Secretoglobin 1A1 and 1A1A Differentially Regulate Neutrophil Reactive Oxygen Species Production, Phagocytosis and Extracellular Trap Formation

Secretoglobin family 1A member 1 (SCGB 1A1) is a small protein mainly secreted by mucosal epithelial cells of the lungs and uterus. SCGB 1A1, also known as club (Clara) cell secretory protein, represents a major constituent of airway surface fluid. The protein has anti-inflammatory properties, and its concentration is reduced in equine recurrent airway obstruction (RAO) and human asthma. RAO is characterized by reversible airway obstruction, bronchoconstriction and neutrophilic inflammation. Direct effects of SCGB 1A1 on neutrophil functions are unknown. We have recently identified that the SCGB1A1 gene is triplicated in equids and gives rise to two distinct proteins. In this study we produced the endogenously expressed forms of SCGBs (SCGB 1A1 and 1A1A) as recombinant proteins, and analyzed their effects on reactive oxygen species production, phagocytosis, chemotaxis and neutrophil extracellular trap (NET) formation ex vivo. We further evaluated whether NETs are present in vivo in control and inflamed lungs. Our data show that SCGB 1A1A but not SCGB 1A1 increase neutrophil oxidative burst and phagocytosis; and that both proteins markedly reduce neutrophil chemotaxis. SCGB 1A1A reduced chemotaxis significantly more than SCGB 1A1. NET formation was significantly reduced in a time- and concentration-dependent manner by SCGB 1A1 and 1A1A. SCGB mRNA in bronchial biopsies, and protein concentration in bronchoalveolar lavage fluid, was lower in horses with RAO. NETs were present in bronchoalveolar lavage fluid from horses with exacerbated RAO, but not in fluid from horses with RAO in remission or in challenged healthy horses. These findings indicate that SCGB 1A1 and 1A1A have overlapping and diverging functions. Considering disparities in the relative abundance of SCGB 1A1 and 1A1A in airway secretions of animals with RAO suggests that these functional differences may contribute to the pathogenesis of RAO and other neutrophilic inflammatory lung diseases.


Introduction
Secretoglobin family 1A member 1 (SCGB 1A1) is a small, secreted protein mainly produced by mucosal epithelial cells in lung and uterus. SCGB 1A1, also known as club (Clara) cell secretory protein (CCSP), was suggested as the standardized nomenclature to define this member of an emerging superfamily of 10 kDa proteins [1]. The SCGB family of proteins exist as disulfide-dependent homodimers that are oriented in an antiparallel superposition [2]. Structurally, this association generates an internal hydrophobic pocket for binding of lipophilic molecules [3][4][5][6]._ENREF_5 SCGB 1A1 dimers sequester calcium and phosphatidylcholine, a cofactor and a substrate required for phospholipase A2 activity, respectively [7,8]. Existence of minor hydrophobic cavities on each side has also been reported, but their functions are unknown. SCGB 1A1 is highly resistant to protease degradation, and stable at high temperatures and pH extremes [9].
The exact roles of SCGB 1A1 in lung physiology and homeostasis are uncertain. Although SCGB 1A1 is not essential for normal lung function, its absence or reduced expression is associated with exacerbation of several inflammatory conditions [10,11]. Development of synthetic antiflammins based on fusion of conserved structures of SCGB 1A1 and lipocortin-1 yielded compounds with in vivo anti-inflammatory properties [12]. Recombinant SCGB 1A1 has been suggested as a therapeutic agent for treating inflammatory diseases [9], and intranasal administration of the protein improved the hospital discharge rate and dependence on supplemental oxygen in premature infants with respiratory distress syndrome [13]. Nevertheless, whether SCGB 1A1 directly affects the function of inflammatory cells is not clearly established [14].
