Pulmonary transplantation of alpha-1 antitrypsin
(AAT)-transgenic macrophages provides a source of functional human
AAT in vivo

Cultivation of cell lines

The mAM cell line was provided by Takuji Suzuki and Bruce Trapnell from Cincinatti Childrens Hospital Medical Center [51]. Cells were cultured in DMEM medium (Gibco, Life Technologies, Paisley, UK) supplemented with 10% FBS superior (Millipore, Billerica, MA, USA), 100 U/ml penicillin/streptomycin (P/S, Thermo Fisher Scientific, Waltham, MA, USA), and 20 mM HEPES (Thermo Fisher Scientific). Cell line was regularly tested for mycoplasma contamination. For lentiviral transduction, 4 μg/ml protamine sulfate was added to the culture medium. U937 cells were cultured in RPMI1640 (Gibco) supplemented with 10% FBS superior (Millipore) and 100 U/ml P/S. Both cell lines were cultured under standard conditions at 37 °C and 5% CO2 and passaged every 3–4 days. Each cell line was only transduced once and samples for the assays were taken at different time points during continuous culture of the lines.

Production and titration of lentiviral vectors

VSV-G pseudotyped third-generation SIN LV were produced in HEK293T cell line and titrated on SC-1 cells as previously described [51]. Briefly, HEK293T cells were transfected with the pcDNA3.GP.4xCTE (encoding HIV-1 gag/pol), pRSV-Rev, pMD.G (encoding the envelope VSV-G), and the desired lentiviral transgene plasmid. After 24 and 48 h the supernatant was filtered through 0.22 µm filter and centrifuged overnight at 10,000×g, 4 °C. The pellet was resuspended in StemSpan medium (Stem Cell Technologies, Vancouver, Canada) and kept at −80 °C. The average titers of the vector preparations obtained for the different constructs were 1.58 ± 0.67  × 108 for Cbx-EFS-GFP, 1.62 ± 1.13 × 108 for Cbx-EFS-AAT, 2.03 ± 2.2 × 108 for Cbx-EF1α-AAT, and 0.4 ± 0.3 × 108 for CAG-AAT.

Vector copy number (VCN) quantification

The VCN was quantified as previously described [52]. Briefly, genomic DNA was isolated using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. The VCN was determined using the Taq-Man based qRT-PCR on StepOne Plus thermocycler (Applied Biosystems, Foster City, CA, USA) targeting the wPRE sequence of the LV vector and the PTPB2 as a house-keeping gene.

Murine lin cell isolation, transduction, and cultivation

Lineage negative (lin) cells were isolated from the bone marrow of C57BL/6J, C57BL/6JHanZtm and B6.SJL-Ptprca-Pep3b/BoyJZtm (Ly5.1; CD45.1) mice using the lineage cell depletion kit mouse (Miltenyi Biotech, Bergisch-Gladbach, Germany) according to the manufacturer’s instructions. Lin cells were cultured in StemSpan medium (Stem Cell Technologies) supplemented with 100 U/ml P/S, 2 mM L-glutamine (Thermo Fisher Scientific), 10 ng/ml SCF, 20 ng/ml TPO, 20 ng/ml IGF-2, and 10 ng/ml FGF-1 (STIF, all Peprotech, Rocky Hill, NJ, USA). The next day, lin cells were transduced with LV vectors at MOI 10 on RetroNectin-coated plates (Takara Bio, Inc., Shiga, Japan) as recommended by the manufacturer. Four days after the transduction, the STIF medium was supplemented with 10 ng/ml IL-3. Cells were sorted for GFP+ cells and transferred to RPMI1640 (Gibco) supplemented with 10% FCS, 100 U/ml P/S, and 30% of conditioned medium from the M-CSF producing L929 cell line. The differentiation medium was supplemented with IL-3 for the first 2 days. Then, 10–14 days after sorting, differentiated MΦ were used for in vitro assays and in vivo experiments. The number of cells that was generated with a single transduction was varying and thus, not all assays could be performed with one cell batch.

Cord blood CD34+ cell isolation, transduction, and cultivation

Umbilical cord blood from donors who gave informed consent was provided from Hannover Medical School. Experiments were approved by the local ethic committee (approval numbers: 1303-2012). CD34+ cells were isolated as previously described [51]. Briefly, after gradient centrifugation, stem and progenitor cells were enriched by magnetic sorting using the CD34 MicroBead Kit (Miltenyi). Cells were cultured in StemSpan (Stem Cell Technologies) supplemented with human cytokines (SCF, Flt3, TPO, all Peprotech), and transduced at the MOI 15 on RetroNectin-coated plates (Takara) as recommended by the manufacturer. Sorted GFP+ cells were differentiated toward MΦ in RPMI1640 containing 10% FCS, human M-CSF, GM-CSF and IL-3 (all Peprotech).

