21. Pulmonary Macrophage Transplantation (PMT) Therapy of Hereditary Pulmonary Alveolar Proteinosis (hPAP) Is Effective with Mature Macrophages and Does Not Require Myeloid Precursor/Progenitor Expansion
Authors: Takuji Suzuki, Paritha Arumugam, Anthony Sallese, Nico Lachmann, Claudia Chalk, Diane Black, Brenna Carey, Thomas Moritz, Bruce C. Trapnell
Journal: Molecular Therapy (2016)

Rationale: Hereditary Pulmonary Alveolar Proteinosis (hPAP) is caused by mutations in the CSF2RA or CSF2RB genes (encoding GM-CSF receptor α or β, respectively) leading to defective GM-CSF-dependent surfactant clearance by alveolar macrophages, resulting in severe respiratory failure. Recently, using a validated model of hPAP (Csf2rb−/− mice), we reported a novel Pulmonary Macrophage Transplantation (PMT) approach as a safe, effective, tissue-specific and durable therapy for hPAP disease (Nature. 2014, 514: 450). Our results showed that wild-type bone marrow-derived macrophages (BMDMs) delivered by PMT successfully engraft, proliferate, gradually replace functionally deficient endogenous alveolar macrophages and efficiently clear surfactant in Csf2rb−/−recipient mice resulting in durable hPAP disease correction, in the absence of myeloablation. However, these studies did not exclude the possibility of an expanded myeloid progenitor population as the effector cell in PMT therapy of hPAP. Because the transplanted cells were capable of clearing surfactant, we hypothesized that the therapeutic efficacy of PMT is mediated by mature macrophages without obligate expansion of any myeloid progenitor/stem cell. Methods: BMDMs or alveolar macrophages were obtained from wild-type mice and intrapulmonary administered into Csf2rb−/− mice by PMT (2.5×105 per mouse). The therapeutic efficacy was evaluated at 2 months after PMT by measuring the optical density (OD λ=600 nm) of bronchoalveolar lavage (BAL) turbidity - an excellent measure of overall PAP disease severity. Results: Characterization prior to transplantation showed that BMDMs were highly purified, mature macrophages: they had the morphologic appearance and surface markers of mature macrophages, clonogenic analysis indicated they contained less than 0.005% CFU-GM and no BFU-E, or CFU-GEMM progenitors, and they were able to clear surfactant in vitro. To further increase macrophage purity, BMDMs were sorted by flow cytometer by applying a conservative gating strategy to isolate CD11cHiF4/80Hi macrophages. PMT using these highly uniform, mature CD11cHiF4/80Hi BMDMs into Csf2rb−/− mice showed extraordinary therapeutic efficacy as evidenced by marked reduction of BAL turbidity compared to untreated, age-matched Csf2rb−/− mice (OD = 0.9±0.1 vs 2.3±0.2; n=4; P<0.01). As a second approach to exclude the possibility of an expanded hematopoietic progenitor as the therapeutic effector cell of PMT therapy, highly purified CD11cHiF4/80Hi alveolar macrophages were delivered by PMT into Csf2rb−/− mice. Mature alveolar macrophages were equally efficacious in hPAP disease correction, as shown by the marked reduction of BAL turbidity compared to untreated, age-matched Csf2rb−/− mice (OD = 1.5±0.1 vs 2.3±0.2; n=4; P<0.01). All donor cells analyzed at 2 months after PMT were of CD11cHiF4/80Hi phenotype. Conclusions: Results demonstrate that mature macrophages are the effector cells of PMT therapy, excluding a requirement for progenitor population expansion in hPAP disease correction. These observations have important implications in clinical trial design for translating PMT therapy to human children with hPAP.