Pulmonary Macrophage Transplantation Therapy

Characterization of macrophages before PMT

Bone marrow derived macrophages (BMDMs) from WT mice had morphology and phenotypic markers (F4/80⁺, CD11b^Hi, CD11c⁺, CD14⁺, CD16/32⁺, CD64⁺, CD68⁺, CD115⁺, CD131⁺, SiglecF^Lo, MerTK⁺, MHC class II⁺, Ly6G⁻, CD3⁻, CD19⁻) of macrophages (Extended Data Fig. 2a-c) and contained <0.0125% lineage negative (Lin⁻) Sca1⁺cKit⁺ (LSK) cells. Clonogenic analysis indicated <0.005% CFU-GM and no BFU-E, or CFU-GEMM progenitors (Extended Data Fig.2d-e). Functional evaluation²³ showed they could clear surfactant (Extended Data Fig. 2f-g). These results demonstrated the cells used for PMT were highly purified, mature macrophages capable of surfactant clearance.

Efficacy of PMT of WT macrophages

To determine the therapeutic potential of PMT, KO mice received WT (Csf2rb^+/+) BMDMs by PMT once (Fig. 1a). One year later, PMT-derived CD131⁺ BAL cells were present (Fig. 1b), alveolar macrophages expressed Csf2rb (Extended Data Fig. 3a), BAL was markedly improved with respect to opacification (Fig. 1c), sediment (Fig. 1c), and microscopic cytopathology (Extended Data Fig. 3b). Importantly, PMT nearly completely resolved the abnormal pulmonary histopathology (Fig. 1d, Extended Data Fig. 3c). Measurement of BAL turbidity and SP-D content (Fig. 1e), which reflect the extent of surfactant accumulation across the entire lung surface, confirmed the improvement in hPAP. BAL fluid biomarkers of hPAP were also improved (Fig. 1f). The effects of PMT were evident early as demonstrated by detection of CD131⁺ alveolar macrophages with Csf2rb mRNA and protein (not shown), reduced BAL opacification and cytopathology (not shown), BAL turbidity (Fig. 1e), SP-D (Fig. 1e), and BAL fluid biomarkers (Fig. 1f) two months after PMT, and reduced lung histopathology 4 months after PMT (not shown). In contrast, PMT of KO BMDMs had no effect on BAL turbidity, SP-D content, or BAL fluid biomarkers (not shown) demonstrating the importance of GM-CSF receptors on transplanted macrophages to the therapeutic effects.

To evaluate the effects of PMT on the alveolar macrophage population, we measured cellular biomarkers after PMT. Results showed alveolar macrophages from PMT-treated KO mice had increased mRNA for PU.1, PPARγ, and ABCG1, improvement was significant by two months, and the effects persisted one year after PMT (Fig. 1g).

Since KO mice develop polycythemia, a secondary consequence of hypoxemiain chronic lung diseases²⁴, the effects of PMT on this systemic clinical manifestation were evaluated. Importantly, PMT corrected polycythemia in KO mice (Fig. 1h).

Finally, the effects of PMT on hPAP-associated mortality were evaluated by comparing the survival of PMT-treated and untreated KO mice. PMT increased the lifespan of KO mice by 107 days, from (median [interquartile range]) 555 [507-592] to 662 [604-692] days (Fig. 1i). In separate studies of KO mice surviving to 617 [604-631] days after PMT of WT BMDMs (561 [548-575] days after PMT), CD131⁺ alveolar macrophages were still present and BAL turbidity remained low compared to untreated KO mice that survived to 631 [631-631] days (OD₆₀₀0.75±0.17 versus 2.63±0.44; n=8, 4, respectively; P<0.001). However, such long-term evaluation of laboratory abnormalities are obfuscated by reduced survival of untreated KO mice.

These results demonstrate PMT had a highly efficacious and durable therapeutic effect on the primary pulmonary and secondary systemic manifestations of hPAP in KO mice.

Macrophage engraftment efficiency

The effects of cell dose (0.5, 1, 2 and 4 million) and repeated administration (one versus four monthly transplantations) on PMT efficacy were evaluated (Extended Data Table 2 and 3, respectively). Neither significantly affected efficacy in the range evaluated and one dose of 2 million cells was used for PMT in the remaining studies.

