with control (*). LXA4 did not reduce production in Fpr2^–/– neutrophils. (C–G) WT, Fpr2^+/–, or Fpr2^–/– littermates were challenged in vivo with MRSA (5 × 10⁷ CFU, i.t.). Bronchoalveolar lavage (BAL), blood, and lung tissue were collected at 24 hours. (D) BAL NETs (NE-DNA complexes), (E) BAL total protein, (F) BAL WBC, and (G) lung bacterial counts were quantified. Data were analyzed using Student’s t test (n = 5–6). *P ≤ 0.05,**P ≤ 0.01. (H) Survival curves for WT, Fpr2^+/–, or Fpr2^–/– littermates challenged with MRSA (5 × 10⁷ CFU, i.t.). Survival curves were analyzed using Gehan-Breslow-Wilcoxon test (n = 10–22). ns, not significant.

Figure 6. NETs are present in ARDS patients with infection.

NETs and NET/DNase I ratio correlate with ARDS severity and mortality. (A–F) Plasma neutrophil extracellular traps (NETs) (neutrophil elastase–DNA complexes) and (G–L) plasma NET/DNase I ratio from patients with (A and G) acute respiratory distress syndrome (ARDS) (n = 104) or with acute cardiac conditions (n = 40), P = 0.0008, P = 0.02; (B and H) pneumonia with and without ARDS (n = 24 and 14, respectively), P = 0.03, P = 0.03; (C and I) nonpulmonary sepsis with and without ARDS (n = 73 and 21, respectively), P = 0.09, P = 0.03; or (D and J) mild, moderate, or severe ARDS according to the Berlin definition (n = 19, 30, or 25, respectively), P = 0.02, P = 0.05. (E and K) Association of NETs with ARDS mortality (n = 64, 40), P = 0.03, P = 0.04 and (F and L) mortality in sepsis/pneumonia (n = 102, 47), P = 0.001, P = 0.0003. Data were analyzed using the Mann-Whitney-Wilcoxon test. ns, not significant.

Figure 7. DNase I plasma levels are associated with ARDS development in patients with nonpulmonary sepsis.

(A) DNase I concentration from patients with pneumonia who never developed acute respiratory distress syndrome (ARDS) (n = 16), who developed ARDS after blood collection (n = 4), or with ARDS at time of blood collection (n = 20), P = 0.06. (B) DNase I concentration from patients with nonpulmonary sepsis who never developed ARDS (n = 83), who developed ARDS after blood collection (n = 15), or with ARDS at time of blood collection (n = 21), *P = 0.04. Data were analyzed using the Mann-Whitney-Wilcoxon test.