P0150Pouch success is correlated to inflammatory mesenteric macrophages producing high levels of calprotectin – potential for biomarker analysis

Abstract Background Alterations in the mesentery of Crohn’s disease (CD) are associated with increased inflammatory activity. Earlier work showed that in CD patients undergoing proctocolectomy, total mesenteric excision improved perineal healing compared with close rectal dissection, due to the mesorectal macrophages. 1,2 CD patients exhibited a predominantly pro-inflammatory (M1, CD206−) macrophage profile, while ulcerative colitis (UC) patients had a more regulatory (M2, CD206+) phenotype. [1 Given the similarity of persistent inflammatory activity in the rectal area in CD patients and UC patients with post-proctocolectomy complications, this study aimed to assess whether rectal mesenteric macrophage profiles are also associated with pouch outcomes. Also, macrophages were profiled both phenotypically and functionally with the aim of identifying a proxy marker for macrophage phenotype suitable for clinical use. Methods Mesenteric specimens were collected from 26 CD and UC patients undergoing surgical resection. The macrophage profile was determined by flow cytometry as CD45+ CD66b− CD14+ cells and divided into regulatory (CD206+) or pro-inflammatory macrophages (CD206−). Sorted cells were analysed by expression profiling and functional analysis. Patient characteristics and postoperative complications (e.g. pouch- and perineal complications), were collected from patients records. Results Patients who developed pouch related complications (pouchitis, CD in the pouch or other dysfunction) showed a significantly reduced ratio of regulatory (CD206+) to pro-inflammatory (CD206−) macrophages compared to those without complications (ratio 0.39 vs 0.05, p 0.001, Fig 1B), indicating a more pro-inflammatory phenotype. Within the macrophage population, two subsets could be identified on flow-cytometry with diverging expression of CD206 and CD11b + (Fig 2). The CD11b− CD206+ population was consistent with a more regulatory M2-like phenotype, while the CD11b+ CD206− was more consistent with the inflammatory M1-type. Among the top upregulated genes in CD11b+ macrophages were S100A8 and S100A9, the two heterodimeric subunits for the pro-inflammatory signalling molecule calprotectin/. Mesenteric calprotectin expression was assessed as proxy for a pro-inflammatory macrophage phenotype, a correlation between calprotectin levels and the M1/M2 ratio was observed (Fig 3). Conclusion In UC patients undergoing pouch surgery, a pro-inflammatory mesenteric macrophage profile is associated with pouch-related complications. Pro-inflammatory macrophages express high levels of calprotectin, identifying calprotectin as a promising biomarker for pro-inflammatory macrophage activity in the mesorectum, with potential utility for perioperative risk stratification. References: 1. de Groof EJ, van der Meer JHM, Tanis PJ, de Bruyn JR, van Ruler O, D’Haens G, van den Brink GR, Bemelman WA, Wildenberg ME, Buskens CJ. Persistent Mesorectal Inflammatory Activity is Associated With Complications After Proctectomy in Crohn’s Disease. J Crohns Colitis 2019;13:285-293. 2. van der Meer JHM, Wasmann K, van der Bilt JDW, Becker MAJ, Boermeester MA, Bemelman WA, Wildenberg ME, Buskens CJ. Anatomical Variation in Mesenteric Macrophage Phenotypes in Crohn’s Disease. Clin Transl Gastroenterol 2020;11:e00198. Conflict of interest: Mrs. Haanappel, Anouck: No conflict of interest Becker, Marte: No conflict of interest Bloemendaal, Felicia: No conflict of interest Van Der Does De Willebois, Eline: No conflict of interest Koelink, Pim: No conflict of interest D’Haens, Geert: No conflict of interest Hompes, Roel: No conflict of interest Bemelman, Willem: 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: No conflict of interest