Neutrophil extracellular traps are indirectly triggered by lipopolysaccharide and contribute to acute lung injury

Abstract

Neutrophil extracellular traps (NETs) facilitate the extracellular killing of pathogens. However, excessive NETs formation and poor degradation are associated with exacerbated immune responses and tissue injury. In this study, we investigated the role of NETs in lipopolysaccharide (LPS)-mediated acute lung injury (ALI) and assessed the use of DNase I, for the treatment of ALI. Additionally, we focused on the controversial issue of whether LPS directly induces NETs release in vitro. NETs formation was detected in murine ALI tissue in vivo and was associated with increased NETs markers, citrullinated-histone H3 tissue levels and NET-DNA levels in BALF. Treatment with DNase I significantly degraded NETs and reduced citrullinated-histone H3 levels, which protected against ALI and ameliorated pulmonary oedema and total protein in BALF. In addition, DNase I significantly reduced IL-6 and TNF-α levels in plasma and BALF. In vitro, LPS-activated platelets rather than LPS alone efficiently induced NETs release. In conclusion, NETs formed during LPS-induced ALI, caused organ damage and initiated the inflammatory response. NETs degradation by DNase I promoted NET-protein clearance and protected against ALI in mice; thus, DNase I may be a new potential adjuvant for ALI therapy. Specifically, LPS induced NETs formation in an indirect manner via platelets activation.

Figure 1. NETs are formed in an LPS-induced ALI mouse model.

(A) Representative immunofluorescence images obtained by performing confocal microscopy of lung sections from mice(n = 6) 24 h after intratracheal LPS treatment, compared to sham and DNase I plus LPS groups; staining depicts Cit-H3 (green), NE(red) and DAPI (blue). The higher-magnification views in the insets (1–6) show NETs formation, demonstrated by the co-localization of Cit-H3 (green), NE (red) and DNA (blue). (B,C) Western blot analysis of Cit-H3 protein levels in the lungs of sham-, LPS-, or DNase I plus LPS-treated mice. Full-length blots/gels are presented in Supplementary Figure S1. (D) BALF levels of NET-DNA were determined using a PicoGreen assay kit. *P < 0.05 vs. the sham group; ^#P < 0.05 vs. the LPS group. The results shown are representative of at least three separate independent experiments.

Figure 2. NETs regulate tissue damage during ALI.

Mice were treated with 20 μl of 3 mg/ml DNase I at time 0 and 10 h after LPS instillation (4 mg/kg in 25 μl of PBS), and lung samples and BALF were collected from the mice 24 h after LPS treatment. (A) H&E staining of lung sections (400×); (B) Lung injury scores; (C) lung water content; (D) total protein concentration in BALF. (E) The number of alveolar neutrophils in BALF; *P < 0.05 vs. the sham group; ^#P < 0.05 vs. the LPS group. n = 6 mice/group. The results are representative of three separate independent experiments.

Figure 3. NETs regulate inflammatory responses both in BALF and plasma.

After DNase I treatment and LPS exposure at predetermined time points, IL 6 and TNF-α levels in the plasma (A,C) and BALF (B,D) were measured. *P < 0.05 vs. the sham group; ^#P < 0.05 vs. the LPS group. n = 6 mice/group. All data shown are representative of at least three separate independent experiments.

Figure 4. Activated platelets, rather than LPS alone, induce NETs release.

Neutrophils (10⁵) from the bone marrow were left untreated or stimulated with 100 nM PMA as negative and positive controls for NETs formation, respectively. In addition, neutrophils were co-cultured with LPS (5 μg/ml), PLT (10⁶), LPS plus PLT, LPS plus PLT plus DNase I(200 U/ml), or PMA plus DNase I for 4 h. (A) Immunofluorescent staining of NETs detected by confocal microscopy in neutrophils. Yellow arrows show NETs formation, white arrows show the remnants of NETs processed by DNase I. Green: Cit-H3; red: NE; blue: nuclei. (B) Fluorescence microscopy images were analysed with Image J software to count the number of NETs remained per one hundred neutrophils; a zero indicates no intact NETs were observed. (C) NET-DNA levels in cell supernatants following neutrophil exposure to various stimuli were quantified using the PicoGreen assay kit. *P < 0.05 vs. the control, LPS or PLT groups; ^#P < 0.05 vs. the control group; ^$P < 0.05 vs. the LPS plus PLT group; ^&P < 0.05 vs. the PMA group. All data shown are representative of at least three separate independent experiments.