AAT sandwich ELISA

To prepare the supernatants, cells were seeded at defined densities (1 × 105 for mAM cells, 5 × 105 for primary murine MΦ) on 12-well tissue culture plates in 0.5 ml of respective cell culture medium. Supernatants were collected after 24 h (mAM and MΦ), 48 h, and 72 h (mAM) and stored at −80 °C until the day of analysis.

High binding 96-well plates (Sarstedt, Nuembrecht, Germany) were coated with 100 μl of 5 μg/ml rabbit anti-human AAT monoclonal antibody (Cat. no. A0409; Sigma-Aldrich) in carbonate-bicarbonate buffer (Medicago, Quebec City, Canada) o/n at 4 °C. Wells were washed four times with 200 μl PBS + 0.05% Tween20 (AppliChem, Darmstadt, Germany) and blocked for 1 h with 1% BSA (Carl ROTH, Karlsruhe, Germany) in PBS at RT. Standard AAT (Calbiochem, San Diego, CA, USA) was prepared in a range of 3–800 ng/ml by serial dilutions in blocking solution. All samples were run in the assay in duplicates. Then, 100 µl of standard and test samples was added to the wells and incubated for 2 h at RT. Wells were washed four times and the secondary, HRP-conjugated mouse anti-human AAT antibody was added (1:5000 in blocking solution, Novus Biologicals, Littleton, CO, USA, Cat. no. NBP1-05147H) for 1 h at RT. Wells were washed four times again and 100 μl of TMB solution (Abcam, Cambridge, UK) was added. To stop the reaction, 50 μl of 2 N H2SO4 (Carl ROTH) was added and the result was immediately analyzed at 405 nm on a Model 680 microplate reader (Bio-Rad, Hercules, CA, USA).

Phagocytosis assay

One day before the assay, cells were seeded at a density of 1 × 105 on a 12-well tissue culture plate. The following day, 10 μl of pHrodo red E. coli bio-particles (Invitrogen, Carlsbad, CA, USA) were added and the cells were incubated for 1.5 h at 37 °C. For each condition, one well without pHrodo particles served as a negative control. After the incubation time, fluorescent pictures were taken and the cells were collected for flow cytometry analysis.

GM-CSF clearance assay

Cells were seeded at a density of 1 × 105 on a 24-well tissue culture plate. On the next day, the medium was changed to 1 ml X-Vivo 15 (Lonza, Basel, Switzerland) containing 1 ng/ml GM-CSF and a 100 μl aliquot was taken as a time point 0 h. The next samples were taken at 24, 48, and 72 h and stored at −20 °C until analysis. The level of GM-CSF was assessed with the Ready-Set-Go!® ELISA Kit (eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Supernatant from a well without cells was used as a negative control. The result was normalized to the time point 0 h.

Western blot

For the preparation of mAM cell and murine MΦ culture supernatants, 2.5 × 105 or 5 × 105 cells were seeded on 12-well tissue culture plates. The next day, the medium was changed to 0.5 ml without FCS (DMEM with 100 U/ml P/S for mAM cells or RPMI with 100 U/ml P/S and 50 ng/ml murine M-CSF for primary MΦ). After 24 h, the supernatant was collected and stored at −80 °C until further analysis. Cellular proteins were isolated in RIPA buffer (Sigma-Aldrich) containing proteinase inhibitor (cOmplete™ Protease Inhibitor cocktail; Sigma-Aldrich). The protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

For western blot analysis, 35 μl of the supernatant or 30 μg of cell lysate proteins was loaded onto 12% acrylamide gels. After blotting the proteins to PVDF membranes, they were incubated with the primary anti-AAT (Invitrogen, Cat. no. MA5-15521) or anti-vinculin (Sigma-Aldrich, Cat. no. V9131) antibodies in 5% milk o/n at 4 °C. After washing, membranes were incubated with the secondary anti-mouse HRP-conjugated antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 45 min at RT. The signal was detected using the SuperSignalTM West Pico or Femto Chemiluminescent Substrate (Thermo Fisher Scientific) according to the manufacturer’s instruction on a ChemiDoc XRS+ (Bio-Rad).