To determine if WT macrophages had a survival advantage over KO macrophages, we measured GM-CSF bioactivity in BAL fluid and found it was detectable in KO but not WT mice (Extended Data Fig. 1h). WT macrophages had increased survival/proliferation compared to KO macrophages in vitro (Fig. 2a) and accumulated to greater numbers after PMT in KO mice than in WT mice (Fig. 2b and Extended Data Fig. 3d). PMT of WT Lys-M^GFP knock-in mouse²⁵ BMDMs into KO mice followed by Ki67 immunostaining revealed PMT-derived cells replicated in vivo (Extended Data Fig. 3e-g). The percentage of Ki67⁺ PMT-derived alveolar macrophages was 32.2 ± 6.05% one month after PMT and declined to 11.29 ± 2.2% by one year (Fig. 2c) similar to baseline Ki67⁺ immunostaining of alveolar macrophages in age-matched, normal WT mice (Extended Data Fig. 3f). To further define this survival advantage, we evaluated the engraftment kinetics after one PMT of WT BMDMs in KO mice. CD131⁺ cells increased steadily from zero to 69.0±2.5% of BAL cells (Fig. 2d) synchronous with a smooth decline in pulmonary GM-CSF to near normal (Fig. 2e). Similarly, BAL turbidity declined with the increase in CD131⁺ alveolar macrophages (Fig. 2f). One year after PMT, CD131⁺ cells were present (Fig. 1b), Csf2rb protein was detectable in alveolar macrophages (Extended Data Fig. 3a), and Csf2rb mRNA in BAL cells from PMT-treated KO mice was only slightly less than in WT and undetectable in untreated KO BAL cells (Fig. 2g). Importantly, numbers of CD131⁺ alveolar macrophages in PMT-treated KO and untreated WT mice were similar one year after PMT (Fig. 2h). These results demonstrate that WT macrophages 1) had a selective survival advantage over KO macrophages, and that after PMT into KO mice they 2) proliferated in vivo at a rate that slowed over time synchronous with reduction in pulmonary GM-CSF, 3) replaced dysfunctional KO alveolar macrophages, and 4) resulted in numbers of CD131⁺, GM-CSF-responsive alveolar macrophages similar to WT mice.

Macrophage characterization after PMT

The fate of macrophages following PMT was evaluated to determine their spatial distribution, phenotype, and gene expression profile. Intra-pulmonary localization was evaluated one year after PMT of WT Lys-M^GFP BMDMs by fluorescence microscopy to identify CD68⁺GFP⁺ (i.e., PMT-derived macrophages), which revealed 88.9 ± 0.87% were intra-alveolar and 11.1 ± 0.87% were interstitial (Extended Data Fig. 3h). GFP-immunohistochemical staining was done to eliminate potential interference from autofluorescence and confirmed these results; 90.5 ± 1.1% PMT-derived macrophages were intra-alveolar and 9.4 ± 1.1% were interstitial (Fig. 3a-b and Extended Data Fig. 3i). Localization was done in similarly-treated mice by flow cytometry to detect GFP⁺ cells two months (not shown) or one year after PMT (Extended Data Fig. 4a-b) and by PCR amplification of Lys-M^GFP transgene-specific DNA (Extended Data Fig. 4c), all of which showed PMT-derived cells were present in the lungs but not detected in blood, bone marrow, or spleen. One year after PMT of CD45.1⁺ WT BMDMs into CD45.2⁺ KO mice, flow cytometric detection of CD45.1⁺ cells confirmed these findings (Extended Data Fig. 4e-g). Results show that the transplanted macrophages remained in the lungs, primarily within the intra-alveolar space.

The effects of the lung milieu on the phenotype of transplanted macrophages were evaluated by measuring cell-surface markers. One year after PMT of WT Lys-M^GFP BMDMs into KO mice, PMT-derived alveolar macrophages comprised 68.7 ± 6.5% of BAL cells and had converted from CD11b^HiSiglecF^Lo to CD11b^LoSiglecF^Hi, similar to the phenotype of WT alveolar macrophages and different from recipient KO mice (CD11b^HiSiglecF^Lo) (Fig. 3c-d). Similarly, one year after PMT of WT CD45.1⁺ BMDMs into CD45.2⁺ KO mice, CD45.1⁺ alveolar macrophages comprised 63.6±12.1% of BAL cells and had undergone the same phenotypic conversion (Extended Data Fig. 4h).

