RESEARCH ARTICLE LUNG DISEASE

Pulmonary transplantation of macrophage progenitors as effective and long-lasting therapy for hereditary pulmonary alveolar proteinosis Christine Happle,1,2* Nico Lachmann,3,4* Jelena Škuljec,1 Martin Wetzke,1 Mania Ackermann,3,4 Sebastian Brennig,3,4 Adele Mucci,3,4 Adan Chari Jirmo,1,2 Stephanie Groos,5 Anja Mirenska,1 Christina Hennig,1,2 Thomas Rodt,6 Jens P. Bankstahl,7 Nicolaus Schwerk,1,2 Thomas Moritz,3,4† Gesine Hansen1,2†‡ Hereditary pulmonary alveolar proteinosis (herPAP) is a rare lung disease caused by mutations in the granulocytemacrophage colony-stimulating factor (GM-CSF) receptor genes, resulting in disturbed alveolar macrophage differentiation, massive alveolar proteinosis, and life-threatening respiratory insufficiency. So far, the only effective treatment for herPAP is repetitive whole-lung lavage, a merely symptomatic and highly invasive procedure. We introduce pulmonary transplantation of macrophage progenitors as effective and long-lasting therapy for herPAP. In a murine disease model, intrapulmonary transplanted macrophage progenitors displayed selective, long-term pulmonary engraftment and differentiation into functional alveolar macrophages. A single transplantation ameliorated the herPAP phenotype for at least 9 months, resulting in significantly reduced alveolar proteinosis, normalized lung densities in chest computed tomography, and improved lung function. A significant and sustained disease resolution was also observed in a second, humanized herPAP model after intrapulmonary transplantation of human macrophage progenitors. The therapeutic effect was mediated by long-lived, lung-resident macrophages, which displayed functional and phenotypical characteristics of primary human alveolar macrophages. Our findings present the concept of organotopic transplantation of macrophage progenitors as an effective and long-lasting therapy of herPAP and may also serve as a proof of principle for other diseases, expanding current stem cell–based strategies toward potent concepts using the transplantation of differentiated cells.

INTRODUCTION Pulmonary alveolar proteinosis (PAP) is a rare, life-threatening disease, characterized by massive protein accumulation in the lungs, progressive respiratory failure, and high susceptibility to severe pulmonary infections (1, 2). The hereditary form of PAP (herPAP) is caused by mutations in the CSF2RA or CSF2RB genes, which encode the a or b subunit of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, respectively (3, 4). Homozygous or compound heterozygous mutations lead to defective GM-CSF signaling and blockade of terminal alveolar macrophage (AM) differentiation (5–8). This results in ineffective phagocytosis and protein degradation by AMs and an extensive accumulation of surfactant proteins within the airways (1, 9). Typical onset for herPAP is preschool age, when affected children suffer from progressive dyspnea, cough, lung infections, and failure to thrive (1, 2). Quality and expectancy of life are significantly reduced (10). So far, the only effective treatment for herPAP is repetitive whole-lung lavage in general anesthesia (5, 6, 10), a merely symptomatic and highly invasive

procedure, associated with significant cardiorespiratory morbidity (10). Although hematopoietic stem cell (HSC) transplantation represents a curative option in other severe congenital hematopoietic diseases, this approach is not applicable to herPAP because the required myeloablation would further aggravate respiratory insufficiency and susceptibility to pulmonary infections (6). Similarly, stem cell–based gene therapy for herPAP is problematic because of the necessity for preconditioning chemotherapy and the inherent risk of leukemogenesis (11, 12). To explore novel therapeutic options for this life-threatening disease, we investigated the feasibility of organotropic transplantation of myeloid progenitor cells in two clinically relevant murine disease models. Because AMs are replenished from a local, long-lived pool of pulmonary macrophage progenitors (13, 14), we hypothesized that intrapulmonary administered healthy myeloid progenitor cells could engraft in the lungs of herPAP recipients and elicit long-term therapeutic effects.

RESULTS 1

Department of Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, 30625 Hannover, Germany. 2Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), 30625 Hannover, Germany. 3Reprogramming and Gene Therapy Group, REBIRTH Cluster-of Excellence, Hannover Medical School, 30625 Hannover, Germany. 4Institute of Experimental Hematology, Hannover Medical School, 30625 Hannover, Germany. 5Institute of Cell Biology in the Center of Anatomy, Hannover Medical School, 30625 Hannover, Germany. 6Department of Diagnostic and Interventional Radiology, Hannover Medical School, 30625 Hannover, Germany. 7Institute for Preclinical Molecular Imaging, Hannover Medical School, 30625 Hannover, Germany. *These authors contributed equally to this work. †These authors contributed equally to this work. ‡Corresponding author. E-mail: [email protected]

Long-term pulmonary cell engraftment after transplantation of macrophage progenitors into herPAP mice Initial experiments were performed in a murine disease model using Csf2rb−/− mice defective for the GM-CSF/IL-3 (interleukin-3)/IL-5 receptor common b chain (15, 16). Phenotypically, these mice have been described to display all hallmark features of the human disease. Indeed, similar to a 3-year-old girl with herPAP due to a homozygous mutation in CSF2RA (R199X) previously reported by our group (17), Csf2rb−/− mice display areas of consolidation and ground glass opacities in chest computed tomography (CCT; Fig. 1, A and B), as well as elevated turbidity

www.ScienceTranslationalMedicine.org

20 August 2014

Vol 6 Issue 250 250ra113

1

RESEARCH ARTICLE

–/– Control CSF2RA

patient

D

***

6

B

Csf2rb–/–

C57Bl/6 WT

Csf2rb–/–

C57Bl/6 WT

4

2 0

–/– Control CSF2RA

** BALF prote in [mg/ml]

C

CSF2RA–/– patient

Control

BALF prote in [mg/ml]