Recurrent airway obstruction (RAO) is an inflammatory airway disease induced by repeated exposure of susceptible horses to inhaled environmental triggers [15]. Influx of neutrophils into the airways is a hallmark of the condition [16]. Horses with RAO have low levels of SCGB1A1 mRNA in the lungs and low protein concentration in bronchoalveolar lavage (BAL) fluid [17]. The recent discovery that the SCGB1A1 gene is triplicated in the equine genome, and that the copies evolved differently over time, suggested that different gene products may play important roles in natural adaptation, biological advantage, and possible functional divergence in health and disease [18]. Three-dimensional modeling of SCGBs suggests that the proteins might have acquired different molecular-binding partners [19] Neutrophils are powerful innate immune cells that rapidly extravasate into injured tissues in response to inflammatory signals. Typically, increased IL-8 in the injured or infected tissue recruit neutrophils via chemotaxis [20]. At the site, activated neutrophils release reactive oxygen species (ROS) or internalize targets by phagocytosis [21]. ROS are also released into the phagocytic vacuoles of neutrophils to kill internalized microbial pathogens [22]. Recent studies show that these innate immune cells also release nuclear components in the form of neutrophil extracellular traps (NETs) to ensnare microbial pathogens [23]. Although the pathways that regulate NET formation, or NETosis, are not clearly defined, this form of cell death occurs at least via two mechanisms: one is dependent on ROS production and the other is dependent on calcium-ion influx [24]. Although NETs are useful, excess NETosis leads to severe tissue damage [25]. SCGB 1A1 appears to reduce neutrophil migration into the lung by highaffinity receptor antagonism and chemoattractant sequestration [26]. It also down-regulates the release of ROS by neutrophils and suppresses activated phagocytosis [27]. However, whether different forms of SCGB 1A1 differentially regulate neutrophil chemotaxis, phagocytosis or NETosis is unknown. Furthermore, neither in vivo formation of NETs in inflammatory lung disease nor the differential expression of SCGBs during the disease is known. Here we tested the hypothesis that equine SCGB 1A1 and 1A1A differentially regulate neutrophil functions. We found that (i) SCGB 1A1A suppressed neutrophil chemotaxis significantly more than SCGB 1A1; (ii) both proteins significantly reduced NETosis in a time and concentration-dependent manner; and (iii) SCGB 1A1A but not SCGB 1A1 significantly enhanced oxidative burst and phagocytic activity. During exacerbated RAO with marked neutrophilic airway inflammation, SCGB1A1 mRNA expression in bronchial epithelium and total SCGB concentration in BAL fluid were significantly reduced in affected animals, and NETs were present in BAL fluid.

Animals
All research involving animals was approved by the University of Guelph Animal Care Committee (protocol R10-031) and conducted in compliance with Canadian Council on Animal Care guidelines. RAO and control horses ranged in age from 6 to 18 years and were selected according to previously established criteria [17]. Clinical challenge, pulmonary function testing (PFT), and BAL procedures were performed as described [17]. Briefly, horses were maintained outdoor to limit dust and mould exposure preceding the study. Prior to assessment, animals were transferred to a dust-free indoor environment for 24 hours. Then, healthy control and RAO horses in remission were exposed to dusty hay twice a day until clinical signs of respiratory impairment were detected in animals with RAO, and pulmonary function testing indicated pleural pressure change of .15 cm H 2 O. For BAL, horses were restrained in stocks and sedated with 0.5 mL of Sedivet (Romifidine 10 mg/mL, IV; Boehringer Ingelheim, Burlington, ON). Briefly, a 13-mm diameter, 180-cm long sterile bronchoscope (Olympus, Tokyo, JP) was inserted through a nostril and passed to the level of the carina. A 0.2% lidocaine solution (AstraZeneca, Mississauga, ON) was intermittently applied via the endoscope to reduce coughing and discomfort. The bronchoscope was wedged in a 2 nd or 3 rd generation bronchus, and 120 mL of warm sterile saline was instilled and re-aspirated. The BAL fluid was filtered, cell concentration was determined with a particle counter (Z2 Coulter counter, Beckman Coulter, Mississauga, ON), and the sample was centrifuged for 10 min at 500 6 g to pellet cells. Protein concentration in cell-free supernatant was determined with using a NanoDrop 2000 photometer (Thermo Fischer Scientific, Cooksville, ON).
Cloning and production of equine recombinant SCGB 1A1 and 1A1A Total RNA was isolated from equine frozen lung tissues (RNeasy, Qiagen, Mississauga, ON) and reverse transcribed into complementary DNA (cDNA) _ENREF_2 [18]. SCGB1A1 and SCGB1A1A coding regions were obtained by PCR amplification using the following primers (Sigma-Aldrich): SCGB1A1-F, 59-GAA ATC TGC CAG AGC TTT GCA GAC ATC ATT CAA GGC C-39; SCGB1A1-R, 59-GTC ACC TGC AGG CTA AGC ACA CAG TGG GCT CTT TGC-39; SCGB1A1A-F, 59-GGA ATC TGC CAG AGA TTG GTA GGC ATC GTT CAA GCC C-39; SCGB1A1A-R, 59-GTC ACC TGC AGG CTA AGC ACA CAG TGG GCT CTC TAC-39. Primers were designed with XmnI and SbfI restriction sites for cloning into the pMAL-c5X expression vector (New England BioLabs, Mississauga, ON). This vector is designed to produce maltose-binding protein (MBP) fusion proteins without adding vector-derived residues. Amplifications were carried out using a Platinum Taq polymerase PCR kit (Invitrogen, Mississauga, ON). Each reaction was performed in a final volume of 25 mL, including 2 mL of 10 6 PCR buffer, 0.2 mM dNTPs, 2 mM MgCl 2 , 0.3 mM of each primer, 2 U of Platinum Taq, and 1 mL of template cDNA (100 ng). Conditions for amplification were 1 min at 94uC followed by 30 cycles of 94uC for 30 sec; 62uC for 30 sec; and 72uC for 90 sec, followed by a final extension at 72uC for 7 min. PCR products were subjected to electrophoresis; bands of appropriate size were excised from the gel, purified (Qiagen) and submitted for automated sequencing (Laboratory Services Division, Guelph, ON). Amplicons were analyzed in duplicate using reverse and forward primer sequencing strategies. SCGB1A1 and SCGB1A1A cDNAs were blunted (New England BioLabs), digested with SbfI, and individually ligated in the XmnI/SbfI site of the pMAL-c5X vector. Both vectors were transformed into NEB Express enhanced competent Escherichia coli BL21 (New England BioLabs), incubated overnight at 37uC, and selected on Luria-Bertani agar plates containing 100 mg/mL ampicillin. Ten clones were grown on agar plates overnight for each construct. Plasmids were screened by PCR using primers pMAL-F, 59-GCG CAG ACT AAT TCG AGC TC-39 and pMAL-R, 59-CCT ACT CAG GAG AGC GTT C-39. PCR products were separated by electrophoresis, excised, purified, and sequenced. Sequences were analyzed with Geneious Pro software (Geneious) to confirm integrity and proper directional insertion of the insert into the vector.
