Recruitment of neutrophils to the airways, and their pathological conditioning therein, drive tissue damage and coincide with the loss of lung function in patients with cystic fibrosis (CF). So far, these key processes have not been adequately recapitulated in models, hampering drug development. Here, we hypothesized that the migration of naïve blood neutrophils into CF airway fluid in vitro would induce similar functional adaptation to that observed in vivo, and provide a model to identify new therapies. We used multiple platforms (flow cytometry, bacteria-killing, and metabolic assays) to characterize functional properties of blood neutrophils recruited in a transepithelial migration model using airway milieu from CF subjects as an apical chemoattractant. Similarly to neutrophils recruited to CF airways in vivo, neutrophils migrated into CF airway milieu in vitro display depressed phagocytic receptor expression and bacterial killing, but enhanced granule release, immunoregulatory function (arginase-1 activation), and metabolic activities, including high Glut1 expression, glycolysis, and oxidant production. We also identify enhanced pinocytic activity as a novel feature of these cells. In vitro treatment with the leukotriene pathway inhibitor acebilustat reduces the number of transmigrating neutrophils, while the metabolic modulator metformin decreases metabolism and oxidant production, but fails to restore bacterial killing. Interestingly, we describe similar pathological conditioning of neutrophils in other inflammatory airway diseases. We successfully tested the hypothesis that recruitment of neutrophils into airway milieu from patients with CF in vitro induces similar pathological conditioning to that observed in vivo, opening new avenues for targeted therapeutic intervention.

Abbreviations

ALI: air–liquid interface
AMPKα1: 5′ adenosine monophosphate-activated protein kinase α1
Arg1: arginase-1
ASN: airway supernatant
CF: cystic fibrosis
CFTR: CF transmembrane conductance regulator
COPD: chronic obstructive pulmonary disease
ECAR: extracellular acidification rate
GRIM: granule-releasing, immunomodulatory, and metabolically active
HC: healthy control
LD: lung disease
LTB4: leukotriene B4
LY: Lucifer Yellow
NE: neutrophil elastase
OCR: oxygen consumption rate
PD-L1: programmed death-ligand 1
PMN: polymorphonuclear neutrophil
PTM: post-transmigration
ROS: reactive oxygen species

1 INTRODUCTION

Cystic fibrosis (CF) is the most frequent fatal recessive disease in Caucasians, caused by mutations in the CF transmembrane conductance regulator (CFTR) gene.1 Most of the morbidity and mortality in CF is due to airway disease, with a triad of obstruction, infection, and inflammation driven by polymorphonuclear neutrophils (PMNs) recruited from blood.2 Inflammation occurs early in CF airway disease and persists through the patients’ lifetime.3 The first symptoms of airway damage are detected in small airways of CF infants,4 with bronchiectasis coinciding with both PMN presence in the lumen, and extracellular neutrophil elastase (NE) activity.5 Extracellular NE activity is also a predictor of lung function in CF adults.6 Currently, no therapy exists to counter PMNs and PMN-derived NE in CF disease.

In prior in vivo studies, we showed that NE is primarily released via exocytosis by live PMNs.7, 8 In the CF airway milieu, PMNs also lose the surface phagocytic receptor CD16,8 and modulate T-cell inhibitory molecules arginase-1 (Arg1) and programmed death-ligand 1 (PD-L1).9 These functional changes occur in the context of anabolic signaling,7 with increased surface Glut1 expression and glucose uptake,10 and track with high extracellular lactate levels,11 high oxygen consumption and reactive oxygen species (ROS) production,12 and redox imbalance.13 Critically, such changes are not discernable in blood PMNs, and PMNs maintain diploid DNA content while undergoing changes in CF airways.7-10

Together, changes undergone by PMNs in the CF airway milieu constitute a distinct fate, characterized by enhanced primary granule release leading to NE exocytosis, immunoregulatory function, and metabolic activities, henceforth dubbed the “GRIM” fate. Because of the major impact on CF airway disease of NE14 and other GRIM PMN-dependent factors, these cells constitute a bona fide target for therapy.15 However, a key limitation has been the absence of models recapitulating the GRIM fate.16, 17

Importantly, PMN-driven inflammation is not only a hallmark of CF, but also of chronic obstructive pulmonary disease (COPD) and other chronic lung diseases (LD), such as severe asthma,18, 19 although the precise functional adaptations that recruited PMNs undergo in these contexts have been described in less details. Here, we describe a simple, yet robust in vitro model based on blood PMN transepithelial migration into airway milieu from patients with CF and other chronic inflammatory airway diseases, which recapitulates the multifaceted functional adaptations corresponding to the GRIM fate in vivo.17 Using this model, drug effects on effector and metabolic pathways relevant to the GRIM fate of human airway PMNs can be tested in vitro, opening new avenues for drug development.