A

1.5 1.0 0.5

0.0

CD11b

100

50 0

–1ctrl –GM

+GM

–1ctrl –GM +GM

SF2RA–/– Control Cpatient

CD11b

CD11b GM-stimulation [%]

WT

C57Bl/6

F

Count

–1ctrl –GM +GM

*** 150

C57Bl/6 Csf2rb–/–

WT

Csf2rb–/–

CSF2RA–/– patient

+GM

CD11b GM -stimulation [%]

–1ctrl –GM

Count

E

Control

patient

***

300

200

100

0

C57Bl/6 Csf2rb–/–

WT

−/−

GM-CSF [% of input]

Fig. 1. Csf2rb mice display all main features of hu- G man herPAP. (A) CCT of healthy proband and 3-year-old 120 Csf2rb –/– CSF2RA−/− herPAP patient with massive opacification due C57Bl/6 WT to alveolar proteinosis (scale bars, 1 cm). (B) RepresentaNo cells 80 tive CCT of wild-type (WT) versus Csf2rb−/− herPAP mouse with areas of consolidation and ground glass opacities 40 (scale bars, 5 mm). (C) High BALF turbidity and protein concentration in the patient compared to healthy control 0 (three samples from independent control probands and 5 10 15 20 25 30 35 four samples from two independent lavages of the Time [hours] herPAP patient). ***P = 0.0003. (D) Elevated BALF turbidity H and proteinosis in Csf2rb−/− mice (n = 4 to 6 mice per group from three experiments). **P = 0.0029. (E) Failed MLB up-regulation of CD11b in patient versus control granuloN cytes in response to GM-CSF, flow cytometric CD11b expression before (nonfilled) and after (gray shaded line) stimulation, minus one control (dashed line); n ≥ 6 b a samples from three experiments. ***P < 0.001. (F) Disturbed CD11b up-regulation in Gr-1+ Csf2rb−/− bone marrow cells (n = 6 to 7 mice per group from two experiments; ***P = 0.0006). (G) Absent GM-CSF uptake in Csf2rb−/− bone marrow cells (three experiments with one to three pooled mice per group). (H) Typical electron microscopic appearance of murine Csf2rb−/− AMs, massive intracellular vacuolation (†), large multilamellar bodies (MLB), and nucleus (N). Scale bars, 2 mm (a) and 500 nm (b). All graphs display means + SEM, and significances were calculated by unpaired two-tailed Student’s t test. MLB

and protein concentration of the bronchoalveolar lavage fluid (BALF; Fig. 1, C and D). Hematopoietic cells from both the herPAP patient and Csf2rb−/− mice fail to adequately respond to GM-CSF and neither upregulate CD11b after in vitro stimulation with GM-CSF (Fig. 1, E and F and fig S8, A and B) nor clear this cytokine from the medium (Fig. 1G). As in herPAP patients (1), the AMs of Csf2rb−/− mice exhibit the characteristic ultrastructural phenotype of large, “foamy” cells with excessive intracellular accumulation of vacuoles and undegraded protein (Fig. 1H). We used this murine model to test our hypothesis whether pulmonary transplantation of GM-CSF–responsive macrophage progenitors could result in long-term

pulmonary cell engraftment and effective treatment of herPAP. To track the fate of the transplanted cells, we isolated lineagenegative (lin−) cells from bone marrow of wild-type C57BL/6 CD45.1+ donor mice. These cells were differentiated in vitro with medium containing macrophage colonystimulating factor (M-CSF). After 4 days, a fourfold increase in cell numbers was observed (fig. S1A). Cells from the adherent fraction of this culture predominantly displayed macrophage morphology and expressed mature myeloid surface markers such as F4/80, CD11b, CD200R, and CD14, with only ∼1% of lin−sca−kit+ myeloid progenitors (figs. S1, B and C and S10). In contrast to undifferentiated lin− cells, these differentiated cells formed extremely few colonies in a methylcellulose-based assay (fig. S1D). CD45.1+ macrophage progenitors (2 × 106) were transplanted intrapulmonary into adult Csf2rb−/− mice (Fig. 2A). These CD45.1+ cells successfully engrafted in the lungs of Csf2rb−/− mice, as assessed by flow and chipcytometry (18, 19), 6 weeks to 9 months after transplantation. Donorderived cells were detected in lungs and BALF of recipient animals 6 weeks after the treatment (Fig. 2, B to D, and figs. S2, A to D and S8C) and persisted at these sites for at least 9 months after a single transplantation, the longest observation period in our experiments (Fig. 2B and figs. S2, B to D and S11A). Donor-derived cells were detected exclusively in BALF and lungs of recipient Csf2rb−/− animals, but not in their bone marrow, spleen, liver, or blood (Fig. 2C and fig. S8D). In lung cryosections of the transplanted mice, donor-derived CD45.1+ cells were F4/80+ and CD11blo (Fig. 2D and fig. S8E).