Transformed E. coli cells were grown overnight in 10 mL of Rich Medium (yeast/tryptone/NaCl/glucose/ampicillin) at 37uC with gentle shaking. Five milliliters of the culture were transferred to flasks containing 500 mL of Rich Medium and incubated 3 hours with gentle shaking. Isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to a concentration of 3 mM and the culture was incubated at 37uC for 2 hours. Cells were pelleted by centrifugation at 4,000 6 g for 20 min at 4uC, resuspended in 25 mL of column buffer (20 mM Tris-HCl pH 7.4, 1 mM EDTA, 200 mM NaCl, 1 mM DTT) and frozen at 220uC overnight. Cells were thawed in an ice-water bath, sonicated on ice, and centrifuged at 20,000 6g for 20 min at 4uC. The supernatant was collected, diluted to 1:6 with column buffer, passed through an amylose column (New England BioLabs), washed with column buffer, and eluted with 10 mM maltose enriched column buffer. The eluate was then incubated with 1% Factor Xa for 44 hours at room temperature. The protein mix was concentrated using 10K Amicon columns (EMD Millipore, Billerica, MA) and purified using Fast Protein Liquid Chromatography (FPLC). A volume of 500 mL of purified proteins were passed through a HiLoad 26/60 SuperDex 200 column attached to Ä KTA explorer FPLC system (GE Healthcare, Baie d'Urfé, QC), using a filtered FPLC buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl). Fractions containing SCGB 1A1 or SCGB 1A1A were passed through an amylose column (New England BioLabs), ran on EndoTrap columns (Hyglos, Bernried, Germany) to remove endotoxin, washed with PBS buffer, and stored at 280uC. Recombinant SCGB 1A1 and SCGB 1A1A preparations tested negative for endotoxin using the Limulus polyphemus amoebocyte lysate assay (Sigma-Aldrich, Burlington, ON).

Phagocytosis and oxidative burst assays
Equine blood was collected with acid citrate dextrose (ACD) as anticoagulant and kept at room temperature for less than one hour. Then, 40 mL of blood diluted 1/3 (v/v) into PBS/EDTA was layered on top of 10 mL of Ficoll-Paque PLUS (GE Healthcare). After centrifugation at 500 6 g for 30 min, erythrocytes were removed by hypotonic lysis for 45 sec in water. Samples were centrifuged again at 300 6 g and the upper layer containing lysed erythrocytes was removed. This step was repeated if the sample still appeared reddish. The resulting white neutrophil pellet was suspended at 5 6 10 6 cells/mL in PBS/FBS 10% (v/v). Neutrophils (5 6 10 5 cells) were incubated in the presence (250, 500, or 1000 ng/mL) or absence of either SCGB 1A1 or SCGB 1A1A (n$5 horses) in a total volume of 200 mL for 30 min at 37uC with gentle agitation, and centrifuged at 300 6 g for 10 min. The supernatant was removed and the pellet was re-suspended in 100 mL of a fresh solution of PBS/FBS 10% (v/v). H 2 DCFDA (Molecular Probes, Eugene, OR, USA) was added to each flow cytometry vial (BD Biosciences, Bedford, MA, USA) at a final concentration of 10 mM and incubated with gentle agitation for 15 min at 37uC. Next, 25 ng/mL of phorbol myristate acetate (PMA, Sigma, St-Louis, MO, USA) was added, and cells were incubated for an additional 15 min. In phagocytosis assays, neutrophils were treated as above. After re-suspension in PBS/ FBS 10% (v/v), neutrophils were incubated with 2.7 6 10 7 fluorescent beads (FluoSpheres sulfate microspheres, 1.0 mm, 505/ 515, Invitrogen) and 1/5 (v/v) filtered normal horse serum (NHS) to opsonize the beads. Samples were incubated 30 min at 37uC with gentle agitation and cells were analyzed by flow cytometry (FACscan) using Cell Quest software (BD Biosciences). Neutrophils were discriminated by forward and side light scatter.