2 METHODS

2.1 Human subjects

The protocol for collection and handling of human samples was approved by the Institutional Review Board at Emory University. Informed consent was obtained from all subjects for collection and use of their samples. CF was diagnosed by sweat chloride (60 mEq/L), using a quantitative iontophoresis test and/or documentation of 2 identifiable cftr mutations. COPD and LD patients were diagnosed using American Lung Association criteria. HC subjects were nonsmokers, with no history of chronic disease. CF patients were assessed at either one or two visits more than 6 months apart. HC, COPD, and LD subjects were assessed at one visit. Supplementary Table 1 presents demographic data for all subjects.

2.2 Sample processing and airway supernatant preparation

Blood was collected by venipuncture. Sputum was collected from CF patients by spontaneous expectoration, and from HC, COPD, and LD subjects by induction. Blood and sputum were processed as previously described.8 In brief, blood was spun at 400 × g to separate cells from platelet-rich plasma, which was further spun at 3,000 × g to generate platelet-free plasma. Sputum was gently dissociated using repeated passage through an 18G needle after addition of 6 mL of PBS with 2.5 mM EDTA. Dissociated sputum was then spun at 800 × g generating cell and fluid fractions. The fluid fraction was further spun at 3,000 × g at 4°C for 10 min to generate a purified, cell and bacteria-free, airway supernatant (ASN), which was stored at –80°C until use. Blood and airway cell fractions were re-suspended in PBS-EDTA and used for flow cytometric analysis. Blood was also used to prepare PMN fractions for in vitro experiments, as previously described.7 In brief, blood layered onto Polymorphprep (PMP, Nycomed Pharma, Vienna, Austria) was centrifuged at 400 × g with minimal brake for 45 min at room temperature. The PMN layer was collected and erythrocytes removed by hypotonic lysis by 30-s incubation in water, after which PMNs were washed and resuspended in RPMI until use.

2.3 Transepithelial migration model

PMN transepithelial migration (TM) and subsequent inflammation occur first in the small airways of CF patients, i.e., the bronchiolar region, which is lined with a microvilli-covered epithelial monolayer dominated by Club cells.3-5 Therefore, to mimic PMN transmigration into the small airway lumen, we selected the H441 human Club cell line20 to grow epithelial monolayers at air–liquid interface (ALI). To enable PMN loading in the lamina propria and transepithelial migration (Fig. 1A), we used Alvetex (ReproCELL, Glasgow, UK) 200 μm-thick inert 3D scaffolds with >90% porosity (pore sizes of 36–40 μm, with interconnects of 12–14 μm). In brief, inserts were activated with 70% ethanol, coated overnight at 37°C with rat-tail collagen I (3 mg/mL, Sigma) and seeded with H441 cells at 2.5 × 10⁵ cells per 12-well insert. Cells were first grown in submerged cultures with DMEM/F12 supplemented with 10% heat-inactivated serum, penicillin, and streptomycin. After 2 days, cells were supplemented basally with serum-free DMEM/F12 with 10% Ultroser G (Crescent Chemical, Islandia, NY) to establish ALI. Cultures were grown for 2 weeks at ALI and supplemented basally with fresh medium every 48 h. For TM experiments, the ALI cultures were placed with the apical compartment exposed to RPMI, leukotriene B4 (LTB4, 100 nM), CXCL8 (100 ng/mL), fMLF (100 ng/mL), LPS (500 ng/mL), or ASN from CF, HC, COPD, and LD subjects. TM experiments with 0.5–1 × 10⁶ PMNs loaded onto the 200 μm-thick basal compartment of the Alvetex scaffold (situated upside), and allowed to migrate at 37°C at 5% CO₂ through the collagen and epithelial layers into the apical compartment (situated downside, and bathed with either control medium with chemoattractant, or ASN). In some experiments, drugs were added to apical ASN and/or basal PMN suspensions. In other experiments, LPS-RS (competitive inhibitor of LPS binding to TLR4) was added to apical LPS or CF ASN. LPS and LPS-RS were purchased as ultrapure reagents from InvivoGen (San Diego, CA).