To determine the effects on gene expression, genome-wide expression profiling was performed on alveolar macrophages from KO mice one year after PMT of WT BMDMs and compared to results for untreated, age-matched WT or KO mice. Unsupervised analysis indicated marked co-clustering between PMT-treated KO and WT mice while KO mice clustered separately (Fig. 4). Expression of genes regulated by GM-CSF was reduced in KO mice and restored by PMT (Fig. 4, Extended Data Fig. 5a). Of 776 genes for which expression was disrupted in KO mice, PMT normalized expression of 600 including 80% of genes up-regulated and 76% of genes down-regulated in KO compared to WT mice (Extended Data Fig. 5b). Supervised Gene Ontology (GO) and detailed KEGG pathway analysis revealed that genes in multiple pathways involved in lipid metabolism, cellular proliferation, apoptosis, and host defense were coordinately down-regulated in KO mice, and normalized by PMT (Extended Data Fig. 5c,d). Results for multiple genes important in lipid metabolism (Abcg1, Nr1h3, Olr1, Lepr, Fabp1, Lipf, Abca1, Apoe, Apoc2, Pla2g7) were validated using separate samples (Extended Data Fig. 5e).

Efficacy of gene therapy by PMT

Since PMT in humans would likely employ autologous, gene-corrected HSPC-derived macrophages, we evaluated PMT of KO macrophages derived from Lin⁻Sca-1⁺c-Kit⁺ (LSK) cells after lentiviral vector (LV)-mediated Csf2rb cDNA expression (Fig. 5a). Csf2rb gene-corrected (GM-R-LV-transduced) or sham-treated (GFP-LV-transduced) KO and non-transduced WT LSK-derived cells all had macrophage morphology, expressed CD68 (Extended Data Fig. 6a) and were F4/80⁺CD11b^HiCD11c⁺ (not shown). In contrast, only WT and GM-R-LV-transduced KO cells were CD131⁺ and only LV transduced cells were GFP⁺ (Extended Data Fig. 6a). GM-R-LV restored GM-CSF signaling in KO macrophages (Fig. 5b and Extended Data Fig. 6b). Two months after PMT into KO mice, GM-CSF receptor-β was detected on alveolar macrophages only from mice receiving gene-corrected KO or WT macrophages (Extended Data Fig. 6c). The efficacy using gene-corrected KO or WT cells was equivalent as demonstrated by a similar degree in improvement in BAL appearance (Extended Data Fig. 6d), BAL turbidity, SP-D, and biomarkers of hPAP (Fig. 5c-d). Further, gene-corrected BMDMs localized to the lung (Extended Data Fig. 4d and Fig. 6e) and underwent phenotypic conversion to CD11b^Lo (Extended Data Fig. 6f). The long-term efficacy of gene-corrected macrophages one year after PMT was demonstrated by marked reduction in BAL turbidity, SP-D, and BAL fluid biomarkers of hPAP (Fig. 5c-d). These results demonstrate that PMT of gene-corrected macrophages had a therapeutic effect on hPAP in KO mice equivalent to that of WT macrophages and was durable, lasting at least one year.

Safety of PMT Therapy in KO Mice

PMT was well-tolerated and without adverse effects. One year after PMT, there were no hematologic abnormalities (Extended Data Table 4), cellular inflammation or pulmonary fibrosis in mice receiving PMT of WT (Fig. 1d) or gene-corrected macrophages (not shown). KO mice had trivial elevations of IL-6 and TNFα in BAL that were reduced by PMT of WT macrophages (Extended Data Table 4). These data identify no safety concerns for PMT therapy of hPAP in KO mice.

Mice

All mice were bred, housed, and studied in the Cincinnati Children’s Research Foundation Vivarium using protocols approved by the Institutional Animal Care and Use Committee. Csf2rb gene-targeted (Csf2rb^−/− or KO) mice¹³, and mice expressing EGFP knocked-into the lysozyme M gene (Lys-M^GFP mice)²⁵ were all generated previously and backcrossed onto the C57Bl/6 background. C57Bl/6 mice (referred to as wild type or WT mice) were purchased from Charles River. B6.SJL-Ptprc^aPepc^b/Boy J (CD45.1⁺) mice were from Jackson Laboratory.