Microchemotaxis assay
Freshly isolated neutrophils (n = 5 horses) were loaded with ng/ mL of calcein AM (Molecular Probes, Life Technologies, Burlington, ON) during 30 min at 37uC with gentle agitation, centrifuged at 300 6 g for 10 min, and re-suspended in a fresh solution of PBS/FBS 10%(v/v). Neutrophils were then pre-treated for 30 min at 37uC with 0, 250, 500, or 1000 ng/mL of SCGB 1A1 or SCGB 1A1A. Twenty mL of sample was plated onto the ChemoTx microplates (Neuro Probe, Maryland, MD). The lower chambers were loaded with 29 mL of a PBS/FBS 10%(v/v) solution supplemented with 0.3 mg/mL of recombinant human IL-8 (R&D Systems, Minneapolis, MN) as chemoattractant. Serial dilutions of the cell suspension (1 610 5 , 7.5 610 4 , 5 610 4 , 2.5 6 10 4 , 1.25 6 10 4 , and 0 cells) were added to the chambers for a calibration curve, and all samples were analyzed in triplicate. Each microplate was incubated at 37uC (5% CO 2 ) for 45 min to allow migration. The filter membrane was then rinsed with PBS to remove non-migrating neutrophils, and fluorescence in wells was measured with a Synergy HT microplate reader using Gen5 analysis software (BioTek, Vermont, USA).

NETosis assays
For NETosis assay, neutrophils were isolated using a modified version of the Polymorphprep protocol (Axis-Shield, Dundee, UK) since EDTA is preferred over ACD anticoagulant for NET assays. Briefly, 20 mL of equine blood was collected into EDTA, layered on an equal volume of Polymorphprep and centrifuged at 500 6 g for 35 min at room temperature. Polymorphonuclear cells (PMN) were harvested, mixed with one volume of HEPES-buffered saline (0.42% NaCl, 5 mM HEPES-NaOH, pH 7.4), and centrifuged for 10 min at 400 6 g. The pellet was rapidly re-suspended in 10 mL of a 0.2% NaCl solution (max 30 sec), mixed with 10 mL of 4 6 HEPES-buffered saline (1.6% NaCl, 20 mM HEPES-NaOH, pH 7.4), and collected by centrifugation. These steps were repeated twice. Cells were washed with 2 6 HEPES-buffered saline solution (0.85% NaCl, 10 mM HEPES-NaOH, pH 7.4), harvested by centrifugation and re-suspended in Roswell Park Memorial Institute (RPMI) medium at a concentration of 7 610 5 cells/mL. Each well of a 96-well special optical plate (Corning, Lowell, MA) was loaded with 50 mL of cells and incubated for 30 min at 37uC with 0, 125, 250, 500, 1000, or 2000 ng/mL of SCGB 1A1 or 1A1A in triplicate wells. After incubation, an additional 50 mL of cells supplemented with 5 mM of cell impermeant Sytox Green Nucleic Acid Stain (Invitrogen) was added to each well. In two replicate plates, triplicate wells with cells were treated with either PMA (20 to 120 nM; ROSdependent) or A23187 ionophore (4 mM; Sigma-Aldrich; ROSindependent) to stimulate NETosis. [24,28] NET formation and subsequent DNA release was monitored by quantifying fluorescence emission generated by the interaction of Sytox Green stain with extracellular DNA. Total fluorescence was measured using a Gemini Analysis of in vivo NET-derived DNA and NET assays BAL fluid from six control and seven age-matched RAOsusceptible horses pre-and post-challenge was analyzed by agarose gel electrophoresis, as described [29]. Briefly, each sample was separated on gels with or without proteinase K (PK; 0.2 mg/mL). Presence of nucleic acids in BAL fluids was verified by DNase (50 mg/ml), RNase (250 mg/ml), or dual treatment for one hour at 37uC in the presence of 3 mM CaCl 2 . Samples were subjected to electrophoresis and DNA was visualized using SYBR Safe DNA gel stain (Invitrogen). Images were acquired with a Chemidoc XRS gel imaging system (Bio Rad, Mississauga, ON) and analyzed using Image Lab 4.1 software (Bio-Rad). Western Blot for citrullinated histone H3 (CitH 3 ) was performed using the antihistone H3 antibody (citrulline R2+R8+R17, Abcam).
Quantitative SCGB1A1 and SCGB1A1A gene expression SCGB1A1 primer specificity and identity of the PCR products was confirmed with a melting curve (95uC for 5 sec; 95 to 45uC; 40uC for 10 sec) and sequence analysis, respectively. For each gene, a series of purified cDNA PCR product dilutions (100, 10, 1, 0.1, 0.01, 0.001 ng/mL) was amplified and the average crossing point of each dilution was used to derive a standard curve. SCGB1A1, SCGB1A1A, GAPDH, and 18S cDNA were amplified in triplicate for each sample along with standard curve calibrators. The cycle threshold (C T ) was determined and normalized to that of the GAPDH, and 18S housekeeping genes (DC T ). The comparative threshold method (DDC T ) was used to measure SCGB gene expression. Data were analyzed using LightCycler 480 SW 1.5 software (Roche).

