Neutrophil extracellular traps are present in the airways of ENaC-overexpressing mice with cystic fibrosis-like lung disease

All animal procedures were approved by the Animal Care and Use Committee at the University of Georgia. The βENaC-Tg mouse line on a C57BL/6 genetic background was obtained from Alessandra Livraghi-Butrico (University of North Carolina, Chapel Hill, NC) with the permission of Marcus Mall (Charité - Universitätsmedizin Berlin, Germany) who generated this model. This mouse strain is also available at Jackson Laboratory: B6.Cg-Tg (Scgb1a1-Scnn1b)6608Bouc/J, (Stock No: 006438). Generation of the transgenic mouse was described previously [21]. Mice heterozygous positive for the overexpressing transgene were confirmed by PCR using forward primer (P1) 5′-CTTCCAAGAGTTCAACTACCG-3′ and reverse primer (P2) 5′-TCTACCAGCTCAGCCACAGTG-3′ to amplify the intron region of Scnn1b. Expected sizes were 254 bp for βENaC-Tg and ~ 350 bp for wild-type (Supplemental Figure 1). Adult βENaC-Tg mice on a C57BL/6 background were studied at 6 and 8 weeks of age, and wild-type (WT), same age C57BL/6 littermates served as controls. All mice in this study had free access to food and water. Mice were bred as hemizygotes. Animals were randomly allocated to experimental groups and randomly assessed. Mice were anesthetized with tribromoethanol TBE (IP; 180–250 mg/kg) administered one time per mouse intraperitoneally. A negative response to a toe pinch confirmed adequate anesthetic depth. Animals were euthanized with CO₂ and subsequent cervical dislocation. Equal numbers of male and female mice were included into the study. The weight of animals at time of their euthanasia was: wild-type male (24.0 +/− 3.7 g), wild-type female (19.3+/− 1.4 g), βENaC-Tg male 23.5 +/− 2.7 g), βENaC-Tg female (19.0 +/− 0.9 g) (mean+/−S.D.). Mice were studied at 6 and 8 weeks of age. For identification purposes, standard ear-tagging and tail-clipping was done. Mice were maintained by breeding on campus in the UGA Veterinary Medical College Central Animal Facility rodent vivarium. Pups were weaned from the mothers at a standard 21 days unless they appeared unable to support themselves, in which case they were weaned at 28 days. The mice were kept in microisolator cages. After euthanasia, lungs were either subjected to bronchoalveolar lavage fluid collection or to fixation followed by pathological examination, immunohistochemistry or immunofluorescence staining. No experimental drugs were used in this study.

For immunohistochemistry with 3,3′-Diaminobenzidine (DAB) amplification of signal, paraffin-embedded lung tissue unstained sections from uninfected WT and βENaC-Tg mice were deparaffinized and rehydrated with xylene and alcohol gradient. Tissue sections were antigen-retrieved with 0.1 M sodium citrate in Pascal Pressure Cooker (DakoCytomation, Aligent Technologies, Santa Clara, CA) for 20 min at 95 °C. Antigen retrieval was followed by endogenous peroxidase blocking with Dual Endogenous Enzyme Block (Dako, Cat# S2003). Sections were then incubated in a blocking buffer (10% Normal Goat Serum) followed by incubation with primary antibody against neutrophil elastase (1:2000 Abcam, Cat# 21595) overnight. After washing with 1X TBS/0.5% Tween, sections were incubated with polymer HRP (GBI Labs, Cat# D13–18) followed by 5 min DAB treatment. Hematoxylin (Vector Laboratories, Cat# H3401) and Acrytol Mounting Medium (EMS, Cat# 13158) were used for counterstaining and mounting, respectively. Pictures were taken with an Olympus BX41 phase contrast and dark field microscope, and analyzed with cellSens Entry software (Olympus, Center Valley, PA).

