Gene therapy of Csf2ra deficiency in mouse fetal monocyte precursors restores alveolar macrophage development and function
Fengqi Li, Katarzyna Maria Okreglicka, Federica Piattini, Lea Maria Pohlmeier, Christoph Schneider, Manfred Kopf
Fengqi Li, Katarzyna Maria Okreglicka, Federica Piattini, Lea Maria Pohlmeier, Christoph Schneider, Manfred Kopf
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Research Article Immunology

Gene therapy of Csf2ra deficiency in mouse fetal monocyte precursors restores alveolar macrophage development and function

Abstract

Tissue-resident macrophage-based immune therapies have been proposed for various diseases. However, generation of sufficient numbers that possess tissue-specific functions remains a major handicap. Here, we showed that fetal liver monocytes cultured with GM-CSF (CSF2-cFLiMo) rapidly differentiated into a long-lived, homogeneous alveolar macrophage–like population in vitro. CSF2-cFLiMo retained the capacity to develop into bona fide alveolar macrophages upon transfer into Csf2ra–/– neonates and prevented development of alveolar proteinosis and accumulation of apoptotic cells for at least 1 year in vivo. CSF2-cFLiMo more efficiently engrafted empty alveolar macrophage niches in the lung and protected mice from severe pathology induced by respiratory viral infection compared with transplantation of macrophages derived from BM cells cultured with M-CSF (CSF1-cBMM) in the presence or absence of GM-CSF. Harnessing the potential of this approach for gene therapy, we restored a disrupted Csf2ra gene in fetal liver monocytes and demonstrated their capacity to develop into alveolar macrophages in vivo. Altogether, we provide a platform for generation of immature alveolar macrophage–like precursors amenable for genetic manipulation, which will be useful to dissect alveolar macrophage development and function and for pulmonary transplantation therapy.

Authors

Fengqi Li, Katarzyna Maria Okreglicka, Federica Piattini, Lea Maria Pohlmeier, Christoph Schneider, Manfred Kopf

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Figure 7

Gene therapy of Csf2ra deficiency by gene transfer to CSF2-cFLiMo.

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Gene therapy of Csf2ra deficiency by gene transfer to CSF2-cFLiMo. (A) I...
(A) Illustration of experimental regimen. Fetal liver monocytes (FLiMo) were purified from E14.5 CD45.2 Csf2ra–/– or CD45.1 WT embryos and spin-infected with a retrovirus encoding for Csf2ra and GFP (RVCsf2ra-gfp) or GFP alone (control RVgfp). Cells were cultured with CSF2 for 7 days. RVCsf2ra-gfp-transduced Csf2ra–/– CSF2-cFLiMo (RVCsf2ra-gfp-FLiMo) or identically treated CD45.1 WT CSF2-cFLiMo were transferred i.n. to neonatal CD45.2+ Csf2ra–/– mice and evaluated after 8 weeks. (B) Efficiency of spin infection (GFP+) and survival of cultured cells at days 3 and 7 after infection. (C) GM-CSF receptor alpha protein expression levels on the cell surface of Csf2ra–/– and WT FLiMo 3 and 7 days after transduction with RVCsf2ra-gfp or RVgfp, respectively. (D) Representative dot plots showing the phenotype of donor-derived cells in the BAL and lung, pre-gated on viable CD45+ single cells. (E and F) Numbers of donor-derived alveolar macrophages (AMs) and WT AMs in the BAL (E) and lung (F). (G) Total protein levels in the BAL. (D–G) Age-matched Csf2ra–/– and CD45.2 WT mice were included as negative and positive controls. (H) Representative histograms showing cell surface expression of characteristic proteins on AMs harvested from GM-CSFRa and GFP on RVCsf2ra-gfp-FLiMo–derived AMs and WT AMs showing fluorescence minus one control (gray) and specific antibodies against indicated markers (black line). (B–H) n = 3–4/group. Data are presented as mean ± SD and the results are representative of 3 experiments. ANOVA (1-way) was used in E–G. NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.