Neue Biomarker zur Überwachung der zellulären Immunität chronisch-entzündlicher Darmerkrankungen

Currently there are no biomarkers which are able to indicate the disease activity or the risk of relapse of chronic inflammatory bowel disease. In this study new biomarkers of immune monitoring were examined for their usefulness in the assessment of disease activity in patients with inflammatory bowel disease (IBD). With the approve by the appropriate ethics committees, the intracellular ATP concentrations of CD4+ cells were measured in blood samples of 100 patients with IBD and set in relation to their disease activity. Previously three separate disease activity indices (CDAI, HBI, SCCAI) were used to assess clinical disease activity of the patients. The scores were obtained with standardized questionnaires from patient’s clinical data. It was noted no significant correlations between the CD4+ cell ATP concentrations and patient’s disease activity neither in the CDAI, HBI nor SCCAI score. This led to the conclusion that individual measurements of intracellular ATP concentrations of lymphocytes do not reflect the disease activity of patients with chronic inflammatory bowel disease. However, a significant difference in the intracellular concentrations of ATP in CD4+ cells has been demonstrated between patients undergoing Infliximab therapy and patients without Infliximab treatment. The patients receiving Infliximab had significantly lower intracellular ATP concentrations of lymphocytes (p <0.01, Mann-Whitney U) compared to those not receiving this drug. This result emphasizes that Infliximab inhibits the immune response as inhibiting the activity of lymphocytes by binding TNF-α. Therefore using the measurements of intracellular ATP concentrations would possibly give a tool to monitor the effectiveness of the inhibition of the immune response by TNF-α-blockers. Furthermore, in 99 patients with IBD the numbers of regulatory T cells were quantified in the peripheral blood. Therefore the cells were stained by using CD4-, CD25-, CD127- and FoxP3 antibodies and quantified by FACS analysis. Then, the determined number of regulatory T cells (CD4+ CD25highCD127-FoxP3+ cells) has been correlated with the disease activity of IBD patients. Again no significant correlation has been detected. By subdividing the patients into groups with increased and decreased disease activity, a difference in the number of regulatory T cells indicated, however, was not significant (p = 0.073, Mann-Whitney U test). These results led to the assumption that the quantification of regulatory T cells is not suitable as a surrogate marker for disease activity of patients with chronic inflammatory bowel disease. In addition, it was postulated that the quantification of Tregs can provide no help in distinguishing between the two diseases Crohn's disease and ulcerative colitis. The tendency of the difference in the number of Tregs between patients with lower and higher disease activity shows that regulatory T cells appear to play a role in the pathogenesis of chronic inflammatory bowel disease. However, there also have to be other pathogenic factors in the complex etiology of chronic inflammatory bowel disease. In 35 of the patients with IBD also another method for the quantification of regulatory T cells has been applied. It was a DNA methylation assay, which determines the regulatory T cells through a specific demethylated region of DNA (TSDR). This TSDR is demethylated in Tregs, whereas it is methylated in all other cells of the blood. However, the results of this assay did not correlate with disease activity of patients and did not correlate well with the results for the number of regulatory T-cells quantified by FACS analysis. This may be due to the fact that in the FACS-analysis in contrast to the DNA methylation assay also activated T effector cells, which only transiently express FoxP3, are quantified. Secondly in the methylation assay also FoxP3+ and CD8+ cells which have no or only low-regulatory properties are quantified, and will not be quantified in flow cytometry. In addition, a lack of correlation between the results of the two methods might be because the Tregs quantified by flow cytometry refer to the totality of CD4+ cells, while the Tregs quantified by the DNA methylation assay refer to the total of the DNA containing cells. For better comparability, a ratio of Tregs and CD4+ cells could be made in future studies. In summary, in this work it has been shown that neither using the intracellular ATP concentrations of lymphocytes nor quantifying the number of regulatory T cells can make a statement regarding the disease activity or the risk of relapse of IBD patients. Since inflammatory bowel diseases are currently incurable, more surrogate markers are needed in order to objectify disease activity and to counteract disease relapse in time.