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 characterize 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 (PAR₂) antagonist. Proteomic analysis revealed IBD FS contained elevated levels of trypsin- and elastase-like serine proteases compared with 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 PAR₂ on colonic afferent nerves, offering a unique insight into luminal contributions to pain and identifying potential therapeutic targets for visceral hypersensitivity in IBD.