Crohn’s disease is a chronic inflammatory disorder of the gastrointestinal tract associated with abdominal pain and diarrhea. Pain caused by Crohn’s disease likely involves neurogenic inflammation which seems to involve the ion channel transient receptor potential ankyrin 1 (TRPA1). Since the promoter methylation of TRPA1 was shown to influence pain sensitivity, we asked if the expression of TRPA1 is dysregulated in patients suffering from Crohn’s disease.
The methylation rates of CpG dinucleotides in the TRPA1 promoter region were determined from DNA derived from whole blood samples of Crohn patients and healthy participants. Quantitative sensory testing was used to examine pain sensitivities. Results
Pressure pain thresholds were lower in Crohn patients as compared to healthy participants, and they were also lower in females than in males. They correlated inversely with the methylation rate at the CpG − 628 site of the TRPA1 promoter. This effect was more pronounced in female compared to male Crohn patients. Similar results were found for mechanical pain thresholds. Furthermore, age-dependent effects were detected. Whereas the CpG − 628 methylation rate declined with age in healthy participants, the methylation rate in Crohn patients increased. Pressure pain thresholds increased with age in both cohorts. Conclusions
The TRPA1 promoter methylation appears to be dysregulated in patients suffering from Crohn’s disease, and this effect is most obvious when taking gender and age into account. As TRPA1 is regarded to be involved in pain caused by neurogenic inflammation, its aberrant expression may contribute to typical symptoms of Crohn’s disease. Background
Inflammatory bowel diseases (IBD) including Crohn’s disease and ulcerative colitis are chronic inflammatory disorders of the gastrointestinal tract resulting from multiple factors that seem to trigger an abnormal activation of the mucosal immune system through commensal microflora. Studies employing animal models on IBD reported that sensory neurons innervating the gastrointestinal tract are likely to be involved in these disorders [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 ]. Activation of sensory neurons results in a release of neuropeptides, including calcitonin gene-related peptide (CGRP) and substance P (SP). Both these peptides induce several effects including vasodilation, an autocrine sensitization of sensory neurons, extravasation and activation of immune cells [ 6 ]. Thus, neuropeptide-induced effects seem to very well correlate with the clinical symptoms of IBD. Certain members of the transient receptor potential (TRP) ion channels, which are expressed in sensory neurons, have been shown to contribute to IBD-like symptoms [ 1 , 3 , 4 ] or colonic distension pain [ 11 ] in rodent models. TRP channels are activated by a wide range of chemical compounds, among them, some possess mechano- or thermosensitivity [ 12 ]. Substances, which are associated with TRPA1 activation, are isothiocyanates from mustard oil, wasabi, and horseradish, Δ(9)-tetrahydrocannabinol from marijuana, allicin from garlic, cinnamaldehyde from cinnamon oil, as well as reactive oxygen species and others [ 13 , 14 , 15 , 16 ]. TRPA1 possesses a species-dependent thermosensitivity [ 17 , 18 , 19 , 20 ], and a recent study suggests that, at least in humans, the channel is able to respond to both cold and heat [ 21 ]. Furthermore, in mice, TRPA1 was shown to be mechanosensitive [ 22 , 23 , 24 , 25 , 26 , 27 ], which correlates with studies on human volunteers showing that TRPA1 agonists are able to induce mechanical hypersensitivity when applied on the skin [ 28 ]. The TRPA1 agonist TNBS (2,4,6-trinitrobenzene-sulfonic-acid), used for induction of colitis, caused IBD-like symptoms in mice due to a release of SP and CGRP [ 3 ]. Also in patients suffering from IBD, an increase in SP was observed [ 29 ]. Similarly, previous studies demonstrated that the TRPA1-agonists isothiocyanate and formalin also induce colitis in rodents [ 30 , 31 ]. Although wild-type mice do not spontaneously develop intestinal inflammation as humans do [ 32 ] and the available animal models of IBD do not fully resemble the human condition [ 33 ], the symptoms and morphology induced by the TRPA1 agonists coincide very well with human IBD. Given the fact that there are several frequently used models based on TRPA1 agonists causing an IBD-like morphology in mice, this ion channel could be involved in the development of IBD in humans as well. In human colon samples from patients suffering from ulcerative colitis or Crohn’s disease, TRPA1 was found to be strongly upregulated [ 34 ], suggesting dysregulation of TRPA1-expression to possibly contribute to the development, maintenance, or typical symptoms of IBD.
