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, 12 (1), e10233

Genetically Engineered Distal Airway Stem Cell Transplantation Protects Mice From Pulmonary Infection

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Genetically Engineered Distal Airway Stem Cell Transplantation Protects Mice From Pulmonary Infection

Yue-Qing Zhou et al. EMBO Mol Med.

Abstract

Severe pulmonary infection is a major threat to human health accompanied by substantial medical costs, prolonged inpatient requirements, and high mortality rates. New antimicrobial therapeutic strategies are urgently required to address the emergence of antibiotic resistance and persistent bacterial infections. In this study, we show that the constitutive expression of a native antimicrobial peptide LL-37 in transgenic mice aids in clearing Pseudomonas aeruginosa (PAO1), a major pathogen of clinical pulmonary infection. Orthotopic transplantation of adult mouse distal airway stem cells (DASCs), genetically engineered to express LL-37, into injured mouse lung foci enabled large-scale incorporation of cells and long-term release of the host defense peptide, protecting the mice from bacterial pneumonia and hypoxemia. Further, correlates of DASCs in adult humans were isolated, expanded, and genetically engineered to demonstrate successful construction of an anti-infective artificial lung. Together, our stem cell-based gene delivery therapeutic platform proposes a new strategy for addressing recurrent pulmonary infections with future translational opportunities.

Keywords: antimicrobial peptide; distal airway stem cells; pulmonary infection; transplantation.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. Constitutive expression of LL‐37 protected mouse lung from bacterial infection

The schematic of human LL‐37(CAMP) transgenic mouse strain.

LL‐37(4‐kD) detection by Western blotting. Prior to loading, samples were centrifuged through 10‐kD ultrafiltration membranes and an equal amount of ultrafiltrate (19 μg/lane) was subjected to immunoblotting. High‐molecular‐weight proteins (GAPDH) were not detected in ultrafiltrate.

The CFU of PAO1 was measured by culturing in ultrafiltrate (upper panel) or retentate (lower panel) of mouse BALF samples from indicated mice. Initial additions of PAO1 were 1 × 103 CFU. Co‐culture duration, 6 h. n = 3. Error bars, SEM.

The bacterial CFU (per gram) in lungs of indicated mice with and without PAO1 infection (5 × 106 CFU). n = 3. Error bars, SEM.

Representative histological sections of indicated lungs with PAO1 infection (5 × 106 CFU) for 6 h. H&E staining. Scale bar, 1,000 μm (upper panel) and 50 μm (lower panel).

Histopathological injury score of indicated mouse lungs with PAO1 infection (5 × 106 CFU) based on blinded expert judgment. n = 3. Error bars, SEM.

Gene expression level of IL‐1β and IL‐6 of indicated mouse lung with PAO1 infection (5 × 106 CFU). n ≥ 3. Error bars, SEM.

Data information: Statistics for graphs: unpaired two‐tailed t‐test (C) and two‐way ANOVA followed by Sidak's test (D, F, G). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.Source data are available online for this figure.
Figure 2
Figure 2. The altered transcriptomic profiles of transgenic mouse lungs before and after PAO1 infection

Heatmap showing transcriptome profile correlation values of indicated lung tissue samples before and after PAO1 infection.

Histogram of selected differentially expressed genes of LL‐37+/+ mouse lung versus wild‐type FVB mouse lung prior to infection. Blue bars indicated genes upregulated in wild‐type FVB mouse lungs, while red bars indicated genes upregulated in LL‐37+/+ mouse lungs.

Enriched Gene Ontology classes of uninfected (C) and PAO1‐infected (D) lungs. Red bar, GO class of upregulated gene in LL‐37+/+ mice. Blue bar, GO class of upregulated gene in wild‐type FVB mouse lung. GO terms were ranked by the enrichment P‐value.

Protein–protein interaction network of selected genes with high expression level in PAO1‐infected wild‐type lung (blue) and PAO1‐infected LL‐37+/+ lung (red), respectively.

Source data are available online for this figure.
Figure 3
Figure 3. Engineered mDASCs possessed normal stem cell properties and enhanced antimicrobial potency

Detection of LL‐37 expression in the engineered mDASCs by immunofluorescence (A), real‐time quantitative PCR (B), and Western blot (C). Scale bar, 50 μm. BF, bright field. n = 10. Error bars, SEM.

Anti‐Krt5 (red) and anti‐P63 (green) immunostaining of WT‐ and LL‐37‐mDASC colonies. Scale bar, 70 μm.

Stem cell colony‐forming efficiency of WT‐ and LL‐37‐mDASCs during five serial passages. n = 6. Error bars, SD.

Representative 3D organoid culture of mDASCs with expression of type I alveolar cell markers (Aqp5 and Pdpn). Left panels, bright‐field imaging of 3D organoids. Right panels, immunofluorescence of organoid sections. Scale bar, 20 μm.