Elastase inhibition assay

mAM cells were seeded at a density of 4 × 105 on a 6-well tissue culture plate. The next day, 2 ml of medium without FBS and phenol red was added (DMEM FluoroBrite + 100 U/ml P/S + 2 mM L-glutamine). For the preparation of the primary MΦ supernatant, 1 × 106 cells were seeded on 6-well tissue culture plates. The next day, 1.5 ml of medium without FBS was added (DMEM FluoroBrite + 50 ng/ml muM-CSF + 100 U/ml P/S + 2mM L-glutamine). After 48 h, the supernatant was collected and concentrated around ten times with 10 kDa Amicon® Ultra 2 ml Centrifugal Filters (Merck, Darmstadt, Germany) according to the manufacturer’s instructions and kept at −80 °C until analysis.

Ten-microliter supernatant concentrate or the AAT protein (Zemaira from CSL Behring, King of Prussia, PA, USA) was incubated in 96-well plates in duplicates with 5 μl of 20 μg/ml PPE (Sigma-Aldrich) and 35 μl of 0.1 M, Tris buffer (pH 8.0) at 37 °C for 20 min. Subsequently, 225 μl Tris buffer and 25 μl of 1 mg/ml PPE substrate (N-Succinyl-Ala-Ala-Ala-p-nitroanilide, Sigma-Aldrich) were added. Blank (without PPE) and an uninhibited reaction (without AAT) were run as controls in concentrated cell culture medium. The OD405 was measured on an InfiniteM200 plate reader (Tecan, Maennedorf, Switzerland) at 37 °C every 30 s for 10 min.

For the determination of the elastase activity, the OD405 value of the blank sample was subtracted, and a slope of the reaction was calculated. Subsequently, the following equation was used to calculate the percentage of elastase inhibition:

$$\% \;{{elsastase}}\;{{inhibition}} = \frac{{{\mathrm{slope}}\;({\mathrm{uninhibited}}\;{\mathrm{reaction}}) – {\mathrm{slope}}\;({\mathrm{test}}\;{\mathrm{sample}})}}{{{\mathrm{slope}}\;{\mathrm{uninhibited}}\;{\mathrm{reaction}}}} \ast 100\% $$

Elastase-AAT complex formation

Ten-microliter supernatant from each condition was incubated with equal volumes of PBS or elastase (Sigma-Aldrich) at a final concentration of 5 ng/ml for 1 h at 37 °C. The reaction was terminated by adding equal volumes of 2X SDS sample buffer and samples were incubated at 95 °C for 5 min. Sample mixtures were shortly spun down and 20 μl were loaded and separated by SDS-PAGE on 10% gels prior to the transfer to a PVDF membrane. Membranes were blocked for 1 h with TBS + 0.01% tween containing 5% BSA (Millipore) followed by overnight incubation at 4 °C with the primary polyclonal rabbit anti-human AAT antibody (Cat. no. A0012, DAKO, Glostrup, Denmark). AAT and elastase complexes were visualized with HRP-conjugated polyclonal anti-rabbit antibody (DAKO) and enhanced by ECL western blotting substrate (Bio-Rad). Images were taken using Chemidoc Touch imaging system (Bio-Rad).

STS-induced apoptosis

Supernatants from culture of murine MΦ were collected and kept at −80 °C. mAM mock cells were seeded at a density of 2 × 104 on 24-well tissue culture plates. The next day, MΦ supernatants were thawed and warmed to 37 °C. The mAM culture medium was aspirated and 330 µl of the respective mock, CAG-AAT or Cbx-EF1α-AAT supernatant medium was added at different dilutions (1–1:8 dilution). As a control, human AAT protein (Zemaira, CSL Behring) at different concentrations was added to the mock supernatant. Cells were incubated at 37 °C for 2 h. Subsequently, STS was added at a final concentration of 25 µM. One well of cells with the mock supernatant without AAT served as a positive control for the apoptosis, a well without AAT and STS as a negative control. After 24 h, cells were trypsinized, stained with 1 µl PI (eBioscience) and analyzed on a CytoFLEX S (Beckman Coulter, Brea, CA, USA). The analysis was performed using the FlowJo v.10 software (Becton Dickinson, Franklin Lakes, NJ, USA).

Flow cytometry

Trypsinized cells or BALF and lung samples were resuspended in 100 μl FACS buffer (PBS with 2% FCS and 1% EDTA), stained with 1 μl antibody and incubated for 45 min at 4 °C (anti-mouse antibodies: CD11c APC (Cat. no. 17-0114-81), CD45.1 PE-Cy7 (Cat. no. 25-0453-81), CD163 PE-Cy7 (Cat. no. 12-1631-82), F4/80 APC (Cat. no. 17-4801-80), CD11b PE (Cat. no. 12-0112-82)—all from eBioscience, Siglec-F from BDBioscience (Cat. no. 552126); anti-human antibodies: CD11b APC from eBioscience (Cat. no. 17-0118-41), CD14 PE (Cat. no. 12-0116-41), and CD163 APC from Invitrogen (Cat. no. 17-1639-41)). After washing with PBS, samples were measured on a CytoFLEX S (Beckman Coulter). The analysis was performed using the FlowJo v.10 software (Becton Dickinson).

