Dysregulated protease activity has long been implicated in the pathogenesis of chronic lung diseases and especially in conditions that display mucus obstruction, such as chronic obstructive pulmonary disease, cystic fibrosis, and non-cystic fibrosis bronchiectasis. However, our appreciation of the roles of proteases in various aspects of such diseases continues to grow. Patients with muco-obstructive lung disease experience progressive spirals of inflammation, mucostasis, airway infection and lung function decline. Some therapies exist for the treatment of these symptoms, but they are unable to halt disease progression and patients may benefit from novel adjunct therapies. In this review, we highlight how proteases act as multifunctional enzymes that are vital for normal airway homeostasis but, when their activity becomes immoderate, also directly contribute to airway dysfunction, and impair the processes that could resolve disease. We focus on how proteases regulate the state of mucus at the airway surface, impair mucociliary clearance and ultimately, promote mucostasis. We discuss how, in parallel, proteases are able to promote an inflammatory environment in the airways by mediating proinflammatory signalling, compromising host defence mechanisms and perpetuating their own proteolytic activity causing structural lung damage. Finally, we discuss some possible reasons for the clinical inefficacy of protease inhibitors to date and propose that, especially in a combination therapy approach, proteases represent attractive therapeutic targets for muco-obstructive lung diseases.

Neutrophil-derived proteases are critical to the pathology of many inflammatory lung diseases, both chronic and acute. These abundant enzymes play roles in key neutrophil functions, such as neutrophil extracellular trap formation and reactive oxygen species release. They may also be released, inducing tissue damage and loss of tissue function. Historically, the neutrophil serine proteases (NSPs) have been the main subject of neutrophil protease research. Despite highly promising cell-based and animal model work, clinical trials involving the inhibition of NSPs have shown mixed results in lung disease patients. As such, the cutting edge of neutrophil-derived protease research has shifted to proteases that have had little-to-no research in neutrophils to date. These include the cysteine and serine cathepsins, the metzincins and the calpains, among others. This review aims to outline the previous work carried out on NSPs, including the shortcomings of some of the inhibitor-orientated clinical trials. Our growing understanding of other proteases involved in neutrophil function and neutrophilic lung inflammation will then be discussed. Additionally, the potential of targeting these more obscure neutrophil proteases will be highlighted, as they may represent new targets for inhibitor-based treatments of neutrophil-mediated lung inflammation.

Epstein-Barr virus (EBV) frequently is measured at high levels in COPD using sputum quantitative polymerase chain reaction, whereas airway immunohistochemistry analysis has shown EBV detection to be common in severe disease. Is valaciclovir safe and effective for EBV suppression in COPD? The Epstein-Barr Virus Suppression in COPD (EViSCO) trial was a randomized double-blind placebo-controlled trial conducted at the Mater Hospital Belfast, Northern Ireland. Eligible patients had stable moderate-to-severe COPD and sputum EBV (measured using quantitative polymerase chain reaction) and were assigned randomly (1:1) to valaciclovir (1 g tid) or matching placebo for 8 weeks. The primary efficacy outcome was sputum EBV suppression (defined as ≥ 90% sputum viral load reduction) at week 8. The primary safety outcome was the incidence of serious adverse reactions. Secondary outcome measures were FEV₁ and drug tolerability. Exploratory outcomes included changes in quality of life, sputum cell counts, and cytokines. From November 2, 2018, through March 12, 2020, 84 patients were assigned randomly (n = 43 to valaciclovir). Eighty-one patients completed trial follow-up and were included in the intention-to-treat analysis of the primary outcome. A greater number of participants in the valaciclovir group achieved EBV suppression (n = 36 [87.8%] vs n = 17 [42.5%]; P < .001). Valaciclovir was associated with a significant reduction in sputum EBV titer compared with placebo (-90,404 copies/mL [interquartile range, -298,000 to -15,200 copies/mL] vs -3,940 copies/mL [interquartile range, -114,400 to 50,150 copies/mL]; P = .002). A statistically nonsignificant 24-mL numerical FEV₁ increase was shown in the valaciclovir group (difference, -44 mL [95% CI, -150 to 62 mL]; P = .41). However, a reduction in sputum white cell count was noted in the valaciclovir group compared with the placebo group (difference, 2.89 [95% CI, 1.5 × 10⁶-7.4 × 10⁶]; P = .003). Valaciclovir is safe and effective for EBV suppression in COPD and may attenuate the sputum inflammatory cell infiltrate. The findings from the current study provide support for a larger trial to evaluate long-term clinical outcomes. ClinicalTrials.gov; No.: NCT03699904; URL: www. gov.

OBJECTIVE: To evaluate the performance of cystatin C as a prognostic and predictive marker in a trial of patients with acute respiratory distress syndrome (ARDS). DESIGN, PATIENTS AND SETTING: A re...

Background: Mucoactives such as hypertonic saline (HTS) and carbocisteine are widely used in the treatment of bronchiectasis, though there is insufficient evidence to support their use. The aim of this mechanistic sub-study, embedded within the CLEAR trial, was to characterise the properties of sputum from patients with bronchiectasis and to assess whether treatment with HTS and/or carbocisteine altered these properties. Methods: In CLEAR, patients were randomised to receive HTS, carbocisteine, HTS plus carbocisteine or standard care, and sputum samples were collected at randomisation (baseline) and at 2 and 8 weeks post-randomisation. Sputum viscoelasticity was determined by rotational plate rheometry. Biomarkers were quantified by ELISA and microbiome composition was assessed by next-generation sequencing. The primary outcome was differences in sputum viscoelasticity between groups at 2 weeks following the commencement of treatment. Results: Sputum viscoelastic properties were not reduced by 2 or 8 weeks of treatment with HTS and/or carbocisteine (n=6–15 per group). Sputum biomarker levels and bacterial community composition were similar across groups at 2 or 8 weeks. At baseline, viscoelasticity (crossover point σc) was positively correlated with IL-8 (r=0.49, p=0.012) and greater relative bacterial dominance (r=0.35, p=0.041). Conclusions: These data do not support the use of HTS or carbocisteine to alter sputum viscoelasticity, inflammatory marker levels or bacterial community composition in patients with bronchiectasis.

The constitutive proteasome and its inflammation-driven derivative, the immunoproteasome (IP), perform important intracellular proteolytic functions. However, the IP is increasingly recognised as being present in the extracellular space during pathology, though its mechanisms of release and functions are unknown. We hypothesised that extracellular IP is a feature of, and plays a role in, the acute respiratory distress syndrome (ARDS). We show that the levels and activity of IP are elevated in bronchoalveolar lavage fluid from patients with ARDS, the human healthy volunteer LPS model and the murine inhaled LPS model. In a series of in vitro experiments, we demonstrate that IP is released constitutively from macrophages, though this release can be exacerbated by activation of the NLRP3 and AIM2 inflammasomes, and that IP release can be abrogated by pharmacological or genetic targeting of the inflammasome pathway. We have found that direct instillation of IP alone into the lungs of mice does not induce inflammation. However, when administered in combination with bacterial endotoxin, IP appears to alter the inflammatory cell population within the lung. We plan to further characterise these changes in order to understand the role of extracellular IP in ARDS. Closely linked to this, since the extracellular substrates of IP are not known, identifying such targets represents a key aspect of our future work. In conclusion, extracellular IP is a feature in human ARDS and murine ARDS-like disease. We have identified a potential mechanism of release of IP, which is closely linked to inflammasome activation, and are exploring the relevance of extracellular IP in the acutely inflamed lung.