SCGB ELISA
BAL fluid protein concentration was measured (Nanodrop) and diluted with 50 nM carbonate buffer (pH 9.6) to 15 ng/mL. The resulting samples (100 mL) were added into triplicate wells of a NUNC MaxiSorp polystyrene ELISA plate (Thermo Fischer Scientific) and incubated overnight at 4uC. Wells were washed with 0.1% (v/v) Tween-20 in 50 mM PBS, pH 7.4 (T-PBS) several times and blocked with 5% (w/v) powdered skim milk in T-PBS for 6 hours at 4uC. Following three washes with T-PBS, 200 mL of SCGB antibody (diluted 1:350 in T-PBS/1% (w/v) skim milk powder) was added to each well, and samples were rotated overnight at 4uC on an orbital shaker. After five washes in T-PBS, 100 mL of polyclonal swine anti-rabbit HRP antibody (Dako) diluted 1:8,000 in T-PBS/1%(w/v) skim milk powder was added to each well. Samples were incubated at room temperature for 30 min and then washed. Finally, 100 mL of 3,39, 5,59-tetramethylbenzidine solution (Thermo Fischer Scientific) was added as substrate to each well, the plates were incubated at room temperature for 15 min, and the reaction was terminated by addition of 100 mL of 2 M sulfuric acid. Absorbance was measured at 450 nm. Incubation of samples with pre-immune rabbit serum served as a negative control, and readings from blank wells were used to determine background signal. Serial dilutions of recombinant equine SCGB (0, 1.25, 2.5, 5, 10, 20, 40 and 280 ng/ mL) in carbonate buffer were used as standards. All samples and standards were tested in triplicate.

Statistical analysis
Values were expressed as means 6 SEM, and data were analyzed as a 2-factor split-plot in a randomized block design with SCGB treatment as the primary factor. Residual plots were constructed, and formal tests of normality of the residuals were applied (SAS, Cary, NC). Two-way ANOVA with repeated measures, and Bonferroni posttests were performed for NETosis data. Graphs were prepared with Prism5 software (GraphPad, San Diego, CA); p#0.05 was considered significant.

Cloning, expression and purification of equine recombinant SCGB 1A1 and SCGB 1A1A
To generate recombinant SCGBs, the cDNAs coding for 1A1 and 1A1A mature proteins, excluding the signal peptide sequences, were obtained by PCR amplification from lung biopsies. For each reaction, a single band of expected size (225bp) was detected by gel electrophoresis (Figure 1A), extracted, purified, and inserted into the pMAL-c5X expression vector ( Figure 1B). Newly generated SCGB1A1 and SCGB1A1A expression vectors were then transformed into competent cells and at least five independent transformants were sequenced to confirm the identity and proper integration of the insert, as shown in Figure 1B. The sequencing chromatograms revealed 100% identity with SCGB1A1 and SCGB1A1A (NCBI JQ906260.1 and JQ906261.1, respectively). A transformant was selected, inoculated, cultured and harvested for each construct. A crude extract was prepared from individual cultures and subjected to affinity and gel filtration chromatography to retrieve the purified fusion proteins. Approximately 30 to 50 mg of purified protein was obtained per liter of culture. To assess purity, multiple fractions were collected at defined steps and loaded on SDS-PAGE gels. SCGB 1A1 and 1A1A fusion proteins (48 kDa) were detected in extracts induced with isopropylthiogalactoside (IPTG) but not in the un-induced fractions ( Figure 1C). Addition of Factor Xa protease to the fusion protein preparations generated two bands of 40 and 7 kDa, indicating proper cleavage of maltose binding protein (MBP) from either SCGB 1A1 or 1A1A. Both recombinant proteins formed homodimers (14 kDa) in non-reducing conditions, and monomers in reducing and denaturing SDS-PAGE ( Figure 1D). Results indicated that structure and conformation of the recombinant proteins were conserved, allowing dimerization via the cysteine residues located at both ends of the polypeptide. Western blot analysis under denaturing and mild reducing conditions was performed to confirm the identity of SCGB 1A1 and SCGB 1A1A monomers and dimers ( Figure 1E).