Co‐culture of bacteria with DASCs shows antimicrobial effects in dose‐dependent manner. Initial additions of PAO1 were 0.1 × , 0.5 × and 1 × 10CFU, respectively. Co‐culture duration, 6 h. n = 4. Error bars, SEM. MOI, multiplicity of infection.

Co‐culture of bacteria with DASCs shows antimicrobial effects in time‐dependent manner. Initial concentration of PAO1 was 1 × 10CFU. MOI = 1. n = 3. Error bars, SEM.

Preincubation of cells with anti‐LL‐37 antibody, but not IgG control, significantly reduced anti‐PAO1 (I) and anti‐Escherichia coli (J) effects of LL‐37‐mDASCs. Initial dose of bacteria was 10CFU. Co‐culture duration, 18 h. n = 4 in (I) and n = 3 in (J). Error bars, SEM.

Data information: Statistics for graphs: unpaired two‐tailed t‐test (B), two‐way ANOVA followed by Sidak's test (G, H) and one‐way ANOVA followed by Tukey's test (I, J). *P < 0.05; **P < 0.01; ****P < 0.0001.Source data are available online for this figure.
Figure EV1
Figure EV1. The antimicrobial effect of LL‐37‐mDASCs in vitro

Cell growth curve of WT‐ and LL‐37‐mDASCs was measured by MTT assay. n = 3–5. Error bars, SD.

Soft agar assay of WT‐ and LL‐37‐mDASCs. Mouse melanoma cell line was included as a positive control.

Histogram showed that LL‐37‐mDASCs conditioned medium (CM) had potent growth inhibitory effect on PAO1. Initial addition of PAO1 was 1 × 104 CFU. n = 5. Error bars, SEM.

Histogram shows that LL‐37‐mDASCs CM had potent growth inhibitory effect on Escherichia coli. Initial addition of E. coli was 1 × 104 CFU. n = 3. Error bars, SEM.

Clone formation unit assay of E. coli following incubation with indicated cellular CM.

Preincubation of CM with anti‐LL‐37 antibody, but not mouse IgG, reduced the antimicrobial effect of LL‐37‐mDASCs against E. coli. n = 3. Error bars, SEM.

Data information: Statistics for graphs: one‐way ANOVA followed by Tukey's test. *P < 0.05; ***P < 0.001.
Figure 4
Figure 4. Lung engraftment of WT‐ and LL‐37‐mDASCs after orthotopic transplantation

Bright‐field and direct fluorescence images of mouse lungs following transplantation of 1 × 106 GFP‐labeled WT‐mDASCs (WT‐Lung) or LL‐37‐mDASCs (LL‐37‐Lung) on indicated days. Scale bar, 1,000 μm.

Engraftment ratio of indicated cells in mouse lungs on indicated days. n = 3. Error bars, SEM. *P = 0.0474. Statistics: two‐way RM ANOVA followed by Tukey's test.

Morphology of engrafted GFP‐labeled cells in lung parenchyma by direct fluorescence. Blue color indicates nucleus DAPI staining. Scale bar, 200 μm.

Anti‐Ki67 immunofluorescence of engrafted GFP‐labeled WT‐ and LL‐37‐mDASCs in lung parenchyma 21 days after transplantation. Scale bar, 50 μm.

Source data are available online for this figure.
Figure EV2
Figure EV2. The antimicrobial effect of LL‐37‐Lung

Left, immunostaining of engrafted LL‐37‐mDASCs cells with anti‐GFP and anti‐AQP5 (type I alveolar cell marker) antibodies; right, amplification of inset in upper panel indicated regenerated alveolar structure. Scale bar, 30 μm.

Distribution of engrafted GFP‐labeled cells in lung parenchyma by immunostaining 21 days after transplantation. WT‐Lung, WT‐mDASCs engrafted; LL‐37‐Lung, LL‐37‐mDASCs engrafted. Scale bar, 40 μm.

Direct fluorescence image of lungs from normal mouse or bleomycin injured mouse 7 days after transplantation of 1 × 106 GFP‐labeled mDASCs.

Direct fluorescence image under stereomicroscope showing mouse lung transplanted with 1 × 106 GFP‐labeled WT‐ and LL‐37‐mDASCs followed by PAO1 infection.

Intratracheal instillation of equal amounts of E. coli (5 × 10CFU per mouse) into WT‐Lung (WT‐mDASCs engrafted) and LL‐37‐Lung (LL‐37‐mDASCs engrafted) followed by bacterial CFU analysis in whole lung homogenates (left panel) and BALF (right panel) 2 days after infection. n = 3. Error bars, SEM.

Arterial blood gas analysis of mice with WT‐Lung and LL‐37‐Lung following E. coli infection 2 days after transplantation. n = 3. Error bars, SEM.

H&E staining showing histology of WT‐Lung and LL‐37‐Lung with E. coli infection after 2 days of cell transplantation. Scale bar, 200 μm.

