Fig. 4: Pulmonary macrophage transplantation (PMT) of AAT MΦ into Csf2rb–/– mice.
A Schematic representation of the experimental procedures. Lineage negative (lin-) cells were isolated from bone marrow of CD45.1 WT mice, transduced with CAG-AAT or Cbx-EF1α-AAT vectors, sorted for eGFP+ cells and differentiated toward MΦ. Cells were administered intratracheally into the lungs of Csf2rb–/– mice. Engraftment and AAT levels in ELF were analyzed 2 months after PMT. Created with BioRender.com. B Representative flow cytometry analysis of donor CD45.1+ cells in the BALF of recipient mice. Untreated WT (CD45.1) and Csf2rb–/– mice were used as positive and negative controls, respectively. The graph represents percentage of engrafted donor MΦ in all experiments. Individual symbols for each mouse are used in all graphs. For the CAG-AAT group, all mice were transplanted with cells from completely independent lin- isolations and transductions. For the Cbx-EF1α and mock groups, two animals each received cells from the same isolation and transduction and one animal received cells from an independent isolation and transduction. Expression of C CD11c and Siglec-F and (D) eGFP on CD45.1+ pre-gated cells. E Immunofluorescent staining of lung cryosections depicting the CD45.1 cells (magenta), nuclei (DAPI) and autofluorescent lung structure (green). Scale bar = 10 µm. F Turbidity (optical density at 600 nm, OD600) of BALF isolated from transplanted (mock, CAG-AAT, Cbx-EF1α-AAT), and untreated control mice (WT, Csf2rb–/–). G Quantification of human AAT in BALF by ELISA. H Concentration of human AAT in ELF after normalization for urea in BALF and serum. Statistical analysis was performed using one-way ANOVA with Tukey’s post-hoc test. Lines represent mean ± SD. *p ≤ 0.05; ns not significant. AF autofluorescence, BALF bronchoalveolar lavage fluid, ELF epithelial lining fluid, PMT pulmonary macrophage transplant, SSC side scatter, Tx transplanted, WT wildtype.
