Urine-derived exosomes from individuals with IPF carry pro-fibrotic cargo
- DeWitt Daughtry Family Department of Surgery, University of Miami Leonard M. Miller School of Medicine, United States
- Department of Medicine, Division of Pulmonary, Critical Care and Sleep, University of Miami, United States
- Medical Director, Grand Health Institute, United States
- Cancer Modeling Shared Resource Sylvester Comprehensive Cancer Center, University of Miami, United States
- ZenBio Inc., United States
- Department of Biochemistry and Molecular Biology, University of Miami, Miller School of Medicine, United States
- Dr. JT Macdonald Foundation Biomedical Nanotechnology Institute, University of Miami Miller School of Medicine, United States
- Miami Clinical and Translational Science Institute, University of Miami Miller School of Medicine, United States
- Wound Healing and Regenerative Medicine Research Program, Dr Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami, United States
- Department of Medicine, Stritch School of Medicine, Loyola University Chicago, United States
Figures
Figure 1
Overview of experimental details and design.
Figure 2
A Transmission electron microscopy of isolated exosomes.
Image magnification Scale bar = 50 nm. 1B. Isolated exosomes express CD63.
Figure 3
Expression of miR-let-7d (A), miR-29a-5p (B), miR 181b-3p (C) and miR-199a-3p (D) in urine-derived exosomes reveals a pattern corresponding to that reported in serum and whole lung of individuals with IPF.
PCR was performed on extracted urine-derived exosomes as described in methods. Data are graphed as relative miRNA expression normalized to U6 and percent of control expression. * p<0.05, **p<0.01, *** p<0.001 compared to control exosomes. Each point represents an individual patient exosome sample. n=5–14 individual samples/group, P values were calculated by Mann-Whitney U test. E. Urine and serum-derived exosomes isolated from the same individuals with IPF have similar miRNA expression. Exosome isolation, RNA preparation and PCR performed as described in methods. n=5 individual samples of urine and serum-derived exosomes. Paired T test analysis was performed. Figure 3—source data 1.
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Figure 3—source data 1
Raw data for Figure 3.
- https://cdn.elifesciences.org/articles/79543/elife-79543-fig3-data1-v1.xlsx
Figure 4 with 1 supplement
Biodistribution of circulating urine-derived exosomes.
Shown are representative in vivo bioluminescence images to study the biodistribution of ExoGlow labeled urine-derived exosomes in mice (n=3/group) at the indicated time points. Panel A=mouse injected with labeled U-IPF exo; Panel B=mouse injected with labeled urine-derived exosomes from age and sex-matched control individuals without lung disease. Panel C=mouse injected with PBS. Intensity of luminescence seen in bar from lowest (red) to highest (blue). n=3 individual exosome preparations/group.
Figure 4—figure supplement 1
Lung fluorescence intensity over time of mice injected with urine-derived exosomes from individuals with IPF (U-IPFexo), urine-derived exosomes from individuals without IPF (Control exosomes), and PBS.
C.Ex-vivo fluorescence imaging of isolated organs at 48 hours following exosome treatment in micee.
Figure 5 with 1 supplement
Representative TEM photos of lung punches.
Panels A-C show mouse lung punches injected with gold nanoparticle labeled urine-derived exosomes from age and sex-matched control subjects (without lung disease) or U-IPFexo (panels D-H). TEM revealed exosomes in alveolar epithelial cells (AEC) type I and type II. Arrows in panels C, F, and G highlight exosomes containing nanoparticles. n=2 individual exosome preparations/group.
Figure 5—figure supplement 1
Histology and trichrome staining of lung punches from C57BL6 mice.
Lung punches from control lungs shown in tissue culture dish (S1A) have normal histology (S1B, Trichrome staining ×10 mag) and structure by TEM (S1C, ×500 mag). Histology of non-injected lung punch (SA1D).
Figure 6
Immunofluorescence staining of lung punches injected with exosomes derived from urine (B–C) or myofibroblasts or fibroblasts (E–F).
Lung punches were fixed four days post injection with either PBS (panels A or D) or control urine-derived exosomes (panel B), U-IPFexo (panel C), control fibroblast (panel E) or MF-IPF exosomes (panel F). Shown are representative merged photographs at 20 x, surfactant protein C (SPC, red), αSMC actin (green) and DAPI (blue). n=3 individual exosome preparations/group. Scale bar 50 µm.
