Activated PMN Exosomes: Pathogenic Entities Causing Matrix Destruction and Disease in the Lung

Abstract

Here, we describe a novel pathogenic entity, the activated PMN (polymorphonuclear leukocyte, i.e., neutrophil)-derived exosome. These CD63⁺/CD66b⁺ nanovesicles acquire surface-bound neutrophil elastase (NE) during PMN degranulation, NE being oriented in a configuration resistant to α1-antitrypsin (α1AT). These exosomes bind and degrade extracellular matrix (ECM) via the integrin Mac-1 and NE, respectively, causing the hallmarks of chronic obstructive pulmonary disease (COPD). Due to both ECM targeting and α1AT resistance, exosomal NE is far more potent than free NE. Importantly, such PMN-derived exosomes exist in clinical specimens from subjects with COPD but not healthy controls and are capable of transferring a COPD-like phenotype from humans to mice in an NE-driven manner. Similar findings were observed for another neutrophil-driven disease of ECM remodeling (bronchopulmonary dysplasia [BPD]). These findings reveal an unappreciated role for exosomes in the pathogenesis of disorders of ECM homeostasis such as COPD and BPD, providing a critical mechanism for proteolytic damage.

Keywords: BPD; COPD; ELA-2; elastase; exosomes; extracellular matrix; extracellular vesicles; inflammation; lung disease; microparticles; neutrophil.

Figure 1.. PMN activation leads to release of exosomes with more surface NE than quiescent PMN exosomes.

(A) Electron micrographs of exosomes derived from fMLP stimulated PMNs (activated, right) or DMSO control in PBS (quiescent, left). (B) Unbiased proteomic analysis of exosomes by mass spectroscopy, representative PMN-associated proteins shown. Exosomes (2.5 × 10⁷) derived from PMNs from healthy donor peripheral blood (n=2) were pulled down on anti-CD66b beads, stained with anti-NE AF647, analyzed by flow cytometry (C), representative histogram shown in D) and percentage staining for NE determined (E). Data for C, E depicted as mean +/− SEM. See also figures S1 and S6.

Figure 2.. PMN activation confers increased expression of enzymatically active, α−1 AT resistant NE.

(A) NE activity of activated and quiescent exosomes measured at 5-minute intervals for production of (BODIPY FL) labelled fluorescent elastin fragments from self-quenching BODIPY FL-conjugated bovine neck ligament elastin. (B) NE activity of activated and quiescent exosomes against specific NE peptidomimetic substrate MeOSucAAPVpNA, by pNA generation. (C) 20ng purified NE coincubated with Human NE Inhibitor II, the endogenous NE inhibitor α−1 AT or PBS control, and NE activity by pNA generation measured as in B. (D) NE activity of an equipotent number of activated exosomes (1 × 10⁹) and 20ng of purified NE measured with Human NE Inhibitor II, α1-AT, or PBS control as in B. Single experiment shown in C. and D. displayed separately for clarity. Data shown as mean +/− SEM. See also figure S2.

Figure 3.. PMN exosomes load NE extracellularly.

(A) 5 × 10⁹ quiescent exosomes coincubated with purified NE (1μg) or left in PBS, filtered and rinsed in PBS to remove unbound NE. Exosome NE activity measured by pNA. (B)1 × 10⁹ activated exosomes coincubated with protamine sulfate or PBS with or without α1-AT and NE activity measured by pNA. Data from A and B. shown as mean +/− SE of 4 experiments. (C-G) Model of hypothesized mechanism of NE loading upon exosomes. (C) Quiescent PMN releases exosomes (yellow spheres) constitutively as NE (blue shapes) is sequestered intracellularly in primary granules. (D) Activated PMN continues to release exosomes constitutively while degranulating, releasing NE molecules into solution which bind to exosome as it passes through locally elevated halo of degranulated NE. (E) Close-up model of activated exosome, to which multiple NE particles are bound via charge-mediated interactions. (F) Activated exosome in an environment rich in α1-AT (crescents). Steric hindrance impedes α1-AT interaction with exosomal NE, whereas free NE readily complexes with α1-AT molecules in an irreversible fashion. (G) Activated exosome after application of cationic molecules such as protamine sulfate to displace NE from exosome surface, now readily complexed and inactivated by α1-AT. See also figure S3.