Lung histology and immunohistochemistry

Animals were sacrificed by intraperitoneal pentobarbital administration and exsanguination by aortic transection. The trachea was exposed by a vertical midline skin incision, cannulated through a small transverse incision in its ventral surface away from the thoracic inlet, inflated with fixative (PBS, pH = 7.4, containing 4% paraformaldehyde) under a hydrostatic head of 25cm and ligated with suture while retracting the cannula to seal the lung under pressure. The sternum and diaphragm were transected sagittally, retracted laterally, and the lungs and heart separated from the chest wall by blunt dissection to avoid puncturing the mediastinal pleura and removed from the chest. The intact tissue block containing the heart, lungs, and ligated trachea was submerged in fixative and kept at 4°C for 24 hours. After fixation, the lung lobes were divided, removed from the tissue block, cut into ~2 mm thick slices along the long axis, washed in cold PBS, dehydrated, embedded in paraffin, and 5 μm thick sections were cut and stained with hematoxylin and eosin (H&E), periodic acid-Schiff reagent (PAS), or Masson’s trichrome as previously described⁴⁰. Immunostaining for surfactant protein B (SP-B) was done by incubating slides with rabbit anti-SP-B polyclonal antibody (diluted 1:500, Seven Hills Bioreagents, Cincinnati, OH) and Vectastain ABC anti–rabbit immunohistochemical horseradish peroxidase kit (Vector Labs, Inc., Burlingame, CA) and counterstaining with hematoxylin as described⁴¹. To prepare frozen lung sections, the lungs were inflation fixed in-situ as described above and then the heart and lungs were removed enbloc and cryoprotected by sequential immersion in PBS containing increasing sucrose concentrations (10%, 15% and 20%; 8-12 hours, 4 °C, at each concentration). The lungs were then embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA), frozen and stored at -80 °C until use. Serial 6 μm sections were prepared for immunostaining or evaluation of GFP⁺ cells. Lung sections and sedimented lung cells were examined by light microscopy using a Zeiss Axioplan 2 microscope (Zeiss) equipped with AxioVision software (Zeiss).

Collection, handling, and evaluation of bronchoalveolar lavage fluid and cells

Epithelial lining fluid and non-adherent cells were collected from lung surface of mice by bronchoalveolar lavage (BAL) as described⁴² and processed immediately. Briefly, five 1 ml aliquots were instilled and immediately recovered per mouse and combined resulting in a BAL recovery of 93.9 ± 1.2% per mouse (BAL recovery data for 10 mice evaluated randomly). The photographs of fresh BAL specimens and the specimen after allowing sediment to be formed by overnight incubation at 4 °C were taken. The turbidity of BAL was determined as described⁴¹. Briefly, after gently mixing to ensure a homogeneous suspension of BAL, a 250 μl aliquot was diluted into 750 μl PBS and the optical density was measured at a wavelength of 600 nm and multiplying the result by the dilution factor. The total number of BAL cells recovered from each mouse was determined by counting cells in an aliquot of known volume using a hemocytometer and multiplying the result by the total volume of BAL and dividing by the volume of the aliquot used for counting. BAL cytology was evaluated in aliquots (~50,000 cells) after sedimentation (Cytospin, Shandon, Inc.; 500 rpm, 7 min, room temperature) onto glass slides and staining with DiffQuick, PAS, or oil-red-O (all from Fisher Scientific) as described⁴¹. The cell differential was determined by microscopic examination of DiffQuick stained cells and the total number of alveolar macrophages per mouse was determined by multiplying the percentage of alveolar macrophages in BAL cells by the total number of BAL cells recovered⁴³. BAL fluid and cells were separated by low speed centrifugation (285 × G, 10 min, room temp) and stored at -80 °C until use (BAL fluid) or immediately evaluated (referred to as BAL cells) or used to isolate alveolar macrophages (see below). Primary alveolar macrophages were purified by brief adherence of BAL cells to plastic as described¹⁸. Viability was evaluated by Trypan blue exclusion and was ≥ 95%.

ELISA

The concentration of surfactant protein D (SP-D) in BAL fluid was measured by enzyme-linked immunosorbent assay (ELISA) as we described⁴¹. The concentration of several cytokines (GM-CSF, M-CSF, MCP-1, IL-1β, IL-6, TNFα) in BAL fluid and erythropoietin in serum was measured by ELISA (Mouse Quantikine Kits, R&D Systems) as described¹.

Quantitative RT-PCR

Total RNA was isolated from alveolar macrophages using TRIzol Reagent (Life Technologies, Carlsbad, CA) and then used to purify mRNA using RNeasy (Qiagen, Valencia, CA), both as directed by the manufacturers. Purified mRNA was used to synthesize cDNA using the Invitrogen SuperScript III First-Strand Synthesis System (Life Technologies). Standard quantitative RT-PCR (qRT-PCR) was performed as previously described¹ on an Applied Biosystems 7300 Real-Time PCR System (Life Technologies) to measure transcript abundance using TaqMan® oligonucleotide primer sets (all from Life Technologies) (Extended Data Table 1). Expression of target genes was normalized to the expression of 18s RNA. Data for each gene were shown as the fold change of the mean of results for wild type mice.

