Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Pulmonary macrophage transplantation therapy

Abstract

Bone-marrow transplantation is an effective cell therapy but requires myeloablation, which increases infection risk and mortality. Recent lineage-tracing studies documenting that resident macrophage populations self-maintain independently of haematological progenitors prompted us to consider organ-targeted, cell-specific therapy. Here, using granulocyte–macrophage colony-stimulating factor (GM-CSF) receptor-β-deficient (Csf2rb−/−) mice that develop a myeloid cell disorder identical to hereditary pulmonary alveolar proteinosis (hPAP) in children with CSF2RA or CSF2RB mutations, we show that pulmonary macrophage transplantation (PMT) of either wild-type or Csf2rb-gene-corrected macrophages without myeloablation was safe and well-tolerated and that one administration corrected the lung disease, secondary systemic manifestations and normalized disease-related biomarkers, and prevented disease-specific mortality. PMT-derived alveolar macrophages persisted for at least one year as did therapeutic effects. Our findings identify mechanisms regulating alveolar macrophage population size in health and disease, indicate that GM-CSF is required for phenotypic determination of alveolar macrophages, and support translation of PMT as the first specific therapy for children with hPAP.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Therapeutic efficacy of PMT in Csf2rb−/− mice.
Figure 2: Pharmacokinetics and pharmacodynamics of PMT in Csf2rb−/− mice.
Figure 3: Localization and phenotype of transplanted macrophages.
Figure 4: Microarray analysis of alveolar macrophages 1 year after PMT.
Figure 5: Effects of PMT of gene-corrected macrophages on hPAP severity and biomarkers.
Figure 6

Accession codes

Accessions

Gene Expression Omnibus

Data deposits

Microarray data are available at Gene Expression Omnibus under accession number GSE60528.

References

  1. 1

    Suzuki, T. et al. Familial pulmonary alveolar proteinosis caused by mutations in CSF2RA. J. Exp. Med. 205, 2703–2710 (2008)

    PubMed  PubMed Central  Google Scholar 

  2. 2

    Martinez-Moczygemba, M. et al. Pulmonary alveolar proteinosis caused by deletion of the GM-CSFRα gene in the X chromosome pseudoautosomal region 1. J. Exp. Med. 205, 2711–2716 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Suzuki, T. et al. Hereditary pulmonary alveolar proteinosis: pathogenesis, presentation, diagnosis, and therapy. Am. J. Respir. Crit. Care Med. 182, 1292–1304 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Tanaka, T. et al. Adult-onset hereditary pulmonary alveolar proteinosis caused by a single-base deletion in CSF2RB. J. Med. Genet. 48, 205–209 (2011)

    PubMed  Google Scholar 

  5. 5

    Suzuki, T. et al. Hereditary pulmonary alveolar proteinosis caused by recessive CSF2RB mutations. Eur. Respir. J. 37, 201–204 (2011)

    CAS  PubMed  Google Scholar 

  6. 6

    Whitsett, J. A., Wert, S. E. & Weaver, T. E. Alveolar surfactant homeostasis and the pathogenesis of pulmonary disease. Annu. Rev. Med. 61, 105–119 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Hawgood, S. & Poulain, F. R. The pulmonary collectins and surfactant metabolism. Annu. Rev. Physiol. 63, 495–519 (2001)

    CAS  PubMed  Google Scholar 

  8. 8

    Ikegami, M. et al. Surfactant metabolism in transgenic mice after granulocyte macrophage-colony stimulating factor ablation. Am. J. Physiol. 270, L650–L658 (1996)

    CAS  PubMed  Google Scholar 

  9. 9

    Kitamura, T. et al. Idiopathic pulmonary alveolar proteinosis as an autoimmune disease with neutralizing antibody against granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 190, 875–880 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Trapnell, B. C., Whitsett, J. A. & Nakata, K. Pulmonary alveolar proteinosis. N. Engl. J. Med. 349, 2527–2539 (2003)

