Collagen proteolysis is important in neutrophilic intestinal auto-inflammation found in inflammatory bowel disease via proline-glycine-proline

Background & Aims: Recently, a collagen derived fragment, Proline-Glycine-Proline (PGP), was shown to have chemotactic properties on neutrophils via CXCR2 in several lung diseases. PGP is formed from collagen by the combined action of matrix metalloproteinase (MMP)- 8 and/or MMP-9 and prolyl endopeptidase (PE). We aimed to investigate the role of PGP in inflammatory bowel disease (IBD). Methods: MMP-8, MMP-9 and PE were detected in intestinal tissue of human IBD patients, colorectal cancer controls, and mice undergoing Dextran Sodium Sulfate (DSS)-induced colitis by ELISA, immunoblot and immunohistochemistry. PGP levels were measured by mass spectrometry in intestinal tissue homogenates and neutrophil infiltration was assessed by immunohistochemistry and myeloperoxidase ELISA. PGP neutralization was realized by using the PGP antagonist, argine-threoninearginine (RTR), and specific antibodies. Protease release from neutrophils isolated from IBD patients and healthy controls was measured and neutrophil-conditioned medium was incubated with collagen type I to study degradation into PGP. Results: In the intestine of IBD patients, MMP-8 and MMP-9 were elevated in inflamed areas, while PE and PGP were both expressed at similar levels in non-inflamed areas and/or control tissue. Neutrophils isolated from IBD patients showed increased release of both MMPs and were far more capable of generating PGP from collagen compared to healthy control neutrophils. In the inflamed intestine of DSS treated mice, similar results were found. Furthermore, PGP neutralization during DSS colitis lead to a significant reduction of intestinal inflammation, especially in the infiltration of neutrophils. Conclusion: The proteolytic cascade that generates PGP from collagen, as well as the tripeptide itself, is present in the intestine of IBD patients and DSS treated mice. PGP neutralization in DSS treated mice showed that PGP guided neutrophilic infiltration plays an important role in intestinal inflammation, and opens new venues in treating the ongoing inflammation of IBD patients.