Bone marrow derived macrophages (BMDMs)

Bone marrow cells were obtained from 6 – 8 week-old WT, KO, or Lys-M^GFP mice by isolating and flushing tibias and femurs with DMEM (Life Technologies) containing 10% heat-inactivated FBS, 50 U/ml penicillin, and 50 μg/ml streptomycin. After red blood cells were removed with BD Pharm Lyse (BD Biosciences), mononuclear cells were isolated by centrifugation on Ficoll-Paque (GE Healthcare) at room temperature for 30 minutes, washed, re-suspended in DMEM containing 10% heat-inactivated FBS, 50 U/ml penicillin, 50 μg/ml streptomycin, 10ng/ml GM-CSF and 5 ng/ml M-CSF (both from R&D Systems), seeded into plastic dishes (Falcon) at a density of ~27 × 10⁶ cells/10 cm dish (1/mouse) and cultured overnight at 37°C in a humidified environment containing 5% CO₂. The next day, non- or weakly-adherent cells were recovered, transferred to a new dish and cultured under the conditions just described to permit differentiation and expansion of macrophages; firmly adherent cells were discarded. After 2 days the culture medium was changed and after 5 days from seeding, adherent bone marrow-derived macrophage were gently washed with PBS, harvested by brief exposure to trypsin-EDTA (Life Technologies), washed, and used for experiments. The cell purity was high as indicated by the percentage of CD68⁺ and F4/80⁺ cells (96.6 ± 0.3%, 95.4 ± 1.3%, respectively, not shown).

Some experiments used lineage-negative (Lin⁻) c-kit⁺Sca-1⁺ (LSK cells) which were obtained from mouse bone marrow as described⁴⁴. Briefly, bone marrow from 6-8 week-old WT or KO mice was collected as above and lineage depleted with biotinylated lineage antibodies CD5 (53-7.3), CD8a (53-6.7), CD45R/B220 (RA3-6B2), CD11b (M1/70), Gr-1 (RB6-8C5), and TER-119 (TER-119) (BD Bioscience), and magnetic beads (Dynabeads sheep anti-rat IgG) (Life-Technologies). After removing lineage-positive cells, the remaining cells were stained with 7-ADD, FITC-Streptavidin (BD Biosciences) and antibodies to Sca-1 (D7) and c-kit (2B8) (BD Biosciences). Then, Lin⁻c-kit⁺Sca-1⁺7-ADD⁻ cells were isolated by cell sorting on a FACSAria (BD Biosciences) and used immediately in experiments. Cell morphology was confirmed by Diff-Quick Staining of sedimented cells (Cytospin, Shandon) and viability was measured by Trypan blue exclusion as described¹⁸ and found to be ≥95%. In some experiments, cells were immunostained for CD68 (FA-11) (AbD Serotec), counter stained with DAPI as described⁴¹, and examined by light microscopy using a Zeiss Axioplan 2 microscope (Zeiss) equipped with AxioVision software (Zeiss).

Colony forming cell (CFC) assay

BMDMs or Lin⁻bone marrow cells were evaluated for the presence of hematopoietic progenitors capable of forming colonies in semisolid medium in response to cytokine stimulation as previously described.⁴⁵ Briefly, fresh Lin⁻ bone marrow cells or BMDMs after induced differentiation into macrophages for five days were seeded into standard mouse methylcellulose media supplemented with insulin, transferrin, SCF, IL-3, IL-6, and erythropoietin (HSC007, R&D Systems, Minneapolis, MN). After seven days in culture, colonies of ≥50 cells were visible and were examined morphologically using whole plate stack images acquired using an AXIO-Z1 microscope and AXIO-vision software (Zeiss, Jena, Germany) to identify and enumerate burst-forming erythroid progenitors (BFU-E), colony-forming myeloid progenitors (CFU-GM), and the multi-potential progenitors (CFU-GEMM).

Surfactant Clearance Assay

BMDMs were evaluated functionally to demonstrate their ability to clear human surfactant as we previously reported⁴¹. Briefly, BMDMs from either WT or KO mice were seeded into 12-well plates (4 × 10⁵ cells/well) in DMEM, 10% FBS, 10ng/ml GM-CSF, 5 ng/ml M-CSF. Human surfactant recovered by lavage of a patient with PAP was added to the media and cells were incubated for 24 hours to permit surfactant uptake into cells and then washed to remove extracellular surfactant. Cells were incubated for 24 hours to permit macrophages to clear internalized surfactant. Cells collected before, immediately after surfactant exposure or 24 hours after the completion of surfactant exposure were sedimented onto slides by cytocentrifugation (Shandon), stained with oil-red-O, and counterstained with hematoxylin. Oil-red-O staining was evaluated in ≥10 random 20x microscopic fields for each sample as described⁴¹.

