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

Litter differences in DTI metrics.

<p>Fractional anisotropy (a); mean (b), radial (c) and axial diffusivity (d) in white matter areas of litter A (grey columns, n = 13), B (black columns, n = 9) and controls (white columns, n = 8) on postnatal day 14. Mean, axial and radial diffusivity are shown in units of mm²/s. Data are presented as mean ± 95% confidence interval. Differences between litters are marked where significant (<i>p</i> < 0.05). Abbreviations: wm: all of white matter; bcc: corpus callosum (body); ec: external capsule; ic: internal capsule; IHH, intermittent hyperoxia-hypoxia; hf: hippocampal fimbriae. </p

Retina.

<p>(a-d): H&E retinal slices from control (a) and IHH animals at P14 (b-d) showing haemorrhage in the ganglion cell layer (b), inner nuclear layer (c) and outer nuclear cell layer (d). (e-h): Retinal wholemounts stained with endothelial-specific Biotinylated <i>Griffonia</i> (<i>Bandeiraea</i>) <i>Simplicifolia</i> Lectin I Isolectin B4 from controls at P28 with a mature vascular bed (e) and no vasculature extending beyond the ora serrata (f). Retinal vascular bed in IHH animal at P28 with less remodelling (g) and areas of vascularization beyond the ora serrata (h). (a-d): x400 magnification; scale bar = 50 μm. (e-h): x100 magnification; scale bar = 200 μm. Abbreviations, IHH, intermittent hyperoxia-hypoxia; P14, postnatal day 14; P28, postnatal day 28.</p

Vascular density.

<p>(a) Vascular density as % of controls (white columns, n = 4) in IHH animals at P14 (grey columns; IHH: n = 10) and P28 (black columns; IHH: n = 12). Data are expressed as mean ± 95% confidence intervals. (b-e) x400 magnification of lectin-stained endothelium in IHH (upper row) and control (lower row) at P14 (b & d) and P28 (c & e). *<i>p</i> = 0.005 IHH vs. control. Scale bar = 50 μm. Abbreviations: IHH, intermittent hyperoxia-hypoxia; P14, postnatal day 14; P28, postnatal day 28; pvwm, periventricular white matter. </p

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

Litter differences in T₂.

<p>T₂-relaxation time in the brain at P14 in litter A (grey columns, n = 13), litter B (black columns, n = 9) and C (white columns, n = 8) in the cortex (a), hippocampus (b), putamen (c) and thalamus (d). T₂-relaxation times are shown in milliseconds. Differences between litters are marked where significant (<i>p</i> < 0.05). Data are presented as mean ± 95% confidence interval. Abbreviations: IHH, intermittent hyperoxia-hypoxia; P14: postnatal day 14; P28: postnatal day 28. </p

DTI on postnatal day 14.

<p>Fractional anisotropy (a); radial (b); mean (c) and axial diffusivity (d) in white matter areas of controls (grey columns, n = 8) and IHH (black columns, n = 22). Mean, axial and radial diffusivity are shown in units of mm²/s. Data are presented as mean ± 95% confidence interval.* <i>p</i> < 0.04. Abbreviations: wm: all of white matter; bcc: corpus callosum (body); ec: external capsule; ic: internal capsule; IHH, intermittent hyperoxia-hypoxia; hf: hippocampal fimbriae. </p

Rotarod testing.

<p>Mean time on a Rotarod on P20 and P27 of IHH (n = 12, black diamonds) and controls (n = 4, grey boxes). *<i>p</i> ≤ 0.03 IHH vs. control. Data are presented as mean ± 95% confidence intervals. Abbreviations: IHH, intermittent hyperoxia-hypoxia; P20, postnatal day 20; P27, postnatal day 27.</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

Albumin.

<p>Albumin immunoreactivity (brown stain) at P14 (a-b) and P28 in controls (c-d) and IHH (e-f). Note positive immunoreactivity for albumin in both experiment groups at P14 and several spots of albumin immunoreactivity in the cortex and thalamus of the IHH animal at P28. Albumin is present in intracellular vesicles in neuroependymal cells in the ventricles (b & d). (g) The sum of positive scores for albumin leakage in the neuropil of the respective brain areas are presented as fractions of the maximum possible score in each experiment group at P28. (a, c & e): scale bar = 2 mm (b, d & f): scale bar = 50 μm at x400 magnification. Abbreviations: IHH, intermittent hyperoxia-hypoxia; P14, postnatal day 14; P28, postnatal day 28. </p