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Pulmonary macrophage transplantation therapy

ARTICLE

doi:10.1038/nature13807 Pulmonary macrophage transplantation therapy

Takuji Suzuki1,Paritha Arumugam2,Takuro Sakagami1,Nico Lachmann3,Claudia Chalk1,Anthony Sallese1,Shuichi Abe1,

Cole Trapnell4,5,Brenna Carey1,Thomas Moritz3,Punam Malik2,Carolyn Lutzko2,Robert E.W ood6&Bruce C.Trapnell1,6,7

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-b-deficient(Csf2rb2/2)mice that develop a myeloid cell dis-order 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 PMTas the first specific therapy for children with hPAP.

Mutations in CSF2RA or CSF2RB,encoding GM-CSF receptor a or b, respectively,cause hPAP by impairing GM-CSF-dependent surfactant clearance by alveolar macrophages,resulting in progressive surfactant accumulation in alveoli and hypoxaemic respiratory failure1–5.Surfac-tant normally comprises a thin phospholipid/protein layer reducing ten-sion on the alveolar surface6that is maintained by balanced secretion by alveolar type II epithelial cells and clearance by these cells and alveolar macrophages7,8.PAP also occurs in people with GM-CSF autoantibodies (,85–90%of all patients with PAP)9,10and mice with disruption of the GM-CSF gene Csf2(refs11,12)or the GM-CSF receptor b-subunit gene Csf2rb(refs13,14)(Csf22/2or Csf2rb2/2mice,respectively).Charac-teristics of PAP caused by disruption of GM-CSF signalling include typical lung histopathology(well preserved alveoli filled with surfactant and‘foamy’macrophages staining positive with periodic acid-Schiff (PAS)or oil red O);turbid,‘milky’appearing bronchoalveolar lavage (BAL)caused by accumulated surfactant and cell debris;and a disease-specific pattern of biomarkers(increased GM-CSF(hPAP),M-CSF/CSF1 and MCP-1in BAL fluid,and reduced mRNA for PU.1,PPARG and ABCG1in alveolar macrophages)1–5,15–20.

Currently,no pharmacological therapy of hPAP exists and surfactant must be removed by whole-lung lavage,an inefficient,invasive procedure to physically remove excess surfactant2–4.In Csf2rb2/2mice,PAP was corrected by bone marrow transplantation(BMT)of wild-type(WT)21 or Csf2rb-gene-corrected Csf2rb2/2haematopoietic stem/progenitor cells(HSPCs)22.However,in humans this approach resulted in death from infection before engraftment2,probably as a result of required myeloabla-tion/immunosuppressive therapy.Since pulmonary GM-CSF is increased in hPAP1–5we hypothesized that macrophages administered directly into the lungs(pulmonary macrophage transplantation or PMT)with-out myeloablation would engraft and reverse the manifestations of hPAP. We first validated Csf2rb2/2mice as a model of human hPAP by dem-onstrating that they had the same clinical,physiological,histopathological and biochemical abnormalities,disease biomarkers and natural history (Fig.1and Extended Data Fig.1)as children with hPAP3. Characterization of macrophages before PMT

Bone-marrow-derived macrophages(BMDMs)from WT mice had mor-phology and phenotypic markers(F4/801,CD11b Hi,CD11c1,CD141, CD16/321,CD641,CD681,CD1151,CD1311,SiglecF Low,MerTK1, MHC class II1,Ly6G2,CD32,CD192)of macrophages(Extended Data Fig.2a–c)and contained,0.0125%lineage negative(Lin2)Sca11c-Kit1(LSK)cells.Clonogenic analysis indicated,0.005%colony-forming units-granulocyte,monocyte/macrophage(CFU-GM)and no burst-forming units-erythrocyte(BFU-E)or colony-forming units-granulocyte, erythrocyte,monocyte/macrophage,megakaryocyte(CFU-GEMM)pro-genitors(Extended Data Fig.2d,e).Functional evaluation23showed that the BMDMs could clear surfactant(Extended Data Fig.2f,g).These results demonstrated that 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,Csf2rb2/2mice received WT(Csf2rb1/1)BMDMs by PMT once(Fig.1a).One year later,PMT-derived CD1311BAL cells were present(Fig.1b),alveolar macrophages expressed Csf2rb(ExtendedDataFig.3a),andBALwasmarkedlyimproved with respect to opacification(Fig.1c),sediment(Fig.1c)and microscopic cytopathology(Extended Data Fig.3b).Importantly,PMT nearly com-pletely resolved the abnormal pulmonary histopathology(Fig.1d and Extended Data Fig.3c).Measurement of BAL turbidity and surfactant protein-D(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 CD1311alveolar macrophages with Csf2rb mRNA and protein(not

1Division of Pulmonary Biology,Perinatal Institute,Cincinnati Children’s Hospital Medical Center,3333Burnet Avenue,Cincinnati,Ohio45229,USA.2Division of Experimental Hematology,Cincinnati Children’s Hospital Medical Center,3333Burnet Avenue,Cincinnati,Ohio45229,USA.3RG Reprograming and Gene Therapy,Institute of Experimental Hematology,Hannover Medical School,Carl Neuberg-Str.1,30625Hannover,Germany.4Department of Stem Cell and Regenerative Biology,Harvard University,Cambridge,Massachusetts02138,USA.5Broad Institute of Massachusetts Institute of Technology and Harvard University,Cambridge,Massachusetts02138,USA.6Division of Pulmonary Medicine,Cincinnati Children’s Hospital Medical Center,3333Burnet Avenue,Cincinnati,Ohio45229, USA.7Division of Pulmonary,Critical Care,and Sleep Medicine,University of Cincinnati Medical Center,3333Burnet Avenue,Cincinnati,Ohio45229,USA.

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shown),reduced BAL opacification and cytopathology(not shown),BAL turbidity(Fig.1e),SP-D(Fig.1e)and BAL fluid biomarkers(Fig.1f) 2months after PMT,and reduced lung histopathology4months after PMT(not shown).In contrast,PMT of Csf2rb2/2BMDMs had no effect on BAL turbidity,SP-D content,or BAL fluid biomarkers(not shown), demonstrating that GM-CSF receptors on transplanted macrophages are important for the therapeutic effects.

To evaluate the effects of PMT on the alveolar macrophage popula-tion,we measured cellular biomarkers after PMT.Results showed that alveolar macrophages from PMT-treated Csf2rb2/2mice had increased mRNA for PU.1,Pparg and Abcg1,improvement was significant by 2months,and the effects persisted1year after PMT(Fig.1g). Since Csf2rb2/2mice develop polycythaemia,a secondary conse-quence of hypoxaemia in chronic lung diseases24,we evaluated the effects of PMT on this systemic clinical manifestation.Notably,PMT corrected polycythaemia in Csf2rb2/2mice(Fig.1h).