Details are in the caption following the image — **Figure 1**
Open in figure viewer PowerPoint

**In vitro model mimicking human PMN transmigration and pathological conditioning in small airways**. (A) We established a model emulating the small airway mucosa by growing the H441 Club-like human small airway cell line at air-liquid interface (upper left insert, arrowheads pointing to apical microvilli typical of Club cells) onto a continuous collagen layer covering a 200 μm thick, porous scaffold (lower left insert). The apical milieu impacts the conditioning of transmigrated PMNs, depending on its origin, e.g., airway supernatant (ASN) from CF or control subjects, or medium containing chemoattractants. Drugs can be tested in the model, via apical and/or basal addition. Transmigrated PMNs conditioned by the apical milieu of choice, exposed or not to candidate drugs, can be retrieved in large numbers and submitted to downstream assays. **(B–D)** Analysis of CD62L, CD16, and CD63 expression, respectively, shown in box plots (left panels) and representative histograms (right panels) for blood PMNs at 2 and 18 h post-transmigration (PTM) into CF ASN (black), compared to PMNs pre-transmigration (grey). *indicates P < 0.05 at 2 or 18 h PTM versus pre-transmigration (n = 8 experiments)

2.4 Flow cytometry

Blood and airway PMNs collected from human subjects and from our transmigration model were assessed by flow cytometry using standard templates for staining, acquisition, and analysis, as described, enabling quantitative comparison of in vivo and in vitro samples throughout the whole study.21 Reagents used for the study included Live/Dead (ThermoFisher, Waltham, MA), and antibodies against Arg1 (6G3, Hycult Biotech, Plymouth Meeting, PA) as well as CD11b (M1/70), CD16 (3G8), CD41a (HIP8), CD45 (HI30), CD63 (H5C6), CD66b (G10F5), CD62L (DREG-56), and PD-L1 (29E.2A3) from BioLegend (San Diego, CA), and the ROS probe CellRox (ThermoFisher), using the gating strategy in Supplementary Fig. 1A. Phosphorylated 5′ adenosine monophosphate-activated protein kinase (AMPK) α1 levels were assessed using an Ab against phospho-Thr172 (Biorbyt, San Francisco, CA) using a previously published protocol.7 For Glut1 detection, we used a specific receptor-binding domain probe (Metafora Biosystems, Evry, France), as detailed before.10 Data were acquired on a LSRII cytometer (BD Biosciences, San Jose, CA) and compensated, gated, and analyzed in Flowjo (Treestar, Ashland, OR). Live PMNs were counted using CountBright beads (ThermoFisher), and expression of all markers is reported as median fluorescence intensities.

2.5 Pinocytosis assay

Pinocytosis was assessed via Lucifer Yellow (LY, Biotium, Fremont, CA) uptake, as described previously,22 with some modifications. For in vivo samples, an NH₄Cl solution (StemCell Technologies, Vancouver, BC, Canada) was used to lyse erythrocytes from whole blood, yielding blood leukocytes. Airway cells were obtained as described above. Blood leukocytes and airway cells were then stained with probes for viability (Live/Dead) and Ab markers for PMNs (CD66b) and exocytosis (CD63), as described above. Cells were then incubated with LY diluted at 1 mg/mL in RPMI for 30 min at 37°C. For in vitro samples, PMNs were transmigrated to CF ASN containing LY (1 mg/mL) for 2, 10, and 18 h, after which the cells were washed with PBS-EDTA and stained with viability probes and Abs, as above. Cells were then washed with RPMI and pinocytosis was measured by flow cytometry.

