In the past two cycles of this grant we have defined what some have called a paradigm shifting pathway of neutrophilic inflammation which, unlike the classic mode associated with IL-8, can become self- propagating in chronic inflammatory diseases such as COPD. Specifically, IL-8 initiates neutrophil (PMN) influx, the PMNs in turn release matrix metalloproteases (MMPs) and prolyl endopeptidase (PE) which degrade collagen and generate the PMN-specific matrikine, proline-glycine-proline (PGP). PGP then propagates further PMN influx and neutrophilic inflammation after IL-8 has subsided. In more common acute inflammatory circumstances, the PGP pathway is terminated by the aminopeptidase activity of leukotriene A4 hydrolase (LTA4H) which destroys PGP. Cigarette smoking (CS) can chemically modify and inactivate LTA4H's aminopeptidase but not hydrolase activity as well as acetylate PGP rendering it immune to LTA4H and markedly increasing the peptides chemotactic activity. This drives persistently elevated PGP levels and chronic neutrophilic inflammation. In COPD, the phenotypes of elevated PGP and inactivated LTA4H aminopeptidase is exacerbated and persists even after smoking cessation and this is likely due to endogenously produced acrolein, a toxic component of CS. One enigmatic aspect of our studies has been an inability to generate PGP in vitro with collagen and the appropriate proteases in solution. The thesis of this renewal application is that this enigma is due to the requirement that PGP generating enzymes, such as PE, be exosome associated. This idea is supported by a number of observations: 1) PE is found in exosomes; 2) PE positive (+) exosomes generate PGP from collagen in vitro; 3) in vivo instillation of PE+ exosomes into mouse airways causes PGP production, PMN influx, alveolar enlargement and right ventricular hypertrophy (RVH); 4) PE+ exosomes are increased in BAL fluids from CS exposed mice; 5) PE+ exosomes are increased in BAL fluids and sputa from COPD patients and cause a COPD-like phenotype when transferred to mice. Collectively these findings led to our hypothesis that proteolytic exosomes constitute a new aspect of the inflammatory process and may participate in chronic inflammatory disorders such as COPD via the PGP pathway. If successful, the results of this project will define a novel proteolytic entity, i.e. PE+ exosome, that drives neutrophilic inflammation via PGP generation and can cause a COPD like disease in mice. In a smoking mouse model of COPD we will characterize the evolution of PE+ exosomes and whether they can transfer disease from smoked to nave animals. In clinical studies, we will delineate whether PE+ exosomes are biomarkers of COPD that correlate with disease parameters and that can transfer pathology from humans to mice.
This project describes a new pathological entity, the proteolytic exosome. In diseases such as COPD this type of exosome drives chronic inflammation and tissue damage. A complete understanding of the proteolytic exosome may lead to new diagnostics and therapeutics for chronic inflammatory diseases such as COPD.
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