    CAS  PubMed  Google Scholar 

  11. 11

    Stanley, E. et al. Granulocyte/macrophage colony-stimulating factor-deficient mice show no major perturbation of hematopoiesis but develop a characteristic pulmonary pathology. Proc. Natl Acad. Sci. USA 91, 5592–5596 (1994)

    ADS  CAS  PubMed  Google Scholar 

  12. 12

    Dranoff, G. et al. Involvement of granulocyte-macrophage colony-stimulating factor in pulmonary homeostasis. Science 264, 713–716 (1994)

    ADS  CAS  PubMed  Google Scholar 

  13. 13

    Robb, L. et al. Hematopoietic and lung abnormalities in mice with a null mutation of the common beta subunit of the receptors for granulocyte-macrophage colony-stimulating factor and interleukins 3 and 5. Proc. Natl Acad. Sci. USA 92, 9565–9569 (1995)

    ADS  CAS  PubMed  Google Scholar 

  14. 14

    Nishinakamura, R. et al. Mice deficient for the IL-3/GM-CSF/IL-5 βc receptor exhibit lung pathology and impaired immune response, while βIL3 receptor-deficient mice are normal. Immunity 2, 211–222 (1995)

    CAS  PubMed  Google Scholar 

  15. 15

    Bonfield, T. L. et al. Peroxisome proliferator-activated receptor-γ is deficient in alveolar macrophages from patients with alveolar proteinosis. Am. J. Respir. Cell Mol. Biol. 29, 677–682 (2003)

    CAS  PubMed  Google Scholar 

  16. 16

    Bonfield, T. L. et al. PU.1 regulation of human alveolar macrophage differentiation requires granulocyte-macrophage colony-stimulating factor. Am. J. Physiol. Lung Cell. Mol. Physiol. 285, L1132–L1136 (2003)

    CAS  PubMed  Google Scholar 

  17. 17

    Seymour, J. F. & Presneill, J. J. Pulmonary alveolar proteinosis: progress in the first 44 years. Am. J. Respir. Crit. Care Med. 166, 215–235 (2002)

    PubMed  Google Scholar 

  18. 18

    Shibata, Y. et al. GM-CSF regulates alveolar macrophage differentiation and innate immunity in the lung through PU.1. Immunity 15, 557–567 (2001)

    CAS  PubMed  Google Scholar 

  19. 19

    Thomassen, M. J. et al. ABCG1 is deficient in alveolar macrophages of GM-CSF knockout mice and patients with pulmonary alveolar proteinosis. J. Lipid Res. 48, 2762–2768 (2007)

    CAS  PubMed  Google Scholar 

  20. 20

    Iyonaga, K. et al. Elevated bronchoalveolar concentrations of MCP-1 in patients with pulmonary alveolar proteinosis. Eur. Respir. J. 14, 383–389 (1999)

    CAS  PubMed  Google Scholar 

  21. 21

    Nishinakamura, R. et al. The pulmonary alveolar proteinosis in granulocyte macrophage colony-stimulating factor/interleukins 3/5 beta c receptor-deficient mice is reversed by bone marrow transplantation. J. Exp. Med. 183, 2657–2662 (1996)

    CAS  PubMed  Google Scholar 

  22. 22

    Kleff, V. et al. Gene therapy of βc-deficient pulmonary alveolar proteinosis (βc-PAP): studies in a murine in vivo model. Mol. Ther. 16, 757–764 (2008)

    CAS  PubMed  Google Scholar 

  23. 23

    Suzuki, T. et al. Use of induced pluripotent stem cells to recapitulate pulmonary alveolar proteinosis pathogenesis. Am. J. Respir. Crit. Care Med. 189, 183–193 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Stradling, J. R. & Lane, D. J. Development of secondary polycythaemia in chronic airways obstruction. Thorax 36, 321–325 (1981)

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Faust, N., Varas, F., Kelly, L. M., Heck, S. & Graf, T. Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. Blood 96, 719–726 (2000)

