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A protease-antiprotease imbalance has long been considered a hallmark of chronic lung diseases (CLDs) such as chronic obstructive pulmonary disease (COPD), whereby abnormally high protease activity overwhelms endogenous anti-proteases resulting in tissue damage. Neutrophil-derived proteases are strongly implicated in pathology, with elevated neutrophil elastase (NE) activity exceeding inhibitory capacity of endogenous antiprotease α-1 antitrypsin (A1AT), and contributing to ECM degradation, inflammation, airway remodeling, and impaired bacterial clearance.1 Research in this space has been invigorated by the recent approval of dipeptidyl peptidase 1 (DPP-1) inhibitors (prevent activation of neutrophil serine proteases including NE) for use in bronchiectasis patients;2 the first immunomodulatory drug to demonstrate clinical benefit in this disease. Accordingly, a fuller understanding of pathways driving protease imbalance and ensuing propagation of pathology in CLDs may yield exciting opportunities for therapeutic intervention. Recently, Blalock and colleagues reported the presence of neutrophil-derived extracellular vesicles (EVs) carrying surface-bound enzymatically active NE, which (unlike soluble NE) was resistant to inhibition by A1AT and possessed a heightened capacity to degrade extracellular matrix (ECM) collagen.3 These NE+-EVs were released by activated neutrophils,3 and were elevated in the airways of mice acutely exposed to cigarette smoke4 or LPS,5 and importantly in bronchoalveolar lavage fluid of COPD patients.3 These EVs could readily induce alveolar enlargement (emphysema), when transferred into lungs of naïve mice.3–5 Remarkably, this NE-dependent induction of emphysema was apparent within days of EV transfer to naïve mice, with EV-bound NE purported to be 10 000-fold more potent than soluble enzyme.3 In this issue of the Journal, Margaroli and colleagues report a novel self-propagating capacity for these neutrophil-derived NE+-EVs, whereby they perpetuate a vicious cycle of neutrophilic inflammation, de novo NE+-EV production, and lung pathology.6 Specifically, administration of human neutrophil-derived NE+-EVs into naive mice elicited pulmonary recruitment of neutrophils, endogenous generation of murine neutrophil-derived NE+-EVs, and emphysematous tissue pathology. This pathology was entirely NE-dependent and was instigated by the de novo generated murine NE+-EVs as opposed to originally transferred human NE+-EVs. Mechanistically, this propagation of pathogenic EVs was mediated by the generation of bioactive ECM fragment proline-glycine-proline (PGP). Initial human-derived NE+-EVs acted to degrade ECM collagen and liberate PGP, which in turn promoted neutrophil recruitment and liberation of mouse NE + EVs that drove ensuing emphysema. Remarkably, newly generated mouse NE+-EVs could be administered to discrete groups of naïve mice and instigate this same cycle of lung pathology, which could then be propagated across multiple passages of mice. This self-propagating cycle of NE+-EV-mediated pathology could also be instigated if original EVs were derived from airways of mice acutely exposed to cigarette smoke. The findings of Margaroli et al. inevitably raise unanswered questions and reveal avenues of clinical research to be explored (Figure 1). Ordinarily, it would take >20 weeks of persistent cigarette smoke exposure to induce emphysema in mice. An extraordinary finding, therefore, is that a singular administration of NE+-EVs derived from in vitro activated neutrophils, or isolated from airways of mice acutely exposed to LPS or cigarette smoke, can induce (and propagate) emphysema within days of being administered to naïve mice.3–6 This would suggest that pathways must ordinarily be induced in the original challenged mice to counteract and restrain the potent pathological capacity of NE+-EVs released from activated neutrophils—and subsequently the transfer of these pathogenic entities to naïve mice, in which protective pathways have not been induced, results in profound and rapid tissue damage. While the identity of this putative pathway that counters the action of NE+-EVs is yet to be elucidated, the central role now described for PGP in perpetuating NE+-EVs may suggest that elevations in PGP-degrading LTA4H (as observed in pulmonary inflammation) could be a prominent countermeasure.7 Furthermore, while current studies have focused upon the robust ability of NE + EVs to degrade ECM and elicit emphysematous disease, it would be intriguing to ascertain the relative capacity of these entities to drive other facets of CLD pathology attributed to soluble NE, such as heightened mucus production/viscosity, reduced cilia beat frequency, and impaired neutrophil anti-microbial responses. Moreover, while there is an unequivocal role for NE in driving EV-mediated emphysema, it would seem pertinent to consider pathological contributions of other EV cargo, such as myeloperoxidase.3 A self-propagating PGP—NE±-EV axis perpetuates pulmonary neutrophilic