Excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis

Abstract

Complications of acute respiratory distress syndrome (ARDS) are common among critically ill patients infected with highly pathogenic influenza viruses. Macrophages and neutrophils constitute the majority of cells recruited into infected lungs, and are associated with immunopathology in influenza pneumonia. We examined pathological manifestations in models of macrophage- or neutrophil-depleted mice challenged with sublethal doses of influenza A virus H1N1 strain PR8. Infected mice depleted of macrophages displayed excessive neutrophilic infiltration, alveolar damage, and increased viral load, later progressing into ARDS-like pathological signs with diffuse alveolar damage, pulmonary edema, hemorrhage, and hypoxemia. In contrast, neutrophil-depleted animals showed mild pathology in lungs. The brochoalveolar lavage fluid of infected macrophage-depleted mice exhibited elevated protein content, T1-α, thrombomodulin, matrix metalloproteinase-9, and myeloperoxidase activities indicating augmented alveolar-capillary damage, compared to neutrophil-depleted animals. We provide evidence for the formation of neutrophil extracellular traps (NETs), entangled with alveoli in areas of tissue injury, suggesting their potential link with lung damage. When co-incubated with infected alveolar epithelial cells in vitro, neutrophils from infected lungs strongly induced NETs generation, and augmented endothelial damage. NETs induction was abrogated by anti-myeloperoxidase antibody and an inhibitor of superoxide dismutase, thus implying that NETs generation is induced by redox enzymes in influenza pneumonia. These findings support the pathogenic effects of excessive neutrophils in acute lung injury of influenza pneumonia by instigating alveolar-capillary damage.

Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Effects of macrophage or neutrophil depletions on animal weight, leukocyte recruitment, and virus titer. Depletion of macrophages was achieved by intranasal administration of clodronate-liposome (50 μL/dose/mouse) at days −4 and −1 before virus challenge. For depletion of neutrophils or polymorphonuclear leukocytes (PMN), animals were injected intraperitoneally with anti-Ly6G monoclonal antibody (1A8) at 200 μg/dose/mouse a day before infection and every 48 hours thereafter. Animals were challenged with a sublethal dose of PR8 virus (100 PFU) through intranasal inoculation. A: Changes in body weight of BALB/c mice after infection. Animal weights were recorded daily for 10 dpi and expressed as means ± SE. At 10 dpi, the CL-I group showed more than 30% weight loss, whereas the 1A8-I and INF groups displayed 20% and 10% weight loss, respectively (n = 10). B: Differential cell counts of BALF measured by modified Giemsa staining. Neutrophil-dominant infiltration was observed in the CL-I and INF groups. Macrophage numbers were significantly decreased in the CL-I group on 5 dpi. The 1A8-I group showed significantly decreased neutrophil numbers but increased macrophage numbers. Results are expressed as means ± SE, with n = 5 per group. C: Determination of virus titers in mouse lung homogenates at 5 and 10 dpi. MDCK cells were infected with serial 10-fold dilutions of lung homogenates from uninfected or virus-infected animals. The numbers on the y axis represent virus titers expressed as 10y 50% tissue culture infectious dose (TCID50) per gram of total protein (means ± SE), with n = 5. CL, clodronate-liposome–treated; INF, infected; CL-I, clodronate-liposome–treated, infected; Con, normal allantoic fluid–inoculated; 1A8-I: anti-Ly6G monoclonal antibody–treated, infected group. *P < 0.05 versus the CL-I group; †P < 0.05 versus the INF and 1A8-I groups.

Figure 2

Effects of macrophage or neutrophil depletion on histopathological changes and NETs induction in lungs of mice challenged with sublethal influenza virus infection. A: Animals administered clodronate-liposome displayed normal architecture of airway (BR) and alveolar epithelia (AV) with minimal neutrophilic infiltration. B: Infected mice showed mild peribronchial infiltration and damage of bronchiolar epithelium at 5 dpi. C and D: Macrophage-depleted infected mice at 5 dpi exhibited severe alveolar damage and extensive neutrophil infiltration (arrows). E and F: Neutrophil-depleted infected animals at 5 dpi displayed mild bronchiolitis and infiltration of macrophages in alveolar spaces (arrows), but neutrophils were rarely seen. G: Lungs from the INF group at 10 dpi exhibited mild alveolitis and bronchiolar epithelial regeneration with mild peribronchial inflammation. H: Neutrophil-depleted infected animals at 10 dpi revealed mild alveolar damage. I–K: Histopathological changes in lungs of mice from the CL-I group on 10 dpi. I: Lung sections show prominent pathological signs of diffuse alveolar damage with thickened interstitium, and alveolar spaces filled with protein exudates (asterisks). J: Extensive NETs formation at the terminal bronchioles opening into alveoli (white arrows). K: Bundles of NETs with endothelial damage (asterisk) were observed in large blood vessels. Magnifications: ×200 (A, B, C, E, G, H, I), and ×1000 (D, F, J, K). Scale bars: 50 μm. Semiquantitative histopathology scoring was performed (n = 5), with the scores shown in Table 1.