SCGB 1A1 and 1A1A differentially modulate neutrophil oxidative burst and phagocytosis
To test the effect of SCGB 1A1 and 1A1A on ROS production and phagocytosis, flow cytometric assays were performed on blood-derived neutrophils freshly isolated from healthy horses. Neutrophil activation and ROS production was initiated with phorbol myristate acetate (PMA), which increased fluorescence intensity .6 fold relative to PBS-treated controls. Cell preparations treated with PMA had characteristic morphological changes of activated neutrophils such as membrane ruffling and cytoplasmic vacuolation (Figure 2A). Addition of either the fluorescent substrate dichlorodihydrofluorescein diacetate (H 2 DCFDA) or SCGB 1A1 or SCGB 1A1A had no effect on neutrophil morphology. A marked and dose-dependent increase in fluorescence was observed in SCGB 1A1A treated neutrophils ( Figure 2B), whereas increasing amounts of SCGB 1A1 had minimal effect. SCGB 1A1A-induced pro-oxidant effects differed significantly from those of SCGB 1A1 at 500 (3386123 vs 6556143, p = 0.0012) and 1000 ng/mL (288642 vs. 9406216; p,0.0001).
The effect of SCGB 1A1 and 1A1A on neutrophil phagocytic activity was analyzed by quantifying fluorescent bead internalization after incubation of neutrophils and beads with or without recombinant proteins. Compared to SCGB 1A1, SCGB 1A1A pre-incubation (250 ng/mL) significantly increased fluorescence (631611 vs. 694627; p = 0.0028; Figure 2C). Significant differences were not detected at 500 and 1000 ng/mL of SCGB 1A1A.

SCGB 1A1 and 1A1A inhibit IL-8-driven neutrophil chemotaxis
The effect of recombinant SCGB 1A1 and SCGB 1A1A on chemotactic activity of equine neutrophils was characterized using a microchemotaxis plate assay. Cells were pre-treated with serially diluted concentrations of SCGB 1A1 or SCGB 1A1A (0, 250, 500, 1000 ng/mL) and deposited on IL-8 filled chemotaxis chambers. Both recombinant proteins significantly attenuated IL-8 induced migration of neutrophils in a dose-dependent manner (Figure 3), but SCGB 1A1A inhibited chemotaxis significantly more than SCGB 1A1. SCGB effects were most different at 250 ng/mL concentration, where 91.864.0% of SCGB 1A1 pre-treated cells had migrated, but only 71.2613.1% of SCGB 1A1A pre-treated cells showed chemotaxis function (p,0.02). At higher concentrations, there was progressively greater inhibition of neutrophil migration, and SCGB 1A1A continued to have a more pronounced effect than SCGB 1A1 (p,0.04).

SCGB 1A1 and 1A1A inhibit NET formation ex vivo
To determine whether SCGBs affect NETosis, we investigated the kinetics of NET formation in neutrophils treated with different concentrations of recombinant SCGBs by determining fluorescence in plate reader assays and confocal microscopy [29]. Plate reader assays detect extracellular DNA because Sytox green is   impermeable to live cells and apoptotic cells. Both SCGB 1A1 and 1A1A inhibited calcium ionophore A23187-induced NET formation by equine neutrophils ex vivo ( Figure 4A and 4B). A dosedependent (0-2000 ng/mL) and time-dependent (0-4 h) inhibition of NET formation was observed in plate reader assays (n = 7 different samples in triplicate; p,0.05). There was a statistically significant interaction between the concentration of SCGB 1A1A and time (p,0.05). Higher concentrations of SCGB 1A1 and 1A1A reduced NETosis by approximately 22% and 30%, respectively. Although SCGB 1A1A appeared to inhibit NETosis better than 1A1, differences in the abilities of these two proteins up to 2000 ng/mL were statistically not different. Of note, PMA concentrations of 20 to 160 nM failed to trigger NET formation in neutrophils (data not shown), suggesting alteration or loss of this ROS-dependent NETosis pathway in horses.
At the end of the NETosis assays, neutrophils were fixed in the presence of Sytox Green, washed, permeabilized and immunostained to evaluate the presence of SCGBs and two established NET markers, CitH 3 and MPO, in the cells and on the NETs. During fixation, Sytox Green enters the cells and also stains intracellular DNA. Ionophore A23187 stimulation yielded strong and diffuse nuclear staining suggestive of chromatin decondensation and NET release, while untreated cells had typical segmented horseshoe-shaped nuclear morphology ( Figure 5). SCGB 1A1 and 1A1A staining was detected only in SCGB treated cells, consistent with lack of endogenous expression of SCGBs in neutrophils ( Figure 5, top panel, SCGB antibody recognizes an epitope shared by both recombinant proteins). Detection of CitH 3 staining was restricted to A23187 stimulated cells, consistent with ongoing nuclear DNA externalization and histone deimination ( Figure 5, middle panel). MPO staining was detected rarely in neutrophil preparations prior to stimulation, but was prominent on string-like DNA following A23187 treatment ( Figure 5, bottom panel). Importantly, there was a substantial reduction in extracellular DNA filaments (green dye) in A23187-stimulated cells pre-treated with SCGB 1A1 and 1A1A relative to positive control cells. Altogether, immunofluorescence analysis showed inhibition of NETosis by SCGB 1A1 and 1A1A proteins.