Significant improvement of the herPAP phenotype after intrapulmonary transplantation of macrophage progenitors To determine the clinical benefit of the intrapulmonary transplantation, we compared the phenotype of transplanted Csf2rb−/− mice to that of age-matched untreated Csf2rb−/− and healthy wild-type mice. Transplanted mice displayed a significant reduction of BALF turbidity 6 weeks and 9 months after transplantation. Moreover, BALF protein concentrations in treated mice were significantly lower when compared to those of nontransplanted Csf2rb−/− mice (Fig. 2F and fig. S2E). Six weeks after transplantation, BALF proteinosis in treated Csf2rb−/− mice was reduced by ∼45%. This beneficial effect lasted at least 9 months after transplantation (longest observational time point), when treated animals displayed a ∼30% reduction of BALF

www.ScienceTranslationalMedicine.org

20 August 2014

Vol 6 Issue 250 250ra113

2

RESEARCH ARTICLE

Healthy donor

Murine herPAP model

C57Bl/6 WT

Bone marrow

Csf2rb–/–

B 6 weeks

Analysis

A

47.7% 79.1% 68.8% 3.5%

F4/80

0.4%

81.7%

AF

0.7%

0.3%

Podoplanin AF

0.3%

CD45.1

2.2%

Csf2rb–/–

D

CD11b

Blood 0.3%

AF

CD45.1

WT

Liver 0.7%

AF

Csf2rb–/– PCT

C57Bl/6

0.2%

Spleen BM 0.3% 0.3%

AF (FITC)

Csf2rb–/– PCT

C57Bl/6 WT

F

9 months

6 weeks

*

1.5

1.0

n.s.

0.5

BALF protein [mg/ml]

Csf2rb–/–

BALF protein [mg/ml]

E

0.0

Csf2rb–/– PCT

H Radiodensity/MW [HU]

C57Bl/6 WT

13

* n.s.

12 11 10 10 0

–/– T b CT f2r –/– P 6 W Cs 2rb 7Bl/ f 5 s C C

1.5

1.0

n.s.

0.5

0

*

Expiratory

n.s.

*n.s.

– 400 – 350 – 300 – 250 – 200 – 150 0

–/– –/– T T b b CT CT f2r –/– P 6 W sf2r –/– P l/6 W C 2rb 7B Cs 2rb 7Bl/ f f 5 5 s s C C C C

9 months

*

–/– T b CT f2r –/– P 6 W Cs 2rb 7Bl/ f 5 C Cs

End-expir. pause (ms)

14

Enhanced pause (PenH)

End-expir. pause (ms)

15

0.5

Inspiratory – 450

7 months

I

## 1.0

0.0

16

*

15 14

n.s.

13 12 11 10 10 0

Enhanced pause (PenH)

Csf2rb–/–

**

–/– T b CT f2r –/– P 6 W Cs 2rb 7Bl/ f 5 s C C

–/– T b CT f2r –/– P 6 W Cs 2rb 7Bl/ f 5 s C C

G

1.5

** 1.5

n.s. 1.0

0.5

–/– T b CT f2r –/– P 6 W Cs 2rb 7Bl/ f 5 s C C

protein levels. Furthermore, CCT scans of untreated and treated Csf2rb−/− and wild-type mice revealed a substantial reduction of herPAPtypical radiographic findings after the treatment (Fig. 2G). Nine months after transplantation, a normalization of inspiratory and expiratory

0

–/– T b CT f2r –/– P 6 W Cs 2rb 7Bl/ f 5 s C C

9 months

Csf2rb–/–

Lung

Csf2rb–/–

C

Csf2rb–/– PCT

1.5–9 months

0.7%

2.7%

0.1%

5.1%

FSC

Merge

CD45.1 C57Bl/6 WT

pulmonary cell transplantation

CD45.1

PCT

Myeloid differentiation

Csf2rb–/– PCT

Fig. 2. Long-term pulmonary engraftment and significant improvement of the herPAP phenotype in recipient Csf2rb−/− mice after intrapulmonary macrophage progenitor transplantation. (A) Experimental design. (B) Engraftment of CD45.1+ donor–derived cells after pulmonary cell transplantation (PCT) in the BALF of recipient Csf2rb−/− mice (flow cytometry 6 weeks and 9 months after transplantation; one representative analysis of n = 6 to 8 mice per group from three experiments per time point). (C) Exclusive pulmonary engraftment of transplanted cells (6 weeks after PCT; AF, autofluorescence; BM, bone marrow; one representative animal of n = 8 mice from three experiments) (D) Representative lung cryosection of Csf2rb−/− recipient [chipcytometry staining for CD45.1 (yellow), CD11b (red), F4/80 (purple), podoplanin (green), and autofluorescence (blue); overlay far right; scale bars, 100 mm; two independent experiments]. (E) Reduced BALF turbidity in treated compared to nontreated Csf2rb−/− and WT mice (representative photography 6 weeks after transplantation). (F) Reduced BALF protein concentration (6 weeks: n = 8 to 11 mice per group, and 9 months: 6 to 7 mice per group from three experiments per time point; 6 weeks, *P = 0.0166; 9 months, **P = 0.0037, ##P = 0.0024; n.s., not significant). (G and H) Representative CCT (G) and inspiratory and expiratory CCT lung densities (H) in untreated and treated Csf2rb−/− mice 9 months after transplantation and WT controls (n = 6 to 7 mice per group from two experiments; inspiratory lung density, *P = 0.017; expiratory lung density, *P = 0.038). (I) Lung function measurements of untreated and treated Csf2rb−/− and WT mice 7 and 9 months after transplantation (n = 6 to 9 mice per group for 7 months and n = 8 to 10 mice per group for 9 months from two experiments per time point; end-expir. pause 7 months, *P = 0.0347; enhanced pause 7 months, *P = 0.0119; end-expir. pause 9 months, *P = 0.0493; enhanced pause 9 months, *P = 0.0034 compared to WT). All graphs display means + SEM; significances were calculated by one-way analysis of variance (ANOVA) with Tukey post hoc testing.

