CC16 augmentation reduces exaggerated COPD-like disease in Cc16-deficient mice

Low Club Cell 16 kDa protein (CC16) plasma levels are linked to accelerated lung function decline in patients with chronic obstructive pulmonary disease (COPD). Cigarette smoke–exposed (CS-exposed) Cc16–/– mice have exaggerated COPD-like disease associated with increased NF-κB activation in their lungs. It is unclear whether CC16 augmentation can reverse exaggerated COPD in CS-exposed Cc16–/– mice and whether increased NF-κB activation contributes to the exaggerated COPD in CS-exposed Cc16–/– lungs. CS-exposed WT and Cc16–/– mice were treated with recombinant human CC16 (rhCC16) or an NF-κB inhibitor versus vehicle beginning at the midpoint of the exposures. COPD-like disease and NF-κB activation were measured in the lungs. RhCC16 limited the progression of emphysema, small airway fibrosis, and chronic bronchitis-like disease in WT and Cc16–/– mice partly by reducing pulmonary inflammation (reducing myeloid leukocytes and/or increasing regulatory T and/or B cells) and alveolar septal cell apoptosis, reducing NF-κB activation in CS-exposed Cc16–/– lungs, and rescuing the reduced Foxj1 expression in CS-exposed Cc16–/– lungs. IMD0354 treatment reduced exaggerated lung inflammation and rescued the reduced Foxj1 expression in CS-exposed Cc16–/– mice. RhCC16 treatment reduced NF-κB activation in luciferase reporter A549 cells. Thus, rhCC16 treatment limits COPD progression in CS-exposed Cc16–/– mice partly by inhibiting NF-κB activation and represents a potentially novel therapeutic approach for COPD.

