Rescue from Pseudomonas aeruginosa Airway Infection via Stem Cell Transplantation

Abstract

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which lead to impaired ion transport in epithelial cells. Although lung failure due to chronic infection is the major comorbidity in individuals with cystic fibrosis, the role of CFTR in non-epithelial cells has not been definitively resolved. Given the important role of host defense cells, we evaluated the Cftr deficiency in pulmonary immune cells by hematopoietic stem cell transplantation in cystic fibrosis mice. We transplanted healthy bone marrow stem cells and could reveal a stable chimerism of wild-type cells in peripheral blood. The outcome of stem cell transplantation and the impact of healthy immune cells were evaluated in acute Pseudomonas aeruginosa airway infection. In this study, mice transplanted with wild-type cells displayed better survival, lower lung bacterial numbers, and a milder disease course. This improved physiology of infected mice correlated with successful intrapulmonary engraftment of graft-derived alveolar macrophages, as seen by immunofluorescence microscopy and flow cytometry of graft-specific leucocyte surface marker CD45 and macrophage marker CD68. Given the beneficial effect of hematopoietic stem cell transplantation and stable engraftment of monocyte-derived CD68-positive macrophages, we conclude that replacement of mutant Cftr macrophages attenuates airway infection in cystic fibrosis mice.

Keywords: Pseudomonas aeruginosa; airway macrophages; chronic lung infection; cystic fibrosis; hematopoietic stem cell transplantation; mouse model; non-epithelial cells.

Graphical abstract

Figure 1

Transplanted Cells Engrafted Successfully in Chimeric CF Mice (A–C) Cells were tracked via flow cytometry in (A) bone marrows, (B) lung lysates, and (C) bronchoalveolar lavage fluids (BALFs) of uninfected CF^B6.CD45.1 (dark gray symbols) and CF^CF mice (white symbols). Most cells harvested from CF^B6.CD45.1 chimeric mice express CD45.1, whereas CF^CF mice solely express CD45.1⁻ cells. Macrophage-like cells (CD68⁺, circles) can be differentiated into donor-derived (CD68⁺CD45.1⁺) or recipient-derived cells (CD68⁺CD45.1⁻). Scatter dot plots are shown as mean ± SEM. (D) Representative immunofluorescence micrographs from lungs (bronchial and alveolar regions) of CF^B6.CD45.1 and CF^CF chimeric mice. Wild-type B6.CD45.1 and untreated CF mice served as controls. CD45.1⁺CD68⁺ cells (arrows) could be detected in the lungs of wild-type B6.CD45.1 and CF^{B6 CD45.1} chimeras. The overlay of red and purple fluorescence signals resulted in a pink labeling of the double-positive cells (seen in the uppermost and third row), whereas the CD45.1⁻CD68⁺ CF cells indicated by asterisks show pure red fluorescence signals (seen in the second and fourth row). Antibodies: CD45.1 (1:100, allophycocyanin [APC], purple), CD68 (1:100, phycoerythrin [PE]-Cy7, red), EpCam (1:200, fluorescein isothiocyanate [FITC], green), nuclei counterstained with DAPI (blue). Original magnification, ×40; scale bars, 25 μm. Long-term engraftment of transplanted cells in chimeric CF mice is displayed in Figure S2.

Figure 2

Attenuation of Acute P. aeruginosa Airway Infection in CF Mice Transplanted with Wild-type Hematopoietic Stem Cells (A–D) The graphs show (A) survival, (B) disease score, (C) body weight, and (D) rectal temperature of chimeric mice infected with 1–2 × 10⁶ CFU of P. aeruginosa PAO1. Left panel: CF chimeric mice (CF^B6: dark gray line, bars, and symbols; CF^CF: dotted line, white bars, and symbols). Right panel: wild-type chimeric mice (B6^B6: black line, bars, and symbols; B6^CF: light gray line, bars, and symbols). CF^CF, n = 15; CF^B6, n = 27; three independent experiments. B6^CF, n = 16; CF^B6, n = 16; two independent experiments. Symbols and bars display mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 3

Lung Function of Chimeric Mice during Infection (A–E) The graphs show (A) tidal volume, (B) minute volume, (C) time of inspiration plus expiration, (D) expiratory flow at 50% of expiration (EF50), and (E) breathing rate of chimeric mice infected with 1–2 × 10⁶ CFU of P. aeruginosa PAO1. Left panel: CF chimeric mice (CF^B6: dark gray circles; CF^CF: white circles). Right panel: wild-type chimeric mice (B6^B6: black squares; B6^CF: light gray squares). CF^CF, n = 15; CF^B6, n = 27; three independent experiments. B6^CF, n = 16; CF^B6, n = 16; two independent experiments (see also Figure S4). Symbols display mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 4

Lung Bacterial Numbers and Histopathology of Chimeric Mice CF^B6 (dark gray circles), CF^CF (white circles), B6^B6 (black squares), and B6^CF chimeric mice (light gray squares). (A) CFU in the lungs of bone marrow transplanted mice 24 h post-infection (p.i.) with 1–2 × 10⁶ CFU of P. aeruginosa PAO1. CF^B6 mice displayed significantly reduced bacterial load in their lungs compared to CF^CF controls, whereas no differences were seen between B6^B6 and B6^CF mice (mean ± SEM; ∗p < 0.05). Caudal and accessory lobes of the right lungs were taken for CFU determination; bacterial numbers were calculated per gram. (B) Representative micrographs of infected lungs. Upper row: overviews of infected lungs. Lower row: detailed views of marked areas. H&E stain. Original magnification, ×20; scale bars, 500 and 50 μm, respectively. (C) Quantitative evaluation of infected lungs. Inflammation was assessed separately for alveolar air space (alveoli) and lung tissue. The pathohistological signs of inflammation in the infected lungs manifested similarly in all four groups.

Figure 5

Expression of Inflammatory and Activation Markers of CD68⁺ Cells in BALF of Chimeric Mice Alveolar macrophages identified by CD68-high expression were harvested from BALF of infected chimeric mice and analyzed via iterative chip-based cytometry (iCBC) regarding their expression of representative markers for inflammation and cell activation. The analysis is based on 85–100 cells/mouse, n = 3 per condition. (A–F) Relative expression of CD68 (A), CD44 (B), CD206 (C), CD11b (D), CD36 (E), and major histocompatibility complex class I (MHC-II) (F) of CF chimeric mice (upper panel: CF^B6, dark gray dots; CF^CF, white dots; lower panel: B6 chimeric mice, B6^B6, black dots; B6^CF, light gray dots; mean ± SEM) ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. (G–J) Heatmaps (G and I) and constellation cluster analysis (H and J) of the expression of markers CD68, CD11b, CD206, CD44, CD36, and MHC-II in individual cells of CF^B6 and CF^CF (G and H) or of B6^B6 and B6^CF (I and J) chimeric mice.