2.6 Bacteria killing assay

Pseudomonas aeruginosa strain PAO1 was grown in LB broth (BD Biosciences) at 37°C with aeration to an optical density (600 nm) of 0.5. Bacteria were pelleted by centrifugation at 14,000 × g for 5 min at room temperature, resuspended in 500 μL 10% autologous serum in RPMI and opsonized for 30 min at 37°C. Opsonized bacteria were added to PMNs at a multiplicity of infection of 0.1. After 1 h of incubation at 37°C, the cultures were lysed using 0.1% Triton for 2 min, after which the bacteria were serially diluted and plated in triplicate on LB plates and incubated at 37°C in a non-CO₂ incubator overnight. Viable bacterial CFUs were counted the following day and bacterial viability was calculated as a percentage of bacterial counts at time 0.

2.7 Metabolic assays

Real-time analysis of the extracellular acidification rate (ECAR) as a measure of glycolysis, and oxygen consumption rate (OCR), were performed using a Seahorse XFp Extracellular Flux Analyzer (Agilent, Santa Clara, CA). In brief, PMNs were collected after transmigration, resuspended in Seahorse assay medium (DMEM-based, without serum, glucose, or bicarbonate) and plated at 7.5 × 10⁵ cells/well on CellTak (BD Biosciences)-coated Seahorse assay plates. PMNs were attached to the bottom of the wells by centrifugation at 350 × g (without brake), and incubated for 45 min before the assay in a non-CO₂ incubator at 37°C. The glycolysis stress test kit (Agilent) was used to obtain real-time measurements of ECAR and OCR upon sequential injections of glucose (10 mM) to induce glycolytic activity, oligomycin (complex V inhibitor, used at 3 μM) to shut down mitochondrial contribution to glucose consumption, 2-deoxyglucose (2-DG, 0.1 M) to competitively inhibit glucose use by hexokinase, and when indicated diphenyleneiodonium (DPI, 10 μM), an NADPH oxidase inhibitor. Extracellular lactate was measured in culture supernatants after Seahorse runs using the lactate assay kit (Biovision, Milpitas, CA), according to manufacturer's instructions.

2.8 Murine PMN isolation and transmigration

Bone marrow PMNs were collected from the tibias and femurs of wild-type mice by flushing with RPMI with 10% FBS using a 25-gauge needle. Cells were then washed with PBS and allowed to migrate in our model to either CF ASN or LTB4 for 5 or 10 h. Recruited cells were stained with antibodies and analyzed by flow cytometry as detailed above for CD11b (M1/70) and Ly6G (1A8) expression to identity mature PMNs, and for CD63 (NVG-2) expression to assess primary granule release. Anti-mouse antibodies were from BioLegend.

2.9 Data analysis

Statistical analyses were performed using JMP12 (SAS Institute, Cary, NC). Between-group and matched-pair statistical analyses used the Wilcoxon rank sum and signed rank tests, respectively. Correlations were tested using the non-parametric Spearman test. A threshold of P < 0.05 was used to determine significance.

3 RESULTS

3.1 Core features of CF airway PMNs are recapitulated upon blood PMN transmigration in vitro

We designed a model (Fig. 1A) in which purified ASN from CF patients leads to the transepithelial migration of blood PMNs, yielding large numbers of “airway-like” PMNs that can be identified by flow cytometry (Supplementary Fig. 1A). PMNs transmigrated into CF ASN shed the CD62L receptor, as expected after transmigration (Fig. 1B). Remarkably, these cells also recapitulated core phenotypes of PMNs recruited to the CF airway lumen in vivo, namely down-modulation of the phagocytic receptor CD16 (Fig. 1C), and high CD63 expression (Fig. 1D), reflecting active release of NE-rich granules.18 While in vivo samples contain a mixed population of PMNs recruited to CF airways over hours/days, our model enables collection of transmigrated PMNs at precise intervals, so that the dynamic course of pathological conditioning can be resolved (Supplementary Fig. 1B). Next, we assessed the status of the epithelial layer after PMN transmigration induced by LTB4 and CF ASN. First, using confocal imaging (Supplementary Fig. 2A), we observed that both conditions grossly preserved the integrity of the epithelial layer based on nuclear staining, and ZO-1 expression (reflecting junctionality). Second, using an extracellular lactate dehydrogenase assay (Supplementary Fig. 2B), we observed that epithelial cell viability was only slightly decreased after PMN transmigration compared to baseline conditions, and did not differ between LTB4 and CF ASN conditions. Third, we checked viability of EpCAM+ epithelial cells after detachment from the filters using flow cytometry (Supplementary Fig. 2C), and confirmed that they were highly viable, with no difference between LTB4 and CF ASN conditions. Finally, we observed that epithelial permeability to 4 kDa (significant) and 70 kDa (non-significant trend) dextrans increased after LTB4- and CF ASN-induced PMN transmigration compared to baseline (Supplementary Fig. 2D). However, it remained lower in LTB4 and CF ASN conditions than in the Triton-X maximum permeability control.

