A serpinB1 regulatory mechanism is essential for restricting neutrophil extracellular trap generation

Abstract

NETosis (neutrophil extracellular trap [NET] generation), a programmed death pathway initiated in mature neutrophils by pathogens and inflammatory mediators, can be a protective process that sequesters microbes and prevents spread of infection, but it can also be a pathological process that causes inflammation and serious tissue injury. Little is known about the regulatory mechanism. Previously, we demonstrated that serpinb1-deficient mice are highly susceptible to pulmonary bacterial and viral infections due to inflammation and tissue injury associated with increased neutrophilic death. In this study, we used in vitro and in vivo approaches to investigate whether SerpinB1 regulates NETosis. We found that serpinb1-deficient bone marrow and lung neutrophils are hypersusceptible to NETosis induced by multiple mediators in both an NADPH-dependent and -independent manner, indicating a deeply rooted regulatory role in NETosis. This role is further supported by increased nuclear expansion (representing chromatin decondensation) of PMA-treated serpinb1-deficient neutrophils compared with wild-type, by migration of SerpinB1 from the cytoplasm to the nucleus of human neutrophils that is coincident with or preceding early conversion of lobulated (segmented) nuclei to delobulated (spherical) morphology, as well as by the finding that exogenous human recombinant SerpinB1 abrogates NET production. NETosis of serpinb1-deficient neutrophils is also increased in vivo during Pseudomonas aeruginosa lung infection. The findings identify a previously unrecognized regulatory mechanism involving SerpinB1 that restricts the production of NETs.

FIGURE 1

Increased production of NETs and expansion of nuclei of serpinb1^−/− lung neutrophils. A-C, NET production. WT (+/+) and serpinb1-, deficient (−/−) lung neutrophils were cultured in poly-L-lysine coated tissue culture wells with no additive, PMA, or PMA plus DPI. Staining with Sytox green, a plasma membrane impermeable DNA dye, revealed NETs as Sytox-positive structures emanating from cells with length greater than 2x cell diameter. A, Representative fluorescence images of PMA-treated neutrophils; examples of NETs are indicated by arrows. B, NETs enumerated in micrographs. C, NETs quantified by DNA fluorimetry. D,E, Nuclear size. D, Distribution of PMA-treated Sytox-stained WT and serpinb1^−/− neutrophils on the basis of nuclear area (as percent of cell area). E, Frequency of neutrophils with expanded nuclei (>50% of cell area). Means ± SEM for 3-4 experiments (each 2-3 mice/group); *, P<0.05; **, P<0.001, ***, P<0.0001.

FIGURE 2

Production of NETs and ROS by WT and serpinb1^−/− bone marrow neutrophils. A, Neutrophils were cultured in uncoated glass chamber slides with the indicated agents, and the resulting NETs were quantified in fluorescence images after Sytox staining. B, ROS. Neutrophils pre-labeled with dihydrorhodamine were stimulated for 15 min, and accumulated ROS was measured as MFI. C,D, NETs produced by neutrophils treated with H₂O_2. C, Representative WT and serpinb1^−/− neutrophils stained with Gr-1 antibody (green) and Sytox orange. D, Quantitation of NETs. (A,B,D) Means ± SEM for 2 experiments (each 3 mice/genotype). *, P<0.05; **, P<0.001, ***, P<0.0001; two symbols show the results for the WT with WT comparison followed by the KO with KO comparison. (D)

FIGURE 3

Translocation of SerpinB1 to the nucleus during NETosis of human neutrophils. Resting neutrophils (A) and neutrophils treated with PMA for 1h (B,C) or 4h (E) or with PMA for 1h after DFP pretreatment (D) were stained as indicated for SerpinB1 (SB1), elastase (ELA), lamin B receptor (LBR), or DAPI. Cells were examined by fluorescence microscopy, original magnification 40X (A,B) or confocal microscopy, original magnification 63X (C-E). Bars indicate 10 microns.

FIGURE 4

Increased in vivo NET production in Pseudomonas aeruginosa-infected serpinb1^−/− mice. WT (solid symbols) and serpinb1-deficient (open symbols) mice were intranasally inoculated with P. aeruginosa (3 × 10⁶ CFU/mouse) and were sacrificed 24h later. (A) Lung bacteriology. CFU/mouse. Symbols represent individual mice; horizontal lines indicate the medians. (B) Elastase and (C) MPO per mouse in cell-free bronchoalveolar lavage fluid (BALF). (D) Free DNA per mouse in cell-free lavage fluid. (E, F) In vivo generated NETs in non-clarified lavage fluid detected in the presence of DFP. (E) Representative Sytox-stained images; examples of NETs are indicated by arrows. In vivo generated NETs were not all cell-associated. (F) Quantitation. (G) Additional NETs generated ex vivo (in the absence of DFP) by neutrophils in equal volumes of lavage fluid. Means ± SEM for representative experiment out of 2 (4–5 mice/group per experiment) *, P<0.05; **, P<0.001, ***, P<0.0001. ND, not detected (<0.002 μg DNA/ml BALF/mouse)

FIGURE 5

Abrogation of NET generation by exogenous rSERPINB1. Bone marrow neutrophils of WT (+/+) and serpinb1-deficient (−/−) mice (A-E) or human blood neutrophils (F) were pre-incubated without or with rSERPINB1 (rSB1) (10µg/ml) for 1 h (A-C,F) or the indicated time (D,E) and stimulated with PMA, PAF and MIP-2 (A) or PMA (B-F). NETs were quantified by microscopy after Sytox staining. A, NET generation by both genotypes of murine neutrophils stimulated with PMA, PAF or MIP-2 and its inhibition by rSERPINB1. B, Lack of inhibition of PMA-stimulated NET generation by α1-antitrypsin (AAT, SERPINA1) and ovalbumin (ova, serpinB14). Protocol as in A except ovalbumin or AAT replaced rSerpinB1. C, ROS generation by PMA-stimulated neutrophils and lack of inhibition by rSERPINB1. ROS detected as in Fig. 2B. D,E, Time course of inhibition by rSERPINB1 of PMA-stimulated NET generation. -60’ indicates the start of preincubation; 0’ indicates the time of PMA addition. NETs were evaluated at 4h (D) or 5h (E). F, NET production by human neutrophils treated with PMA and inhibition by rSERPINB1 but not by AAT or ovalbumin. A-E, Means ± SEM for 2-4 experiments (each 3 mice/group). Data in A and B are from the same series of experiments; data in E and F are from another series. *, P<0.05; **, P<0.001, ***, P<0.0001. Two symbols show the results for the WT with WT comparison followed by the KO with KO comparison. F, Means ± SEM for cells of 3-4 normal healthy donors.

FIGURE 6

Schematic of proposed NETosis pathways induced by PMA and other mediators, represented by MIP-2, PAF and S. aureus as described in text. Four upstream pathways are shown, but additional diversity is likely. Dashed lines indicate uncertainties. SerpinB1 (SB1) is exclusively in the cytoplasm of resting neutrophils and translocates into the nucleus early during NETosis. Both PMA-induced nuclear envelope disintegration (11) and non-lytic vesicular exocytosis (26) are compatible with the proposed stage 1 and 2 schematic.