Finally,we evaluated the effects of PMT on hPAP-associated mortality by comparing the survival of PMT-treated and untreated Csf2rb2/2 mice.PMT increased the lifespan of Csf2rb2/2mice by107days,from 555(median;interquartile range507–592)days to662(604–692)days (Fig.1i).In separate studies of treated Csf2rb2/2mice surviving to617(604–631)days(561(548–575)days after PMT of WT BMDMs),CD1311 alveolar macrophages were still present and BAL turbidity remained low compared to untreated Csf2rb2/2mice that survived to631(631–631) days(optical density at600nm(OD600)50.7560.17versus2.6360.44; n58,4,respectively;P,0.001).However,such long-term evaluation of laboratory abnormalities is obfuscated by reduced survival of untreated Csf2rb2/2mice.

These results demonstrate that PMT had a highly efficacious and durable therapeutic effect on the primary pulmonary and secondary sys-temic manifestations of hPAP in Csf2rb2/2mice.

Macrophage engraftment efficiency

We next evaluated the effects of cell dose(0.5,1,2and4million)and repeated administration(one versus four monthly transplantations) on PMT efficacy(Extended Data Tables2and3,respectively).Neither treatment significantly affected efficacy in the range evaluated,and one dose of2million cells was used for PMT in the remaining studies. To determine whether WT macrophages had a survival advantage over Csf2rb2/2macrophages,we measured GM-CSF bioactivity in BAL fluid and found that it was detectable in Csf2rb2/2but not WT mice (Extended Data Fig.1h).WT macrophages had increased survival/pro-liferation compared to Csf2rb2/2macrophages in vitro(Fig.2a)and accumulated to greater numbers after PMT in Csf2rb2/2mice than in WT mice(Fig.2b and Extended Data Fig.3d).PMT of WT Lys-M GFP knock-in mouse25BMDMs into Csf2rb2/2mice followed by Ki67immu-nostaining revealed that PMT-derived cells replicated in vivo(Extended Data Fig.3e–g).The percentage of Ki671PMT-derived alveolar macro-phageswas32.266.05%1month afterPMT and declined to11.2962.2% by1year(Fig.2c)similar to baseline Ki671immunostaining of alve-olar macrophages in age-matched,normal WT mice(Extended Data Fig.3f).To define this survival advantage further,we evaluated the engraftment kinetics after one PMT of WT BMDMs in Csf2rb2/2mice. CD1311cells increased steadily from zero to69.062.5%of BAL cells (Fig.2d)synchronous with a smooth decline in pulmonary GM-CSF to

–

(

–

)

(

)

(

–

)

}PMT{

WT KO

PU.1 Pparg

Abcg1

h i

Age (days)

d WT KO KO + PMT

–

– +

–

Figure1|Therapeutic efficacy of PMT in Csf2rb2/2mice.a,Schematic of

the method used.WT HSPCs(1)were isolated,expanded(2),differentiated

into macrophages(3),and administered by endotracheal instillation into

2-month-old Csf2rb2/2(KO)mice(4)and evaluated after2months(2M)

(e–g)or one year(1Y)(b–h)with age-matched,untreated WT or Csf2rb2/2

mice(KO1PMT,WT or KO,respectively).b,CD131-immunostained BAL

cells.c,Appearance of BAL fluid(left)or sediment(right).d,Lung histology

after staining with haematoxylin and eosin(H&E),PAS,Masson’s trichrome

(MT),or surfactant protein B(SP-B).Scale bar,100m m;inset,50m m.

e,BAL turbidity and SP-D concentration.f,BAL biomarkers.g,Alveolar

macrophage biomarkers.h,Effects of PMT on blood haemoglobin(Hb),

haematocrit(Hct)and serum erythropoietin(Epo).i,Kaplan–Meier analysis of

PMT-treated(n543)and untreated Csf2rb2/2mice(n548).Images are

representative of6mice per group(b–d).Numeric data are mean6s.e.m.of

7(2M)or6(1Y)mice per group.*P,0.05,**P,0.01,***P,0.001,

****P,0.0001.h

+PMT

(

)

(

)

Time (days)

+PMT

–

Time after PMT (months)

Months

Figure2|Pharmacokinetics and pharmacodynamics of PMT in Csf2rb2/2

mice.a,Competitive proliferation of WT and Csf2rb2/2BMDMs co-cultured

with GM-CSF and M-CSF(n53plates per point).b,Quantification of

GFP1BAL cells2months after PMT of Lys-M GFP BMDMs into WT(n53)or

Csf2rb2/2(n56)mice.c,Quantification of Ki671Lys-M GFP cells in Csf2rb2/2

mice(n53)1or12months after PMT.d–f,Csf2rb2/2mice received PMT

of WT BMDMs and were evaluated at the indicated times to quantify CD1311

BAL cells(d),BAL GM-CSF concentration(e)and BAL turbidity(f).

Exponential regression(6prediction bands),R250.943(d),R250.819(e),

R250.958(f).Data are mean6s.e.m.for3–7mice per group.g,Csf2rb mRNA

in BAL cells from Csf2rb2/2mice1year after PMT,or untreated,age-matched

control mice(n56).h,Number of BAL cells(open bars)or CD1311

alveolar macrophages(filled bars)in Csf2rb2/2mice1year after PMT(n55)

or untreated WT mice(n510).Data are mean6s.e.m.*P,0.05,

***P,0.001;NS,not significant.

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near normal (Fig.2e).Similarly,BAL turbidity declined with the increase in CD1311alveolar macrophages (Fig.2f).One year after PMT,CD1311cells were present (Fig.1b),CD131protein (encoded by Csf2rb )was detectable in alveolar macrophages (Extended Data Fig.3a),and Csf2rb mRNA in BAL cells from PMT-treated Csf2rb 2/2mice was only slightly less than in WT and undetectable in untreated Csf2rb 2/2BAL cells (Fig.2g).Importantly,numbers of CD1311alveolar macrophages in PMT-treated Csf2rb 2/2and untreated WT mice were similar 1year after PMT (Fig.2h).These results demonstrate that WT macrophages had a selective survival advantage over Csf2rb 2/2macrophages and that after PMT into Csf2rb 2/2mice,they proliferated in vivo at a rate that slowed over time synchronous with reduction in pulmonary GM-CSF,replaced dysfunctional Csf2rb 2/2alveolar macrophages,and resulted in numbers of CD1311,GM-CSF-responsive alveolar macrophages similar to WT mice.