CCT lung densities was observed in the treated mice (Fig. 2H). To further assess the benefit of our treatment, we performed lung function measurements using whole-body plethysmography. Here, Csf2rb−/− mice displayed prolonged expiratory intervals and enhanced pauses,

www.ScienceTranslationalMedicine.org

20 August 2014

Vol 6 Issue 250 250ra113

3

RESEARCH ARTICLE typical for the mucoid airway obstruction in herPAP. This pathological breathing pattern was normalized 7 and 9 months after a single PCT, indicating a clear improvement of airway resistance (Fig. 2I). Differentiation of transplanted cells into functional macrophages within herPAP recipient lungs Subsequently, we investigated the in situ differentiation status of the intrapulmonary administered cells that accounted for this remarkable therapeutic effect. On the basis of the elevated pulmonary GM-CSF levels in Csf2rb−/− mice (5), we expected a differentiation propensity of donor-derived cells along the monocyte/ macrophage lineage. Donor-specific CD45.1+ cells, sorted from BALF and lung homogenates of recipient mice, were compared to primary wild-type AMs, in vitro differentiated wild-type bone marrow–derived macrophages (BMDMs), or in vitro differentiated wild-type bone marrow–derived dendritic cells (BMDDCs). As assessed by cytospin preparations and in-well photography, CD45.1+ donor–derived cells, sorted from recipient lungs 6 weeks to 9 months after PCT treatment, displayed typical macrophage-like morphology, whereas lymphocyte- or dendritic cell–like and polymorphonuclear cells were absent (Fig. 3A). Further analysis showed that these cells were highly autofluorescent and CD11chi, F4/80+, CD14lo, CD11blo, MHC-IIlo, and Siglec-Fhi (Fig. 3B), an Fig. 3. Transplanted cells undergo in vivo differentiation into functional macrophages. (A) Morpholexpression pattern most similar to AMs ogy of donor-derived cells from Csf2rb−/− recipient lungs. Representative cytospins (upper panel) and in(13, 20, 21). Functional evaluation of the well microscopy (lower panel) of donor-derived cells, WT AMs, and in vitro differentiated BMDMs and transplanted cells sorted from the Csf2rb−/− BMDDCs (scale bars, 40 mm; three or more independent experiments). (B) Surface marker expression of recipient lungs revealed poor antigen pre- donor-derived versus control cells [chipcytometry, mean fluorescence intensity (MFI), n = 5 pooled mice sentation capacity and high phagocytic per group from one experiment 9 months after PCT, means ± SD]. (C) Representative antigen presentation and phagocytosis of donor-derived cells versus control cells. Upper panel: Flow cytometry of antigen preactivity (Fig. 3C and figs. S3, A and B sentation, unpulsed T cells (blue) and T cells incubated with specific antigen and respective cells (red). and S9, A and B), recapitulating AM- Gate denotes proliferating cells [carboxyfluorescein diacetate succinimidyl ester (CFSE)]; n = 2 to 5 pooled like rather than BMDM- or BMDDC-like mice per group, two experiments. Lower panel: Flow cytometry of phagocytic activity, cells without (blue) properties. In addition, the recovered and after incubation with latex beads (red). Gate denotes phagocytic cells (n = 2 to 5 pooled mice per CD45.1+ cells were clearly able to take group, two experiments). (D) GM-CSF uptake in donor-derived versus control cells (one experiment with up GM-CSF in an in vitro clearance assay n = 5 pooled mice per group, 9 months after PCT). (E) Light and electron microscopy of AMs sorted from −/− (Fig. 3D). GM-CSF is essential for lyso- BALF of untreated and treated Csf2rb mice versus control cells [a: periodic acid–Schiff (PAS) light microscopy, b/c: representative electron microscopy; MLB, multilamellar body; scale bars, 20 mm (a), somal degradation of surfactant proteins, and AMs from herPAP patients display 2 mm (b), and 500 nm (c); one experiment with n = 5 to 6 mice per group 9 months after PCT]. characteristic intracellular accumulation of undegraded protein (“foamy macrophages”) (1). We compared the ul- of treated Csf2rb−/− mice demonstrated a rather normal morphology trastructural appearance of macrophages recovered from the BALF of with significantly smaller cell diameters and less vacuolation than untreated and treated Csf2rb−/− mice (9 months after transplantation) Csf2rb−/− AMs (Fig. 3E and fig. S3C). Together, these data illustrate to that of wild-type AMs, BMDMs, and BMDDCs (Fig. 3E). In light and that intrapulmonary transplanted myeloid cells engraft exclusively in electron microscopy, Csf2rb−/− AMs appeared as large, foamy cells with the lungs of Csf2rb−/− recipients, differentiate into functional macromultiple intracellular vacuoles and large multilamellar bodies. By phages, and mediate significant and long-term improvement of the contrast, the CD45.1+ donor–derived cells sorted from the BALF herPAP phenotype. www.ScienceTranslationalMedicine.org

20 August 2014

Vol 6 Issue 250 250ra113

4

RESEARCH ARTICLE

NSG control CD34+ cells

PCT

Myeloid differentiation

pulmonary cell transplantation

huPAP

2–6.5 months huPAP PCT

huPAP

C

DDC Lung

BM

Liver

Spleen H2O

D

huPAP

B 2 months

Analysis

Humanized herPAP model

6.5 months

Healthy human donor

huPAP PCT

0.3%

5.2%

0.6%

5.4%

huCD45

A

AF (FITC)

huPAP huPAP PCT NSG

huGAPDH muß-actin

***

1

0

BALF protein [mg/ml]

BALF protein [mg/ml]

6.5 months 3

*

P T G PA C S hu AP P N P u h

huPAP

2

* 1

2 months

200 150 100 50

T G P PA PC NS hu AP P u h

NSG

huPAP

huPAP PCT

NSG

*

250 200 150 100 50 0

T P PA PC hu PAP hu

H

T P PA PC hu PAP hu

# – 450

*

– 400 – 350 – 300 – 250 – 200 – 150

0

P CT SG PA N hu AP P P hu

*

J Volume [ml]

I

6.5 months

n.s.

