OP07Epithelial calprotectin contributes to the refractory nature of Crohn’s disease related fistula

Abstract Background Crohn-related perianal (CD) fistulas are often refractory to therapy and not amenable for surgical interventions, in particular in comparison to cryptoglandular fistula. However, the etiology of this difference remains largely unclear. We previously identified epithelial redifferentiation into keratinized epithelium as a physiological response in fistula healing. Although present, this activity was decreased in CD fistulas. In this study we aimed to identify the biology underlying this phenomenon. Methods The epithelial compartment of 30 fistulas (20 CD, 10 cryptoglandular) was analyzed using single cell RNA-sequencing and pseudotime analysis. Data was confirmed using IHC and functional effects validated in cell line and organoid cultures. Analysis included chromatin immunoprecipitation (CHIP), rt-pcr and protein analysis. Results The fistula tracts contained all stages of normal intestinal epithelium (LGR5+ stem cells to BEST4+ late enterocytes) as well as the keratinized epithelium we described previously (KRT5+, KRT13+, Fig 1a). Pseudotime analysis indicated differentiation from intermediate OLFM4+ cells to the keratinized subset (Fig 1b). Interestingly, the developmental trajectory showed a branching point with divergent tracts for cells derived from cryptoglandular versus CD fistulas with the CD-derived trajectory expressing more inflammatory genesets (Fig 1c). Close-up analysis of the branching point indicated epithelial calprotectin (S100A8/9) as a key factor in the aberrant development of keratinized tissue in CD (Fig 1d). Immunohistochemistry not only confirmed calprotectin protein expression in intestinal epithelium, but also showed that in particular in CD, expression was largely intranuclear. This was further validated using epithelial cell lines and organoids, where a mix of cytokines (TNF-a, IFN-g, IL22 and IL17), all known to be present in fistula tracts, was able to induce intracellular but not secreted calprotectin. The nuclear localization suggested a potential role in transcription, and CHIP analysis indeed showed binding of calprotectin to the loci of various inflammatory genes including C3, CXCL17 and LCN2 as well as their subsequent upregulation in response to the cytokine mixture. Knockdown of either S100A8 or A9 abrogated this response, showing calprotectin is a crucial mediator of inflammatory signals in inflammatory epithelium in CD related fistula. Conclusion These data show a novel role for calprotectin in IBD. In fistula derived epithelium, calprotectin functions as a transcriptional regulator, relaying cytokine signals resulting in an inflammatory phenotype of epithelium not amenable to surgical closure. Interventions in this pathway may improve therapy responsiveness in Crohn-related fistula. Conflict of interest: Becker, Marte: No conflict of interest Koelink, Pim: No conflict of interest Ouahoud, Sarah: No conflict of interest Meisner, Sander: No conflict of interest Buskens, Christianne J.: Grant: C. Buskens has received an unrestricted grant from Boehringer Ingelheim and Roche Personal Fees: C. Buskens has received consultancy fees and/or speaker’s honoraria from Tillotts, Takeda, MSD and Janssen Wildenberg, Manon: Received research grant support from Hoffman-La Roche, Boehringer-Ingelheim