    CAS  Google Scholar 

  26. 26

    Yoshida, M., Ikegami, M., Reed, J. A., Chroneos, Z. C. & Whitsett, J. A. GM-CSF regulates surfacant Protein-A and lipid catabolism by alveolar macrohpages. Am. J. Physiol. Lung Cell. Mol. Physiol. 280, L379–L386 (2001)

    CAS  PubMed  Google Scholar 

  27. 27

    Lachmann, N. et al. Gene correction of human induced pluripotent stem cells repairs the cellular phenotype in pulmonary alveolar proteinosis. Am. J. Respir. Crit. Care Med. 189, 167–182 (2014)

    CAS  PubMed  Google Scholar 

  28. 28

    Seymour, J. F. et al. Mice lacking both granulocyte colony-stimulating factor (CSF) and granulocyte-macrophage CSF have impaired reproductive capacity, perturbed neonatal granulopoiesis, lung disease, amyloidosis, and reduced long-term survival. Blood 90, 3037–3049 (1997)

    CAS  PubMed  Google Scholar 

  29. 29

    Wu, M. et al. Genetically engineered macrophages expressing IFN-γ restore alveolar immune function in scid mice. Proc. Natl Acad. Sci. USA 98, 14589–14594 (2001)

    ADS  CAS  PubMed  Google Scholar 

  30. 30

    Guilliams, M. et al. Alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via GM-CSF. J. Exp. Med. 210, 1977–1992 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Hashimoto, D. et al. Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes. Immunity 38, 792–804 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Yona, S. et al. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity 38, 79–91 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Schulz, C. et al. A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Science 336, 86–90 (2012)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    van Furth, R. & Cohn, Z. A. The origin and kinetics of mononuclear phagocytes. J. Exp. Med. 128, 415–435 (1968)

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Godleski, J. J. & Brain, J. D. The origin of alveolar macrophages in mouse radiation chimeras. J. Exp. Med. 136, 630–643 (1972)

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Matute-Bello, G. et al. Optimal timing to repopulation of resident alveolar macrophages with donor cells following total body irradiation and bone marrow transplantation in mice. J. Immunol. Methods 292, 25–34 (2004)

    CAS  PubMed  Google Scholar 

  37. 37

    Murphy, J., Summer, R., Wilson, A. A., Kotton, D. N. & Fine, A. The prolonged life-span of alveolar macrophages. Am. J. Respir. Cell Mol. Biol. 38, 380–385 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Guth, A. M. et al. Lung environment determines unique phenotype of alveolar macrophages. Am. J. Physiol. Lung Cell. Mol. Physiol. 296, L936–L946 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Gautier, E. L. et al. Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages. Nature Immunol. 13, 1118–1128 (2012)

    CAS  Google Scholar 

  40. 40

    Wert, S. E. et al. Increased metalloproteinase activity, oxidant production, and emphysema in surfactant protein D gene-inactivated mice. Proc. Natl Acad. Sci. USA 97, 5972–5977 (2000)

    ADS  CAS  PubMed  Google Scholar 

  41. 41

    Sakagami, T. et al. Patient-derived granulocyte/macrophage colony-stimulating factor autoantibodies reproduce pulmonary alveolar proteinosis in nonhuman primates. Am. J. Respir. Crit. Care Med. 182, 49–61 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    LeVine, A. M., Reed, J. A., Kurak, K. E., Cianciolo, E. & Whitsett, J. A. GM-CSF-deficient mice are susceptible to pulmonary group B streptococcal infection. J. Clin. Invest. 103, 563–569 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Berclaz, P. Y. et al. GM-CSF regulates a PU.1-dependent transcriptional program determining the pulmonary response to LPS. Am. J. Respir. Cell Mol. Biol. 36, 114–121 (2007)

    CAS  PubMed  Google Scholar 

  44. 44

    Perumbeti, A. et al. A novel human gamma-globin gene vector for genetic correction of sickle cell anemia in a humanized sickle mouse model: critical determinants for successful correction. Blood 114, 1174–1185 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Kaufman, D. S., Hanson, E. T., Lewis, R. L., Auerbach, R. & Thomson, J. A. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc. Natl Acad. Sci. USA 98, 10716–10721 (2001)