Pulmonary macrophage transplantation (PMT)

BMDMs or LSK cell-derived macrophages were administered directly into the lungs of 8 week-old mice using a relatively non-invasive endotracheal instillation method described previously⁴⁶. Briefly, mice received light anesthesia by isoflurane inhalation and were suspended on a flat board by a rubber band across the upper incisors and placed in a semi-recumbent (45-degree) position with the ventral surface and rostrum facing upwards. Using a curved blade Kelly forceps, the tongue was gently and partially retracted rostrally, and 50μl of PBS containing the macrophages to be administered was placed in the back of the oral cavity using a micropipette. The PBS and cells were inhaled into the lungs by subsequent respiratory efforts under direct visualization. Mice were then observed while recovering from anesthesia to ensure continued retention of the administered fluid and cells and then returned to their cages for routine care and handling. Because the efficacy of PMT at a dose of two million macrophages was optimal, this dose given as one administration was used throughout the study except where noted (Extended Data Table 2-3). Age-matched mice were used in all experiments to control for the degree of lung disease severity.

Flow cytometry

BAL cells were purified by centrifugation on Percoll to remove surfactant and debris ³¹. BAL cells or BMDMs were immunostained to detect CD115 (AFS98), F4/80 (BM8) (eBioscience), CD3 (145-2C11), CD11b (M1/70), CD11c HL3), CD16/32 (2.4G2), CD19 (1D3), CD64 (X54-5/7.1.1), CD131 (JORO50), Ly6G (1A8), CD45.1 (A20), CD45.2 (104), MHC class II (I-A/I-E) (M5/114.15.2), SiglecF (E50-2440) (BD Biosciences), CD14 (Sa14-2) (BioLegend), CD68 (FA-11) (AbD Serotec), and MerTK (108928) (R&D Systems), as previously described¹, evaluated by flow cytometry using a FACSCaliber or FACSCanto flow cytometers (both from BD Biosciences, San Jose, CA), and the results were analyzed using CellQuest, FACSDiva (BD Biosciences), or FlowJo software (Tree Star). For intracellular staining of CD68, Leucoperm (AbDSerotec) was used as directed by the manufacturer.

Quantification of CD131⁺ alveolar macrophages

The percentage of BAL cells expressing the GM-CSF receptor β subunit was determined by immunostaining. Briefly, aliquots of BAL cells were sedimented onto glass slides and incubated (10 minutes, room temperature) in fixative (PBS containing 4% paraformaldehyde), washed with PBS and incubated (4°C, overnight) with anti-mouse GM-CSF receptor-β (CD131) antibody (sc-678) (Santa Cruz) diluted 1:400 in PBST (PBS containing 2.5% (w/v) Triton X-100 and 5% (v/v) goat serum). After incubation, slides were rinsed 5 times in PBST and incubated (room temperature, 1 hour) with the secondary detection antibody (Alexa Fluor 594-conjugated, anti-rabbit IgG (Life Technologies)) and counter stained with 4’,6-diamidino-2-phenylindoledihydrochloride (DAPI) (Vector Labs, Burlington, CA). Cells were examined using a Zeiss Axioplan 2 microscope (Zeiss) equipped with AxioVision software (Zeiss). The percentage of CD131⁺ BAL cells was determined by first counting the CD131⁺ and DAPI⁺ cells in 5 (or more) random 20x microscopic fields for each BAL sample. Then, the number of CD131⁺ cells in each field was divided by the number of DAPI⁺ cells in the same field and results for all fields examined were averaged and multiplied by 100. The total number of CD131⁺ cells per mouse was calculated by multiplying the percentage of CD131⁺ cells by the total number of BAL cells recovered from each mouse.

STAT5 phosphorylation index assay

GM-CSF bioactivity in BAL fluid and GM-CSF receptor function in transduced or WT macrophages was evaluated by measuring GM-CSF-stimulated phosphorylation of STAT5 in BMDMs or LSK cell-derived macrophages using anti-phospho STAT5 antibody (47/Stat5(pY694)) (BD Biosciences) by flow cytometry as previously reported¹. The STAT5 phosphorylation index (STAT5-PI) was calculated as the mean fluorescence intensity of phosphorylated STAT5 staining in GM-CSF-stimulated cells minus that of non-stimulated cells, divided by that of non-stimulated cells, and multiplied by 100. In experiments to quantify GM-CSF bioactivity, WT BMDMs were incubated in BAL fluid containing anti-GM-CSF (22E9, eBioscience) or isotype control antibody (10 μg/ml) for 30 minutes and then evaluated.

