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Chapter 5 112 0 1 2 3 4 5 0.00 0.02 0.04 0.06 Mean abdominal pain score (2-week diary) TRPM8 mRNA expression relative to GAPDH (2 - ' Ct ) Sigmoid Proximal Colon 0.00 0.05 0.10 0.15 0.20 TRPM8 mRNA expresstion relative to GAPDH (2 - ' Ct ) * A B C IL-8 IL-10 IL-1β IL-6 TNF-α Control Icilin (1µM) 0 10 20 30 40 50 pg/mL ** Control Icilin (1µM) 0 20 40 60 80 100 pg/mL * Control Icilin (1µM) 0 20 40 60 pg/mL * Control Icilin (1µM) 0 50 100 150 pg/mL Control Icilin (1µM) 0 50 100 150 200 250 pg/mL A B C D E Based on the experiments performed on the localization of TRMP8-IR, these data suggested that stimulation of TRPM8 may influence cytokine release by mucosal immune cells. Incubation of paired IBS biopsies with TRPM8 agonist icilin (1 μM) compared to buffer control, significantly reduced release of cytokines IL-1 β , IL-6, and TNF- α but not IL-10 or IL-8 ( Figure 5.3A-5.3E ). Figure 5.2 Colonic TRPM8 mRNA expression is increased in IBS patients. A: mRNA expression of TRPM8 in sigmoid biopsy tissue is significantly increased in IBS patients (IBS) compared to healthy controls (C) ( N =30 IBS, N =23 C, * P <0.001). Data shown as median and IQR, corrected for age and gender, with P <0.05 deemed significant. B: Differential expression of TRPM8 mRNA in right-sided colon (proximal) vs. sigmoid (distal colon) in mucosal samples from IBS patients ( N =24, * P <0.001). C: TRPM8 mRNA expression in sigmoid biopsies is significantly associated to pain symptom scores in IBS ( N =15, P =0.015, corrected for age and gender, with P <0.05 deemed significant). Figure 5.3 TRPM8 agonism reduces inflammatory cytokine release from IBS patient biopsies. Stimulation of IBS patient biopsies (paired) with the selective TRPM8 agonist icilin (1 μM) reduced release of inflammatory cytokines IL-1 β , IL-6, and TNF α but not IL-8 or anti-inflammatory cytokine IL-10. P <0.05 is deemed significant.