The 3D images were reconstructed using an ordered-subsets expectation-maximization algorithm (10), and consisted of 80 x 80 (transaxial) x 200 (axial) isotropic voxels each having 0.4 x 0.4 x 0.4 mm 3 dimensions. The reconstructed image volumes taken at baseline, and 1 h and 3 h later were analyzed using ImageJ (https://imagej.nih.gov/ij/), creating a new image from "sum slices" z-projection over 80 coronal images. The regions of interest were drawn to exclude the trachea and gastrointestinal tract. Retention at each time point was subtracted from 1.00 and multiplied by 100% to obtain the percent mucociliary clearance (9,10).
Lymphocyte subsets in enzymatic lung digests: WT and Cc16 -/mice were exposed to CS for 4 weeks to induce an acute pulmonary inflammatory response in the lungs or to air for 8 weeks as a control. CSexposed mice were treated thrice weekly with rhCC16 protein solution or vehicle during an additional 4 weeks of CS exposures (8 weeks of CS exposure in total). The right lungs were digested in a 1 mg/mL collagenase D (Sigma Aldrich, St. Louis, MO) solution at 37 o C for 30 min, as described previously (11). Samples were passed through a 70 μm strainer and the resulting single-cell suspension was washed twice with cold PBS. Cell viability was quantified with trypan blue dye, and was >90% in all experiments. Surface staining for markers of leukocytes and leukocyte subsets was performed for 20 min at 4°C in PBS containing 2% fetal calf serum. Intracellular staining for Foxp3 was performed using the Foxp3 Staining Buffer Set (eBioscience, San Diego, CA). T and B lymphocyte panels and antibodies used are shown in Table E4. Flow cytometry was performed using a BD LSRFortessa Cell Analyzer (San Jose, CA) and analyzed using FlowJo software (Tree Star, Inc., Ashland, OR).
Quantification of the expression of mediators of inflammation, mucins, senescence markers, and activation of the Wnt-β catenin pathway in whole lung samples: WT and Cc16 -/mice were exposed to CS for 4 weeks (to induce an acute pulmonary inflammatory response in the lungs) or to air for 8 weeks as a control. CS-exposed mice were treated thrice weekly with rhCC16 protein solution or vehicle during an additional 4 weeks of CS exposures (8 weeks of CS exposure in total). RNA was extracted from murine lungs using a SurePrep TrueTotal RNA Purification Kit (Fisher Scientific, Fair Lawn, NJ). RNA (1 μg) was reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, Carlsbad, CA). Real-time RT-PCR was performed to quantify the expression of Ccl-2, Ccl-3, Ccl-5, Il-6, Tnf-α, Il-10, Tgf-β, Foxj1, Muc5ac, Muc5b, Muc1, Mmp-9, Mmp-12, p16, p21, Sirtuin, Wisp, and Tcf-7, using Ppia as the housekeeping gene (see Table E5 for primer sequences), and an AriaMx Real time PCR device (Agilent technologies, Santa Clara, CA). Primers and SYBR green dye were obtained from ThermoFisher Scientific (Waltham, MA). The results were expressed as fold change relative to expression of the gene of interest in samples from mice exposed to air. Alveolar septal cell apoptosis: Sections of lungs from mice were immunostained for active caspase-3. Lung sections were deparaffinized, and antigen retrieval was performed by heating the slides in a microwave in 10 mM citrate buffer (pH 6.0) for 10 min. The sections were blocked overnight at 4°C in PBS containing 1% albumin and 5% normal goat serum. The sections were incubated at 4°C overnight rabbit anti-murine cleaved caspase-3 IgG at a 1:50 dilution (Cell Signaling, Danvers, MA) or non-immune rabbit IgG (Agilent, Carpinteria, CA) applied at the same concentration. After washing the lung sections with PBS, the sections were incubated for 1 h at 37°C with goat anti-murine F(ab')2-conjugated to Alexa 488 (Invitrogen, Carlsbad, CA). After washing the slides twice with PBS, the sections were incubated with Sudan Black buffer for 25 min at room temperature. After washing the slides twice with PBS, nuclei were counterstained with DAPI mounting gel (Abcam, Cambridge, MA). Images were captured using a digital camera, and the number of active-caspase-3-positive cells was quantified. Six 200 X magnification images per mouse were randomly acquired using a Leica microscope and a digital camera. Images were processed using ImageJ software (https://imagej.nih.gov/ij/). Images of the DAPI (stained blue) and active caspase-3-positively-stained cells (stained green) were merged, and active caspase-3 positively-stained alveolar septal cells were counted in ~5,000 alveolar septal cells per mouse (identified by their DAPIstained nuclei). The results were expressed as % of alveolar septal cells that were positively-stained for active caspase-3. Alveolar septal cell proliferation: Lung sections from WT and Cc16 -/mice were immunostained for Ki-67, a marker of cellular proliferation. Lung sections were deparaffinized, and antigen retrieval was performed by heating the slides in a microwave in 10 mM citrate buffer (pH 6.0) for 10 min. The sections were blocked overnight at 4°C in PBS containing 1% albumin and 5% normal goat serum. After washing the slides with PBS, a rabbit polyclonal anti-murine Ki67 IgG (Abcam, Cambridge, MA) was added to the slides at a 1:100 dilution and incubated overnight at 4°C. A non-immune rabbit IgG (Invitrogen, Carlsbad, CA) was applied to other slides at the same concentration as the immune antibody. After washing the slides twice in PBS, goat anti-murine F(ab')2-conjugated to Alexa 488 (Invitrogen, Carlsbad, CA) at 1:100 dilution was added to the slides, and the slides were incubated at 37°C for 1 h. After washing the slides twice with PBS, the sections were incubated with Sudan Black buffer for 25 min at room temperature. After washing the slides twice with PBS, nuclei were counterstained with DAPI mounting gel (Abcam, Cambridge, MA). Six 200 X magnification images per mouse were randomly acquired using a Leica microscope and a digital camera. Images were processed using ImageJ software (https://imagej.nih.gov/ij/). Images of the DAPI (stained blue) and Ki-67 positively-stained cells (stained green) were merged, and Ki67 positively-stained alveolar septal cells were counted in ~ 5,000 alveolar septal cells per mouse identified by their DAPI-stained nuclei. The results were expressed as % of alveolar septal cells that were positively-stained for Ki-67.

Airway epithelial Muc5ac staining:
To identify the Muc5ac-positive cells in bronchial epithelial cells, sections of lung from WT and Cc16 -/mice were immunostained for Muc5ac. Lung sections were deparaffinized, and antigen retrieval was performed by heating the slides in a microwave in 10 mM citrate buffer (pH 6.0) for 10 min. The sections were blocked overnight at 4°C in PBS containing 1% albumin and 5% normal rabbit serum. The sections were incubated at 4°C overnight with a murine IgG to Muc5ac (diluted 1:50, ThermoFisher Scientific, Waltham, MA), and non-immune murine IgG applied at the same concentration. After washing the lung sections with PBS, the sections were incubated for 1 h at 37°C with F(ab')2-rabbit anti-mouse conjugated with Alexa 488 (Invitrogen, Carlsbad, CA) for Muc5ac. After washing the slides twice with PBS, the sections were incubated with Sudan Black buffer for 25 min at room temperature. After washing the slides twice with PBS, nuclei were counterstained with DAPI (4′,6diamidino-2-phenylindol) mounting gel (Abcam, Cambridge, MA). Images were captured using a digital camera, the number of Muc5ac-positive cells was counted and normalized for the bronchial epithelial cell areas using MetaMorph software (Molecular Devices, San Jose, CA).