TRPA1 was demonstrated to mediate pain-like behavior in rodent models of visceral pain, and also visceral hyperalgesia developing in models of IBD was reported to be mediated by TRPA1 [ 30 , 35 , 36 , 37 , 38 ]. There is little doubt that TRPA1 is an important determinant of pain sensitivity in humans, as well. A rare hereditary gain-of-function mutation is associated with an intensive pain phenotype [ 39 ], and a SNP (single-nucleotide polymorphism) in the TRPA1 gene was reported to be associated with paradoxical heat sensations in neuropathic pain patients [ 40 ]. Here, we refer to the relevance of the epigenetic regulation of TRPA1 . The promoter methylation of TRPA1 was initially reported to be strongly associated with thermal pain thresholds [ 41 ]. We and others could demonstrate that the methylation rate of the TRPA1 promoter also seems to predict pressure pain thresholds in healthy individuals as well as neuropathic pain symptoms in patients suffering from chronic pain [ 42 , 43 ]. Hypothesizing that the previously reported elevated expression of TRPA1 in individuals suffering from Crohn’s disease might be due to epigenetic regulation, we used DNA from whole blood of Crohn patients and healthy participants to analyze the methylation status of individual CpG sites in the TRPA1 promoter with regard to pain sensitivity. Results Quantitative sensory testing and postoperative morphine consumption in Crohn patients
Mean values for QST parameters and postoperative morphine consumption of female and male Crohn patients are shown in Table 1 . Deviance from normal distribution was checked according to Shapiro-Wilk. HPT was shown to be the only normally distributed variable of the QST data ( P = 0.052). Comparison between female and male Crohn patients by calculation with Mann-Whitney U test (random sample selection) revealed only significant differences for HPT ( P = 0.020) and PPT ( P = 0.039), with females showing lower thresholds as compared to males. Table 1 Patient-controlled analgesia and quantitative sensory testing in Crohn patients Full size table Comparison of the pressure pain thresholds in healthy participants as compared to Crohn patients
The measurement of PPT via algometer revealed significant differences between healthy participants and Crohn patients (Mann-Whitney U , p 20 for Trace Score in the Quality Control Report were included for further analyses (11 of 147 sequences were excluded due to low quality). All statistical calculations were performed using the Statistical Package for the Social Sciences (SPSS 24, IBM, Armonk, NY, USA). We used GraphPad Prism for Windows 5.03 for data illustration (Graphpad Software Inc., La Jolla, CA, USA). Single CpGs with less than 95% sequencing success among the samples were excluded, which applied to CpG − 161, − 40, and − 38 ahead of the first exon, as well as CpG + 147, + 333, and + 335 of the first exon. Samples with less than 95% sequencing success of overall CpGs, which applied to 6 samples, and CpG sites with less than 5% inter-individual variability, which applied to CpG − 480, + 13, + 15, and + 191 were also not further analyzed. After applying the criteria for data exclusion as mentioned previously, the remaining number of samples dropped from 147 (88 healthy participants, 59 Crohn patients) to 130 (75 healthy participants, 55 Crohn patients), the number of analyzed CpGs dropped from 55 to 45. Methylation levels for individual CpG sites are provided in Fig. 5 . No apparent differences in methylation rate at single CpG sites between healthy participants and Crohn patients were visible in this figure at first sight. Deviance from normal distribution was checked according to Shapiro-Wilk. In case of normally distributed variables, parametric methods were used; for all other cases, nonparametric tests were applied. Multiple linear regression (BACKWARD method) was conducted to identify significant predictors for values of PCA, QST, and demographic and disease-associated data. In each analysis, a p value of
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