Expression of CCL20 and Its Corresponding Receptor CCR6 Is Enhanced in Active Inflammatory Bowel Disease, and TLR3 Mediates CCL20 Expression in Colonic Epithelial Cells

<div><p>Background</p><p>The chemokine CCL20 and its receptor CCR6 are putative drug targets in inflammatory bowel disease, and CCL20 is a novel IBD predilection gene. Previous findings on the CCL20 response in these diseases are divergent. This study was undertaken to examine CCL20 and CCR6 during active and inactive disease, and mechanisms for CCL20 regulation by the innate immune system. As TLR3 has recently emerged as a possible mediator of CCL20 production, we hypothesised that this TLR plays an important role in enterocytic CCL20 production.</p><p>Methods</p><p>A large microarray study on colonic pinch biopsies from active and inactive ulcerative colitis and Crohn’s disease provided background information. CCL20 and CCR6 were localized and their expression levels assessed in biopsies using <i>in situ</i> hybridization and immunohistochemistry. Regulation of CCL20 was studied in the HT29 cell line using a panel of pattern recognition receptor ligands followed by a TLR3 siRNA assay.</p><p>Results</p><p><i>CCL20</i> and <i>CCR6</i> mRNA abundances were increased during active inflammation (<i>CCL20</i> 5.4-fold in ulcerative colitis and 4.2-fold in Crohn’s disease; <i>CCR6</i> 1.8 and 2.0, respectively). <i>CCL20</i> and <i>CCR6</i> mRNA positive immune cells in lamina propria were more numerous, and CCL20 immunoreactivity increased massively in the epithelial cells during active inflammation for both diseases. TLR3 stimulation potently induced upregulation and release of CCL20 from HT29 cells, and <i>TLR3</i> silencing reduced CCL20 mRNA and protein levels.</p><p>Conclusions</p><p>The CCL20-CCR6 axis is involved during active inflammation in both ulcerative colitis and Crohn’s disease. The epithelial cells seem particularly involved in the CCL20 response, and results from this study strongly suggest that the innate immune system is important for activation of the epithelium, especially through TLR3.</p></div

Localization of <i>CCR6</i> mRNA in colonic biopsies.

<p>In situ hybridization of <i>CCR6</i> mRNA in colonic inflammatory bowel disease tissue. Sections are taken from biopsies active (UCa) or inactive (UCi) ulcerative colitis, and active (CDa) or inactive (CDi) Crohn’s disease. High and low expression of <i>CCR6</i> in inactive disease is shown. Scale bars as indicated.</p

CCL20 gene expression and protein release in TLR3 transfection assay.

<p><b>A:</b><i>TLR3</i> and <i>CCL20</i> mRNA abundance in poly (I:C) stimulated HT29 cells with and without TLR3 small interfering RNA(siRNA) transfection. Non-signalling siRNA is designated nsRNA, two different TLR3 specific siRNAs (TLR3.6 and TLR3.8) were used alone or in combination. The cells were transfected for 24 hours using TLR3 siRNAs or nsRNA and then stimulated with the TLR3 ligand poly (I:C) for 20 hours. Controls were unstimulated untranfected cells, poly (I:C) stimulated cells and cells treated with nsRNA stimulated with poly (I:C). ** p< 0.01 versus nsRNA, ## p<0.01 versus unstimulated control. Mean ± SD of triplicated is shown. <b>B:</b> The poly (I:C) effect on CCL20 release in HT29 cells after transfection with TLR3 siRNAs as described above. *** p< 0.001 versus nsRNA, #### p<0.0001 versus unstimulated control. Mean ± SD of triplicates is shown.</p

Counting of CCR6 positive cells in Immunohistochemistry and <i>in situ</i> hybridization.

<p>Number of positive staining cells given in mean ± SD. N = controls, UC = Ulcerative Colitis, CD = Crohn's Disease, a = active, i = inactive.</p><p>* p<0.05 versus control.</p><p>** p<0.01 versus control.</p><p>^# p<0.05 versus inactive disease.</p><p>^## p<0.01 versus inactive disease.</p><p>Counting of CCR6 positive cells in Immunohistochemistry and <i>in situ</i> hybridization.</p

<i>CCL20</i> and <i>CCR6</i> gene expression in colonic biopsies.

<p><b>A and B:</b> Microarray gene expression results of <i>CCL20</i> and <i>CCR6</i> in colonic biopsies from healthy controls (N), active (UCa) or inactive (UCi) ulcerative colitis, and active (CDa) or inactive (CDi) Crohn’s disease. Individual values (Log₂) and mean are plotted. <b>C and D:</b> qRT-PCR gene expression results of <i>CCL20</i> and <i>CCR6</i> in colonic biopsies from N, Ulcerative Colitis (UC) and Crohn’s disease (CD), n = 5 in each group. Individual values (foldchange 2^-ΔΔCt) and mean are plotted. *p<0.05 versus N, **p<0.01 versus N, ***p<0.001 versus N, ###p<0.001 versus inactive disease.</p

CCL20 release from HT29 cells in PRR stimulation assay.

<p>Stimulation assay in the HT29 cell line. CCL20 release after 20 hour stimulation with ligands for pattern recognition receptors (PRRs) from left to right, TLR-1–9 and NOD2; and IL-10 and IL-1β for 20 hours. Control is medium alone. Abbreviations, PRR ligands and concentrations used are given in material and methods. * p<0.05 versus medium. Mean ± SD is shown.</p

CCR6 protein and mRNA in serial sections.

<p>Immunohistochemistry (IHC) and in situ hybridization (ISH) show CCR6 protein and mRNA in colonic biopsies from active UC and inactive CD. Serial sections from the same biopsy were used to compare the localization of mRNA and protein. Arrows show that there is no overlap of protein and mRNA in active disease (a, b), while a clear overlap is seen in inactive disease(c). Scale bars as indicated.</p

Characteristics of subjects enrolled in microarray analysis.

<p>Age and duration of disease are given as mean and gender and medication as numbers. UC = Ulcerative colitis. CD = Crohn's disease.</p><p>* Significantly higher use of 5-ASA/S-ASA in UC vs CD subjects.</p><p>Characteristics of subjects enrolled in microarray analysis.</p