inflammation and tissue pathology in COPD. Activated neutrophils release NE+-EVs which degrade ECM collagen to liberate bioactive fragment PGP, which in turn promotes further neutrophil recruitment and activation. Newly recruited and activated neutrophils release NE+-EVs, perpetuating the cycle and driving ECM destruction and lung pathology. Opportunities for therapeutic intervention include targeting neutrophil elastase and/or NE+-EVs using DPP-1 inhibitors, NE-inhibitors or protamine sulfate, or targeting PGP using RTR. Areas for future investigation are highlighted in boxes: Exploration of the role of NE+-EVs in the context of COPD presentation and progression; Evaluation of the capacity of NE+-EVs to drive facets of lung pathology in addition to ECM degradation; Interrogation of NE+-EV prevalence and contribution to tissue pathology in other CLDs defined by neutrophilic inflammation and protease imbalance; Evaluation of the capacity to transfer NE+-EVs between individuals to passively transmit disease. Abbreviations: EV, extracellular vesicle; NE, neutrophil elastase; ECM, extracellular matrix; PGP, proline-glycine-proline; DPP-1, dipeptidyl peptidase-1; RTR, arginine-threonine-arginine; COPD, chronic obstructive pulmonary disease; CLDs, chronic lung diseases. Figure created in BioRender (https://biorender.com/shortURL). In a clinical context, what proportion of the NE instigating lung damage in COPD patients is A1AT-resistant EV-bound NE as opposed to A1AT-sensitive soluble NE? Soluble NE has been demonstrated to display modest correlation with COPD progression,8 but would a more pronounced relationship with disease outcomes be apparent if EV-bound NE activity was assessed? While NE+-EVs have been reported in the airways of COPD patients,3 it would be prudent to more fully interrogate their numbers and activity in the context of disease heterogeneity and severity, and align with inflammatory/remodeling parameters, clinical outcomes, and surrogates of tissue pathology. Given the capacity of a relatively small number of NE+-EVs to amplify and autonomously self-perpetuate inflammation and lung damage, it is intriguing to speculate whether this axis is a mechanism by which COPD-associated inflammation/pathology can continue to progress and escalate in individuals many years after smoking cessation. Does the extent to which the PGP/NE+-EV axis is induced (relative to putative counter-acting protective pathway) define the likelihood of smokers developing COPD, and does this axis continue to perpetuate inflammation/pathology in those with established COPD that have stopped smoking? In this regard, it has been demonstrated that PGP persists in airways of COPD patients after smoking cessation owing to deficiencies in PGP-degrading LTA4H.9 Furthermore, given that elevated neutrophils, NE, and PGP are observed in COPD exacerbations,10,11 is there a role for NE+-EVs in driving these devastating episodes? While much of the work pertaining to the PGP/NE+-EV axis has focused upon COPD and its capacity to drive emphysematous disease,3 it would be rational for this pathway to be abundant and operational in other CLDs such as severe asthma, cystic fibrosis, and bronchiectasis, where neutrophils, NE, and PGP are elevated.9,11–14 Exploration of this axis in other CLDs should consider whether EV cargo is distinct and if patient-derived EVs impart disparate lung pathologies when administered to naïve mice. A fascinating consideration raised by the authors is the potential of these EVs to act as “quasi-infectious”, sub-cellular entities with the capacity to transmit inflammation and pathology between individuals. Given that EVs can be detected in exhaled breath condensate, is it feasible that they may be transmitted via respiratory droplets of smokers and COPD patients into the airways of proximal individuals, and passively transfer disease? Whether sufficient NE+-EVs can be transmitted via this route to then cause notable inflammation and pathology in the donor is currently purely hypothetical but is certainly an intriguing proposition. The potency of the PGP/NE+-EV axis and capacity to readily impart substantial lung damage ultimately raises questions as to how it could be therapeutically targeted. Both NE-specific and DPP-1 inhibitors could seemingly prove beneficial through targeting EV-bound NE. Given findings from the current study, neutralizing PGP (eg, with antagonist RTR) may also disrupt the NE+-EV pathogenic axis. Finally, protamine sulfate, approved for use in humans, has been demonstrated to drive dissociation of NE from the surface of EVs, making it amenable to inhibition by A1AT.15 Supplementary material is available at American Journal of Respiratory and Critical Care Medicine online. Please see the ICMJE disclosure forms, which have been provided as supplementary material. The authors acknowledge financial support from the Biotechnology and Biological Sciences Research Council (BBSRC; MR/Z50404X/1). No artificial intelligence tools were used in writing this manuscript.
Baldi et al. (Wed,) studied this question.