Macrophage characterization after PMT

The fate of macrophages after PMT was evaluated to determine their spa-tial distribution,phenotype and gene expression profile.Intra-pulmonary localization was evaluated 1year after PMT of WT Lys-M GFP BMDMs by fluorescence microscopy to identify CD681GFP 1(that is,PMT-derived)macrophages,which revealed that 88.960.87%were intra-alveolar and 11.160.87%were interstitial (Extended Data Fig.3h).GFP immuno-histochemical staining was done to eliminate potential interference from autofluorescence and confirmed these results;90.561.1%PMT-derived macrophages were intra-alveolar and 9.461.1%were interstitial (Fig.3a,b and Extended Data Fig.3i).Localization was done in similarly treated mice by flow cytometry to detect GFP 1cells 2months (not shown)or 1year after PMT (Fig 3c and Extended Data Fig.4a,b)and by PCR amplification of Lys-M GFP transgene-specific DNA (Extended Data Fig.4c),all of which showed that PMT-derived cells were present in the lungs but not detected in blood,bone marrow,or spleen.One year after PMT of CD45.11WT BMDMs into CD45.21Csf2rb 2/2mice,flow cytometric detection of CD45.11cells confirmed these findings (Extended Data Fig.4e–g).Results show that the transplanted macro-phages remained in the lungs,primarily within the intra-alveolar space.The effects of the lung environment on the phenotype of transplanted macrophages were evaluated by measuring cell-surface markers.One year after PMT of WT Lys-M GFP BMDMs into Csf2rb 2/2mice,PMT-derived alveolar macrophages comprised 68.766.5%of BAL cells and had converted from CD11b Hi SiglecF Low to CD11b Low SiglecF Hi ,sim-ilar to the phenotype of WT alveolar macrophages and different from Csf2rb 2/2mice at the point of PMT (CD11b Hi SiglecF Low )(Fig.3c,d).Similarly,one year after PMT of WT CD45.11BMDMs into CD45.21Csf2rb 2/2mice,CD45.11alveolar macrophages comprised 63.6612.1%

of BAL cells and had undergone the same phenotypic conversion (Ex-tended Data Fig.4h).

To determine the effects on gene expression,we performed genome-wide expression profiling on alveolar macrophages from Csf2rb 2/2mice 1year after PMT of WT BMDMs and compared to results for untreated,age-matched WT or Csf2rb 2/2mice.Unsupervised analysis indicated marked co-clustering between PMT-treated Csf2rb 2/2and WT mice while Csf2rb 2/2mice clustered separately (Fig.4).Expression of genes regulated by GM-CSF was reduced in Csf2rb 2/2mice and restored by PMT (Fig.4and Extended Data Fig.5a).Of 776genes for which expres-sion was disrupted in Csf2rb 2/2mice,PMT normalized expression of 600including 80%of genes upregulated and 76%of genes downregu-lated in Csf2rb 2/2compared 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 defence were coordinately down-regulated in Csf2rb 2/2mice,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 humanswould probably employ autologous,gene-corrected HSPC-derived macrophages,we evaluated PMT of Csf2rb 2/2macro-phages derived from 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)Csf2rb 2/2and non-transduced WT LSK-derived cells all had macrophage morphology,expressed CD68(Extended Data Fig.6a)and were F4/801CD11b Hi CD11c 1(not shown).In contrast,only WT and GM-R-LV-transduced Csf2rb 2/2cells were CD1311and only lentiviral-vector-transduced cells were GFP 1(Extended Data Fig.6a).GM-R-LV restored GM-CSF signalling in Csf2rb 2/2mac-rophages (Fig.5b and Extended Data Fig.6b).Two months after PMT into Csf2rb 2/2mice,GM-CSF receptor-b was detected on alveolar mac-rophages only from mice receiving gene-corrected Csf2rb 2/2or WT macrophages (Extended Data Fig.6c).The efficacy using gene-corrected Csf2rb 2/2or WT cells was equivalent as demonstrated by a similar degree of improvement in BAL appearance (Extended Data Fig.6d),BAL turbidity,SP-D and biomarkers of hPAP (Fig.5c,d).Furthermore,gene-corrected BMDMs localized to the lung (Extended Data Fig.4d and Fig.6e)and underwent phenotypic conversion to CD11b Low (Ex-tended Data Fig.6f).The long-term efficacy of gene-corrected macro-phages 1year 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 Csf2rb 2/2mice equivalent to that of WT macrophages and was durable,lasting at least 1year.

Safety of PMT therapy in Csf2rb 2/2mice

PMT was well tolerated and without adverse effects.One year after PMT,

there were no haematological abnormalities (Extended Data Table 4),cellular inflammation or pulmonary fibrosis in mice receiving PMT of WT (Fig.1d)or gene-corrected macrophages (not shown).Csf2rb 2/2

P M T c e l l s (%)

FSC

SSC

Fluorescence

K O +P M T

Untreated PMT- treated 67.7%

1.8% Alveolar

Interstitial

Figure 3|Localization and phenotype of transplanted macrophages.

Lys-M GFP BMDMs were transplanted into Csf2rb 2/2mice and evaluated after 1year.a ,Immunostained lung showing GFP 1cells.Scale bars,left,200m m;right,20m m.b ,Localization of GFP 1macrophages to intra-alveolar (A)and interstitial (I)spaces (n 56).c ,GFP 1BAL cells identified by flow cytometry.d ,Phenotypic analysis of F4/801BMDMs before PMT,and alveolar

macrophages from PMT-treated Csf2rb 2/2mice,or untreated,age-matched WT or Csf2rb 2/2mice (n 56per group).Data are mean 6s.e.m.

1 – PCC

PMT

Figure 4|Microarray analysis of alveolar macrophages 1year after PMT.Unsupervised hierarchical clustering dendrogram and heat map of selected GM-CSF-regulated genes in PMT-treated Csf2rb 2/2mice or untreated,age-matched WT or Csf2rb 2/2mice (3per group).Pearson correlation coefficient (PCC).

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mice had trivial elevations of IL-6and TNF-a in BAL that were reduced by PMT of WT macrophages (Extended Data Table 4).These data iden-tify no safety concerns for PMT therapy of hPAP in Csf2rb 2/2mice.

Discussion

Multiple lines of evidence indicate that the high efficacy of PMT ther-apy of hPAP in Csf2rb 2/2mice was attributable to a selective survival advantage conferred by increased pulmonary GM-CSF to alveolar mac-rophages bearing functional GM-CSF receptors.However,pulmonary surfactant remained slightly increased 1year after a single PMT.This could be because the treatment time was too short or exceeded the dura-bility of the clinical benefit,or due to the continued presence of Csf2rb 2/2alveolar macrophages despite engraftment of GM-CSF-responsive mac-rophages.The latter is likely due to ongoing Csf2rb 2/2myelopoiesis,pulmonary recruitment of monocytes and local proliferation,and GM-CSF-independentsurvivalasoccursinuntreated Csf2rb 2/2mice.Csf2rb 2/2macrophages may provide a ‘protected intracellular niche’for surfac-tant accumulation since without GM-CSF,alveolar macrophages inter-nalize but cannot clear surfactant 26.Another factor may be reduction of the survival advantage over time,that is,reduced pulmonary GM-CSF (driving WT cell proliferation)and reduced surfactant burden (driving surfactant-engorgement-related Csf2rb 2/2cell death).Notwithstanding these points,a single PMT of GM-CSF responsive cells cleared ,90%of the abnormal surfactant accumulation for at least one year.