    ADS  CAS  PubMed  Google Scholar 

  46. 46

    Walters, D. M., Breysse, P. N. & Wills-Karp, M. Ambient urban Baltimore particulate-induced airway hyperresponsiveness and inflammation in mice. Am. J. Respir. Crit. Care Med. 164, 1438–1443 (2001)

    CAS  PubMed  Google Scholar 

  47. 47

    Maeda, Y. et al. Kras(G12D) and Nkx2-1 haploinsufficiency induce mucinous adenocarcinoma of the lung. J. Clin. Invest. 122, 4388–4400 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Tusher, V. G., Tibshirani, R. & Chu, G. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl Acad. Sci. USA 98, 5116–5121 (2001)

    ADS  CAS  PubMed  MATH  Google Scholar 

  49. 49

    Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57, 289–300 (1995)

    MathSciNet  MATH  Google Scholar 

  50. 50

    Richard, E. et al. Gene therapy of a mouse model of protoporphyria with a self-inactivating erythroid-specific lentiviral vector without preselection. Mol. Ther. 4, 331–338 (2001)

    CAS  PubMed  Google Scholar 

  51. 51

    Mizushima, S. & Nagata, S. pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 18, 5322 (1990)

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    Zufferey, R., Donello, J. E., Trono, D. & Hope, T. J. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J. Virol. 73, 2886–2892 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53

    Arumugam, P. I. et al. The 3′ region of the chicken hypersensitive site-4 insulator has properties similar to its core and is required for full insulator activity. PLoS ONE 4, e6995 (2009)

    ADS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Arumugam, P. I. et al. Improved human β-globin expression from self-inactivating lentiviral vectors carrying the chicken hypersensitive site-4 (cHS4) insulator element. Mol. Ther. 15, 1863–1871 (2007)

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the NIH (R01 HL085453, R21 HL106134, R01HL118342, 8UL1TR000077-05, AR-47363, DK78392, DK90971), American Thoracic Society Foundation Unrestricted Research Grant, CCHMC Foundation Trustee Grant, Deutsche Forschungsgemeinschaft (DFG; Cluster of Excellence Rebirth; Exc 62/1), the Else Kröner-Fresenius Stiftung, the Eva-Luise Koehler Research Prize for Rare Diseases 2013, and by the Pulmonary Biology Division, CCHMC. Flow cytometric data were acquired within the Research Flow Cytometry Core in the Division of Rheumatology, CCHMC. We thank our hPAP patients and their family members in the United States and internationally for their collaboration; J. Whitsett (CCHMC) and F. McCormack (UCMC) for critical reading of the manuscript; J. Krischer (University of South Florida) and Y. Maeda (CCHMC) for helpful discussions; S. Wert for help with lung histology; and D. Black, K. Link and C. Fox (CCHMC), and S. Brennig and H. Kempf (Hannover Medical School) for their technical help.

Author information

Affiliations

Authors

Contributions

T.Su., P.A., N.L., S.A., T.M., P.M. and B.C.T. designed research. T.Su., P.A., T.Sa., N.L., C.C., A.S., S.A., B.C. and B.C.T. performed research. T.Su., P.A., T.Sa., N.L., S.A., C.T., T.M., P.M. and B.C.T. analysed data. T.Su., P.A., N.L., P.M., C.L., R.E.W. and B.C.T. wrote the paper.