Foxj1 staining:
To identify the Foxj1-positive cells in bronchial epithelial cells, sections of lung from WT and Cc16 -/mice were immunostained for Foxj1. Lung sections were deparaffinized, and antigen retrieval was performed by heating the slides in a microwave in 10 mM citrate buffer (pH 6.0) for 10 min. The sections were blocked overnight at 4°C in PBS containing 1% albumin and 5% normal goat serum. The sections were incubated at 4°C overnight with a murine IgG to Foxj1 (diluted 1:200, ThermoFisher Scientific, Waltham, MA), and non-immune murine IgG was applied at the same concentration. After washing the lung sections with PBS, the sections were incubated for 1 h at 37°C with F(ab')2-goat antimouse conjugated with Alexa 488 (Invitrogen, Carlsbad, CA). After washing the slides twice with PBS, the sections were incubated with Sudan Black buffer for 25 min at room temperature. After washing the slides twice with PBS, nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindol) mounting gel (Abcam, Cambridge, MA). Images were captured using a digital camera, the number of Foxj1-positive cells was counted and normalized for the bronchial epithelial cell areas using MetaMorph software (Molecular Devices, San Jose, CA).

Statistical Analyses:
Statistical analysis was performed using SigmaPlot™ software (Systat, San Jose, CA). The Shapiro-Wilk test was used to determine whether the data were normally distributed, and Brown-Forsythe test was used to assess equal variance. Data were analyzed using one-way ANOVAs for continuous data, followed by post-hoc testing with 2-sided Student's t-tests (normally-distributed data) or Mann-Whitney U tests (not normally-distributed data). Normally-distributed data are presented as mean ± SEM, and not normally-distributed data are presented as box plots showing medians and 25th and 75th percentiles and error bars showing 10th and 90th percentiles. ANOVA for repetitive measures was used to analyze changes in body weight from baseline values. P < 0.05 was considered statistically significant. Table E1. Gender differences in COPD-like disease features in cigarette smoke-exposed WT and Cc16 -/mice treated with rhCC16 protein solution. § WT and Cc16 -/mice were exposed to air (5-6 mice/group) or CS (12-14 mice/group) for 24 weeks, and rhCC16 protein solution (75 µg of rhCC16; 6-7 mice/group) or vehicle (6 mice/group) was delivered thrice weekly by the intranasal route to CS-exposed mice for the last 12 weeks of the CS exposures. Sections of inflated and fixed lungs were stained with either Gill's stain and alveolar chord lengths as a measure of airspace size were quantified. ‡ WT and Cc16 -/mice were exposed to air or CS for 24 weeks, and rhCC16 protein solution (75 µg of rhCC16) or vehicle was delivered thrice weekly by the intranasal route to CS-exposed mice for the last 12 weeks of the CS exposures. Sections of inflated and fixed lungs were stained with Masson Trichrome stain (which stains extracellular matrix [ECM] proteins blue) and the thickness of the layer of ECM deposited around the small airways was quantified.
*Data were analyzed using one-way ANOVAs followed by pair-wise testing with Student's t tests.  Table 2. Respiratory mechanics Parameters in WT and Cc16 -/exposed to 6 months of Air or Cigarette Smoke (CS), treated with rhCC16 or vehicle.
Respiratory mechanics were evaluated using a Flexivent device. Data are expressed in medians and interquartile ranges (IQR) for data that are not normally distributed.
Ұ Data were analyzed with One-way ANOVAs followed by pair-wise comparisons using Mann Whitney U tests.
WT and Cc16 -/mice were exposed to air (7-8 mice/group) or CS for 24 weeks (20-28 mice/group). CSexposed mice were treated with rhCC16 protein solution (75 µg of rhCC16; 10-15 mice/group) or vehicle (13-15 mice/group) thrice weekly for the last 12 weeks of the exposures and bronchoalveolar lavage (BAL) was performed. BAL total leukocytes, macrophages, PMNs and lymphocytes were counted, and expressed as mean ± SD (standard deviation). § All results are expressed as x 10 4 cells.
*Data were analyzed using one-way ANOVAs followed by pair-wise testing with Student's t tests. The table shows the isotype of the antibodies and fluorochromes that were used to quantify lymphocyte subsets in enzymatic digests of lungs from WT and Cc16 -/mice. The table shows the sequences of the primers that were used to perform quantitative real-time RT-PCR assays to quantify the expression of chemokines (Ccl-2, Ccl-3, and Ccl-5), mediators of inflammation (Tnfα, Il-6, Il-10, and Tgf-β), mucins (Muc1, Muc5ac, and Muc5b), ciliary motility (Foxj1), Wnt-beta catenin