The feasibility of translating PMT therapy to humans with hPAP is supported by the safety and efficacy of PMT in Csf2rb 2/2mice and the Macrophages could endotracheal intuba-which are required myeloablation would

be unnecessary and use of autologous,gene-corrected cells would elim-inate the need for immunosuppression,which are required for BMT.PMT may also be possible with gene-corrected,inducible pluripotent-cell-derived macrophages recently prepared from children with hPAP 23,27.However,formal preclinical toxicology studies related to PMT and to gene transfer will be needed before this approach can be tested in humans.Since pulmonary GM-CSF is critical to lung host defence and clearance of a broad range of microorganisms 28,PMT may also be useful in treat-ing serious lung infections.Indeed,

pulmonary administration of mac-rophages constitutively expressing IFN-c improved host defence in SCID mice 29.In such applications,inhaled GM-CSF could be used to promote survival of transplanted macrophages 30.

Identification of a homeostatic mechanism by which

pulmonary GM-CSF regulates alveolar macrophage population size (Fig.6)was an unexpected but important finding.Its existence is supported by recent fate-mapping studies indicating that tissue-resident alveolar macrophages derive before birth and self-maintain by local replication independent of circulating monocytes

at steady state 30–33.

The concept of alveolar macrophages (and other tissue-resident mac-rophages)as short-lived,terminally differentiated,non-dividing repre-sentatives of a unified mononuclear phagocyte system replenished via monocyte intermediates has evolved considerably since its inception 34.Alveolar macrophage half-life was initially estimated at 2weeks based on studies of repopulation after lethal irradiation

and allogeneic BMT 35.Improved detection methods using GFP 1cells increased the estimate to 30days 36.Shielding the thorax during irradiation increased it further to 8months 37.Our data,obtained without irradiation or myeloablation,show that macrophages transplanted directly into the respiratory tract persisted for one-and-a-half years.A caveat of such estimates is

their inability to discern if persistence is due to prolonged survival or replication.Normally,alveolar macrophages are phenotypically CD11b Low SiglecF Hi while other macrophage populations are CD11b Hi SiglecF Low .Surpris-ingly,WT BMDMs cultured in GM-CSF and M-CSF were CD11b Hi SiglecF Low in vitro but converted to CD11b Low SiglecF Hi after PMT.In contrast,

BMDMs instilled in the peritoneum adopt the CD11b Hi phe-notype of peritoneal macrophages 38.These changes agree with gene-expression profiling studies 39and indicate that local microenvironments provide critical ‘phenotypically instructive’cues that direct development of

tissue-resident macrophage populations.Our results show for alveolar macrophages that GM-CSF provides one such phenotypic cue while the lung environment provides another critical,albeit unidentified,cue.The limitations of our study include the fact that it did not establish a minimum effective dose,a maximum tolerated dose,or a significant dose–response relationship.BMDMs were capable of clearing surfactant before transplantation but results did not determine whether ‘lung-conditioning’further increased their clearance capacity.While the macro-phages used for PMT contained very few progenitors,it is theoretically possible that clonal expansion of a progenitor subpopulation may have contributed to therapeutic efficacy and,if so,potential clonal shrinkage may have contributed to loss of benefit at later times.Thus,additional studies are needed to further confirm the identity of effector cells and precise pharmacokinetics and durability of the therapeutic benefit.

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Alveolar macrophages ?Survival

?Population size ?Differentiation Lungs

?Surfactant homeostasis ?Alveolar stability ?Oxygen uptake ?Host defence

Pulmonary GM-CSF

Precursor

GM-CSF

Blood Alveolus

Mature

macrophages

|Proposed mechanism by which pulmonary GM-CSF size in vivo.

M-CSF

MCP-1

p g m l –1 B A L

GM-CSF

KO, GM-R-LV, 1Y

SP-D O D 600

Turbidity S T A T 5-P I (×100)

+ GFP-LV:

+ GM-R-LV:––––Figure 5|Effects of PMT of gene-corrected macrophages on hPAP severity and biomarkers.Csf2rb 2/2mice received PMT of non-transduced WT or lentiviral-vector-transduced Csf2rb 2/2macrophages and were evaluated after 2months (2M)or 1year (1Y)(with untreated,age-matched Csf2rb 2/2mice).The key indicates PMT cells used,previous lentiviral vector treatment,and time after PMT analysis was performed.a ,Lentiviral vector schematics.b ,GM-CSF signalling measured by the STAT5phosphorylation index (STAT5-PI)in the indicated cells before PMT.c ,BAL turbidity and SP-D concentration.d ,BAL biomarkers.Mean 6s.e.m.of n 53(b )or 5–10(c ,d )mice per group.*P ,0.05,**P ,0.01.

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Acknowledgements This work was supported by grants from the NIH(R01HL085453, R21HL106134,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;Exc62/1),the Else Kro¨ner-Fresenius Stiftung,the

Eva-Luise Koehler Research Prize for Rare Diseases2013,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 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.

Author Information Reprints and permissions information is available at

https://www.wendangku.net/doc/d28995753.html,/reprints.Microarray data are available at Gene Expression Omnibus under accession number GSE60528.The authors declare no competing financial interests.Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to

B.T.(Bruce.Trapnell@https://www.wendangku.net/doc/d28995753.html,)or T.Su.(Takuji.Suzuki@https://www.wendangku.net/doc/d28995753.html,).

ARTICLE RESEARCH

00M O N T H2014|V O L000|N A T U R E|5

METHODS

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(Csf2rb2/2)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/6background.C57BL/6mice(referred to as wild type or WT mice)were purchased from Charles River.B6.SJL-Ptprc a Pepc b/BoyJ(CD45.11)mice were from Jackson Laboratory.