Figure 7
Fibrotic pathways are activated in lung punches after injection with urine (U-IPFexo) or myofibroblast-derived (MF-IPFexo) exosomes.
Human (A–C) and mouse lung (D–M) punches were injected with PBS alone, U-IPFexo (D–I) or MF-IPFexo (J–M) or age and sex-matched control urine exosomes or lung fibroblast exosomes from control subjects (without lung disease). Punches were collected 4 days later and processed as described in Methods. Human lung punches were injected with MF-IPFexo or fibroblast cell derived exosomes (panels A and B) or urine-derived control exosomes and U-IPFexo (panels B and C). n=2 human lung punch isolates, 2 biological exosome preparations/group. Panels D-M, n=3 mouse lung replicates/group, n=3–5 biological exosome isolates/group Data are graphed as percent PBS control. αv-integrin (panels A, D and J, Figure 7—source data 1) and collagen type 1 (panels A, E and K, Figure 7—source data 1) mRNA expression increased in punches injected with IPFexo (derived from urine or myofibroblasts). Downstream fibrotic pathways; ERα (C, F and L), activated AKT (H), c-Jun (G and M), protein expression and MMP-9 activity (I) were also stimulated by exosomes from individuals with IPF. * p<0.05, **p<0.01. p Values were calculated by Mann Whitney U test.
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Figure 7—source data 1
Raw data for Figure 7.
- https://cdn.elifesciences.org/articles/79543/elife-79543-fig7-data1-v1.xlsx
Figure 8
Epithelization in ex vivo wound healing is decreased by urine-derived IPF exosomes (U-IPFexo).
Human skin was wounded, injected with U-IPFexo or control (age and sex-matched from individuals without lung disease) exosomes and maintained at the air-liquid interface. Wound healing was assessed at day 4 post-wounding, a time point when exponential epithelialization occurs. (A) Data are graphed as mean with each data point representing a single wound. Experiments were performed using triplicate technical replicates and two to three biological replicates (Figure 7—source data 1). p<0.005 PBS and control compared to IPF, PBS vs control = 0.05 p values were calculated by Mann Whitney U test. (B). Photos of gross skin show visual signs of closure and correspond to the histology assessments. Black arrows point to the initial site of wounding, while white arrows point to the wound edge of the migrating epithelial tongue. Scale bars, 500 µm proportional to the image size.
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Figure 8—source data 1
Raw data for Figure 8.
- https://cdn.elifesciences.org/articles/79543/elife-79543-fig8-data1-v1.xlsx
Figure 9 with 1 supplement
Assessment of fibrosis in Bleomycin (Bleo) treated mice intravenously infused with exosomes derived from the urine of individuals with IPF (U-IPFexo) compared to infusion with urine exosomes derived from age and sex- matched control subjects without lung disease or urine exosomes derived from subjects with non-CF bronchiectasis or asthma (non-fibrotic lung disease).
Histological sections of lung tissue were stained with Masson’s-Trichrome as described in Materials and Methods. Representative photomicrographs (4 x, 10 x, and 20 x) of lung sections from Bleo +vehicle (panels A-C), Bleo +control exosome injected mice (panels D-F), from Bleo +U-IPFexo injected mice (panels G-I) or from non-fibrotic inducing exosomes (Bronchiectasis, panels J-L). Fibrotic score (M), collagen content (N), αvintegrin (O) increased after Bleo +U-IPFexo treatment. (M) Ashcroft scores were used to evaluate the degree of fibrosis. Data are graphed as the mean score of 32 fields/section of lung. (N) Collagen content was estimated by hydroxyproline assay as described in Methods. Data are graphed as μg/mg of lung tissue. (O) αv-integrin mRNA expression was determined by RT-PCR as a marker of fibrosis. Data are graphed normalized for 18 S content. Each data point represents an individual mouse, n=4–11 technical replicates/group and two biological replicates/group (Figure 9—source data 1) *p<0.05 compared to control exosome treatment or compared to Bleo +vehicle treatment. Data were analyzed using one-way analysis of variance (ANOVA) and Mann-Whitney U test. Scale bar panels A, D, G, J, 200 µm; panels B, E,H, K,100 µm; panels C, F,I,L, 50 µm.
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Figure 9—source data 1
Raw data for Figure 9.
- https://cdn.elifesciences.org/articles/79543/elife-79543-fig9-data1-v1.xlsx
Figure 9—figure supplement 1
Collagen content increases in mice receiving urine derived exosomes from individuals with IPF.