Evaluation of macrophage proliferation

Western blotting

Detection of GM-CSF receptor β and actin by Western blotting was done as previously described¹ with the following modifications. Briefly, primary alveolar macrophages (0.5 × 10⁶/condition) or cultured BMDMs (1 × 10⁶/condition, after culture with either GM-CSF or M-CSF at 10 ng/ml for 30 min) were collected by low speed centrifugation (285 × G, 4 °C, 10 min) and the pellets incubated on ice for 30 min in 200 μl of lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 % (v/v) nonidet p-40, 0.5 % (w/v) sodium deoxycholate, 0.1 % (w/v) sodium dodecyl sulfate (SDS), 0.004 % (w/v) sodium azide) containing 2 % (v/v) proteinase inhibitor cocktail (phenyl-methyl-sulfonyl-fluoride and sodium orthovanadate; Santa Cruz). Insoluble debris was removed by centrifugation 10,000 × G, 4 °C, 15 min and the supernatant transferred to a clean polypropylene tube. An equal volume of Laemmli sample loading buffer (Bio-Rad, CA) was added and the tubes were capped tightly, vortexed briefly, boiled for 5 min, and separated by electrophoresis on SDS- polyacrylamide gradient (4-12%) gels (Invitrogen) under reducing conditions. Separated proteins were transferred to PVDF membranes by electro-blotting, incubated in blotting solution (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 % (w/v) Non-fat dry milk (Kroger, Cincinnati, OH), 0.1 % (v/v) Tween 20; 4°C, overnight) to block non-specific binding. Diluted primary detection antibody (see below) was added and the membranes were incubated for 2 hours at room temperature and then washed in TBST (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1 % [v/v] Tween 20). Membranes were then incubated with the secondary HRP-conjugated detection antibody in blotting solution for 1 hour at room temperature and then washed as above and then incubated with ECL-Plus (GE Healthcare) as directed by the manufacturer. Anti-mouse GM-CSF receptor β antibody (sc-678) (Santa Cruz) diluted 1:500 and anti-actin (sc-1616) (Santa Cruz) diluted 1:1000 were used for primary antibodies.

Hematologic analysis

Blood was obtained from the superior vena cava from mice and 20 μl was used to measure complete blood counts on a fully automated Hemavet 850 (Drew Scientific). Data for the precision and linearity of measurements made with the Hemavet850 can be found online at www.drew-scientific.com/product_hemavet850.htm.

Microarray analysis

Alveolar macrophages were obtained from age-matched mice (3 per condition) and analyzed individually as follows. Total RNA was isolated as described above and microarray analysis was performed using the Mouse Gene 1.0 ST Array (Affymetrix, Santa Clara, CA) in the CCHMC Affymetrix Core using standard procedures as described⁴⁷. Data (available at www.ncbi.nlm.nih.gov/geo/GSE60528) were analyzed using the Affymetrix package in the R statistical programming language (Bioconductor; www.bioconductor.org). Probes were corrected for background using the Microarray Analysis Suite algorithm, quantile normalized, and probe sets were summarized using the average difference of perfect matches only. Differential expression tests were performed using significance analysis of microarrays⁴⁸ with Benjamini-Hochberg correction for multiple testing ⁴⁹. Significant gene lists were selected with a Δ that constrained the false discovery rate to less than 10%. Cluster dendrogram was generated from unsupervised hierarchical clustering analysis of microarray data from probes for all 28,853 genes represented on the chip (Spearman correlation; 3 mice/group). In Venn diagrams, numbers of genes whose expression was altered in alveolar macrophages from KO compared to WT mice (WT→KO) or PMT-treated compared to untreated KO mice (KO→KO+PMT) were shown. Only genes with statistically significant changes (false detection rate < 10%) of at least two-fold were marked as increased (up arrows) or decreased (down arrows). The numbers of genes for which expression was disrupted in KO mice and normalized by PMT (or unchanged in both comparisons) is shown in the overlap regions. In gene ontology analysis, data show the coordinate increases (red) or decreases (blue) in expression of genes in all gene sets significant at or below a false detection rate of 10% calculated by the Gene Set Test with correction for multiple testing.

