Electrophysiological and proteomic evidence of protease-mediated pro-nociceptive signaling in inflammatory bowel disease patients

Abdominal pain is a debilitating symptom of inflammatory bowel disease (IBD). Despite advances in understanding IBD pathology, the mechanisms underlying pain remain poorly defined. While studies of tissue biopsies from IBD patients and rodent models have highlighted the roles of proinflammatory cytokines and proteases in pain signaling, these approaches predominantly capture host-derived mediators, overlooking the broader luminal environment influenced by the microbiota. Given the compromised barrier in IBD leading to increased mucosal permeability, examining the luminal milieu would characterise a novel source of factors involved in pain modulation in IBD patients with active disease. Fecal supernatants (FS) from healthy volunteers (HV) of either sex had no effect on ex vivo colonic afferent nerve mechanosensitivity or in vitro dorsal root ganglia (DRG) neuron excitability. In contrast, FS from Crohn's disease (CD) and ulcerative colitis (UC) patients of either sex significantly excited colonic afferent nerves and increased mechanosensitivity ex vivo, and increased DRG neuronal excitability in vitro . These were blocked by the serine protease inhibitor and a protease-activated receptor 2 (PAR 2 ) antagonist. Proteomic analysis revealed IBD FS contained elevated levels of trypsin- and elastase-like serine proteases compared to HV FS. Proteomics identified CELA3B, ELA2A, and PRSS1 as key proteases enriched in IBD FS, with distinct activity profiles in UC and CD. These findings establish that proteases within FS from IBD patients directly modulate pain-sensing pathways by activation of PAR 2 on colonic afferent nerves, offering a unique insight into luminal contributions to pain and identifying potential therapeutic targets for visceral hypersensitivity in IBD. Significance Statement: Abdominal pain is a debilitating symptom of inflammatory bowel disease (IBD), yet its underlying mechanisms remain poorly understood. Given the compromised mucosal barrier in IBD, the luminal milieu could play a crucial role in pathophysiology. This study reveals a novel role for luminal serine proteases in fecal supernatants from IBD patients as mediators of peripheral pain sensitization. Prior studies focus on tissue biopsies, which predominantly reflect host-derived mediators, this work highlights how proteases in the gut lumen, shaped by both host and microbial factors, sensitize nociceptive colonic afferent nerves via protease-activated receptor 2 activation. By revealing how luminal proteases contribute to pain signaling in IBD, this research underscores the therapeutic potential of targeting luminal protease signaling to alleviate abdominal pain.