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Chapter 5 110 Wilcoxon matched-pairs signed rank test was used. Significance was defined as P <0.05. Considering the exploratory nature of the experiments, no formal sample size calculation or correction for multiple testing was applied. All data is presented as median and interquartile range (IQR) and P <0.05 was deemed significant. Results In human, uninflamed colonic tissue obtained from surgical resection, TRPM8 immunoreactivity (IR) was observed to colocalize with cell bodies within the myenteric plexus and co-labelled with CGRP ( Figure 5.1A ), corroborating findings in mouse myenteric ganglia. 10 Majority of TRPM8-IR in the inflamed mucosa (of Crohn’s disease patients) was found to colocalize with CD11c+ immune cells with a small population colocalizing with CD45+ (expressed in all differentiated haematopoietic cells) immune cells ( Supplementary Figure S5.1 ), and not with CGRP-IR mucosal nerve endings as previously reported in mice 13 ( Figure 5.1B ). TRPM8-IR CD11c+ cells were however, in close apposition to mucosal CGRP-IR nerve fibers ( Figure 5.1B ). As CD11c identifies a sub-population of dendritic cells (DC) in addition to monocytes 16 , we further explored the DC phenotype using CD103, a marker of a DC subset termed ‘conventional DC1’ commonly found in gastrointestinal mucosa. 17 In a representative IBS patient biopsy, TRPM8-IR was colocalized on both CD11c+ and CD103+ cells in colonic mucosa ( Figure 5.1C & 5.1D ). We also assessed TRPM8-IR in relation to mast cells and macrophage/monocytes and found absence of colocalization on these cell types ( Supplementary Figure S5.3 ). These findings suggested a possible pathway mediating IBS symptom relief following TRPM8 agonism considering the apparent colocalization of TRPM8-IR with dendritic cells in close proximity to CGRP-IR mucosal nerve fibers. We therefore examined TRPM8 expression in IBS patient and control biopsies from cohorts at Maastricht University Medical Centre+ (Netherlands) and The Royal London Hospital (U.K.) (56 IBS patients and 30 controls in total, demographic details in Table 5.1 ). mRNA expression of TRPM8 in the sigmoid colon was significantly higher in IBS patients compared to healthy controls ( P <0.001) ( Figure 5.2A , examined in 30 IBS patients, 23 controls, all obtained from the Maastricht Cohort). This increased expression was also found when counting TRPM-IR cells within the mucosa of IBS patients compared to controls ( P =0.045) ( Figure 5.1E , examined in 28 IBS patients (5/28 obtained from the Maastricht Cohort) and 7 controls (all obtained from the London Cohort)). IBS subtype analysis showed no differences in sigmoid TRPM8 mRNA expression between IBS subtypes. When comparing immunoreactivity, TRPM8-IR cells