250

0

0

huPAP PCT

F

Radiodensity/ MW [HU]

G

**

BALF GM-CSF [pg/ml]

2 months 2

BALF GM-CSF [pg/ml]

E

n.s

1.0

0.5

0.0

P C T SG PA N hu AP P P u h

Effective treatment of herPAP in the humanized model after pulmonary transplantation of human macrophage progenitors After demonstrating the beneficial effect of pulmonary transplanted wild-type macrophage progenitors in a murine herPAP model, we next used humanized herPAP model and assessed the therapeutic

Fig. 4. Intrapulmonary transplantation of human macrophage progenitors leads to long-term engraftment and significant amelioration of the phenotype in humanized herPAP mice. (A) Experimental protocol. (B) Engraftment of human CD45+ (hCD45+) cells in the BALF of huPAP recipients. Flow cytometry 2 and 6.5 months after transplantation (n = 9 to 10 mice per group from three experiments per time point). (C) Donor-derived cells (DDC) engraft in lungs, but not in spleen, liver, or bone marrow of recipient mice [2 months after transplantation; semiquantitative RT-PCR of human GAPDH (glyceraldehyde-3phosphate dehydrogenase) and murine b-actin (mub-actin) mRNA; DDC, hCD45+ donor-derived cells purified from recipient lungs; BM, bone marrow; two representative mice from one experiment]. (D) Reduced BALF turbidity in transplanted huPAP mice (6.5 months after PCT, n = 9 mice per group from three experiments). (E) Decreased BALF protein concentration 2 and 6.5 months after intrapulmonary transfer of human cells (2 months, n = 8 to 9 mice per group, ***P < 0.0001, *P = 0.0194; 6.5 months, n = 10 to 11 mice per group, *P = 0.0308, **P = 0.0025, mice from three experiments per time point). (F) Reduced BALF GM-CSF levels 2 and 6.5 months after transplantations (2 months: n = 4 to 5 mice per group, one experiment, 6.5 months: n = 7 to 8 mice per group from three experiments, *P = 0.0447, unpaired t test). (G) Representative CCT images of one mouse per group (6.5 months after PCT). (H) Inspiratory CCT lung densities in untreated versus treated huPAP and WT mice (*P = 0.0057, #P = 0.011). (I) Representative three-dimensional (3D) rendering of CCT data depicting lung density and structural changes. (J) Inspiratory lung volumes as assessed by CCT values 6.5 months after PCT (*P = 0.0375) (G to J: n = 8 to 10 mice per group from two independent experiments). All graphs display means + SEM, and significances were calculated by one-way ANOVA with Tukey post hoc testing.

potential of human macrophage progenitors. Humanized herPAP mice (129S4-Rag2tm1.1Flv Csf2/Il3tm1.1(CSF2,IL3)Flv Il2rgtm1.1Flv/J; huPAP) were generated on the immunodeficient Rag2−/− Il2rg−/− background and carry a targeted replacement of the regions coding for murine IL-3 and GM-CSF (mIL-3 and mGM-CSF) with those encoding for human IL-3 and human

www.ScienceTranslationalMedicine.org

20 August 2014

Vol 6 Issue 250 250ra113

5

RESEARCH ARTICLE

Fig. 5. In vivo differentiation of transplanted human progenitors into functional macrophages within the lungs of humanized herPAP mice. (A) Representative cytospins (upper panel) and in-well microscopy (lower panel) of donor-derived hCD45+ cells, sorted from huPAP recipient lungs 6.5 months after transplantation, primary human AMs, and in vitro differentiated CD34+-derived macrophages and dendritic cells (scale bars, 40 mm; n = 9 mice from two experiments). (B) Cell surface marker expression in hCD45+ cells, sorted from huPAP recipient lungs 2 months after intrapulmonary cell transfer. Heatmap depicts cells in rows and analyzed markers in columns (unbiased hierarchical clustering at left margin; yellow, high expression; blue, low expression; z score scale far right; one experiment with n = 4 mice 2 months after PCT). (C) Primary human BALF cells. Main cell populations are marked by color blocks on the right margin (brown, T helper cells; green, alveolar monocytes/AMs; yellow, cytotoxic T cells; red, B cells; purple, AMs). (D) Representative phagocytic capacity of hCD45+ cells sorted from huPAP recipient lungs 6.5 months after transplantation versus control cells. Flow cytometry of cells without (blue) and after incubation (red) with fluorescent latex beads (donor-derived cells: n = 4 mice from two experiments). (E) In vitro GM-CSF uptake by hCD45+ cells, sorted from huPAP recipient lungs and control cells (one experiment with n = 4 pooled mice).

GM-CSF (hIL-3 and hGM-CSF) (22). Because hGM-CSF and hIL-3 are not cross-reactive with mGM-CSF and mIL-3 (23), the loss of mGM-CSF leads to a block of inherent AM differentiation, resulting in the fully developed herPAP phenotype. This results in high serum and BALF levels of hGM-CSF, facilitating engraftment and differentiation of transplanted human cells in huPAP mice (22). To test the therapeutic effect of human macrophage progenitors, we cultured healthy human CD34+ cells for 4 days in a cytokine cocktail promoting proliferation and macrophage differentiation (Fig. 4A). This led to a fivefold increase in cell numbers (fig. S4A). On day 4, most cells displayed a monocyte/macrophage morphology and

