Fig 6. Human BALF-derived CD63⁺/CD66b⁺ exosomes confer a COPD-like phenotype to mice in a NE-dependent manner.

(A) Photomicrographs of H&E stained lung tissue of mice exposed to various exosomes. (B) Exosomes obtained from BALF of healthy never smokers (N/S) (n=10), and subjects with COPD (n=10). Exosomes pooled from BALF of all 10 healthy never smoker subjects (N/S), all 10 pooled COPD subjects with and without Human NE Inhibitor II preincubation, current smoker COPD subjects (n=5) (current smoker COPD”), or former smoker COPD subjects (n=5) (former smoker COPD), administered i.t. (4.0 × 10⁸ exosomes/dose, 6 doses over 12 days) to 8–10 week old mice (n ≥ 15 per group), sacrifice at 14 days and Lms were determined. (C) RV/(LV+S) shown for PBS, N/S, and COPD experiments of panel B. Dot plot displayed with line delineating mean measurement. (D) Exosomes from BALF of pooled healthy N/S subject (n=10) and pooled COPD subjects (n=10) captured on anti-CD66b or anti-MUC4 antibody coated beads and resulting population depleted and/or purified for expression of CD66b or MUC4 used for i.t. mouse exposure; COPD subject BALF purified for CD66b expression (with and without Human NE Inhibitor II) and MUC4 expression, NS subject BALF purified for CD66b expression, and MUC4⁺ purified exosomes from pooled COPD patient BALF administered to mice i.t. as in (B), n ≥ 4 per group. The dose of exosomes used for these experiments correspond to the entire population of exosomes given in B. after depletion of or purification for the respective marker. Shown for comparison are the Lm of pooled COPD subject and N/S BALF exosome treated animals from experiment in panel B. Representative photomicrographs of these experiments shown in panel A. (E) BALF exosomes from separate individual N/S or COPD subject BALF administered i.t. to mice (n=4 per individual) and Lm measured as in B. Each data point shown represents mean increase of Lm over control (i.e., intra-experimental PBS treated mice) of mice treated with the exosomes from a single N/S or COPD individual subject, shown as mean +/− SEM for the two groups. The standard deviation of control PBS treated mice in these experiments was +/− 0.87μm. (F) Exosomes (2.5 × 10⁷) derived from individual N/S or COPD subjects (n=3/group) were pulled down on anti-CD66b beads, stained with anti-NE AF647, analyzed by flow cytometry and MFI of anti-NE staining of exosomes determined, shown as representative histogram, with quantitative display of mean in inset. (G) Percentage of exosomes that stained for NE analyzed as in F. Data for B. and D. presented as follows: center line, median; box limits, upper and lower quartiles; whiskers, minimum and maximum values. ** P<0.01, *** p<0.001 ****p<0.0001. See also figures S1, S4, S5, and S6 and Table S1.