SCGB mRNA and protein are reduced in animals with RAO SCGB1A1 and SCGB1A1A transcripts and total SCGB protein concentrations were measured by quantitative real-time PCR of cDNA preparations obtained from bronchial biopsies and by ELISA in BAL fluid, respectively. Previous studies showed that SCGB1A1 was reduced in horses with RAO compared to those without lung disease, but did not assess expression of individual  genes [17]. Here, SCGB expression was compared to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA and 18S ribosomal RNA (RN18S) as internal controls. SCGB1A1 mRNA concentration was significantly lower in animals with RAO compared to controls (p = 0.016, Figure 6A), while expression of SCGB1A1A was minimally changed. Total SCGB concentration in BAL was significantly lower in horses with RAO pre-and postchallenge than in control horses ( Figure 6B).
In vivo NET formation in the airways of horses with RAO NET formation during neutrophilic inflammation in RAO has not been investigated. We determined whether there are NETderived DNA-protein complexes in BAL fluid of horses without lung disease and with RAO using agarose gel electrophoresis. BAL fluid samples were collected from each horse pre-and postexposure to dusty hay (which triggered exacerbated RAO in susceptible animals). After electrophoresis, pre-and post-challenge samples from control horses lacked high molecular weight nucleic acid (HMW) bands in BAL fluid, whereas such bands were present in samples from four of six animals with exacerbated RAO ( Figure 7A). HMW bands in cell free preparations suggested the presence of nucleic acid-protein complexes. These complexes were detected only in samples from animals with exacerbated RAO (Table 1; Figure 7A). To evaluate the nature of these HMW bands, samples were treated with PK alone or in tandem with DNAse and/or RNAse. As shown in Figure 7B, incubation with PK alone detached the nucleic acid-protein complexes, highlighted by a shift in the molecular weight of the band. Disappearance of HMW bands following DNAse, but not RNAse, treatment confirmed that these complexes were made of DNA and proteins. Western blotting showed that the samples that had DNA-protein complexes also contained CitH 3 . Furthermore, the samples that lacked HMW DNA-protein complexes also lacked CitH 3 ( Figure 7C). These data are consistent with the interpretation that RAO BAL samples contained NETs. Notably, NETs were detected only in samples with high BAL neutrophil and total leukocyte concentrations, and low SCGB1A1 and SCGB1A1A expression. These data suggest that during RAO, horse neutrophils release NETs into the airways.

Discussion
SCGB 1A1 is the prototypic member of the secretoglobin family produced by specialized epithelial cells at the mucosal surface of the lungs and uterus. SCGB 1A1 has anti-inflammatory and immunomodulatory properties due to inactivation of PLA2, sequestration of pro-inflammatory cytokines and interference with leukocyte chemotaxis [7,30,31]. Inhibition of PLA2 by SCGB 1A1 limits generation of neutrophil activating arachidonic acid metabolites, and is considered a mechanism of reducing inflammation and limiting neutrophil-induced lung injury in acute respiratory distress syndrome [32]. In addition to indirect effects on neutrophils, direct interaction was suggested by detecting SCGB 1A1 in lung neutrophils of animals with neutrophilic inflammation by immunoelectron microscopy [27]. SCGB 1A1 is depleted in bronchial epithelium and airway fluid of human asthmatics [33,34] and horses with stable RAO, an asthma-like condition characterized by intense neutrophilic inflammation of the airways [17]. It was recently identified that SCGB1A1 genes are triplicated in the equid family, and that two of the genes are differentially expressed and give rise to distinct proteins [18,19]. SCGB 1A1 and 1A1A differ by 13 of 70 amino acids, with 7 of the13 substitutions in the region that forms the hydrophobic pocket [19]. The binding pocket of SCGB 1A1A is smaller than that of SCGB 1A1, and substitutions include amino acids with different biochemical properties. Theoretical isoelectric points (pI) of SCGB 1A1 and A1A1 are 4.90 and 6.48, further suggesting potentially different biological functions. Hence, we here sought to determine whether the different SCGB proteins have unique effects on functional properties of neutrophils and NET formation in a naturally occurring model of inflammatory lung disease.
Incubation of neutrophils with recombinant SCGB 1A1A but not SCGB 1A1 enhanced ROS production and increased phagocytosis. Receptors whereby SCGB 1A1 directly interacts with cells to affect function have not clearly been identified. It was reported that SCGB 1A1 enters renal tubular cells via binding to cubilin, a large peripheral membrane protein, and that subsequently this complex interacts with the endocytic lipocalin receptor megalin [35]. Expression of these receptors by nonepithelial cells has not been identified; hence interaction of SCGB with other molecules on neutrophils appears more likely. The structurally related SCGB 3A2 binds to macrophage scavenger receptor with collagenous structure (MARCO) present on alveolar macrophages, and competes with LPS for binding to MARCO and bacteria [36]. Similar innate pattern recognition receptors are plausible candidate SCGB 1A1 receptors. SCGB 1A1 had been reported to inhibit neutrophil oxidative burst, but studies preceded identification of multiple expressed SCGB1A1 genes in horses [27]. The in vivo significance of functional effects of either protein will further depend on concentration and local conditions such as pH, but in vitro assays suggest specific properties that functionally distinguish between the isoforms. Future studies will need to determine in situ concentration of each protein during active disease.