Figure 3

Assessment of acute lung injury by determining alveolar–capillary damage and release of MMP-9 into the alveolar air spaces. A: Damage of the thin microvascular barrier was determined using Western blot analyses of BALF samples for T1-α and thrombomodulin, which are membrane proteins present on alveolar type I pneumocytes and capillary endothelium. Lanes depict BALF samples from five groups: uninfected control (lanes 1 and 6); CL (lanes 2 and 7); INF (lanes 3 and 8); CL-I (lanes 4 and 9); and 1A8-I (lanes 5 and 10). T1-α and thrombomodulin levels were significantly elevated in the BALF of the CL-I group at both 5 and 10 dpi (lanes 4 and 9) compared to other groups. To determine the impact of neutrophil accumulation on lung injury, pro- and active MMP-9 levels were also assayed using Western blot analysis. Significantly increased MMP-9 levels were observed in CL-I compared to other groups, thus alluding to the contribution of MMP-9 to basement membrane degradation and promoting tissue damage. B and C: Densitometric analyses of Western blot bands, each expressed as fold change versus the control group. Data are represented as means ± SE, with n = 5 per group. *P < 0.05 versus other groups.

Figure 4

Effects of macrophage or neutrophil depletion on protein leakage into the air spaces and on MPO enzyme activity. A: Significant increase in BALF protein content was observed in the CL-I group compared to other groups, indicating alveolar capillary damage. B and C: MPO activities measured in both BALF samples and lung homogenates from the CL-I group were significantly increased compared to the other groups, correlating with intense neutrophil recruitment. Data are represented as means ± SE, with n = 5. *P < 0.05 versus other groups.

Figure 5

In vivo evidence for NETs formation after lethal challenge of influenza virus in mice. Paraffin-embedded lung tissues from mice challenged with lethal doses of influenza A/PR/8 H1N1 virus (500 PFU) were stained with hematoxylin-eosin or by immunofluorescence. A: Histopathology of lungs showing tissue consolidation and alveolar destruction. B: Extensive induction of NETs was detected within the alveoli (white arrows). C: Bundles of NETs were also observed in large blood vessels showing damaged endothelium (arrows). D and E: Nuclei stained with DAPI showing alveoli (white arrows) or occlusion in the bronchiole (defined by the white line). F and G: NETs were identified by close localization of DNA (blue) with histone H2B (F, green), and neutrophil granule marker MMP-9 (G, green). White arrows indicate NETs formation. Scale bars: 20 μm (A); 50 μm (B–G).

Figure 6

In vitro NETs induction by co-incubation of neutrophils with infected alveolar epithelial cells, and the role of redox enzymes. Neutrophils isolated from lungs were incubated with influenza virus–infected alveolar epithelial cells (at a multiplicity of infection of 20 for 5 hours) in the presence of inhibitors of specific enzymes, including 10 μg/mL anti-MPO, 20 μmol/L diphenyleneiodonium chloride (DPI, inhibitor of NADPH oxidase), 2 mmol/L diethyldithiocarbamate (DETC, inhibitor of SOD), or 2 μmol/L H2O2. After 150 minutes of incubation, the DNA release was visualized by fluorescence microscopy with DAPI staining. A and B: Induction of NETs when neutrophils were co-incubated with infected alveolar epithelial cells (white arrows). C: Double staining with anti-influenza antibody showing infected epithelial cells embroiled by NETs (white arrows). Influenza virus was stained with anti-rabbit polyclonal antibody and secondary anti-rabbit Alexa Fluor 546 (orange). D: NETs stained with anti-MPO (arrowheads), FITC-phalloidin (for F-actin) and DAPI (arrow) to detect the MPO localization within NETs. E: Neutrophils co-incubated with uninfected alveolar epithelial cells revealed minimal NETs generation in the absence of viral stimulation. F: Neutrophils incubated in the presence of anti-MPO antibody resulted in decreased NETs induction. G: Neutrophils alone showing intact nuclei. H: Quantitative assessment of the percentage of NETs-forming cells revealed significant NETs induction following co-incubation of neutrophils (PMN) with infected alveolar epithelial cells (IEP), but not with uninfected control epithelial cells (CONEP). NETs induction was significantly diminished in the presence of anti-MPO and DETC, but not by DPI or anti–ENA-78 (anti–CXCL-5). The presence of H2O2 also significantly induced NETs formation. Values are means ± SE of three independent experiments. *P < 0.05 versus PMN+IEP group.

Figure 7

NETs-induced endothelial cell damage. Neutrophils (PMN) were incubated with HUVECs (ENDO) in the presence or absence of 2 or 5 μmol/L of H2O2. Endothelial damage was assessed by propidium iodide staining followed by immunofluorescence analysis. A: Neutrophils alone do not induce endothelial damage, but the presence of H2O2 significantly induced NETs to cause significant endothelial damage as indicated by increased propidium iodide–positive cells. Statistical significance at *P < 0.05 versus other groups. B: Western blot analysis for release of thrombomodulin into the culture supernatant. Lanes 1 and 2: Supernatants from neutrophil-endothelium cocultures without H2O2 or in the presence of (lane 3) 2 μmol/L H2O2 or (lane 4) 5 μmol/L H2O2. Supernatants of HUVECs alone in the presence of (lane 5) 2 μmol/L H2O2 or (lane 6) 5 μmol/L H2O2. The data are expressed as means ± SE of three independent experiments.