To characterize myeloid cell subsets, bronchoalveolar lavage fluid (BAL) was collected from adult, uninfected βENaC-Tg and WT mice at 6 and 8 weeks of age (n = 6/group). The airways of euthanized mice were washed with 1 ml of sterile 1X PBS. Retained BAL was centrifuged at 400 x g for 10 min. The cells recovered from the wash were suspended in 1 ml sterile 1X PBS, counted and stained to detect the number of cells from each of the following myeloid cell subsets: neutrophils (CD11b⁺, CD115⁻, Ly6G⁺), eosinophils (CD11b⁺, CD115⁻, CD11c⁻, Ly6G⁻, Ly6c^lo); monocytes (CD11b⁺, CD115⁺, Ly6G⁺); inflammatory monocytes (CD11b⁺, CD115⁺, Ly6G^High), macrophages (CD11b⁺, F4/80⁺): dendritic cells (CD11b⁺,CD11c⁺,F4/80⁻); alveolar macrophages (CD11b⁺, F4/80⁻, CD115⁺, CD11c⁻) and inflammatory macrophages (CD11b⁺, F4/80⁺, CD115⁺, CD11c⁺). All antibodies were purchased from Biolegend (San Diego, CA). Cells were suspended in 100 μl PBS and incubated with Zombie Aqua fixable viability dye (1:1000, Biolegend, San Diego, CA) at room temperature in the dark for 15 min to distinguish between live and dead cells collected from the BAL. After centrifugation, cells were resuspended in 100 μl PBS/1% BSA. All subsequent cell processing was performed on ice, protected from light. Cells were blocked with TruStain FcX™ (Biolegend, San Diego, CA) for 10 min. All antibodies were added to the cells at a 1:100 dilution at the same time. Cells were incubated for 1 h, then washed with PBS/1% BSA and re-suspended in 500 μl Stabilizing Fixative (BD Biosciences, San Jose, CA), and stored at 4 °C until analysis. Samples were read at the University of Georgia College of Veterinary Medicine Cytometry Core Facility on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA) within 24 h of staining. Data were analyzed with the BD FACsDiva™ software (BD Biosciences, San Jose, CA). The gating strategy is shown in Supplemental Figure 2.

To measure the amount of histone citrullination occurring in the airways of adult βENaC-Tg and WT mice at 6 and 8 weeks of age, BAL was harvested as described above. Within each group, BALs were pooled to make a total of 1 × 10⁶ cells/sample (n = 5–6/group). Citrullinated histone H3-positive neutrophils were defined as CD11b⁺, CD115⁻, Ly6G⁺, histone H3 (citrulline R2+R8+R17)⁺. The gating strategy is shown in Supplemental Figure 4. Cells were suspended in 100 μl PBS and incubated with Zombie Aqua fixable viability dye (1:10,000, Biolegend, San Diego, CA) at RT in the dark for 15 min to distinguish between live and dead cells collected from the BAL. Cells were washed with PBS/1% BSA, and then fixed and permeabilized with the Fix & Perm/ Cell Fixation and Permeabilization Kit (Abcam, Cambridge, MA) following manufacturer’s instructions. All subsequent cell processing was performed on ice, protected from light. Cells were blocked with TruStain FcX™ (Biolegend, San Diego, CA) for 10 min. The primary anti-histone H3 (citrulline R2+R8+R17) (Abcam, Cambridge, MA) antibody was incubated with the cells for 30 min. Cells were washed with PBS/ 1% BSA followed by incubation with donkey-anti-rabbit-PE (Biolegend, San Diego, CA) for 30 min. Cells were washed with PBS/ 1% BSA. The conjugated antibodies for CD11b, CD115, Ly6G (Biolegend, San Diego, CA) were added together and incubated for 30 min. Cells were washed with PBS/1% BSA and resuspended in 500 μl Stabilizing Fixative (BD Bioscience, San Jose, CA), and stored at 4 °C until analysis. Flow cytometry was performed at the University of Georgia CTEGD Shared Resources Laboratory on a Cyan ADP cytometer (Beckman Coulter, Hialeah, Florida, NIH grant # 1S10RR027814) within 24 h of staining. Data were analyzed using FlowJo™ Software for Windows, version 10 (Becton Dickinson and Company, Ashland OR).

Cells were harvested from the BAL of adult βENaC-Tg and WT mice at 6 and 8 weeks of age as described above. 1.5X10 [5] cells, pooled from multiple mice/cohort were adhered to precleaned microscope slides using Double Cytofunnel Sample Chambers (Thermo Scientific, Waltham, MA) and a cytospin preparations were performed with a Shandon Cytopsin 2 centrifuge (Thermo Scientific, Waltham, MA). Cells were fixed with 4% paraformaldehyde for 15 min and washed with PBS. Cells were permeabilized with 0.1% Triton X100 while blocking in PBS with 5% BSA and 10% goat or horse serum for 1 h. Primary antibody staining to detect myeloperoxidase (RD Systems, Minneapolis, MN) and/or histone H3 (citrulline R2 + R8 + R17) (Abcam, Cambridge, MA) was performed at a 1:250 dilution in PBS with 1% BSA, 1% goat or horse serum overnight at 4C. Slides were washed in PBS. Secondary antibody staining was performed at a 1:500 dilution in PBS with 1% BSA, 1% goat or horse serum for 1 h with horse- anti-rabbit IgG Dylight™ 488 for citrullinated histone, or horse-anti-goat Dylight™ 594 for myeloperoxidase (Vector Laboratories, Burlingame, CA). Slides were washed with PBS. Vectashield™ anti-fade mounting medium with DAPI (Vector Laboratories, Burlingame, CA) was applied to the cells prior coverslip addition. For immunofluorescence assay of histology sections, paraffin-embedded lung tissues were de-paraffinized as described above. Antigen unmasking and antibody staining was performed following the protocol described by Abed and Brinkmann, 2019 [29]. Both the myeloperoxidase (RD Systems, Minneapolis, MN) and histone H3 (citrulline R2 + R8 + R17) (Abcam, Cambridge, MA) primary antibodies were used at 1:500 dilution. Secondary antibody staining was performed at a 1:1000 dilution in PBS with 1% BSA, 1% goat or horse serum for 1 h with horse-anti-rabbit IgG Dylight™ 488 for citrullinated histone, or horse-anti-goat Dylight™ 594 for myeloperoxidase (Vector Laboratories, Burlingame, CA). Vector® Laboratories TrueView® autofluorescence quenching kit (Vector Laboratories, Burlingame, CA) was used prior to coverslip addition. All digital images were acquired at the University of Georgia College of Veterinary Medicine Cytometry Core on a Nikon A1R confocal microscope (Nikon Eclipse Ti-E inverted microscope) and examined with NIS Element software (Nikon, Version 6.4).