Naïve mice were treated with PBS, control or IPF urine-derived exosomes. Mice were sacrificed 21 days later as described in methods. Data are graphed as mean ± SEM. Each data point represents an individual mouse (n=2 exosome preps/group, Figure 9—figure supplement 1—source data 1). p<0.05 IPF compared to control and PBS, Data were analyzed using Mann Whitney test.
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Figure 9—figure supplement 1—source data 1
Raw data for Figure 9—figure supplement 1.
- https://cdn.elifesciences.org/articles/79543/elife-79543-fig9-figsupp1-data1-v1.xlsx
Figure 10
Potential microRNA regulated pathways leading to fibrosis.
The genes and biological processes in the network are generated from the IPF vs Control Lung dataset from NCBI GEO (GS21369) of 11 IPF samples and 6 healthy lung samples.
Tables
Table 1
Male IPF (A group), non‐CF bronchiectasis (B group), or asthma (C group) urine-derived exosomes (Age of subject at collection).
| Subject number | Age of subject at collection | Ethnicity | FEV1 (liters) | FVC (liters) | FEV/FVC (%) | FEV/FVC(predicted) | DLCO (% reference) |
|---|---|---|---|---|---|---|---|
| A4 | 72 | Caucasian | 2.93 | 3.75 | 78 | 77 | 11.8 (47) |
| A6 | 79 | Caucasian | 2.36 | 2.91 | 81 | 67 | 16.2 (101) |
| A26 | 69 | Hispanic | 1.67 | 1.98 | 84 | 72 | NT |
| A35 | 69 | Hispanic | 1.71 | 2.22 | 70 | 73 | NT |
| A37 | 69 | Hispanic | 2.7 | 3.21 | 84 | 77 | 10.5 (44) |
| A62 | 67 | Hispanic | 1.94 | 2.33 | 82 | 77 | 8.7 (33) |
| A74 | 75 | Hispanic | 2.48 | 2.77 | 80 | 74 | 9.9 (49) |
| A77 | 55 | Hispanic | 1.14 | 1.27 | 90 | 78 | 6.7 (32) |
| A80 | 70 | Caucasian | 2.09 | 2.62 | 78.9 | 87.49 | 11.8 (48) |
| A83 | 68 | Hispanic | 2.39 | 2.69 | 86 | 75 | 10.5 (49) |
| A84 | 67 | Caucasian | 1.78 | 2.12 | 84 | 84 | 14.1 (57) |
| A88 | 66 | Hispanic | 1.36 | 1.39 | 98 | 75 | 2.6 (11) |
| A90 | 67 | Caucasian | 2.48 | 2.85 | 88.9 | 74 | 12.8 (45) |
| A103 | 72 | Hispanic | 1.95 | 2.64 | 74 | 75 | 13.4 (59) |
| A104 | 76 | Caucasian | 2.95 | 3.27 | 90 | 65 | 14.6 (58) |
| A105 | 62 | Hispanic | 1.47 | 1.69 | 87 | 78 | 11.7 (41) |
| B1 | 73 | Caucasian | 1.04 | 2.06 | 50 | 79.91 | 9.6 (48) |
| B10 | 70 | Hispanic | 1.33 | 2.89 | 46 | 74 | 21.29 (69.5) |
| B13 | 86 | Hispanic | 2.54 | 3.59 | 70.7 | 74 | 23.08 (76.78) |
| C1 | 36 | Hispanic | 3.67 | 4.75 | 77.16 | 82.89 | 31.49 (92) |
| C4 | 61 | Caucasian | 3.38 | 4.72 | 71.65 | 79.09 | 27.77 (96) |
| C7 | 44 | Caucasian | 3.08 | 4.57 | 67.3 | 70 | 27.83 (96) |
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NT, not tested.
Table 2
Male control (D group) urine-derived exosomes (Age of subject at collection).
