Zsa Zsa Weerts

Chapter 5 114 Certain limitations apply to our findings. First, we did not have the statistical power to differentiate findings according to IBS-subtype. Subtype-specific changes in neuro- immune activation have indeed been shown previously. 18 Second, patients were not specifically matched to controls. This resulted in an older IBS population, in particular in the Maastricht cohort (presumably due to a bias towards older patients undergoing colonoscopy). There were also more females in the IBS group compared to the control group, which was not significantly different in the aggregate cohort. We corrected for these factors by using multivariate linear regression corrected for age and gender. Third, diary scores were only completed by a subset of patients (15/30 of the Maastricht cohort). Although the subset was representative for the Maastricht IBS cohort and a significant association was shown between pain score and TRPM8 mRNA expression, the found association does not necessarily reflect a causal relationship. Fourth, the immunoreactivity identified need not necessarily reflect the exact expression of the TRPM8 protein molecule. The finding of potential TRPM8 expression on immune cells is a novel finding and should therefore warrant the necessary conformation from future studies. Nevertheless, our previous experiments using this same antibody supports the specificity of our findings. 25 Aside from the TRPM8-specific agonist, no additional antagonist was applied to further confirm TRPM8 specificity of the effects observed in the functional experiments. Therefore, future experiments with healthy controls and TRPM8 antagonists will need to be performed to corroborate findings. Finally, due to limited availability of biopsies from controls, cytokine release experiments were only conducted on IBS patient biopsies. Although the number of TRPM8-IR cells was significantly higher in IBS compared to healthy controls, release of inflammatory cytokines may be reduced in control biopsy tissue. The mechanism by which TRPM8 influences IBS symptom generation therefore remains to be established. We were not able to directly ascertain the expression and function of TRPM8 on dendritic cells, the presumed neuro-immune interaction is therefore primarily based on surrogate parameters derived from the current experimental setup and therefore have an exploratory rather than a confirmatory nature. As this was a small, preliminary study, findings will need to be substantiated in a larger IBS patient cohort. Nevertheless, fundamental understanding of the role of TRPM8 in the regulation of neuro-immune interactions, and in particular the identification of the endogenous agonist(s) of TRPM8, may provide further mechanistic insight and improve therapeutic targeting in IBS and other disorders characterized by chronic abdominal pain.