Complexes of MPO-DNA were detected in the BAL of adult βENaC-Tg and WT mice at 6 and 8 weeks of age using a published protocol adapted to murine samples [15, 30]. Supernatants from the BAL of mice (n = 6/group) were diluted 1:50 in PBS. Diluted samples were added to a 96-well plate, pretreated overnight at 4 °C with capture anti-MPO (1:200 RD Systems, Minneapolis, MN), and blocked with 5% BSA in PBS at RT for 2 h. Samples were incubated overnight at 4 °C. Following 3X washes with PBS/Tween 20, the secondary anti-DNA-POD (1:500, Roche, Basal, Switzerland) was added for 1 h at room temperature. Samples were washed 4X with PBS/Tween 20. TMB substrate (Thermo Scientific, Waltham, MA) was added. The reaction was stopped by the addition of a 1 M HCl solution. Absorbance was measured at 450 nm with an Eon microplate spectrophotometer (BioTek, Winooski, VT). Background absorbance values of the medium and untreated neutrophils were subtracted. All samples being compared were run on the same plate in two trials. Differences between optical densities were compared.

Results between two groups were analyzed by Student’s t-test and data among more than two cohorts were compared by One-Way ANOVA, or the nonparametric Kruskal-Wallis test. Analysis of data sets comparing more than two variables for more than two cohorts were analyzed using a Two-Way ANOVA. Data are expressed as mean plus-minus standard error of the mean (SEM). Statistically significant differences were considered as *, p < 0.05; **, p < 0.01; ***, p < 0.001. Exact p values for each test are listed in the figure legends. Statistical analysis was carried out with GraphPad Prism version 6.07 for Windows software.

To characterize lung pathology in βENaC-Tg mice at the ages of 6 and 8 weeks, hematoxylin and eosin staining was performed on fixed lung tissue sections (n = 8–13 mice per group) (Fig. 1). Heavy inflammatory infiltrates characterize βENaC-Tg lung tissues (Fig. 1.) It was also noted that many of the βENaC-Tg mice had acidophilic macrophage pneumonia and eosinophilic crystal accumulation (Fig. 1). This observation is consistent with earlier studies characterizing the βENaC-Tg mouse lung environment at earlier time points [22, 31].