Corresponding authors

Correspondence to Takuji Suzuki or Bruce C. Trapnell.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Validation of Csf2rb−/− mice as an authentic model of human hPAP.

a, Typical lung pathology showing surfactant-filled alveoli with well-preserved septa in a child homozygous for CSF2RBS271L mutations and identical pulmonary histopathology in a Csf2rb−/− mouse. PAS stain. Scale bar, 100 μm. b, Photographs of ‘milky’-appearing BAL from a 14-month-old Csf2rb−/− mouse and normal-appearing BAL from an age-matched WT mouse (representative of n = 6 mice per group). c, Increased BAL turbidity and SP-D concentration in 4-month-old Csf2rb−/− mice compared to age-matched WT mice. d, BAL fluid biomarkers of hPAP (GM-CSF, M-CSF and MCP-1) are increased in 4-month-old Csf2rb−/− mice compared to age-matched WT mice. e, Alveolar macrophage biomarkers (PU.1, Pparg, Abcg1 mRNA) are reduced in 4-month-old Csf2rb−/− compared to age-matched WT mice. f, Progressive increase in BAL turbidity in Csf2rb−/− mice but not age-matched WT mice (linear regression: Csf2rb−/−, slope = 0.1271 ± 0.16 (r2, 0.311); WT, slope = 0.031 ± 0.005). g, Progressive increase in BAL fluid GM-CSF level in Csf2rb−/− mice but not age-matched WT mice (linear regression: Csf2rb−/−, slope = 0.89 ± 0.016 (r2, 0.249); WT, slope = 0). h, GM-CSF bioactivity in BAL fluid from 10-month-old Csf2rb−/− or WT mice (or 1 ng ml−1 murine GM-CSF) measured in the presence of anti-GM-CSF antibody (GM-CSF Ab) or isotype control (Control Ab) using the GM-CSF-stimulated STAT5 phosphorylation index (STAT5-PI) assay. Data are mean ± s.e.m. of n = 7 mice per group (ce), n = 4 (h) or symbols representing individual WT (n = 38) or Csf2rb−/− (n = 84) mice and regression fit ± 95% CI (f-g). *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

Extended Data Figure 2 Characterization of BMDMs before PMT.

a, b, Photomicrographs of WT BMDMs before transplantation phase-contrast (a) or DiffQuick staining (b) (representative of n = 7 BMDM preparations). Scale bar, 20 μm. c, Flow cytometry evaluation of cell-surface phenotypic markers on WT BMDMs before PMT. d, Photographs of methylcellulose cultures of Lin cells (5,000 per dish) from bone marrow (left) and BMDMs (50,000 per dish) prepared as described in the Methods (right) and typical colonies (below) (representative n = 3 per condition). e, Colony counts of BFU-E, CFU-GEMM and CFU-GM showing that BMDMs contained <0.005% CFU-GM and no BFU-E or CFU-GEMM progenitors, corresponding to 93 CFU-GM per dose of BMDMs administered (n = 3 determinations per condition). f, g, Evaluation of surfactant clearance capacity. Representative photomicrographs of BMDMs from WT (left) or Csf2rb−/− (right) were examined before (top) or immediately after incubation with surfactant for 24 h (middle), or after exposure, removal of extracellular surfactant and culture for 24 h in the absence of surfactant (lower) after oil-red-O staining (representative of n = 3 per condition). Scale bar, 20 μm. g, Measurement of surfactant clearance by BMDMs after exposure as just described (f) and quantified using a visual grading scale (the oil-red-O staining index) to measure the degree of staining. Bars represent the mean ± s.e.m. (n = 3 per condition) of oil-red-O staining score for 10 high-power fields for each group. ND, not detected; ns, not significant; ***P < 0.001.

Extended Data Figure 3 Efficacy of PMT in Csf2rb−/− mice and characterization of macrophages after PMT.