Lentiviral vectors, LSK-cell transduction, and differentiation and expansion of transduced macrophages

Gene transfer vectors were constructed using routine methods⁴⁴ from the vector backbone of (Ery-GFP), a human immunodeficiency virus-based, self-inactivating (SIN) lentiviral vector (LV) harboring a 398-bp U3 deletion eliminating the strong viral promoter/enhancer element⁵⁰. GM-R-LV contains a chimeric transgene comprised of the human elongation factor 1-alpha (ELF-1α) promoter (a 1189 bp fragment containing intron 1 ending 20 bp upstream of the ATG codonisolated from the pEF-BOS plasmid⁵¹) followed by the mouse Csf2rb cDNA (nucleotides -80 to 2691, GenBank accession no. M34397.1) located 3’ of the lentiviral central poly-purine tract and followed by an internal ribosome binding site (IRES) and then an enhanced green fluorescent protein (GFP) transgene (Figure 5A). GFP-LV is a LV of similar design except that the Csf2rb and IRES were omitted and the GFP transgene is driven from the ELF1α promoter (Figure 5A). Both vectors contain a viral splice donor site, packaging sequence, splice acceptor site, and central polypurine tract (cPPT) 5’ of the ELF1α promoter and a woodchuck hepatitis post-transcriptional regulatory element (WPRE) (nucleotides 1093 to 1684; GenBank accession no. J04514)⁵² located 3’ of the GFP stop codon as described⁵⁰. LVs were produced by transient transfection as vesicular stomatitis virus-G (VSVG) virions, concentrated, and tittered as described⁴⁴. KO LSK cells were isolated, transduced and expanded as described⁵³ except that transductions were done at a multiplicity of infection (MOI) of 20 for two 12 hour periods, IL-11 was omitted, GM-CSF (10 ng/ml) and M-CSF (5 ng/ml) were included, SCF and Flt-3 ligand were sequentially reduced (50, 1, 0 ng/ml), and IL-3 was present early.

Transduction, expansion, and differentiation of LSK cells into gene-corrected macrophages was done by adjusting the cytokine ‘cocktail’ mixture to optimize the culture conditions for each of four sequential stages, which included: 1) LSK transduction –murine SCF (R&D) 50ng/ml, mIL-3 (PeproTech) 10 ng/ml, hFlt3-L (PeproTech) 50 ng/ml and GM-CSF (R&D) 10 ng/ml), culture time – two 12 hour periods; 2) progenitor expansion - mSCF 50 ng/ml, hFlt3-L 50 ng/ml and GM-CSF 10 ng/ml, culture time - 4 days; 3) macrophage lineage commitment - mSCF 1 ng/ml, hFlt3-L 1 ng/ml, GM-CSF 10 ng/ml, M-CSF (R&D) 5 ng/ml, culture time - 3 days; and 4) macrophage differentiation - GM-CSF 10 ng/ml and M-CSF 5 ng/ml, culture - 4 days. StemSpan (STEMCELL Technologies) containing 2% FBS, 1% penicillin/streptomycin, 10 mM dNTP, and low density lipoprotein was used as the culture medium for the LSK transduction and DMEM with 10% FBS, 50 U/ml penicillin and 50 ug/ml streptomycin was used for all other stages. Phenotype markers (F4/80, CD11b, CD11c) were analyzed by flow cytometry at each stage to monitor macrophage differentiation. Only adherent macrophages at the end of this procedure were used for PMT.

Localization of PMT-derived cells after transplantation

Several approaches were utilized to identify and localize PMT-derived cells within the lung parenchyma and in different organs.

Statistical Analysis

Numeric data were evaluated for normality and variance using the Shapiro-Wilk and Levene median tests, respectively, and presented as mean± SEM (parametric data) or median and interquartile range (nonparametric data). Statistical comparisons were made with Student t test, one-way analysis of variance, or Kruskal-Wallis rank-sum test as appropriate; post-hoc pairwise multiple comparison procedures were done using the Student-Newman-Keuls or Dunn’s method as appropriate. P-values of ≤ 0.05 were considered to indicate statistical significance. Based on use of BAL turbidity (the primary outcome variable for efficacy) measured two months after PMT of WT BMDMs into KO mice and compared to age-matched, untreated KO mice, 6 mice per group had a power 0.8 to detect a difference of 1.4 O.D. 600 nm using a two-tailed Student’s t-test and a P value of 0.05. All studies used male and female mice by randomly assigning mice housed in the same cage to separate experimental groups but without formal randomization or blinding. Results from all mice were included in the final analysis without exclusion. Analyses, including Kaplan-Meyer survival analysis, were performed with SigmaPlot, Version 12.5 (Systat Software, San Jose, CA). All experiments were repeated at least twice, with similar results.