3.2 Healthy control airway milieu promotes transmigration, but not pathological conditioning, of PMNs

PMNs transmigrated to HC and CF ASN showed similar loss of CD62L, while CD16 downregulation and CD63 up-regulation were less pronounced at all time points for PMNs transmigrated to HC ASN (Fig. 2A–C). In addition, HC ASN recruited PMNs in lesser numbers than CF ASN at 10 and 18 h PTM (Fig. 2D). These patterns recapitulate differences between HC and CF airway PMNs observed in vivo8 (independently reproduced in Supplementary Fig. 3A-C). We also showed previously9 that the immunomodulatory receptor PD-L1 is uniformly increased in vivo on HC airway PMNs compared to HC blood PMNs, as also seen in our model, while CF airway PMNs cluster into PD-L1^Lo and PD-L1^Hi subsets, a bimodal pattern also recapitulated in our model (Supplementary Fig. 4A). In both HC and CF subjects, airway PMNs increase surface expression of Arg1 compared to blood PMNs,9 a pattern again recapitulated in vitro (Supplementary Fig. 4B).

3.3 Pathological conditioning induced by CF ASN on transmigrated PMNs is independent of their origin

Next, we investigated whether the origin of blood PMNs affected transmigration and pathological conditioning. We found that blood PMNs from HC and CF subjects do not markedly differ in their recruitment and exocytosis profile after transmigration to CF ASN (Supplementary Fig. 3D). Murine PMNs (from wild-type animals) also undergo transmigration and primary granule hyperexocytosis in the context of CF ASN (Supplementary Fig. 3E). Thus, our data demonstrate a dominant role for CF ASN in causing pathological conditioning of transmigrated PMNs, irrespective of their origin.

3.4 CF airway milieu and transmigration are both critical to pathological conditioning of PMNs

A low dilution of CF ASN (1:3) leads to levels of primary granule exocytosis in transmigrated PMNs matching those seen in CF patients in vivo, while intermediate (1:30) and high (1:300) dilutions lead to 2- and 4-fold lower levels, respectively (Fig. 2E). Diluting CF ASN also decreases transmigrated PMN count (Fig. 2F). CF ASN contains multiple factors able to recruit and activate PMNs, including host chemoattractants23 like LTB4 and CXCL8, and bacterial products,14 like fMLF and LPS. In our model, LTB4, CXCL8, and fMLF alone failed to induce primary granule exocytosis, despite promoting transmigration (Fig. 2G and H). Importantly, direct incubation of blood PMNs in CF ASN without transmigration failed to induce primary granule exocytosis, and delayed the down-regulation of CD16 (Fig. 2I and J). Thus, pathological conditioning of blood PMNs by CF ASN requires transmigration, and can be modulated by experimental dilution of the ASN.

3.5 Inhibition of LPS and LTB4 signaling does not impact the pathological conditioning of PMNs by the CF airway milieu

LPS alone failed to induce primary granule exocytosis or CD16 down-regulation when used as an apical stimulus in the model (Fig. 3C and D). LPS signals through TLR4, and blockade of this signaling cascade in PMNs using LPS-RS24 did not affect primary granule exocytosis and CD16 modulation, whether in the context of CF ASN or LPS (Fig. 3C and D). LPS-RS down-modulated surface CD11b expression and intracellular ROS accumulation in PMNs in the context of both LPS and CF ASN at 2 h PTM, confirming the activating potential of LPS on airway PMNs, and the inhibitory mode of action of LPS-RS on LPS-induced activation. LTB4 is a critical host-derived chemoattractant orchestrating PMN migration through the lamina propria.25 Acebilustat blocks LTB4 production by inhibiting LTA4 hydrolase, the rate-limiting enzyme for LTB4 synthesis.26 In our model, acebilustat decreased the apical PMN count at 2, 10, and 18 h PTM (Fig. 3E), but it did not affect primary granule exocytosis or CD16 down-regulation in PMNs (Fig. 3F).