Lung histology and immunohistochemistry.Animals were killed by intraper-itoneal 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,pH7.4,containing4%paraformaldehyde)under a hydrostatic head of25cm 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 at4u C for24h.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,and5-m m-thick sections were cut and stained with haematoxylin 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 haematoxylin 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 en bloc and cryoprotected by sequential immersion in PBS containing increasing sucrose concentrations(10%,15%and20%;8–12h,4u C, at each concentration).The lungs were then embedded in Tissue-Tek OCT com-pound(Sakura Finetek,Torrance,CA),frozen and stored at280u C until use.Serial 6m m sections were prepared for immunostaining or evaluation of GFP1cells.Lung sections and sedimented lung cells were examined by light microscopy using a Zeiss Axioplan2microscope(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 described42and processed immediately. Briefly,five1-ml aliquots were instilled and immediately recovered per mouse and combined resulting in a BAL recovery of93.961.2%per mouse(BAL recovery data for10mice evaluated randomly).The photographs of fresh BAL specimens and the specimen after allowing sediment to be formed by overnight incubation at4u C were taken.The turbidity of BAL was determined as described41.Briefly,after gently mixing to ensure a homogeneous suspension of BAL,a250m l aliquot was diluted into750m l PBS and the optical density was measured at a wavelength of600nm 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 haemocytometer 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(,50,000cells)after sedimentation(Cytospin,Shandon,Inc.; 500r.p.m.,7min,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(285g, 10min,room temperature)and stored at280u C until use(BAL fluid)or imme-diately 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 mea-sured by enzyme-linked immunosorbent assay(ELISA)as we described41.The concentration of several cytokines(GM-CSF,M-CSF,MCP-1,IL-1b,IL-6,TNF-a) in BAL fluid and erythropoietin in serum was measured by ELISA(Mouse Quan-tikine 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 described1on an Applied Biosystems7300 Real-Time PCR System(Life Technologies)to measure transcript abundance using TaqMan oligonucleotide primer sets(all from Life Technologies)(Extended Data Table1).Expression of target genes was normalized to the expression of18s 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 from6–8-week-old WT,Csf2rb2/2,or Lys-M GFP mice by isolating and flushing tibias and femurs with DMEM(Life Technologies)containing10%heat-inactivated FBS,50U ml21penicillin,and50m g ml21streptomycin.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 for30min, washed,re-suspended in DMEM containing10%heat-inactivated FBS,50U ml21 penicillin,50m g ml21streptomycin,10ng ml21GM-CSF and5ng ml21M-CSF(both from R&D Systems),seeded into plastic dishes(Falcon)at a density of,273106 cells per10cm dish(1per mouse)and cultured overnight at37u C in a humidified environment containing5%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.After2days the culture medium was changed and after5days from seed-ing,adherent bone-marrow-derived macrophages 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 CD681and F4/801cells(96.660.3%,95.461.3%,respectively,not shown). Some experiments used lineage-negative(Lin2)c-Kit1Sca-11(LSK)cells which were obtained from mouse bone marrow as described44.Briefly,bone marrow from 6–8-week-old WT or Csf2rb2/2mice 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 Bio-science),and magnetic beads(Dynabeads sheep anti-rat IgG)(Life-Technologies). After removing lineage-positive cells,the remaining cells were stained with7-ADD, FITC-Streptavidin(BD Biosciences)and antibodies to Sca-1(D7)and c-Kit(2B8) (BD Biosciences).Then,Lin2c-Kit1Sca-117-ADD2cells were isolated by cell sort-ing on a FACSAria(BD Biosciences)and used immediately in experiments.Cell morphology was confirmed by DiffQuick Staining of sedimented cells(Cytospin, Shandon)and viability was measured by Trypan blue exclusion as described18and found to be$95%.In some experiments,cells were immunostained for CD68(FA-11)(AbD Serotec),counterstained with DAPI as described41,and examined by light microscopy using a Zeiss Axioplan2microscope(Zeiss)equipped with AxioVision software(Zeiss).

Colony forming cell(CFC)assay.BMDMs or Lin–bone marrow cells were eval-uated for the presence of haematopoietic progenitors capable of forming colonies in semisolid medium in response to cytokine stimulation as previously described45. Briefly,fresh Lin2bone marrow cells or BMDMs after induced differentiation into macrophages for5days were seeded into standard mouse methylcellulose media supplemented with insulin,transferrin,SCF,IL-3,IL-6and erythropoietin(HSC007, R&D Systems,Minneapolis,MN).After7days in culture,colonies of$50cells were visible and were examined morphologically using whole-plate stack images acquired using an AXIO-Z1microscope 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 Csf2rb2/2mice were seeded into12-well plates(43105cells per well)in DMEM,10%FBS,10ng ml21GM-CSF,5ng ml21M-CSF.Human surfactant recovered by lavage of a patient with PAP was added to the media and cells were incubated for24h to permit surfactant uptake into cells and then washed to remove extracellular surfactant.Cells were incubated for24h to permit macro-phages to clear internalized surfactant.Cells collected before,immediately after surfactant exposure or24h after the completion of surfactant exposure were sedi-mented onto slides by cytocentrifugation(Shandon),stained with oil red O,and counterstained with haematoxylin.Oil red O staining was evaluated in$10ran-dom203microscopic fields for each sample as described41.

Pulmonary macrophage transplantation(PMT).BMDMs or LSK cell-derived macrophages were administered directly into the lungs of8-week-old mice using a relatively non-invasive endotracheal instillation method described previously46. Briefly,mice received light anaesthesia by isoflurane inhalation and were suspended on a flat board by a rubber band across the upper incisors and placed in a semi-recumbent(45u)position with the ventral surface and rostrum facing upwards. Using a curved blade Kelly forceps,the tongue was gently and partially retracted rostrally,and50m l 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 anaesthesia to ensure continued

RESEARCH ARTICLE

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 mac-rophages was optimal,this dose given as one administration was used throughout the study except where noted(Extended Data Tables2and3).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 debris31.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 analysed using CellQuest,FACSDiva (BD Biosciences),or FlowJo software(Tree Star).For intracellular staining of CD68, Leucoperm(AbD Serotec)was used as directed by the manufacturer. Quantification of CD1311alveolar macrophages.The percentage of BAL cells expressing the GM-CSF receptor b-subunit was determined by immunostaining. Briefly,aliquots of BAL cells were sedimented onto glass slides and incubated(10min, room temperature)in fixative(PBS containing4%paraformaldehyde),washed with PBS and incubated(4u C,overnight)with anti-mouse GM-CSF receptor-b(CD131) antibody(sc-678)(Santa Cruz)diluted1:400in PBST(PBS containing2.5%(w/v) Triton X-100and5%(v/v)goat serum).After incubation,slides were rinsed five times in PBST and incubated(room temperature,1h)with the secondary detection antibody(Alexa-Fluor-594-conjugated,anti-rabbit IgG(Life Technologies))and counterstained with49,6-diamidino-2-phenylindole dihydrochloride(DAPI)(Vector Labs,Burlington,CA).Cells were examined using a Zeiss Axioplan2microscope (Zeiss)equipped with AxioVision software(Zeiss).The percentage of CD1311 BAL cells was determined by first counting the CD1311and DAPI1cells in five(or more)random203microscopic fields for each BAL sample.Then,the number of CD1311cells in each field was divided by the number of DAPI1cells in the same field and results for all fields examined were averaged and multiplied by100.The total number of CD1311cells per mouse was calculated by multiplying the percen-tage of CD1311cells by the total number of BAL cells recovered from each mouse. STAT5phosphorylation index assay.GM-CSF bioactivity in BAL fluid and GM-CSF receptor function in transduced or WT macrophages was evaluated by measur-ing GM-CSF-stimulated phosphorylation of STAT5in BMDMs or LSK cell-derived macrophages using anti-phospho STAT5antibody(47/Stat5(pY694))(BD Bio-sciences)by flow cytometry as previously reported1.The STAT5phosphorylation index(STAT5-PI)was calculated as the mean fluorescence intensity of phosphor-ylated STAT5staining in GM-CSF-stimulated cells minus that of non-stimulated cells,divided by that of non-stimulated cells,and multiplied by100.In experi-ments to quantify GM-CSF bioactivity,WT BMDMs were incubated in BAL fluid containing anti-GM-CSF(22E9,eBioscience)or isotype control antibody(10m g ml21) for30min and then evaluated.