No evidence of documented lung disease or abnormal PFTs.
| Subject number | Age of subject at collection | Ethnicity |
|---|---|---|
| D8 | 66 | Caucasian |
| D9 | 70 | Caucasian |
| D12 | 77 | Caucasian |
| D28 | 77 | Caucasian |
| D31 | 55 | Caucasian |
| D32 | 73 | Caucasian |
| D38 | 72 | Caucasian |
| D41 | 65 | Caucasian |
| D50 | 75 | Caucasian |
| D101 | 57 | Caucasian |
Table 3
Myofibroblast and control fibroblast-derived exosomes (Age of subject at collection).
| Male myofibroblasts IPF (Age of subject at collection) | Male fibroblast Control (Age of subject at collection) |
|---|---|
| 1 (52) | 5 (70) |
| 2 (83) | 6 (69) |
| 3 (73) | 7 (67) |
| 4 (74) |
Table 4
Mouse lung punch miRNA expression.
| Mouse lung punch miRNA expression (% of PBS) | miR-let-7d | miR-29 | miR-181b | miR-199 fibrotic | miR-34a fibrotic | miR-142 antifibrotic |
|---|---|---|---|---|---|---|
| Urine exosomes (n=6–8/group) | ||||||
| PBS | 100±0.5 | 100±0.3 | 100±0.2 | 100±0.1 | 100±.3.1 | 100±0.2 |
| Control exosomes | 74±6.7 | 96±13.3 | 137±18 | 92±5 | 88±15 | 105±20 |
| U-IPFexo | 32±6.5 @ | 55±12.1 *& | 52±17 + | 169±18 + & | 131±10 *+ | 74±14 = 0.5 |
| Fibroblast exosomes (n=3–4/group) | ||||||
| PBS | 100±0.1 | 100±0.3 | 100±0.5 | 100±0.5 | 100±0.2 | 100±0.2 |
| Control exosomes | 114±5 | 110.8±9.2 | 110±8.2 | 53±5 | 101±16.5 | 78±14 |
| MF-IPFexo | 57±5.9 @ | 56±7.9 *& | 42±12.7@ | 115±14 NS | 100.±27 NS | 47±12 NS |
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*
+p = 0.05 compared to PBS, @<0.01 compared to PBS and control exosomes, * p<0.05 compared to control exosomes, &p<0.01 compared to control exosomes, p values were calculated by Mann-Whitney U test. NS = not significant.
Table 5
Lung tissue microRNA expression.
| Lung tissue microRNA expression/U6 | miR-let-7d | miR-29 | miR-181b | miR-199fibrotic | miR-34a fibrotic | miR-142 antifibrotic |
|---|---|---|---|---|---|---|
| PBS | 0.18±0.03 | 2.1±0.4 | 0.26±0.06 | 0.03±0.001 | 0.07±0.02 | 0.23±0.016 |
| Control U-exo | 0.3±0.01 ## | 2.8±0.7 | 0.3±0.07 | 0.06±0.003 | 0.09±0.01 | 0.19±0.03 |
| U-IPFexo | 0.14±0.02 | 0.48±0.1 *& | 0.15±0.05 | 0.05±0.006 † | 0.13±0.03 * | 0.16±0.02 * |
| Bleomycin (Bleo) | 0.057±0.009 ^^ | 0.17±0.02 | 0.009±0.001 | 0.19±0.02 | 0.18±0.05 | 0.23±0.04 |
| Bleo +Control U- exo | 0.24±0.05 † | 0.17±0.02 | 0.008±0.001 | 0.35±0.11 | 0.18±0.02 | 0.29±0.05 |
| Bleo +U-IPFexo | 0.10±0.011 $$^^ | 0.20±0.17 | 0.004±.001 † | 1.68±0.44 *$$^^ | 0.43±0.08 *$$^ | 0.16±0.01 &$^ |
| Bleo +U-bronchiectasis exo | 0.17±.03 | 0.14±0.03 | ND | 0.13±0.35 | 0.06±0.02 | 0.3±0.07 |
| Bleo +U-asthma exo | 0.39±0.1 | 0.11±0.01 | ND | 0.06±0.01 | 0.06±0.02 | 0.29±0.04 |
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*
p<0.05 compared to control exosomes, &p<0.001 compared to control exosomes.
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†
p<0.05 compared to Bleo, ##p<0.001 compared to Bleo, $p<0.05, $$ p<0.01 compared to bronchiectasis exosomes ^p<0.01 ^^ p<0.001compared to asthma exosomes. p values were calculated by Mann-Whitney U test. ND = not detected.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/79543/elife-79543-mdarchecklist1-v1.pdf
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Source data 1
Source file for antibodies and primers.
- https://cdn.elifesciences.org/articles/79543/elife-79543-data1-v1.pdf
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Source data 2
Raw unedited gels.
- https://cdn.elifesciences.org/articles/79543/elife-79543-data2-v1.zip
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Urine-derived exosomes from individuals with IPF carry pro-fibrotic cargo
eLife 11:e79543.
https://doi.org/10.7554/eLife.79543