Cytospin

A total of 20,000–40,000 cells in 150 μl PBS were spun onto a glass slide at 700×g for 10 min in a Cytofuge (Medite, Burgdorf, Germany). After allowing the slides to air-dry the slides were incubated in the May–Grünwald solution for 5 min, washed in deionized water, and incubated in Giemsa solution (1:20 in water). After washing the slides with water, they were left to dry and mounted in ROTI™ mounting medium (Carl ROTH).

Clonogenic assay

After sorting of the lin cells, 1.5 × 103 cells/ml were cultured in mouse methylcellulose complete media (R&D systems, Minneapolis, MN, USA) on 8.8 cm2 cell culture dishes with a 2 mm grid (Thermo Fisher Scientific) and kept in a wet chamber in a cell culture incubator. The number of colonies composed of more than 50 cells was counted 10–14 days after seeding.

qRT-PCR

Total RNA was extracted from cells using TRIsure (Bioline, London, UK) according to the manufacturer’s instructions. Then, 1  μg of RNA was treated with the DNAse I (Thermo Fisher Scientific) and reverse transcribed using Oligo (dT) primers with the RevertAid H Minus Reverse Transcriptase kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. For the qRT-PCR reaction, 1 μl of the cDNA was mixed with 7.5 μl of SybrGreen PCR master mix (Applied Biosystems), 1 μl of the primer mix for human SERPINA1 (Integrated DNA Technologies, Coralville, IA, USA) or murine Actb (Qiagen, Hilden, Germany) and 5.5 μl H2O. PCR reaction was run in duplicates on a StepOne Plus thermocycler (Applied Biosystems) for 40 cycles at 95 °C for 15 s and 60 °C for 1 min.

Protein preparation, in-gel digest, and mass spectrometry (MS)

Proteins from cell culture supernatants of CAG-AAT mAM cells were precipitated using the the TCA-NLS method [52]. Briefly, 10% N-Lauroylsarcosine was added to the supernatant to a final concentration of 0.1% and mixed. In the next step, 100% TCA was added to a final concentration of 7.5%, mixed and precipitated on ice in dark for at least 2 h. The solution was centrifuged at 10,000×g for 15 min at 4 °C and the pellet was then washed with 1 ml tetrahydrofurane. After centrifuging the sample at the maximum speed for 10 min at 4 °C, the pellet was dried on ice for 30 min and resuspended in sample buffer (30 mM Tris-HCl (pH 8.0), 7 M urea (Carl ROTH), 2 M thiourea (Sigma-Aldrich), and 4% CHAPS (Carl ROTH)).

Samples were separated by SDS-PAGE on 12% gels and stained with InstantBlue protein stain (Novus Biologicals). Bands at the expected molecular weight of AAT (~52 kDa) were excised and cut into small pieces. The gel pieces were then subjected to in-gel digestion with first PNGaseF for liberation of N-glycans and second with trypsin for generation of peptides. Briefly, the gel pieces were dehydrated with acetonitrile (ACN, Merck), proteins in the gel were reduced with 10 mM ditihothreitol (Sigma-Aldrich) in 100 mM ammonium bicarbonate buffer (AmBic) and subsequently carbamidomethylated with 100 mM iodoacetamide (Sigma-Aldrich) in 100 mM AmBic. After dehydrating with ACN, rehydrated with 50 mM AmBic, and dehydrating with ACN, peptide-N-glycosidase F (PNGaseF from Elizabethkingia meningoseptica; BioReagent grade, Sigma-Aldrich) was added at a final concentration of 0.1 U/µl and incubated o/n at 37 °C. The supernatant containing the N-glycans was dried in a vacuum centrifuge. Subsequently, the same gel pieces were dehydrated with ACN, rehydrated with 50 mM AmBic containing 20 ng/µl sequencing-grade trypsin (Promega, Madison, WI, USA) and incubated for 16 h at 37 °C. The peptides were extracted using ACN and dried in a vacuum centrifuge. In the next step, dried peptides were dissolved in 15 µl 2% ACN and 0.1% formic acid and the supernatant was subjected to LC-MS/MS analysis. Reverse phase chromatography using acetonitrile as an eluent was performed on a nanoACQUITY UPLC (Waters, Milford, MA, USA) device equipped with an analytical column (Waters, BEH130 C18, 100 µm × 100 mm, 1.7 µm particle size) coupled online to an ESI Q-TOF Ultima (Waters). Spectra were recorded in positive reflection mode and peptides were automatically subjected to fragmentation. ProteinLynxTM Global Server software V2.1 (Waters) was used to identify proteins by searching against human database. Carbamidomethylation was set as fixed modification and oxidation of methionine as variable modification. Up to one missed cleavage was allowed. Peptide tolerance was set to 100 ppm and fragment tolerance to 0.1 Da and the validation filter was selected in the ProteinLynxTM Global Server software.

Multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection (xCGE-LIF)

Extracted N-glycans were labeled with 8-aminopyrene-1,3,6-trisulfonic acid (APTS, Sigma-Aldrich) and excess APTS was removed by hydrophilic interaction liquid chromatography. Labeled glycans were separated and monitored by xCGE-LIF applying a remodeld ABI PRISM® 3100-Avant Genetic Analyser (Thermo Fisher Scientific). xCGE-LIF data were assessed and processed using GeneMapperTM Software v.3.7 [53]. We determined the relative intensity of all N-glycan peaks by calculating the relative signal height of an individual peak in relation to the sum of signal intensities of all peaks.

In vivo experiments

C57BL/6J, C57BL/6JHanZtm, and B6.SJL-Ptprca-Pep3b/BoyJZtm (CD45.1) mice were used for isolation of bone marrow lin cells. B6;129P2-Csf2rb2tm1Mur (Csf2rb–/–) mice at the age of 15–21 weeks were used as recipients of the MΦ transplantation. Mice were obtained from and housed in the central animal facility of Hannover Medical School where they were kept in specific-pathogen-free or individually ventilated cage conditions. All in vivo experiments were approved by the Lower Saxony State animal welfare authorities.

Terminally differentiated MΦ derived from CD45.1 lin cells were harvested and 2.5–4 × 106 MΦ were resuspended in 50 μl PBS. Csf2rb–/– mice were anesthetized, a 22G Introcan Safety® IV catheter (B. Braun, Melsungen, Germany) without the sharp end of the needle was inserted into the trachea and the cell solution was applied into the catheter. The delivery of the cells into the lung was confirmed by the quick inhalation of the solution by the mouse.

Analysis of in vivo experiments

Two months after PMT, mice were sacrificed with an overdose of Ketamine (10 mg per mouse) and Rompun (1 mg per mouse) by intraperitoneal injection.

To obtain the BALF, the trachea was cannulated with a 20 G Introcan Safety® IV catheter (B. Braun). The left lung was pinched off and the right lung lobes were flushed twice with PBS (600 + 500 μl–three times in and out). BALF was centrifuged to pellet the cells and the supernatant was aliquoted and stored at –80 °C for further analysis. The cells were distributed into 5 ml round-bottom tubes for staining. The left lung lobe was filled with the Tissue-Tek OCT compound solution (Sakura Finetek, Torrance, CA, USA), and kept at –80 °C until further processing.

Blood was collected via cannulation of the inferior vena cava and left at room temperature for 20 min to clot. After 10 min centrifugation at 1800×g, 4 °C, the serum phase was collected, aliquoted, and stored at –80 °C.

Immunofluorescence analysis of lung slices

Left lung lobes frozen after filling them with Tissue-Tek OCT compound solution were cryosectioned into 6 µm slices and fixed and permeabilized in ice-cold acetone for 10 min. Dried slides were rehydrated in PBS for 10 min and subsequently blocked with the 10% swine serum in PBS for 30 min at room temperature. After removing the blocking solution, staining with the APC-conjugated CD45.1 antibody (eBioscience, Cat. no. 17-0453-82) was performed at dilution of 1:100 in the blocking solution and incubated o/n at 4 °C in a wet chamber. Following washing, nuclei were stained with DAPI (1:10000). The slides were covered in the mounting medium (DAKO). Pictures of the lung sections were taken with a fluorescent microscope Leica DM6000B (Leica Microsystems).

Urea quantification in serum and BALF

To calculate the AAT concentration in ELF, the dilution of the ELF with PBS was determined by measuring the urea concentration in serum and BALF with the QuantiChrom™ Urea Assay Kit (BioAssay Systems, Hayward, CA, USA) following the manufacturer’s instructions. The fold difference in the concentration in serum and BALF represents the dilution factor.

Statistics

GraphPad Prism version 8 (Graphpad Software, San Diego, CA, USA) was used to perform one-way or two-way ANOVA. If not stated otherwise, data are represented as mean plus/minus standard deviation.

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