Evaluation of macrophage proliferation.In vitro mixed-cell proliferation assay. CD45.11WT LSK-derived cells and CD45.21Csf2rb2/2LSK-derived cells were isolated,seeded into dishes at an initial ratio of1:3,respectively,and cultured in DMEM containing10%bovine calf serum,1%penicillin/streptomycin,GM-CSF (10ng ml21)and M-CSF(5ng ml21).Cells were collected at1,7,14and18days, immunostained with anti-murine CD45.1,anti-CD45.2and evaluated by flow cytom-etry to determine the percentage of each cell type at these times.

In vivo evaluation of transplanted macrophage proliferation.Frozen lung tissue sections were immunostained with anti-Ki67antibody(Roche)and examined using a Zeiss Axioplan2microscope(Zeiss).The percentage of proliferating PMT-derived cells was determined by enumerating GFP1Ki671cells among total GFP1cells in$7random203microscopic fields for each sample.To confirm the specificity of Ki67immunostaining,paraffin-embedded sections or WT alveolar macrophages isolated by BAL and adherence were also stained with Ki67and examined by light microscopy.

Western blotting.Detection of GM-CSF receptor-b and actin by western blot-ting was done as previously described1with the following modifications.Briefly, primary alveolar macrophages(0.53106per condition)or cultured BMDMs (13106per condition)were collected by low-speed centrifugation(285g,4u C, 10min)and the pellets incubated on ice for30min in200m l of lysis buffer(50mM Tris-HCl pH8.0,150mM NaCl,1%(v/v)nonidet p-40,0.5%(w/v)sodium deox-ycholate,0.1%(w/v)sodium dodecyl sulphate(SDS),0.004%(w/v)sodium azide) containing2%(v/v)proteinase inhibitor cocktail(phenyl-methyl-sulphonyl-fluoride and sodium orthovanadate;Santa Cruz).Insoluble debris was removed by centri-fugation at10,000g,4u C,15min and the supernatant transferred to a clean poly-propylene tube.An equal volume of Laemmli sample loading buffer(Bio-Rad,CA) was added and the tubes were capped tightly,vortexed briefly,boiled for5min, 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(50mM Tris-HCl pH8.0,150mM NaCl,5%(w/v)non-fat dry milk(Kroger,Cincinnati,OH), 0.1%(v/v)Tween20;4u C,overnight)to blocknon-specific binding.Diluted primary detection antibody(see below)was added and the membranes were incubated for2h at room temperature and then washed in TBST(50mM Tris-HCl pH8.0,150mM NaCl,0.1%(v/v)Tween20).Membranes were then incubated with the secondary HRP-conjugated detection antibody in blotting solution for1h 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-b antibody(sc-678) (Santa Cruz)diluted1:500and anti-actin(sc-1616)(Santa Cruz)diluted1:1,000 were used for primary antibodies.

Haematological analysis.Blood was obtained from the superior vena cava from mice and20m l was used to measure complete blood counts on a fully automated Hemavet850(Drew Scientific).Data for the precision and linearity of measure-ments made with the Hemavet850can be found online at https://www.wendangku.net/doc/d28995753.html,/product_hemavet850.htm.

Microarray analysis.Alveolar macrophages were obtained from age-matched mice(three per condition)and analysed individually as follows.Total RNA was isolated as described above and microarray analysis was performed using the Mouse Gene1.0ST Array(Affymetrix,Santa Clara,CA)in the CCHMC Affymetrix Core using standard procedures as described47.Data(available at Gene Expression Omnibus accession GSE60528)were analysed using the Affymetrix package in the R statis-tical programming language(Bioconductor;https://www.wendangku.net/doc/d28995753.html,).Probes were corrected for background using the Microarray Analysis Suite algorithm,quan-tile normalized,and probe sets were summarized using the average difference of perfect matches only.Differential expression tests were performed using signifi-cance analysis of microarrays48with Benjamini–Hochberg correction for multiple testing49.Significant gene lists were selected with a D that constrained the false dis-covery rate to less than10%.Cluster dendrogram was generated from unsuper-vised hierarchical clustering analysis of microarray data from probes for all28,853 genes represented on the chip(Spearman correlation;3mice per group).In Venn diagrams,numbers of genes for which expression was altered in alveolar macro-phages from Csf2rb2/2compared to WT mice(WT R KO)or PMT-treated com-pared to untreated Csf2rb2/2mice(KO R KO1PMT)were shown.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 Csf2rb2/2mice 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 of10%calcu-lated 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 rou-tine methods44from the vector backbone of(Ery-GFP),a human immunodeficiency virus-based,self-inactivating(SIN)lentiviral vector(LV)harbouring a398-bp U3 deletion eliminating the strong viral promoter/enhancer element50.GM-R-LV con-tains a chimaeric transgene comprised of the human elongation factor1-a(ELF-1a) promoter(a1,189-bp fragment containing intron1ending20bp upstream of the ATG codon isolated from the pEF-BOS plasmid51)followed by the mouse Csf2rb cDNA(nucleotides280to2,691,GenBank accession number M34397.1)located 39of the lentiviral central poly-purine tract and followed by an internal ribosome binding site(IRES)and then an enhanced green fluorescent protein(GFP)trans-gene(Fig.5a).GFP-LV is a lentiviral vector of similar design except that the Csf2rb and IRES were omitted and the GFP transgene is driven from the ELF1a promoter (Fig.5a).Both vectors contain a viral splice donor site,packaging sequence,splice acceptor site,and central polypurine tract(cPPT)59of the ELF1a promoter and a woodchuck hepatitis post-transcriptional regulatory element(WPRE)(nucleotides 1093to1684;GenBank accession number J04514)52located39of the GFP stop codon as described50.Lentiviral vectors were produced by transient transfection as vesicular stomatitis virus-G(VSVG)virions,concentrated,and titred as described44.Csf2rb2/2 LSK cells were isolated,transduced and expanded as described53except that transduc-tions were done at a multiplicity of infection(MOI)of20for two12-h periods,IL-11 was omitted,GM-CSF(10ng ml21)and M-CSF(5ng ml21)were included,SCF and Flt-3ligand were sequentially reduced(50,1,0ng ml21),and IL-3was 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 ml21,mIL-3(PeproTech)10ng ml21,hFlt3-L (PeproTech)50ng ml21and GM-CSF(R&D)10ng ml21,culture time of two12-h periods;(2)progenitor expansion:mSCF50ng ml21,hFlt3-L50ng ml21and GM-CSF10ng ml21,culture time of4days;(3)macrophage lineage commitment:mSCF 1ng ml21,hFlt3-L1ng ml21,GM-CSF10ng ml21,M-CSF(R&D)5ng ml21,culture