Phagocytosis was significantly increased by incubation with 250 ng/mL of SCGB 1A1A but not SCGB 1A1. Increased neutrophil phagocytosis of opsonized 1 mm beads suggests that SCGB 1A1A may interact with phagocytic receptors, immunoglobulins or complement products such as C3bi. Intravenous administration of SCGB 1A1 reduced complement deposition and glomerulonephritis in a mouse model, and mice lacking SCGB 1A1 developed IgA nephropathy, suggesting that SCGBs in vivo regulate function of these immune mediators [37,38]. Phagocytosis of inert particulate does not necessarily initiate a subsequent proinflammatory neutrophil response, and may rather reflect removal of apoptotic cells and cell debris [39]. Since in vitro concentrations of SCGB corresponded to estimates of physiologic levels in airway secretions, effects on oxidative burst and phagocytosis should also be further investigated in vivo (38,39).
Numerous reports have identified effects of SCGB 1A1 on cell migration and chemotaxis of leukocytes, fibroblasts, smooth muscle and neoplastic cells [26,31,[40][41][42][43]. In agreement with these observations, our findings showed both SCGB 1A1 and 1A1A strongly reduced IL-8-stimulated chemotaxis of bloodderived neutrophils in a dose-dependent manner. SCGB 1A1A was a more potent inhibitor of cell migration. A postulated mechanism whereby SCGB 1A1 affects chemotaxis is binding to the formyl-met-leu-phe (fMLP) receptor FPR2, which blocks association with bacterial-derived fMLP. SCGBs may also interfere with binding of IL-8 to its CXCR1 or CXCR2 receptor. Since despite much effort receptors for SCGB 1A1 have not been conclusively identified and the mechanisms for chemotaxis inhibition remain elusive. Hall and colleagues [44] reported that BAL fluid from RAO-affected animals enhanced chemotaxis. However, BAL fluid contains many mediators produced by bronchial epithelium or leaked into the airways from vasculature, and such assay does not allow dissection of individual protein effects. SCGBs are highly abundant in secretions of the lower bronchi and bronchioles, and animals with RAO have overall reduced club cells and SCGB production [17], which may facilitate neutrophil chemotaxis to these sites of most intense inflammation. Hence, SCGB-mediated reduction of neutrophil influx is likely a protective anti-inflammatory property of these proteins.
SCGB pre-incubation of neutrophils activated by calcium flux significantly reduced ex vivo NETosis. NETs trap microbes or particulate matter, and in turn enhance their phagocytosis and elimination, but constituents of NETs such as citrullinated histones and extracellular MPO are also immunogenic and cytotoxic [45]. NETs in the equine male reproductive tract were associated with neutrophil activation and impaired fertility [46], and excessive NETs may have deleterious effects in the lung [25]. SCGBs likely contribute to balancing NETosis in the lung, a function to be further explored. NETs have previously been identified in the asthmatic airways of humans but not horses [47]. In humans, both neutrophilic and eosinophilic extracellular traps occur [46], but neutrophilic inflammation characterizes types of human asthma less responsive to therapy, which is similar to equine RAO. Measurement of SCGB1A1 and SCGB1A1A transcripts in bronchial biopsies, and total SCGB in BAL fluid, showed that SCGBs were decreased in animals with RAO, and in particular transcripts of SCGB1A1 were barely detectable in RAO horses. SCGB 1A1 may be more important for modulating neutrophil responses in the airway, but depletion in chronic inflammation abrogates in vivo effects. We identified NETs in BAL samples of horses with acutely exacerbated RAO and severe neutrophilic inflammation, but not in the same horses during disease remission or in control horses exposed to the same stimulus. The in vivo stimuli or significance of NETs are incompletely defined, but a wide range of microbial components and inflammatory mediators such as GM-CSF, ROS, IL-8 and MIP-2 may induce NETosis [25]. Since agents that incite asthmatic responses in horses include LPS and fungal spores [48], and the bronchial epithelium responds with abundant IL-8 production [49], it is not surprising that neutrophils are activated in vivo and form NETs. From data presented here it appears that NETs are present in the most severe the inflammatory states, but the precise role of NETs in RAO remains to be determined.
RAO is a severe neutrophilic lung inflammatory disease induced by air containing dust, fungal spores and bacterial components. Bronchial epithelial club cells and their SCGB products are markedly reduced in affected animals. We here show that SCGB 1A1 and 1A1A have differential effects on neutrophils, and that duplication and evolution of the SCGB1A1 gene may have resulted in differential effects on neutrophil responses. A global understanding of the molecular targets and pathways affected by SCGB proteins may help development of pharmacological interventions to counteract pathogenic neutrophilic inflammation, and will be the subject of future studies.