12. GM-CSF But Not M-CSF is Critical to the Therapeutic Mechanism of Action and Efficacy of Pulmonary Macrophage Transplantation (PMT) Therapy of Hereditary Pulmonary Alveolar Proteinosis (hPAP)
Authors: Takuji Suzuki, Anthony Sallese, Claudia Chalk, Brenna Carey, Carolyn Lutzko, Bruce Trapnell
Journal: Molecular Therapy (2015)

Rationale: Hereditary pulmonary alveolar proteinosis (hPAP) is a disorder caused by recessive mutations in the GM-CSF receptor α or β gene (CSF2RA or CSF2RB), which is characterized by surfactant accumulation and hypoxemic respiratory failure, and for which no pharmacologic therapy exits. Using a murine hPAP model (Csf2rb–/–) phenotypically identical to children with hPAP, we recently reported a new gene/cell transplantation therapy approach – pulmonary macrophage transplantation (PMT) (Nature. 2014, 514: 450), with spontaneous engraftment – without preparative myeloablation, extraordinary therapeutic efficacy, a durable treatment effect, and no safety signal. Interestingly, both GM-CSF and M-CSF are elevated in the lungs of hPAP and provide a possible explanation for the remarkable survival advantage observed for transplanted macrophages compared to endogenous cells. The present study was undertaken to determine the potential relative contribution of these factors to the therapeutic mechanism of action of PMT therapy in preclinical studies in Csf2rb–/– mice. Methods: Csf2rb–/– mice received a single PMT 'treatment' – intrapulmonary instillation of wild type (WT) or (as a control) Csfr2b–/– bone marrow derived macrophages (BMDM) (2×106 per mouse) in the absence or presence of a specific M-CSF receptor kinase inhibitor (GW2580) or vehicle. Two months later, therapeutic efficacy was evaluated by measuring both disease severity (bronchoalveolar lavage (BAL) turbidity, BAL surfactant protein D (SP-D) levels) and pulmonary biomarkers of hPAP (BAL GM-CSF, M-CSF levels). Results: We first demonstrated the activity of GW2580 in vivo by measuring splenic macrophage numbers in Csf2rb–/– mice, which were reduced in GW2580-treated compared to untreated mice (2.8±0.2% vs 6.5±0.5%, n=13, 10 /group; p<0.001). Using an in vitro cell proliferation assay, we also demonstrated that BMDM from WT and Csf2rb–/– mice both responded to M-CSF (19.9±0.3-fold and 18.6±0.2-fold increase in cell numbers, respectively, after 3 days in culture; n=4 determinations/group; P=0.04) and that WT but not Csf2rb–/– BMDM responded to GM-CSF (22.8±0.5-fold and 0.1±0.0-fold increase, respectively; p<0.001). The therapeutic efficacy of PMT of WT BMDM was demonstrated by marked reductions in BAL turbidity and concentrations of SP-D, GM-CSF, and M-CSF compared to untreated age-matched Csf2rb–/– mice (n=7 /group; p<0.05; all comparisons). In contrast, PMT of Csf2rb–/– BMDM had no effect on BAL turbidity, SP-D content, or BAL fluid biomarkers demonstrating the importance of GM-CSF receptors on transplanted macrophages to the therapeutic effects. Importantly, the degree of therapeutic improvement was similar in the absence or presence of GW2580 as demonstrated by similar changes in BAL turbidity, SPD, GM-CSF, and M-CSF (n=4-7/group; P>0.05; all comparisons). Conclusions: Results demonstrate that GM-CSF but not M-CSF contributes to the efficacy of PMT therapy that restores surfactant homeostasis in mice with hPAP. Funding: ATS, CCHMC, NIH UL1TR000077 (TS); NIH R01HL085453, R21HL106134, R01HL118342 (BCT)