Data information: Statistics for graphs: unpaired two‐tailed t‐test. *P < 0.05; **P < 0.01.
Figure 5
Figure 5. LL‐37‐expressing lung had enhanced host defense ability

Distribution of engrafted GFP‐labeled cells in lung parenchyma by immunofluorescence 7 days after transplantation. WT‐Lung, WT‐mDASCs engrafted; LL‐37‐Lung, LL‐37‐mDASCs engrafted. Scale bar, 200 μm. Arrows show the representative cells with overlapping fluorescence of GFP and LL‐37.

Intratracheal instillation of equal amount of PAO1(5 × 106 CFU per mouse) into WT‐Lung (WT‐mDASCs engrafted) and LL‐37‐Lung (LL‐37‐mDASCs engrafted) followed by bacterial CFU analysis in whole lung homogenates 6, 24, and 48 h after infection. n = 3. Error bars, SEM.

Intratracheal instillation of equal amount of PAO1 into WT‐Lung and LL‐37‐Lung followed by bacterial CFU analysis in BALF 6, 24, and 48 h after infection. n = 3. Error bars, SEM.

Downregulated gene in LL‐37‐Lung enriched in Gene Ontology and KEGG pathways.

Protein–protein interaction network of selected genes with high expression level in WT‐Lung (red) and LL‐37‐Lung (blue), respectively.

Arterial blood gas analysis of mice with WT‐Lung and LL‐37‐Lung following PAO1 infection. n = 6. Error bars, SEM.

Data information: Statistics for graphs: two‐way ANOVA followed by Sidak's test (B, C) and unpaired two‐tailed t‐test (F). *P < 0.05; **P < 0.01; ****P < 0.0001.Source data are available online for this figure.
Figure 6
Figure 6. LL‐37‐mDASCs transplantation protected infected mouse from pulmonary inflammation

H&E staining showing histology of injured lung infected by PAO1 with WT‐mDASCs or LL‐37‐mDASCs transplantation. Scale bar, 100 μm.

Histopathological examination according to the lung injury scoring system based on blinded expert judgment. n = 5 mice per group. Error bars, SEM.

CD68 immunochemistry (brown) in infected lung with WT‐mDASCs or LL‐37‐mDASCs transplantation. Scale bar, 50 μm.

Quantification of brown‐stained (CD68+) area by Image J software. n = 5. Error bars, SEM.

Gene expression level of indicated pro‐inflammatory cytokines of lung infected by PAO1 with WT‐mDASCs or LL‐37‐mDASCs transplantation. n = 3. Error bars, SEM.

Data information: Statistics for graphs: one‐way ANOVA followed by Tukey's test. **P < 0.01; ***P < 0.001; ****P < 0.0001.Source data are available online for this figure.
Figure 7
Figure 7. Lung scaffold recellularized by LL‐37 engineered human DASCs has enhanced anti‐bacterial ability

Anti‐Krt5 (red) and anti‐P63 (green) immunofluorescence of isolated hDASCs. Scale bar, 90 μm.

The mRNA expression levels of LL‐37 were measured by real‐time qPCR for WT‐ and LL‐37‐hDASCs. n = 5. Error bars, SEM.

H&E staining of native rat lung tissue and decellularized scaffold showed the absence of nuclei following decellularization. Scale bar, 40 μm.

Ex vivo biomimetic culture of LL‐37‐hDASCs recellularized lungs with constant media perfusion.

Direct fluorescence image showing GFP‐labeled LL‐37‐hDASCs before (left) and 7 days after (right) being engrafted onto the decellularized scaffold. Scale bar, 50 μm (right panel) and 2,000 μm (right panel).

Immunofluorescence of recellularized lung with indicated antibodies. Blue color indicates nuclear DAPI staining. Scale bar, 50 μm.

The recellularized lung by LL‐37‐hDASCs displayed growth inhibitory effect on PAO1 and Escherichia coli. Initial dose of bacteria was 2 × 10CFU. Culture duration, 24 h. n = 6. Error bars, SEM.

Data information: Statistics for graphs: unpaired two‐tailed t‐test (B) and one‐way ANOVA followed by Tukey's test (G). *P < 0.05; ***P < 0.001; ****P < 0.0001.Source data are available online for this figure.
Figure EV3
Figure EV3. Lung scaffold recellularized by engineered human DASCs

The expression of LL‐37 in engineered human DASC (hDASCs) was detected by immunostaining (A) and Western blot (B). Scale bar, 20 μm.

Cryosections of native and decellularized rat lung with nuclei counterstain. Blue color indicates nucleus DAPI staining Scale bar, 50 μm.

Immunofluorescence staining showed that major cells preserved KRT5 + hDASCs phenotype (red). Scale bar, 50 μm.

Immunofluorescence staining showed that a few grafted cells of elongated shape had AQP5 (type I alveolar cell marker) expression. Scale bar, 50 μm.

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