Figure 4.. Exosomes associate with collagen fibrils selectively via Mac-1 and possess NE dependent collagenase activity

(A) EM image of collagen fibrils after 24 hour-incubation. (B) Collagen fibrils and quiescent exosomes imaged immediately after being combined. (C) Collagen fibrils and activated exosomes imaged immediately after being combined. (D) Collagen fibrils and quiescent exosomes after 24 hour co-incubation. (E) Collagen fibrils imaged after 24 hours of co-incubation with activated exosomes. (F) Closer view of activated exosome with collagen fibril. Arrow denotes area where a collagen fibril appears to be nicked and frayed near exosome. (G) PBS or exosomes of varying quantities coincubated with type I collagen-coated plates. ELISA for exosome associated lactoferrin performed using horse-radish peroxidase-conjugated anti-lactoferrin. (H) ELISA performed as in G. in the presence of MP-9, or RGD peptide at varying concentrations. (I) Activated and quiescent exosomes measured for production of fluorescein isothiocyanate (FITC) from FITC-labelled type I collagen. (J) Activated exosomes with and without Human NE Inhibitor II as well as purified NE measured for type I collagen degradation as in I. See also figure S2.

Figure 5.. Activated neutrophil exosomes confer a COPD-like phenotype to mice in an NE-dependent fashion and express CD66b and CD63

(A) Hematoxylin/eosin (H&E) stained photomicrographs of lung tissue of female A/J mice exposed i.t. to PBS,1.67×10⁸ activated or quiescent PMN derived exosomes (n= 3–5 mice per group repeated 8 times with exosomes representing 8 separately tested donors) and sacrificed on day 7. (B) Alveolar size measured as mean linear intercept (Lm). (C) Mice (n=6) exposed to exosomes or PBS control as in A. and pulmonary function testing performed and resistance measured. (D) Weight ratios of right ventricle: left ventricle + septum (RV/LV+S) measured, shown as dot plot with mean indicated by line. (E) Activated exosomes administered as in A, animals sacrificed at days 1–7 with Lm measured as in B. (F) Varying doses of exosomes given as in A., animals sacrificed on day 7, and Lm measured as in B., n ≥ 4 per group. (G) Exosomes from activated PMNs with and without preincubation with Human NE Inhibitor II delivered i.t. into mice as in A. and Lm measured as in B., n ≥ 10 per group. (H) Purified human NE instilled i.t at varying doses, animals sacrificed at day 7, Lm measured as in B., n≥ 4 per group (I) Exosomes from neutrophils stimulated with the CXCR2 ligand PGP or control peptide PGG administered to mice with Lm measured as in B., n ≥ 4 per group. (J) Exosomes delivered to mice as in A., Lm measured as in B., after antibody coated magnetic bead capture and release to purify for CD63, CD66b or MUC4 (negative control), or after bead depletion for CD63 and CD66b, n ≥ 4 per group. Data shown as follows: center line, median; box limits, upper and lower quartiles; whiskers, minimum and maximum values. * p<0.05, ** P<0.01, *** p<0.001. See also figures S3, S4 and S5.

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.

Figure 7.. Tracheal aspirate exosomes from BPD subjects confer a BPD-like phenotype to exposed neonatal mice.

(A) Exosomes from tracheal aspirate samples of subjects (n=5/condition) with BPD and non-BPD controls were purified and pooled into BPD and Non-BPD groups. Mice (n=4/group) treated i.n. with 4 × 10⁸ exosomes in 15μL on postnatal days 3, 6, and 12 and sacrificed on postnatal day 14. Lungs stained by H&E for alveolar morphometry and representative histology of treated animals shown. (B) Radial alveolar counts measured for animals treated in A. (C) Mice (n=5) treated i.n. with exosomes as in A., pulmonary function testing was performed, and resistance is shown. (D) RV/(LV+S) of animals treated in A. was measured. See also Table S1.