ARTICLE RESEARCH

time of3days;and(4)macrophage differentiation:GM-CSF10ng ml21and M-CSF5ng ml21,culture time of4days.StemSpan(STEMCELL Technologies) containing2%FBS,1%penicillin/streptomycin,10mM dNTP,and low-density lipoprotein was used as the culture medium for the LSK transduction and DMEM with10%FBS,50U ml21penicillin and50m g ml21streptomycin was used for all other stages.Phenotype markers(F4/80,CD11b,CD11c)were analysed 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 used to identify and localize PMT-derived cells within the lung parenchyma and in different organs.

Intra-pulmonary localization of PMT-derived cells.CD131immunostaining and fluorescence microscopy or flow cytometry was used to detect and quantify transplantation-derived donor macrophages among BAL cells from the lungs of Csf2rb2/2mice that previously received PMT of WT(C57BL/6)BMDMs,Lys-M GFP BMDMs,CD45.11WT BMDMs,or GM-R-GFP-LV Csf2rb gene-corrected Csf2rb2/2LSK-derived macrophages.

To localize PMT-derived macrophages to intra-alveolar space or interstitium of the lung,frozen lung sections from mice that received PMT of Lys-M GFP BMDMs 1month or1year earlier were immunostained with CD68,counterstained with DAPI (Vector Labs)and evaluated by fluorescence microscopy to identify macrophages, PMT-derived cells,and nucleated cells,respectively.PMT-derived macrophages (that is,GFP1CD681cells)located within the intra-alveolar space or the intersti-tium were then enumerated.To eliminate the possibility of any interference from non-specific auto-fluorescence of alveolar macrophages,paraffin-embedded lung sections from these mice were immunostained with anti-GFP antibody(Life Tech-nologies)and examined by light microscopy to enumerate immunohistochemi-cally marked macrophages located within the intra-alveolar space or interstitium. Organ-specific localization of PMT-derived cells.In one approach,Csf2rb2/2 mice received PMT of Lys-M GFP BMDMs and1year later,cells isolated from the BAL,blood,bone marrow,and spleen were evaluated by flow cytometry to detect GFP1cells as a marker for PMT-derived cells.

In a second approach,CD45.21Csf2rb2/2mice received PMT of CD45.11 BMDMs and1year later,cells isolated from the BAL,blood,bone marrow and spleen were evaluated by flow cytometry to detect CD45.11cells as a marker for PMT-derived cells.

In a third approach,Csf2rb2/2mice received PMT of Lys-M GFP BMDMs and 1year later,DNA was extracted from the BAL cells(lung),blood leukocytes,bone marrow cells,and spleen using a DNeasy Blood&Tissue Kit(Qiagen).Organ-specific DNA was subjected to PCR amplification using oligonucleotide primers (Extended Data Table1)specific for the Lys-M GFP knock-in transgene or the unmod-ified Lysozyme M gene to detect PMT-derived and endogenous cells,respectively, as previously reported25.

A fourth approach was conducted using a specific operating procedure(TSL 6-13)and Good Laboratory Practice(GLP)conditions within the CCHMC Trans-lational Core Laboratory.Here,DNA was extracted from the BAL cells(lung),blood leukocytes,bone marrow cells,and spleen of Csf2rb2/2mice that had received PMT of GM-R-GFP-LV Csf2rb gene-corrected,Csf2rb2/2LSK-derived macrophages 1year earlier and subjected to quantitative PCR amplification with oligonucleo-tide primers specific for the R-U5of GM-R-GFP-LV using Applied Biosystems ABI7900HT Fast Real-Time PCR System(Life Technologies).The number of GM-R-GFP-LV vector copies per microgram of organ-specific DNA was quantified and normalized to the level of mouse apolipoprotein

B gene as described previously54.Statistical analysis.Numeric data were evaluated for normality and variance using theShapiro-Wilk and Levenemediantests,respectively,andpresented asmean6s.e.m. (parametric data)or median and interquartile range(nonparametric data).Statis-tical comparisons were made with Student’s t-test,one-way analysis of variance,or Kruskal–Wallis rank-sum test as appropriate;post-hoc pairwise multiple compar-ison procedures were done using the Student–Newman–Keuls or Dunn’s method as appropriate.P values of#0.05were considered to indicate statistical significance. Based on the use of BAL turbidity(the primary outcome variable for efficacy) measured2months after PMT of WT BMDMs into Csf2rb2/2mice and compared to age-matched,untreated Csf2rb2/2mice,6mice per group had a power0.8to detect a difference of1.4OD600nm using a two-tailed Student’s t-test and a P value of0.05.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 exclu-sion.Analyses,including Kaplan–Meyer survival analysis,were performed with SigmaPlot,Version12.5(Systat Software,San Jose,CA).All experiments were repeated at least twice,with similar results.

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RESEARCH ARTICLE

Extended Data Figure1|Validation of Csf2rb2/2mice as an authentic model of human hPAP.a,Typical lung pathology showing surfactant-filled alveoli with well-preserved septa in a child homozygous for CSF2RB S271L mutations and identical pulmonary histopathology in a Csf2rb2/2mouse.PAS stain.Scale bar,100m m.b,Photographs of‘milky’-appearing BAL from a

14-month-old Csf2rb2/2mouse and normal-appearing BAL from an

age-matched WT mouse(representative of n56mice per group).c,Increased BAL turbidity and SP-D concentration in4-month-old Csf2rb2/2mice compared to age-matched WT mice.d,BAL fluid biomarkers of hPAP (GM-CSF,M-CSF and MCP-1)are increased in4-month-old Csf2rb2/2mice compared to age-matched WT mice.e,Alveolar macrophage biomarkers (PU.1,Pparg,Abcg1mRNA)are reduced in4-month-old Csf2rb2/2compared to age-matched WT mice.f,Progressive increase in BAL turbidity in Csf2rb2/2mice but not age-matched WT mice(linear regression:Csf2rb2/2,

slope50.127160.16(r2,0.311);WT,slope50.03160.005).g,Progressive increase in BAL fluid GM-CSF level in Csf2rb2/2mice but not age-matched WT mice(linear regression:Csf2rb2/2,slope50.8960.016(r2,0.249); WT,slope50).h,GM-CSF bioactivity in BAL fluid from10-month-old Csf2rb2/2or WT mice(or1ng ml21murine GM-CSF)measured in the presence of anti-GM-CSF antibody(GM-CSF Ab)or isotype control(Control Ab)using the GM-CSF-stimulated STAT5phosphorylation index(STAT5-PI) assay.Data are mean6s.e.m.of n57mice per group(c–e),n54(h)or symbols representing individual WT(n538)or Csf2rb2/2(n584)mice and regression fit695%CI(f-g).*P,0.05,**P,0.01,***P,0.001.ns, not significant.