To characterize leukocyte infiltration in the lungs of βENaC-Tg mice at the age of 8 weeks, flow cytometry was performed to assess myeloid cell populations in the BAL using surface markers to distinguish among neutrophils, monocytes, inflammatory monocytes, eosinophils, dendritic cells, macrophages, alveolar macrophages, and inflammatory macrophages (Supplemental Figure 2). BAL was collected from uninfected mice at 6 and 8 weeks of age (n = 6 per group), and the number of cells/ml was determined for each cell type listed above. While there was no significant difference in cell populations present in βENaC-Tg and WT mice at 6 weeks of age, there was a trend towards higher numbers of neutrophils and inflammatory macrophages in transgenic animals (Fig. 2a). This trend became significant in 8 week-old mice as there were 2 times more total myeloid cells (p < 0.0001) and 66 times more neutrophils in the βENaC-Tg mice compared to WT (p = 0.0049) (Fig. 2b). Most likely the enhanced neutrophilic recruitment accounted for most of the overall increase in total myeloid cell counts. Additionally, there was a significant 4.5-fold decrease in total macrophages present in the BAL of the βENaC-Tg mice compared to WT animals (p = 0.0002) (Fig. 2b). To demonstrate the increased presence of myeloid cells, inflammation and tissue damage in the lungs of the βENaC-Tg animals, cytospin preparations of equal volumes of BAL fluids followed by staining with Hema 3 stain™ were generated from mice in each group (Fig. 2c). These results show increased number of cells and inflammatory debris in the βENaC-Tg BAL fluid compared to an equal volume of BAL fluid from WT mice at 6 and 8 weeks, indicating heavy ongoing inflammation. Additionally, while –as expected- macrophages dominate in the lungs of WT mice, neutrophils are the dominant cell type in the airways of βENaC-Tg animals (Fig. 2c). To further confirm neutrophil dominance in the βENaC-Tg mouse lung, immunofluorescence staining of myeloperoxidase (MPO) of equal cell counts (1.5 × 10⁵) from BAL of both mouse strains shows the increased presence of neutrophils in the lungs of the βENaC-Tg mice (Fig. 2d). Lastly, immunohistochemistry for neutrophil elastase (NE) shows increased neutrophil recruitment in the airways of βENaC-Tg mice at both 6 and 8 weeks of age (Fig. 2e). These results reporting neutrophil-dominant leukocyte infiltration in the lungs of βENaC-Tg animals at later time points (6 and 8 weeks) are consistent with previous characterizations of the βENaC-Tg mouse model at earlier time points and with human CF lung disease [3, 19, 21].

PMNs are driven to the airway space by chemoattractants. In CF, several PMN-guiding cytokines and chemokines were shown to be elevated. To explore the cytokine levels in the BAL of βENaC-Tg mice, a bead-based, multiplex cytokine/chemokine array measuring 23 target molecules was applied. The results of all analytes measured are shown in Supplemental Figure 3. Data showed significant increases ranging from 9 to 200-fold in neutrophil-attracting chemokines, KC, MIP-1α and MIP-1β in βENaC-Tg at both 6 and 8 weeks of age (p < 0.006, p = 0.0006, p < 0.0001 (Fig. 3a). Additionally, BAL levels of the proinflammatory cytokines IL-5 and IL-6, the monocyte chemoattractant MCP-1, and the neutrophil proliferation cytokine G-CSF were significantly elevated by 44-, 9-, 140-, and 25-fold, respectively, in β-ENaC-Tg mice compared to control animals at 8 weeks of age (p = 0.03, p < 0.006) (Fig. 3b). These data suggest that βENaC-Tg mice develop chronic airway inflammation characterized by elevated levels of cytokines known to be also increased in human CF patients.

Although the βENaC-Tg mouse model has been proposed as a model for CF airway disease, no study has investigated whether NETs are formed in the airways of these mice. To explore this, cell-free supernatants of BAL collected from βENaC-Tg mice and WT littermate controls were examined for the presence of MPO-DNA complexes, indicative of NETs, using an in-house generated ELISA assay as described [15, 30]. The absorbance values in BAL supernatants of βENaC-Tg mice were significantly higher than that of the WT littermate controls at both 6 and 8 weeks (Fig. 4a) indicating that NETs are present in the airways of βENaC-Tg animals.

To detect NETs by another method, immunofluorescence imaging of equal numbers of BAL cells isolated from both mouse strains was used. Co-localization of MPO, citrullinated histone 3 (CitH3), a hallmark of PAD4-dependent NET release [8], and DNA (DAPI) was observed that is indicative of NET release in the βENaC-Tg mouse (Fig. 4b). Only minimal MPO and CitH3 staining was detected in BAL cells from WT mice using the same microscope settings (Fig. 4b).

As a third approach to study NETs, flow cytometry was used to quantify the presence of neutrophils undergoing PAD4-mediated NET release. The gating strategy for this assay is shown in Supplemental Figure 4. The number of CitH3 positive neutrophils (CD11b⁺, CD115⁻, Ly6G⁺, CitH3⁺) was quantified using 1 × 10⁶ BAL cells, pooled from multiple mice from either βENaC-Tg or WT background at 6 and 8 weeks of age (Fig. 4c-e). There were significantly more CitH3 positive cells/ml in the BAL of βENaC-Tg mice at both ages compared to the WT controls. Lastly, immunofluorescence imaging of lung sections was performed on 8 week-old βENaC-Tg and WT mice. Granular, intracellular MPO staining, indicative of resting neutrophils, was detected in the tissues of both mouse strains (Fig. 5a). Large NETs marked by co-localization of MPO, citrullinated histone and DNA were detected in the lung of the βENaC-Tg mice but where absent in the lungs of their wild-type littermate controls (Fig. 5a and b). Overall, this data demonstrates the presence of NETs in the lungs of the βENaC-Tg mice.