C-C Motif Ligand 20 (CCL20) and C-C Motif Chemokine Receptor 6 (CCR6) in Human Peripheral Blood Mononuclear Cells: Dysregulated in Ulcerative Colitis and a Potential Role for CCL20 in IL-1β Release

Abstract

The chemokine C-C motif ligand 20 (CCL20) is increased in the colonic mucosa during active inflammatory bowel disease (IBD) and can be found both in the epithelium and immune cells in the lamina propria. The present study investigated CCL20 and C-C motif Chemokine Receptor 6 (CCR6) in peripheral blood mononuclear cells (PBMCs) (n = 40) from IBD patients and healthy controls, to identify inductors of CCL20 release encountered in a local proinflammatory environment. CCL20 release from PBMCs was increased when activating TLR2/1 or NOD2, suggesting that CCL20 is part of a first line response to danger-associated molecular patterns also in immune cells. Overall, ulcerative colitis (UC) had a significantly stronger CCL20 release than Crohn’s disease (CD) (+242%, p < 0.01), indicating that the CCL20-CCR6 axis may be more involved in UC. The CCL20 receptor CCR6 is essential for the chemotactic function of CCL20. UC with active inflammation had significantly decreased CCR6 expression and a reduction in CCR6+ cells in circulation, indicating chemoattraction of CCR6+ cells from circulation towards peripheral tissues. We further examined CCL20 induced release of cytokines from PBMCs. Stimulation with CCL20 combined with TNF increased IL-1β release from PBMCs. By attracting additional immune cells, as well as inducing proinflammatory IL-1β release from immune cells, CCL20 may protract the inflammatory response in ulcerative colitis.

Intestinal Epithelial Cells Express Immunomodulatory ISG15 During Active Ulcerative Colitis and Crohn’s Disease

“…Colonic pinch biopsies [Sample set I] and peripheral blood mononuclear cells [PBMCs] [Sample set II], were collected as described previously. 21 , 25 …”

“…PBMCs from non-IBD controls [ n = 7], inactive CD [CDi, n = 8], and inactive UC [UCi, n = 9], and from active CD [CDa, n = 8] and active UC [UCa, n = 7], were randomly selected from Sample set I, as described. 25 PBMCs isolated from buffy coat from six healthy blood donors at the Department of Immunology and Transfusion Medicine, St Olav’s University Hospital, were included for additional functional assays, as described below.…”

“…Human PBMCs [Sample set II] were prepared and thawed as previously described. 25 For stimulation experiments, recombinant human ISG15 [500 ng/mL] and/or recombinant human IL12 [20 ng/mL] were added for 48 h before conditioned medium was harvested and stored at -80°C.…”

“…PBMC subpopulations in 35 samples [Sample set II] had previously been identified by flow cytometry analysis, 25 and the data were used for correlation analyses in the present study. Detailed description of antibody staining and compensation controls are given in Skovdahl et al .…”

“…Detailed description of antibody staining and compensation controls are given in Skovdahl et al . 25 Flow cytometry was performed on a BD FACS Canto II flow cytometer with FACS Diva software from BD Bioscience [10 000 events per sample] and samples were analysed using FlowJo v10 software from Flow Jo, LLC.…”