a, Detection of CD131 (top) or actin (bottom) in BAL cells by western blotting 1 year after PMT (each lane represents one mouse of 6 per group). b, Representative cytology of BAL obtained 1 year after PMT after staining with PAS or oil red O (ORO) (6 mice per group). Scale bar, 25 μm. Oil-red-O positive cells were seen rarely in WT mice and occasionally in PMT-treated Csf2rb−/− mice (insets). Cytological abnormalities in BAL from untreated Csf2rb−/− mice including large, ‘foamy’, PAS- and oil-red-O-stained alveolar macrophages and PAS-stained cellular debris, were corrected by PMT. c, Representative photomicrographs of PAS-stained whole-mount lung sections 1 year after PMT. Note that some residual disease remained at 1 year (original magnification, ×1). d, GFP+ cells in BAL cells from WT or Csf2rb−/− mice 2 months after PMT of Lys-MGFP BMDMs (representative of n = 3 (WT) or n = 6 (Csf2rb−/−) mice) (original magnfication, ×20). e, Macrophage replication after PMT. Csf2rb−/− mice received Lys-MGFP BMDMs by PMT and paraffin-embedded lung was immunostained for Ki67 1 month or 1 year later. Scale bar, 50 μm; inset, 10 μm. f, Ki67 staining of BAL cells from untreated WT mice (e). Inset shows positive (left) or negative (right) staining. Scale bar, 50 µm; inset 10 µm. Graph shows the per cent Ki67+ BAL cells in age-matched WT mice (n = 5). g, Representative immunofluorescence photomicrographs of frozen lung sections 1 year after PMT of Lys-MGFP into Csf2rb−/− mice identifying GFP+ cells (top), Ki67+ cells (middle) and GFP+Ki67+ (replicating, PMT-derived) cells (bottom) (representative of n = 3 mice). Scale bar, 20 μm; inset scale bar, 10 μm. Quantitative summary data are shown in Fig. 2c. h, Localization of macrophages within the lungs 1 year after PMT of Lys-MGFP BMDMs into Csf2rb−/− mice and visualization in frozen lung sections after CD68 immunostaining, DAPI counter staining, and fluorescence microscopy to detect CD68+GFP+ cells (that is, PMT-derived macrophages) or CD68+GFP cells (that is, non-PMT-derived endogenous macrophages). Graph shows quantitative data for n = 6 mice. i, Localization of macrophages in these same mice (h) by detecting GFP by immunohistochemical staining of paraffin-embedded lung sections using light microscopy to eliminate potential interference from autofluorescence (representative of n = 6 mice). Quantitative summary data are shown in Fig. 3b.

Extended Data Figure 4 Tissue distribution and characterization of transplanted cells 1 year after PMT.

ad, Two-month-old Csf2rb−/− mice (4 per group) received one PMT of Lys-MGFP BMDMs. Twelve months later, untreated, age-matched WT Lys-MGFP or Csf2rb−/− mice and PMT-treated Csf2rb−/− mice were evaluated using flow cytometry to detect GFP+ cells in the indicated organs. Representative data (a) and the percentage of GFP+ cells in the gated region are shown (b). Similar results were observed in Csf2rb−/− mice 2 months after PMT of Lys-MGFP BMDMs except the percentage of GFP+ BAL lung cells was not quantified (not shown). c, Detection of Lys-MGFP PMT cells by PCR. PCR of genomic DNA from BAL cells (Lung), white blood cells (Blood), bone marrow (BM) cells and splenocytes (Spleen) 1 month or 1 year after Lys-MGFP BMDM PMT was performed to detect EGFP and Lysozyme M gene. BAL cells (Lung) from WT and Lys-MGFP were shown as negative and positive control for EGFP. EGFP was only detected in lung. d, Vector copy number analysis after gene-corrected BMDM PMT. Quantitative PCR with vector-specific primers (R-U5) was performed using genomic DNA from BAL cells (Lung), white blood cells (Blood), bone marrow (BM) cells and splenocytes (Spleen) obtained 1 year after PMT of gene-corrected macrophages. Note that the viral vector was only detected in lung. eh, CD45.2+ Csf2rb−/− mice received one PMT of CD45.1+ BMDMs from congenic WT mice (e) and 1 year later, untreated, age-matched WT (CD45.1+) or Csf2rb−/− (CD45.2+) mice and PMT-treated Csf2rb−/− mice were evaluated by flow cytometry to detect CD45.1+ cells in the indicated organs. Representative data (f) and the percentage of CD45.1+ cells in the gated regions are shown (g). Phenotypic characterization of PMT-derived (CD45.1+) cells (as shown in the gated region (f)). Results are similar to those for PMT of Lys-MGFP BMDMs (Fig. 3d). Numeric data are mean ± s.e.m. of n = 4 mice per group (b, d) or n = 5 mice per group (g). ND, not detected. *P < 0.05. ns, not significant.

