Toll-Like Receptor 4 Engagement Mediates Prolyl Endopeptidase Release from Airway Epithelia via Exosomes

Abstract

Proteases are important regulators of pulmonary remodeling and airway inflammation. Recently, we have characterized the enzyme prolyl endopeptidase (PE), a serine peptidase, as a critical protease in the generation of the neutrophil chemoattractant tripeptide Pro-Gly-Pro (PGP) from collagen. However, PE has been characterized as a cytosolic enzyme, and the mechanism mediating PE release extracellularly remains unknown. We examined the role of exosomes derived from airway epithelia as a mechanism for PE release and the potential extracellular signals that regulate the release of these exosomes. We demonstrate a specific regulatory pathway of exosome release from airway epithelia and identify PE as novel exosome cargo. LPS stimulation of airway epithelial cells induces release of PEcontaining exosomes, which is significantly attenuated by small interfering RNA depletion of Toll-like receptor 4 (TLR4). These differences were recapitulated upon intratracheal LPS administration in mice competent versus deficient for TLR4 signaling. Finally, sputum samples from subjects with cystic fibrosis colonized with Pseudomonas aeruginosa demonstrate elevated exosome content and increased PE levels. This TLR4-based mechanism highlights the first report of nonstochastic release of exosomes in the lung and couples TLR4 activation with matrikine generation. The increased quantity of these proteolytic exosomes in the airways of subjects with chronic lung disease highlights a new mechanism of injury and inflammation in the pathogenesis of pulmonary disorders.Keywords: protease; Toll-like receptor 4; exosome; cystic fibrosis; pulmonary Cell-to-cell communication via secreted soluble mediators is critical to the maintenance of cellular homeostasis in complex metazoans. Much of this intercellular communication is regulated through the classical secretory mechanism, in which proteins containing an endoplasmic reticulum (ER)-signaling sequence are actively released via the ER-toGolgi pathway (1). Despite the broad range of proteins entering this pathway, a number of actively secreted proteins lack a signal sequence for release and are released via alternative mechanisms, including exosomes (2).

Transforming growth factor beta induces fibroblasts to express and release the immunomodulatory protein PD‐L1 into extracellular vesicles

“…We hypothesized that the PD‐L1 induction by TGFβ (Figures and ) might be similarly expressed in EVs as they represent mediators of intercellular communication under both physiological and disease conditions . In that the contribution of EVs to chronic lung disease and the role of serum‐derived EVs as potential biomarkers has been recently reported, studies were undertaken to determine whether the expression of PD‐L1 following fibroblast treatment with TGFβ could function in an analogous paracrine manner to mediate T cell anergy and/or fibroblast activation. This was addressed in Figure where (i) it was first shown that fibroblast‐derived EVs contained increased amounts of PD‐L1 after TGFβ stimulation; and (ii) the secreted EVs decreased T cell proliferation and increased fibroblast migration in a PD‐L1 dependent manner.…”

Bioactive extracellular matrix fragments in lung health and disease

“…ing protein for structural and immune cells, but is now considered the rate-limiting enzyme for free PGP formation and is released from airway epithelia via an exosomal pathway during inflammation (45). PGP-induced signaling is considered a feed-forward inflammatory signal wherein CXCR1 and CXCR2 ligation triggers neutrophil influx, driving the release of collagenase MMP9 from neutrophil tertiary granules, which cleaves more collagen and results in the release of additional PGP and subsequent neutrophil influx (46) (Figure 1).…”

The expression of proline-specific enzymes in the human lung

“…It was reported that PREP is released from airway epithelial cells through exosomes. The sputum of patients with cystic fibrosis colonized with Pseudomonas aeruginosa showed more exosomes and increased PREP protein expression (121).…”

“…PREP activity has been measured in sputum with Z-Gly-Pro-pNA and was 5 times higher in patients with cystic fibrosis compared to healthy controls (93). PREP mRNA, protein expression and enzymatic activity has been shown in multiple airway epithelial cells, both in cell lines and primary cells (121). PREP could be found in the medium of cystic fibrosis bronchial epithelial cells and was increased after stimulation with lipopolysaccharide (LPS), without an increase in PREP mRNA.…”