Activated Cathepsin S is a Biomarker and Therapeutic Target in Experimental Colitis

Inflammatory bowel diseases (IBD), comprising ulcerative colitis and Crohn's disease, are characterised by chronic inflammation in the intestine. Although the aetiology of IBD is not well understood, a number of proteases have been identified as having pro‐inflammatory actions in the gut. Cathepsin S is a cysteine protease that promotes visceral pain through Protease Activated Receptor 2 (PAR 2 )‐dependent mechanisms. We hypothesised that cathepsin S would also be a key driver of intestinal inflammation and that inhibiting its activity would be of therapeutic value. To address this, we used the DSS mouse model of experimental colitis. To measure the activation and localisation of cathepsin S, we used the novel fluorescently quenched activity‐based probe, BMV157, which specifically labels active cathepsin S. We administered BMV157 to mice with colitis and healthy controls. By ex vivo imaging, we found that cathepsin S activity was significantly upregulated in the proximal colon during colitis. By confocal microscopy, we identified the source of cathepsin S to be CD68 + macrophages within mucosal and submucosal layers. Cathepsin S activity was also strikingly increased in luminal fluid and faecal pellets, suggesting increased secretion during colitis. To test this, we incubated normal and inflamed colons in media overnight and measured cathepsin S activity in the supernatant. While we clearly detected secreted cathepsin S activity, as confirmed by immunoprecipitation with a cathepsin S‐specific antibody, no differences were observed between normal and inflamed colons. This suggests that the increased cathepsin S activity in luminal fluids and faecal samples may be derived from microbial species rather than the host. We found that this protease could be labeled by three distinct cathepsin ABPs and was inhibited by multiple cathepsin S‐specific inhibitors. Unlike cathepsin S in colon tissue, however, the luminal protease had limited reactivity with antibodies raised towards mammalian cathepsin S. This protease was also present in a bacteria‐enriched faecal fraction, further suggesting a microbial source. Efforts to purify this protease with biotinylated activity‐based probes are underway, which will allow its identification by mass spectrometry. To determine whether cathepsin S‐like activity (host or microbe‐derived) is required for the induction of colitis and whether blocking this activity would ameliorate symptoms of colitis in mice, we utilised the cathepsin S inhibitor LY3000328. Contrary to our hypothesis, inhibitor treatment worsened symptoms of colitis, including faecal consistency, rectal bleeding, splenomegaly, and inflammation. Biochemical analysis revealed an upregulation of activity and expression of cathepsin S and L in colon tissue after inhibitor treatment, suggesting a compensatory mechanism in response to loss of activity. These results suggest that increased murine cathepsin S and L activity may function to promote colitis. Thus, improved pharmacological strategies that produce sustained inhibition of cathepsin S/L may provide therapeutic relief in colitis. In summary, cathepsin S is activated in macrophages during murine colitis and may function to promote negative symptoms. We have also identified a potentially novel bacterial cathepsin S‐like protease that is upregulated in the lumen during colitis. Cathepsin S‐like proteases may therefore be disease biomarkers and therapeutic targets in colitis. Support or Funding Information LEM is supported by an Early Career Fellowship from the National Health and Medical Research Council (NHMRC, Australia, GNT1091636). NB is supported by NHMRC and ARC Centre of Excellence in Convergent Bio‐Nano Science and Technology. Work in the authors' laboratory is funded in part by Takeda Pharmaceuticals Inc.Cathepsin S Activity is increased during colitis. A) Ex vivo imaging of cathepsin S activity in healthy colon and those with DSS‐induced colitis using the specific activity‐based probe BMV157 (2.2‐fold increase in proximal colon; p=0.0006, n=4). B) Biochemical characterisation of cathepsin S activity in colon tissue and luminal fluids after in vivo labeling with BMV157 (1.5‐fold increase in colon; p=0.005, n=4 and 7.8‐fold increase in luminal fluids; p=0.009, n=4).