Cathepsin S as a mediator of acute lung inflammation

The acute respiratory distress syndrome (ARDS), first described over half a century ago, is a common and catastrophic syndrome that develops in critically ill patients. ARDS is characterised by the flooding of the alveoli with protein- and leukocyte-rich, non-cardiogenic oedema and refractory hypoxaemia, which can be triggered by numerous pro-inflammatory stimuli including pneumonia, sepsis and gastric acid aspiration. The inciting injury may be further exacerbated by the application of supportive mechanical ventilation and the development of nosocomial infections. Overall, with a mortality rate in the region of 30-50 % and contributing substantially to morbidity within Intensive Care Units, the syndrome represents a significant economic and healthcare burden. Unfortunately, despite many years of preclinical testing and clinical trials, no specific pharmacological therapies have been identified that can meaningfully affect outcomes. Proteolytic enzymes, or proteases, are known to play important roles in the maintenance of pulmonary homeostasis. However, during disease, proteolytic activity can become dysregulated and cause damage to the lung, contributing to the pathology of conditions like cystic fibrosis, chronic obstructive pulmonary disease, asthma, pulmonary fibrosis and ARDS. Increased protease activity is thought to be caused, at least in part, by the loss of endogenous antiproteases or their failure to control their cognate proteases. Thus, the use of protease inhibitors may be utilised to suppress aberrant protease activity. To date, protease inhibition strategies in ARDS have focussed on the neutrophil serine protease, neutrophil elastase (NE). Although in clinical use in Japan, NE inhibitors have not proven effective and other protease targets warrant investigation. The cysteine protease, cathepsin S (CTSS), is emerging as an influential mediator of pulmonary disease and may represent a novel target for the treatment of chronic and acute inflammatory lung conditions. In this thesis, we first evaluated the status of CTSS in the context of ARDS and models of ARDS. These investigations revealed that CTSS levels and activity were elevated in the lungs of patients with ARDS, and that elevated CTSS activity was also detectable in the plasma of these patients. Elevated pulmonary CTSS activity was identified in the human inhaled lipopolysaccharide (LPS) model and three separate murine models of ARDS. To assess the potential pathological contribution of CTSS to ARDS, active CTSS was introduced into the lungs of mice. These instillations reproduced typical ARDS hallmarks including neutrophil recruitment, alveolar leakage and pro-inflammatory cytokine release. Interestingly, dysregulated CTSS activity in patients with ARDS did not appear to result from the loss of cystatin C (CST3), the predominant extracellular cysteine cathepsin inhibitor. However, other cystatins, such as cystatin SN (CST1), which was identified here as a novel CTSS inhibitor, were found to be lost from the lungs of patients with ARDS. CST1 also demonstrated anti-inflammatory abilities in vivo, apparently independent of its role as a CTSS inhibitor, intimating that the loss of these other cystatins in ARDS may contribute to acute lung inflammation. Next, we showed that CTSS inhibition using a specific, small molecule inhibitor was effective at reducing neutrophil recruitment, alveolar leakage and epithelial inflammation, as well as promoting endothelial stabilisation in the murine LPS model, even when the inhibitor was administered 2 h after the inciting injury. The CTSS inhibitor also limited neutrophil recruitment to the peritoneum in the murine caecal ligation and puncture model of sepsis, and significantly reduced pulmonary pro-inflammatory cytokine levels in this model. The exact mechanisms by which CTSS causes acute lung injury are not yet clear. However, through a combination of in vivo and in vitro studies, along with previous data generated in our laboratory, we proposed that CTSS mediates some of its pathogenic effects via the activation of protease-activated receptor 1. Finally, we showed that total knockout of CTSS did not detrimentally affect the ability of mice to clear the common ventilator-associated pathogen, Pseudomonas aeruginosa. Altogether, these findings support a role for CTSS in the pathogenesis of ARDS and indicate that the therapeutic inhibition of CTSS may be beneficial in the context of pulmonary neutrophilic inflammation in ARDS.