Our findings encouraged us to investigate T cell subset compositions by flow cytometric analyzes. We used standard staining procedures to identify TH1, TH2, and TH17 cells, whereas for identification of anti-inflammatory Tregs, both classic extracellular staining of CD4 + CD25high and a more specific extraand intracellular staining of CD4 + CD25highCD127lowFoxP3+ was applied. As activated human T cells can transiently express FoxP3 and CD25, differentiation of Tregs from activated effector T cells by only using these two markers may suffer from inaccuracies. CD127 is a newly described surface marker that allows distinguishing regulatory T cells from other CD25 + cells. For TH17 identification, we chose two experimental approaches: determination the mRNA expression of the TH17 specific transcription factor RORcT by qPCR, and FACS analyses of IL-17 production, which has revealed as a very reliable method to identify TH17 cells. However, flow cytometry staining protocols combining IL-17 with further markers, e.g. CD161 or CCR6, may further refine these measurements and thus may be implemented in future studies. Flow cytometry clearly proved the assumed alterations of the TH17/Treg balance, as a significantly increased frequency of Tregs and decreased frequency of TH17 cells was Galanthamine HBr observed in our CLBP patients. Even in flow cytometric analyzes, no differences in the TH1/TH2 ratio were detectable. There are several investigations which point to a beneficial role of anti-inflammatory cells and cytokines together with a detrimental function of a proinflammatory Bisoctrizole immune response in pain patients. In contrast to these findings, our results showing an anti-inflammatory shift on cellular level are in accordance with other chronic diseases like mild depression or chronic fatigue syndrome. A potential explanation for our findings on TH17/Treg balance may therefore be that pain-related, long lasting chronic stress and fatigue induces an ongoing dysregulation of immune cells towards an anti-inflammatory phenotype.On the other hand, it may also be discussed that dysregulation of the TH17/Treg balance may exist first, thus predisposing the affected individuals to experience chronification of pain symptoms.