expressed myeloid cell markers (figs. S4, B and C and S11B). However, the transplanted cell population contained ∼35% of cells still expressing CD34, and the cells were able to form colonies in a methylcellulose assay (fig. S4, C to E). These day 4 human CD34+– derived cells (2 × 106) were subsequently transplanted intrapulmonaryintohuPAPrecipient mice (Fig. 4A). Two to 6.5 months after transplantation, age-matched untreated and treated huPAP mice, as well as control NOD.Cg-Prkdcscid-Il2rgtm1Wjl/SzJ (NSG) animals, were analyzed. Intrapulmonary transplantation led to an exclusive pulmonary engraftment of the transplanted human cells, which were detectable in BALF and lungs of huPAP recipients up to 6.5 months after a single treatment (latest time point of observation; Fig. 4B and figs. S5, A to F, S9, C and D, and S11C). No human cells were detectable in bone marrow, liver, spleen, or other organs of recipient animals by flow cytometry or reverse transcription polymerase chain reaction (RT-PCR) (Fig. 4C and figs. S5, A and B and S12). A significant reduction in BALF turbidity was observed 2 and 6.5 months after transplantation, and BALF proteinosis 2 and 6.5 months after the treatment was reduced by ∼60 and ∼50%, respectively (Fig. 4E and fig. S6A). Furthermore, levels of human GM-CSF were reduced at both time points in the BALF of treated huPAP mice (Fig. 4F). CCT scans, performed 6.5 months after intrapulmonary cell transplantation, revealed a clear resolution of herPAP-related radiographic findings and a reduction of inspiratory lung densities (Fig. 4, G and H). Moreover, treated mice displayed an improvement of overall CCT opacities and a significant reduction of inspiratory lung volumes (Fig. 4, I and J). The obtained CCT values clearly correlated with levels of BALF proteinosis (fig. S7, A and B). Together, intrapulmonary transfer of human CD34+–derived myeloid cells resulted in a significant and long-lasting improvement of the disease phenotype in huPAP mice.

Differentiation of functional human macrophages after PCT into huPAP mice To analyze the phenotype and function of the transplanted human cells, we sorted them from BALF and lungs of huPAP recipient mice based on their expression of hCD45. Two and 6.5 months after transplantation, the cells were compared to primary human BALF cells or in vitro differentiated human CD34–derived macrophages and dendritic cells. Cytospin preparations and in-well photography of hCD45+ cells recovered from the lungs of huPAP recipients displayed typical morphological features of human AMs (Fig. 5A). Moreover, the

www.ScienceTranslationalMedicine.org

20 August 2014

Vol 6 Issue 250 250ra113

6

RESEARCH ARTICLE transplanted cells expressed a homogeneous marker panel characteristic for AMs, with high expression of CD71, CD11c, and MHC-II (major histocompatibility complex II), whereas CD11b, CD14, and CD4 expression was low. Markers specific for T or B cells were absent (Fig. 5B). Next, we compared hCD45+ cells recovered from transplanted huPAP mice to the cells occurring in primary human BALF. In a human BALF sample, clear clusters of T cells, B cells, and macrophages were identified by hierarchical cluster analysis (Fig. 5C). Compared to this, the cells sorted from huPAP recipient lungs displayed a homogeneous surface marker panel most similar to that of human AMs. Moreover, these cells were well able to phagocytose (Fig. 5D and fig. S6B) and to clear GM-CSF in an in vitro uptake assay (Fig. 5E), thus illustrating typical functional characteristics of human AMs. In conclusion, intrapulmonary transplantation of human macrophage progenitors in the huPAP model resulted in long-term engraftment of lung-resident human macrophages, which mediated a significant and sustained clinical improvement of the herPAP phenotype.

DISCUSSION Applying two independent, clinically relevant disease models, we present intrapulmonary transplantation of healthy macrophage progenitors as an effective treatment for herPAP. A single PCT of healthy macrophage progenitors into Csf2rb−/− mice resulted in exclusive pulmonary engraftment, in situ differentiation, and long-term persistence of donor-derived macrophages, with substantial clinical improvement of the disease phenotype. Moreover, human myeloid progenitors, intrapulmonary applied to humanized herPAP mice, differentiated into functional human macrophages and elicited significant long-lasting therapeutic effects. Our data bear important implications for the future treatment of hereditary PAP, as we demonstrate that organotropic transplantation of macrophage progenitors conveys a sustained therapeutic benefit and may represent a feasible alternative to high-risk stem cell–based therapies. So far, no herPAP disease–specific, long-term effective, or curative treatment options exist (1, 5, 9, 10), and the quality of life and life expectancy of herPAP patients remain extremely poor (1, 6, 10). The only currently available treatment, repetitive whole-lung lavage, is highly invasive, hazardous, and purely symptomatic (10). By contrast, our concept of organotropic macrophage progenitor transplantation represents an easily performed, safe, and effective treatment strategy. Alternative approaches like HSC transplantation (24) or other stem cell–based therapies (16) are not applicable in the clinical setting, given the extreme vulnerability of the diseased lung and the required myeloablation. In the only HSC transplantation attempt in herPAP reported so far, the patient died because of an overwhelming pulmonary infection after preconditioning chemotherapy (6). In the clinical setting, autologous, gene-corrected, and in vitro differentiated macrophage progenitors derived from CD34+ cells represent the most promising cell type for transplantation. herPAP-specific, gene-corrected induced pluripotent stem cells (17, 25) may also represent a future source for transplantable macrophage progenitors. Current gene therapy approaches primarily use the transplantation of HSCs, a strategy successfully applied in a variety of diseases (26–29), and we previously described effective HSC-based gene therapy for herPAP in Csf2rb−/− mice (16). However, the use of gene-corrected HSCs requires myelotoxic preconditioning and is associated with the risk of