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Extended Data Figure2|Characterization of BMDMs before PMT.

a,b,Photomicrographs of WT BMDMs before transplantation phase-contrast (a)or DiffQuick staining(b)(representative of n57BMDM preparations). Scale bar,20m m.c,Flow cytometry evaluation of cell-surface phenotypic markers on WT BMDMs before PMT.d,Photographs of methylcellulose cultures of Lin–cells(5,000per dish)from bone marrow(left)and BMDMs (50,000per dish)prepared as described in the Methods(right)and typical colonies(below)(representative n53per 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 to93CFU-GM per dose of BMDMs administered(n53determinations per condition).f,g,Evaluation of surfactant clearance capacity. Representative photomicrographs of BMDMs from WT(left)or Csf2rb2/2 (right)were examined before(top)or immediately after incubation with surfactant for24h(middle),or after exposure,removal of extracellular surfactant and culture for24h in the absence of surfactant(lower)after

oil-red-O staining(representative of n53per condition).Scale bar,20m m. 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 mean6s.e.m. (n53per condition)of oil-red-O staining score for10high-power fields for each group.ND,not detected;ns,not significant;***P,0.001.

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Extended Data Figure3|Efficacy of PMT in Csf2rb2/2mice and characterization of macrophages after PMT.a,Detection of CD131(top)or actin(bottom)in BAL cells by western blotting1year after PMT(each lane represents one mouse of6per group).b,Representative cytology of BAL obtained1year after PMT after staining with PAS or oil red O(ORO)(6mice per group).Scale bar,25m m.Oil-red-O positive cells were seen rarely in WT mice and occasionally in PMT-treated Csf2rb2/2mice(insets).Cytological abnormalities in BAL from untreated Csf2rb2/2mice 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 sections1year after PMT.Note that some residual disease remained at1year(original magnification,31).d,GFP1cells in BAL cells from WT or Csf2rb2/2mice2months after PMT of Lys-M GFP BMDMs(representative of n53(WT)or n56(Csf2rb2/2)mice)(original magnfication,320).e,Macrophage replication after PMT.Csf2rb2/2mice received Lys-M GFP BMDMs by PMT and paraffin-embedded lung was immunostained for Ki671month or1year later.Scale bar,50m m;inset,10m m. f,Ki67staining of BAL cells from untreated WT mice(e).Inset shows positive (left)or negative(right)staining.Scale bar,50m m;inset10m m.Graph shows the per cent Ki671BAL cells in age-matched WT mice(n55).

g,Representative immunofluorescence photomicrographs of frozen lung sections1year after PMT of Lys-M GFP into Csf2rb2/2mice identifying GFP1 cells(top),Ki671cells(middle)and GFP1Ki671(replicating,PMT-derived) cells(bottom)(representative of n53mice).Scale bar,20m m;inset scale bar, 10m m.Quantitative summary data are shown in Fig.2c.h,Localization of macrophages within the lungs1year after PMT of Lys-M GFP BMDMs into Csf2rb2/2mice and visualization in frozen lung sections after CD68 immunostaining,DAPI counter staining,and fluorescence microscopy to detect CD681GFP1cells(that is,PMT-derived macrophages)or

CD681GFP–cells(that is,non-PMT-derived endogenous macrophages). Graph shows quantitative data for n56mice.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 n56mice).Quantitative summary data are shown in Fig.3b.

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Extended Data Figure4|Tissue distribution and characterization of transplanted cells1year after PMT.a–d,Two-month-old Csf2rb2/2mice (4per group)received one PMT of Lys-M GFP BMDMs.Twelve months later, untreated,age-matched WT Lys-M GFP or Csf2rb2/2mice and PMT-treated Csf2rb2/2mice were evaluated using flow cytometry to detect GFP1cells in the indicated organs.Representative data(a)and the percentage of GFP1cells in the gated region are shown(b).Similar results were observed in Csf2rb2/2 mice2months after PMT of Lys-M GFP BMDMs except the percentage of GFP1 BAL lung cells was not quantified(not shown).c,Detection of Lys-M GFP PMT cells by PCR.PCR of genomic DNA from BAL cells(Lung),white blood cells(Blood),bone marrow(BM)cells and splenocytes(Spleen)1month or 1year after Lys-M GFP BMDM PMT was performed to detect EGFP and Lysozyme M gene.BAL cells(Lung)from WT and Lys-M GFP 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)obtained1year after PMT of gene-corrected macrophages.Note that the viral vector was only detected in lung. e–h,CD45.21Csf2rb2/2mice received one PMT of CD45.11BMDMs from congenic WT mice(e)and1year later,untreated,age-matched WT(CD45.11) or Csf2rb2/2(CD45.21)mice and PMT-treated Csf2rb2/2mice were evaluated by flow cytometry to detect CD45.11cells in the indicated organs. Representative data(f)and the percentage of CD45.11cells in the gated regions are shown(g).Phenotypic characterization of PMT-derived(CD45.11)cells (as shown in the gated region(f)).Results are similar to those for PMT of Lys-M GFP BMDMs(Fig.3d).Numeric data are mean6s.e.m.of n54mice per group(b,d)or n55mice per group(g).ND,not detected.*P,0.05.ns, not significant.

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Extended Data Figure5|Global gene expression analysis of alveolar macrophages from age-matched WT,Csf2rb2/2and Csf2rb2/2mice

1year after PMT of WT BMDMs.a,Expression of Spi1(PU.1)and Pparg (PPAR c)were confirmed by qRT–PCR using independent samples(6mice per group).b,Venn diagrams showing numbers of genes whose expression was altered in alveolar macrophages from Csf2rb2/2compared to WT mice (WT R KO)or PMT-treated compared to untreated Csf2rb2/2mice

(KO R KO1PMT).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 Csf2rb2/2mice and normalized by PMT(or unchanged in both comparisons)is shown in the overlap regions.c,Gene ontology analysis identifying pathways disrupted in Csf2rb2/2mice 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 of10% calculated by the Gene Set Test with correction for multiple testing.d,Heat maps showing differentially expressed genes in multiple KEGG pathways including PPAR c-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 mean6s.e.m.(6mice per group).*P,0.05.

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