3.6 ASN from various airway diseases induce the GRIM fate upon PMN transmigration

Severe asthma and bronchiolitis are examples of other diseases in which PMN dysfunction has been implicated as a potential pathogenic process.18 In our model, airway milieu from patients with these conditions leads to PMN transmigration and primary granule exocytosis, again recapitulating in vivo data (Supplementary Fig. 5A–C). In patients with COPD, disease symptoms, airway PMN dysfunction, and release of toxic by-products, such as NE, are similar to those observed in CF.19 In our model, COPD ASN leads to PMN recruitment and exocytosis of primary granules, mimicking in vivo data (Supplementary Fig. 5D and E). Similarly to what we observed for CF ASN, CD16 is also down-regulated upon PMN transmigration to COPD ASN (Supplementary Fig. 5E).

3.7 Increased pinocytosis is a newly identified feature of PMNs transmigrated to the CF airway milieu, in vivo and in vitro

A prior study linked enhanced primary granule release by PMNs to pinocytosis.27 In CF patients, airway PMNs showed enhanced pinocytosis compared to blood PMNs, and pinocytic activity in CF airway PMNs correlated with primary granule release (Fig. 4A). In our model, PMNs transmigrated to CF ASN increased their pinocytic activity over time, with a similar correlation with primary granule release to that observed in vivo (Fig. 4B).

3.8 PMN transmigration to CF airway milieu leads to increased metabolism, but decreased bacterial killing

In vivo, CF airway PMNs undergo metabolic activation.7, 10 In vitro, PMNs transmigrated to CF ASN show increased glycolysis, as reflected by a higher extracellular acidification rate, and increased surface Glut1 expression, compared to those transmigrated to LTB4 (Fig. 5A–D). PMNs transmigrated to CF ASN also showed a strong up-regulation of their oxygen consumption rate, which was not sensitive to the inhibitor of mitochondrial oxidative phosphorylation oligomycin (Fig. 5E and F), but was associated with an increased ROS production (Fig. 5G). This is consistent with the notion that activated PMNs use oxygen for ROS production via either NADPH oxidase or mitochondrial complex I, with support from glycolysis.28, 29 Despite this metabolically activated state, PMNs transmigrated to CF ASN showed lower ability to kill P. aeruginosa than those transmigrated to LTB4 (Fig. 5H). Thus, our model recapitulates a key paradox in CF, namely the inability to kill bacteria,14 despite PMN recruitment and their metabolic activation.

3.9 Metformin inhibits key pathological phenotypes of PMNs recruited to the CF airway milieu

Metformin is a metabolic modulator able to curb down glucose-dependent cell activation, ROS production, and inflammation.29, 30 Metformin had a mild effect on glycolysis, but strongly inhibited oxygen consumption in PMNs transmigrated to CF ASN (Fig. 6A and B). Metformin acts, in part, via activation of the metabolic checkpoint enzyme 5′ adenosine monophosphate-activated protein kinase α1 (AMPKα1), by facilitating its phosphorylation.31 We observed that phosphorylated AMPKα1 levels were indeed reduced in CF airway compared to blood PMNs in vivo, and in PMNs transmigrated to CF ASN compared to LTB4 in vitro, and that treatment with metformin increased phosphorylated AMPKα1 levels in PMNs transmigrated to CF ASN (Fig. 6C). This effect was associated with a reduction of ROS production and primary and secondary granule exocytosis (Fig. 6D). Interestingly, metformin treatment did not rescue the ability of PMNs transmigrated to CF ASN to kill bacteria (Fig. 6E).