insertional leukemogenesis, a major hurdle in current gene therapy (11, 12). Because leukemic transformation typically takes place in the stem cell compartment and is unlikely to occur in differentiated cells (30, 31), our concept to transplant rather mature cells instead of HSCs could significantly reduce the risk of secondary leukemia. However, this hypothesis needs to be addressed in further experiments. Moreover, in the case of GM-CSF receptor gene correction, the possibility of secondary autoimmune PAP due to the development of anti–GM-CSF antibodies needs to be taken into account. The murine models used in our study resemble all hallmark characteristics of the human syndrome and have proven extremely valuable in understanding herPAP pathophysiology and macrophage biology (15, 22, 32). A murine model of GM-CSF deficiency led to the discovery that the deficiency of this cytokine is central to herPAP pathophysiology (33, 34). On the basis of this, effective treatment strategies for nonhereditary PAP were first developed in the murine system and are now successfully applied in the clinical setting (35–38), which further supports the relevance of our data set with regard to its potential clinical translation. Notably, both mouse strains used in our experiments are valid but imperfect models for the human herPAP syndrome, which is mainly caused by mutations in CSF2RA. So far, however, no murine Csf2ra−/− strain is available. Also, murine and human lung architecture and immunology may differ with regard to specific macrophage homing mechanisms, and it remains to be investigated whether the treatment effects achieved in the two mouse models presented here can be translated into human herPAP patients. With regard to the marked and long-term efficacy of the organotropic transplantation in our study, some specific characteristics of AM biology should be taken into account. AMs are long-lived and display a low natural turnover (39, 40). Recent publications indicate that under physiologic conditions, AMs are maintained primarily from a local, long-lived pool of lung-resident progenitors, and this process is critically regulated by GM-CSF (13, 14, 41). In our two models, a single PCT treatment with macrophage progenitors resulted in sustained pulmonary macrophage engraftment and clinical improvement, which was maintained for the complete observation period of 9 months in Csf2rb−/− mice and 6.5 months in the huPAP model. These results support the notion that even a single intrapulmonary transplantation to herPAP patients may provide long-term beneficial effects on their disease course. Because of the relatively normal natural life span of Csf2rb−/−, as well as huPAP mice, we chose to analyze surrogate parameters for PAP disease activity, such as BAL protein content, lung function, and radiographic findings, rather than survival as the primary endpoint for treatment efficacy in our studies. However, we did not address the exact duration of the effect of our treatment by these parameters beyond our longest observation period of 9 months. Another shortcoming of our study is the fact that we cannot clearly say which cell macrophage progenitor cell type was most relevant for the therapeutic effect in our models and whether the transplanted cells proliferated or persisted in the recipient’s lungs. We aimed at addressing the latter point by analyzing the frequency of donor-derived cells within the BALF and lungs of recipient animals at different time points after transplantation by flow cytometry. However, the technical limitations of flow cytometry of murine herPAP lung and BALF cells, given the high autofluorescence and granularity of donor-derived cells and contaminating autofluorescent lipid droplets naturally occurring in herPAP, need to be taken into account when interpreting these results, and we

www.ScienceTranslationalMedicine.org

20 August 2014

Vol 6 Issue 250 250ra113

7

RESEARCH ARTICLE have addressed these limitations by adding other techniques like RT-PCR and chipcytometry. GM-CSF, the key cytokine in herPAP pathophysiology (1, 5, 6), attracts macrophages and stimulates AM differentiation, proliferation, and survival (32, 34, 42). Because of the dysfunctional negative feedback loop governing AM proliferation in herPAP, GM-CSF levels are markedly elevated in serum and even higher in the lungs of affected patients and mice (5, 9, 22). The GM-CSF responsiveness of the transplanted cells may explain the selective and long-term pulmonary engraftment of donor-derived macrophages in our models. Most likely, also in the clinical setting, high pulmonary GM-CSF would favor a selection advantage and exclusive pulmonary residence of GM-CSF– responsive macrophages. Together, we introduce intrapulmonary transplantation of macrophage progenitors as a simple and effective treatment for herPAP, achieving prolonged and substantial therapeutic effects in this life-threatening disease. Our concept of organotropic transplantation of rather mature cells may not only be relevant for herPAP but also could serve as a proof of principle for other diseases, expanding current stem cell–based treatment strategies toward potent macrophage-based strategies.

MATERIALS AND METHODS Study subjects This study was approved by the institutional review board of the Hannover Medical School. All study subjects or their legal guardians gave written informed consent, and minors gave assent. Blood samples were collected in heparinized tubes. Human CD34+ cells were isolated from healthy individual cord blood mononuclear cells. Mice Csf2rb−/− mice (15) and huPAP (129S4-Rag2tm1.1Flv Csf2/Il3tm1.1(CSF2,IL3)Flv Il2rgtm1.1Flv/J) mice (22) were obtained from the Jackson Laboratory and housed in the central animal facility of Hannover Medical School. NSG and B6.SJL-Ptprca-Pep3b/BoyJ (CD45.1) donor mice were obtained from the central animal facility of Hannover Medical School. OT-II mice were provided by O. Pabst from Hannover Medical School, now Aachen University, Germany. Mice were kept under specific pathogen–free conditions, and NSG and huPAP mice were kept in individually ventilated cages. All mice had free access to food and water, and all animal experiments were approved by the Lower Saxony State animal welfare committee and performed according to their guidelines. Study design The objective of the study was to analyze the therapeutic potential of intrapulmonary transplanted macrophages in herPAP mice over time. For this purpose, two different mouse strains, Csf2rb−/− and huPAP mice, were observed for 6 weeks up to 9 months after intrapulmonary transplantation of either murine or human macrophage progenitors and compared to untreated control animals. Readout was engraftment of donor-derived cells in the lungs of recipient animals, BALF protein content, CCT, and lung function measurements. Experiments were performed two to three times with age- and gender-matched mice within respective groups. The analysis of mice was performed in a blinded way for CCT, protein measurements in the BALF, and determination of cell sizes based on electron microscopy images.

