## 3 Discussion  
Our previous studies, using both immunohistochemistry and in situ hybridization showed a high density of CCL20 positive immune cells in the lamina propria during active IBD [18]. In the present study PBMCs had low basal CCL20 gene expression and release with no difference between disease groups, suggesting that the regulation of CCL20 expression in immune cells mainly occurs after immune cells have reached the colonic mucosa. CCL20 was highly inducible in PBMCs, in particular through TLR2/1, NOD2 and IL-1\(\beta\). NOD2 induced release of CCL20 is supported by [PERSON] et al., who showed that CCL20 release from macrophages is NOD dependent [29]. We showed for the first time that PBMCs from the UC group tend to have a stronger NOD2, TLR2/1 and IL-1\(\beta\) induced CCL20 release than PBMCs from the CD group, suggesting a disease dependent difference in CCL20 involvement. Response to IL-1\(\beta\) stimulation and reduced release following IL-10 stimulation indicated that CCL20 release from PBMCs is a trait of a proinflammatory environment. We proceeded to show that CCL20 release from PBMCs after TLR2/1 and NOD2 activation is not only secondary to IL-1\(\beta\) production but also a direct response to stimulation of these PRRs. It has been established that CCL20 has antimicrobial actions and release of CCL20 in response to bacterial components could be part of an antibacterial response, both directly and through immune cell recruitment. Cells recruited to the mucosa along a CCL20 gradient may commence CCL20 production upon arrival, further accelerating cell recruitment, a process common in chemokine biology [30]. In the colonic mucosa, _CCR6_ is increased during active IBD and in our previous study [18] we found an increase in CCR6 mRNA positive immune cells in lamina propria in IBD during active inflammation, compared to IBD in remission and healthy controls. As the main recognized function of  
Figure 4: CCR6\({}^{+}\) populations in PBMCs Flow cytometry measuring CCR6\({}^{+}\) cells in the main cell populations (CD4\({}^{+}\) T cells, CD8\({}^{+}\) T cells, CD16\({}^{+}\) NK cells and CD19\({}^{+}\) B cells and CD14\({}^{+}\) monocytes), in 10,000 PBMCs from each subject (\(n\) = 40), in the groups healthy controls (N), active ulcerative colitis (UCa) inactive ulcerative colitis (UCi), active Crohn’s disease (CDa) and inactive Crohn’s disease (CDi). Bars show mean frequency (%) with standard deviation of CCR6\({}^{+}\) cells in different cell populations and individual values are plotted. Statistical comparison was performed using unpaired \(t\)-test with levels of significance denoted by * \(p\) < 0.05. CCL20 is recruitment of CCR6+ immune cells, investigating the expression of CCR6 on immune cells is relevant. We found reduced CCR6 mRNA and CCR6+ cells in PBMCs from active UC compared to inactive UC, active CD and healthy controls. [PERSON] et al. have shown that frequency of circulating CCR6+ cells is reduced during active UC and suggested that CCR6+ cells migrate from the circulation to the inflamed bowel mucosa and are thus reduced in peripheral blood [21]. An alternative explanation is downregulation of \(CCR6\) in response to an increase in CCL20 production in the mucosa. The expression of chemokine receptors is readily turned on and off through stages of maturation and activation [12; 31; 32], allowing traffic between and within tissues. However, we found no effect of CCL20 stimulation on \(CCR6\) expression in PBMCs, indicating that migration is a likely explanation. We found novel differences between UC and CD for CCL20 release and in CCR6 expression in PBMCs. As the pair is responsible for recruitment of both Treg and Th17 cells, recognised as important factors in IBD [33], this is an interesting discrepancy. Skewed sampling may lead to systematic group differences, for example, if the UC group tended to be more diseased than the CD group. However, there were no indications that this was the case in our patient material.