4 DISCUSSION

Massive airway recruitment of PMNs, and their pathological conditioning resulting in heightened granule release, immunoregulatory, and metabolic activities (“GRIM” fate), and depressed bacterial killing, drive early and chronic lung damage in CF and other intractable diseases. These processes are incompletely understood and remain untapped as potential therapeutic targets, in large part due to the lack of adequate models. The model described here enables transepithelial recruitment and pathological conditioning yielding GRIM PMNs that feature characteristic hyperexocytosis of NE-rich granules, but also decreased surface CD16, increased Arg1 expression, bimodal PD-L1 expression, increased metabolic activity, and decreased bacterial killing, all hallmarks of CF airway GRIM PMNs in vivo. In addition, we describe a new metabolic and functional feature of these cells in vivo and in vitro, which relates to their enhanced pinocytosis.

A dominant conditioning effect of CF airway secretions was noted in studies where these were acutely added to healthy or CF airway epithelium,32 and macrophages,33 although their effect on PMNs was not explored. In our model, apical CF ASN was necessary and sufficient to recruit and condition blood PMNs to adopt the GRIM fate, regardless of whether these PMNs originate from CF or HC subjects. Thus, our data demonstrate a dominant environmental effect of factors present in CF airway milieu onto recruited PMNs. However, they do not exclude a contribution of endogenous CFTR in PMNs,34, 35 although such an effect appears to be secondary relative to the induction of the pathogenic GRIM fate. Further studies are needed to fully delineate the still debated issue of whether endogenous CFTR is key to the function of PMNs recruited to CF airways in vivo, and in this model.

Our data show that PMN transmigration toward ASN from COPD and LD patients also lead to substantial primary granule exocytosis, similar to the results obtained with CF ASN, while HC ASN induced transmigration but not primary granule exocytosis. Thus, PMN pathological conditioning into the GRIM fate is not unique to CF. Murine PMNs also undergo transmigration and hyperexocytosis of NE-rich granules, suggesting that factors in CF ASN leading to PMN pathological conditioning are cross-specific. This result is reminiscent of the cross-specific recruitment of murine PMNs in human CF small airway xenografts.36 It also opens potential avenues for future studies using relevant mouse strains in our model to assess mechanisms of PMN plasticity and conditioning,14 and further delineate the role of endogenous CFTR using PMNs from wild-type versus CFTR knockout or mutant mice.

CF ASN is characterized by an abnormal molecular composition (including altered lactate,11 small metabolite,37 and protein38 contents), itself caused by altered epithelial function, and the sustained presence of bacteria and PMNs in the CF airway lumen.14 It is interesting to note that at concentrations similar to those used to promote PMN transmigration and conditioning in our model, CF ASN induces T cell apoptosis.9 At lower concentrations, T cells survive but their ability to activate and proliferate is dramatically reduced, due in part to Arg1 activity originating from GRIM PMNs. Both Arg1 and PD-L1, another prominent T-regulatory molecule, are modulated in the model in similar fashion to in vivo.9 Thus, CF ASN exerts paradoxical effects on PMNs and T cells, promoting the former while demoting the latter, which further contributes to the buildup of a PMN-dominated inflammatory environment in the CF airway lumen.

The ability of CF ASN to license recruited PMNs metabolically toward increased Glut1 expression and glycolytic activity is of particular significance, since PMNs are generally thought of as short-lived, yet thrive and undergo transcriptional changes10 in this pathological milieu. In addition, GRIM PMNs obtained by transmigration into CF ASN show decreased killing of P. aeruginosa as compared to those transmigrated to LTB4, despite their metabolic licensing, which is consistent with CF pathology in vivo.14, 15 Further studies will focus on this paradox and screen for drugs capable of rewiring GRIM PMNs to enact proper bacterial clearance. The correlation between pinocytic activity and primary granule exocytosis in PMNs migrated to the CF airway lumen in vivo and in vitro also suggests a link between metabolic activity and abnormal granule mobilization,27 which warrants further study.

In typical Transwell models, PMNs are forced to squeeze one-by-one through pre-drilled 3 μm-wide cylindrical pores before reaching the epithelial basement membrane. By contrast, our model includes a highly porous lamina propria-like scaffold with 36–40 μm-wide pores that can each accommodate 20+ cells, thus enabling PMNs to congregate and migrate in physiologically relevant manner.25 Confocal imaging confirmed that LTB4- and CF ASN-induced PMN transmigration largely preserved the integrity of the epithelial layer, including typical peripheral ZO-1 staining pattern. In addition, we ascertained that epithelial viability was preserved, based on extracellular lactate dehydrogenase assay and flow cytometry. Dextran permeability assays showed that LTB4- and CF ASN-induced PMN transmigration led to an expected increase in transepithelial permeability, which remained well below that observed in Triton X-treated monolayers (maximum permeability control). These results emphasize the physiological relevance and robustness of this experimental model.