Murine BMDMs/BMDDCs For preparation of murine BMDMs and BMDDCs, bones from the hind legs of adult CD45.1+ donor mice were flushed. Bone marrow was rinsed through a 70-mm cell strainer (Becton Dickinson) followed by red blood cell depletion (0.83% NH4Cl, 0.5% KHCO3, 0.5 mM EDTA). Lin− bone marrow cells were counted and seeded at a density of 2 × 105 cells in a 2.5-cm-diameter petri dish. To obtain BMDMs and cells for transplantation, cells were cultured in standard RPMI medium (Lonza) supplemented with 10% fetal bovine serum, 1% penicillin/ streptomycin (Biochrom AG), and 30% L929/mM-CSF supernatant, as previously described (43). Medium was replaced every second day. After 4 days, adherent cells were washed and disattached by trypsinization. Cells were then washed, counted, and further analyzed or used for transplantation. Mature BMDMs were harvested at day 8 of culture. For BMDDCs, lin− bone marrow cells were seeded in standard medium with mGM-CSF (10 ng/ml) (Sigma-Aldrich). Medium was changed every second day, and BMDDCs were harvested on day 8. Human CD34 cell–derived macrophages and dendritic cells and primary human BALF cells Human cord blood CD34+ cells were purified and differentiated in 12-well plates in standard medium containing hIL-3 (20 ng/ml), hIL-6 (20 ng/ml), hM-CSF (100 ng/ml), hGM-CSF (20 ng/ml), and hFlt-3 ligand (100 ng/ml; all from PeproTech). Cells were taken at day 4, washed, and transplanted intrapulmonary or further characterized. To obtain mature CD34-derived macrophages, cells were taken after 11 days of culture. For dendritic cell differentiation of CD34+, cells were sorted and cultured 11 days in standard medium containing hGM-CSF (50 ng/ml), hTNF-a (human tumor necrosis factor–a) (10 ng/ml), and hFlt-3 ligand (100 ng/ml). Medium was changed every 3 days. Human bronchoalveolar lavage collection and cell isolation were conducted according to local standard operation procedures. To obtain various cell types of BALF leukocytes (Fig. 5C), a sample from a patient with lung inflammation was analyzed. Pulmonary transplantation of murine and human macrophage progenitors At the age of 10 to 14 weeks, mice were pulmonary transplanted with 2 × 106 cells per animal. Before transplantation, mice were anesthetized with ketamine/Rompun or propofol (Bayer/Gräub or Braun). When fully unconscious, they were carefully intubated orotracheally with a 20-gauge cannula (Braun). After confirming successful tracheal intubation (careful ventilation maneuvers with a 2-ml Pasteur pipette fitted to distal end of cannula), the cells were applied in phosphatebuffered saline (PBS) in a total volume of 50 to 80 ml, followed by three careful ventilation maneuvers. Thereafter, mice were extubated and kept under an infrared lamp until full recovery from anesthesia. Cell engraftment was confirmed postmortem by flow cytometry 6 weeks to 9 months after transplantation. Three mice for the 6-week time point in the Csf2rb−/− model were excluded from analyses based on nonengraftment. Rodent CCT imaging methodology and data processing Micro-CCT images were acquired on a small-animal scanner (eXplore CT120, TriFoil Imaging) in spontaneously breathing, continuously warmed animals anesthetized with 1.0 to 1.5% isoflurane. Respiratory monitoring was performed to detect maximal inspiration and expiration for gating. CCT acquisition parameters were set to 80 kV, 50 mA, 16-ms

www.ScienceTranslationalMedicine.org

20 August 2014

Vol 6 Issue 250 250ra113

8

RESEARCH ARTICLE exposure time, 386 views, and 0.5° increment angle. For reconstruction, filtered back-projection with a binning of 2 was performed, resulting in isotropic voxel dimensions of 98.3 mm. Postprocessing of inspiratory and expiratory micro-CCT data was performed by use of Visage 7.1.3 (Visage Imaging). A region growing algorithm was used for segmentation of lung parenchyma and interstitial structures, applying a threshold of −205.63 Hounsfield units and two dilatations of the volume. Segmented parts of the airways cranial to the tracheal bifurcation were removed manually (3D rendering of micro-CCT and segmented volume; fig. S5C). Resulting segmented volumes and average densities were calculated with Visage 7.1.3 and further processed in Prism V5 (GraphPad). Measurement of lung function Lung function was assessed as described previously (44). Briefly, conscious mice, breathing ambient air, were placed in a whole-body plethysmograph (emka TECHNOLOGIES). Mice were kept for 10 min in a body plethysmographic chamber to accommodate before measurements. Then, breathing parameters were recorded for at least 5 min, and the data were further processed with Prism V5 (GraphPad). Collection of murine BALF and measurement of protein amount and GM-CSF levels Murine bronchoalveolar lavage was performed by cannulating the murine trachea postmortem, and the right lung was lavaged three times with 400 ml of PBS. Aliquots of fresh BALF were stained for flow cytometry or immediately frozen to −80°C and stored until further analysis. Protein concentrations from BALF samples were measured using Pierce BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer’s instructions. BALF GM-CSF levels in the huPAP model were determined by means of cytometric bead array technology [BD Cytometric Bead Array (CBA) Flex Set human GM-SCF, BD Biosciences]. Preparation of lung homogenates Lung homogenates were prepared by means of the gentleMACS system (Miltenyi Biotec). Briefly, lungs were blended into