DOP028 The resolution of fecal calprotectin configurations and their biological functions in gut inflammation
Authors: Jukic A , Hilbe R , Zundel L , Willeit P , Vouk K , Faserl K , Zollner A , Meyer M , Kozsar A , Schwärzler J , Marteau V , Vich Vila A , Grabherr F , Mayr L , Aden K , Weersma R K , Trajanoski Z , Tilg H , Adolph T E 
Published: January 2025

Abstract

Background
Fecal calprotectin (CP) serves as a worldwide established biomarker used for the diagnosis and management of inflammatory bowel diseases (IBD). However, the biological function of CP in the human gut is still unknown and numerous studies obtained contradictory results. Calprotectin is mainly described to be present as a tetrameric protein complex (S100A8/S100A9)2, while the quaternary protein structure (configuration) of S100A8 and S100A9 in the human gut lumen remains enigmatic.1 In this study we resolved the fecal protein configurations of calprotectin and their biological functions in IBDs.

Methods
We defined biological functions of S100A8 and S100A9 dimers by antibody-mediated immunoprecipitation of S100A8 and S100A9 from stool obtained from IBD patients coupled with subsequent LC-MS/MS analysis. Next, we analysed human colonic organoids by single-cell RNA sequencing after S100A8 or S100A9 stimulation. Moreover, we orally exposed mice to recombinant human S100A8 and S100A9 protein during experimental gut inflammation and genetically inactivated S100a9 in toxic and genetic mouse models. To identify the protein configurations of S100A8 and S100A9 in the human gut lumen we examined stool samples from IBD patients using size-exclusion chromatography and LC-MS/MS analysis.

Results
We demonstrate pro-inflammatory actions for S100A8 and S100A9 dimers as oral administration of human S100A8 and S100A9 homodimers, but not CP, worsened experimental enteritis and colitis. In accordance, multi-omic phenotyping revealed that human S100A8 and S100A9 homodimers induce the NLRP1 inflammasome in intestinal epithelium. In turn, genetic deletion of S100a9 ameliorated experimental gut inflammation. S100A8 and S100A9 dimers were frequently detectable in stool from IBD patients from three independent IBD cohorts, but not in healthy individuals. More specifically, fecal S100A9 detection was associated with clinical and endoscopic disease activity in IBD patients with low fecal CP levels (≤150µg/g). Importantly, four fecal CP assays in clinical use were not able to detect S100A8 and S100A9 homodimers.

Conclusion
Our study is the first to reveal the presence of S100A8 and S100A9 dimers in stool from IBD patients. Moreover, we demonstrate a diagnostic gap for fecal CP assays in clinical use. We establish that fecal S100A9 detection is associated with endoscopic disease activity in IBD patients in which fecal CP fails to identify active disease. We reveal disease-modifying functions for S100A8 and S100A9 homodimers by inducing the NLRP1 inflammasome, while the same effects were not observable for calprotectin. Taken together, our findings may reflect a major advance in the IBD biomarker field.

References
Jukic, A., Bakiri, L., Wagner, E. F., Tilg, H. & Adolph, T. E. Calprotectin: from biomarker to biological function. Gut 70 , 1978-1988 (2021). https://doi.org/10.1136/gutjnl-2021-324855