While we plan to test the use of primary epithelial cells from CF patients in our model in future studies, the present setup including a commercial 3D substrate, a small airway epithelial cell line, standard culture media, and human blood PMNs, provides a highly robust and reproducible platform to mass-produce airway-like PMNs for research. The CF ASN used to induce PMN migration and pathological conditioning is a very abundant material easily collected from expectorations. Our model is advantageous not only because it integrates patient airway milieu as the apical stimulus; but also because blood PMNs from patients can be integrated as target cells; and because drugs can be readily added to blood and/or airway sides, mimicking systemic and airway routes of administration. These combined features provide the ability to de-risk drugs at an early stage, in conditions close to those seen in vivo.

Because of the long-held belief that PMNs die rapidly after migration into CF airways, PMN-targeted therapies have focused on reducing their recruitment via corticosteroids or non-steroidal anti-inflammatories.39 Because GRIM PMNs found in CF airways are alive when they release their toxic contents,7, 8 the pathological conditioning of PMNs, rather than their recruitment, may be a more effective target for new therapeutic agents to benefit patients with CF and other similar diseases.40 Our data obtained with the metabolic regulator metformin illustrate that point. Indeed, combined effects of metformin on GRIM PMN metabolism (i.e., oxygen consumption), ROS production, and degranulation, suggest that pathogenic functions in these cells are fueled by dysregulated metabolism, and that targeting metabolism in GRIM PMNs may be a relevant strategy for therapeutic intervention. Whether metformin is a relevant drug in the context of CF is questionable, however, since it did not rescue but rather seemed to further depress the ability of PMNs transmigrated to CF ASN to kill bacteria. Further studies are required to determine whether this adverse effect of metformin treatment may be linked to its ability to counter ROS production and degranulation, an otherwise beneficial effect of metformin considering the self-damaging impact of ROS and granule effectors unleashed by pathologically conditioned PMNs in CF airways.

Together, this study emphasizes the complex roles of GRIM PMNs, which represent a promising target for immunotherapy in CF and other human airway diseases. We also introduce a simple, robust, and flexible in vitro model for basic studies and drug development efforts focused on this key pathogenic subset.

ACKNOWLEDGMENTS

The authors would like to thank Dr. J. Alvarez, as well as Ms. W. Si Hassen for assistance; the Emory + Children's Pediatric Flow Core (A. Rae and Dr. D. Archer), and Integrated Confocal Imaging Core for access to flow cytometry and confocal imaging systems; Children's Healthcare of Atlanta and Emory University Pediatric CF Discovery Core, Mouse Models Core, and Experimental Models Core (M. Maliniak, J. Flores, J. Wu, B. Imhoff, and Drs. S. Molina, N. McCarty and A. Stecenko) for access to CF patient samples, mouse tissues, and confocal microscopy expertise; Drs. A. Fitzpatrick and S. Stevenson for HC sputum collection; and Dr. E. Springman (Celtaxsys, Inc.) for his generous donation of the acebilustat drug.

FUNDING SOURCES

This study was supported by the Emory Pediatrics Startup, EECR Seed, and URC Programs (R.T.), Cystic Fibrosis Foundation (TANGPR12A0 to V.T.; MCCART15RC, TIROUV13P0, and TIROUV15A0 to R.T.), and National Institutes of Health (T32 AA013528 to S.I.; R01HL102371 to A.G.; UL1TR000454 to V.T. and R.T.; and R01HL126603 to R.T.).

DISCLOSURE

The authors declare no conflict of interest.

Supporting Information

REFERENCES

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Citing Literature

Volume104, Issue4

Special Focus Issue: Development of Tissue‐ and Pathogen‐Specific Cellular Innate Immunity

October 2018

Pages 665-675

Frontline Science: Pathological conditioning of human neutrophils recruited to the airway milieu in cystic fibrosis