Extended Data Figure 5 Global gene expression analysis of alveolar macrophages from age-matched WT, Csf2rb−/− and Csf2rb−/− mice 1 year after PMT of WT BMDMs.

a, Expression of Spi1 (PU.1) and Pparg (PPARγ) were confirmed by qRT–PCR using independent samples (6 mice per group). b, Venn diagrams showing numbers of genes whose expression was altered in alveolar macrophages from Csf2rb−/− compared to WT mice (WT→KO) or PMT-treated compared to untreated Csf2rb−/− mice (KO→KO+PMT). Only genes with statistically significant changes (false detection rate <10%) of at least twofold were marked as increased (up arrows) or decreased (down arrows). The numbers of genes for which expression was disrupted in Csf2rb−/− mice and normalized by PMT (or unchanged in both comparisons) is shown in the overlap regions. c, Gene ontology analysis identifying pathways disrupted in Csf2rb−/− mice and restored by PMT. 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. d, Heat maps showing differentially expressed genes in multiple KEGG pathways including PPARγ-regulated genes, glycophospholipid metabolism, peroxisome function apoptosis, cell cycle control, and immune host defence. Genes with increased or decreased transcript levels are shown by red and blue colours, respectively. e, Confirmation by qRT–PCR for selected genes important in lipid metabolism, using independent samples. Data are mean ± s.e.m. (6 mice per group). *P < 0.05.

Extended Data Figure 6 Effects of PMT of gene-corrected macrophages on hPAP.

a, Macrophages derived from Csf2rb−/− LSK cells transduced with GM-R-LV or GFP-LV, or from non-transduced WT LSK cells (indicated) were examined by light microscopy after DiffQuick staining (top), or by immunofluorescence microscopy after staining with anti-CD131 (GM-CSF-R-β) and DAPI (upper middle), DAPI alone (lower middle), or anti-CD68 and DAPI (bottom). Images are representative of three experiments per condition. b, Evaluation of GM-CSF receptor signalling in the indicated cells (before PMT) by measurement of GM-CSF-stimulated STAT5 phosphorylation by flow cytometry. Representative of n = 3 experiments per condition. Quantitative summary data are shown in Fig. 5b. c, Western blotting to detect GM-CSF receptor-β (CD131) (top) or actin (bottom, as a loading control) in BAL cells from age-matched Csf2rb−/− mice 2 months after PMT as indicated (each lane represents one mouse of n = 10, 8, 10 per group, respectively). d, Appearance of BAL from age-matched Csf2rb−/− mice 2 months after PMT as indicated (representative of n = 10, 8, 10 per group, respectively). e, f, One year after PMT of GM-R-LV transduced Csf2rb−/− LSK cell-derived macrophages in Csf2rb−/− mice, GFP+ cells were identified (e) and evaluated for cell surface markers by flow cytometry (f) (representative of n = 7 mice).

Extended Data Table 1 Oligonucleotide primers used to quantify mRNA transcripts by qRT–PCR and detection of PMT-derived cellular DNA by PCR
Extended Data Table 2 Effect of the number of macrophages transplanted on the efficacy of PMT therapy of hPAP in Csf2rb−/− mice
Extended Data Table 3 Comparison of the effects of single versus repeated macrophage administrations on the efficacy of PMT therapy of hPAP in Csf2rb−/− mice
Extended Data Table 4 Effect of PMT of WT or gene-corrected macrophages on haematological indices and lung proinflammatory cytokine levels

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Suzuki, T., Arumugam, P., Sakagami, T. et al. Pulmonary macrophage transplantation therapy. Nature 514, 450–454 (2014). https://doi.org/10.1038/nature13807

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing