Abstract
Background
Chromogranin-A (CgA) is elevated in inflammatory bowel disease (IBD) and in murine models of colitis. CgA produced by enteroendocrine cells can be cleaved in several peptides including the immunoregulatory pancreastatin (PTS: CgA273-301). Macrophages (Mo) play a major role in IBD through an impaired transition from a pro-inflammatory (classical activated Mo (CAMs)) to anti-inflammatory (alternative activated Mo (AAMs)) phenotypes. Previously we demonstrated that in mouse, the lack of CgA (CgA-/-) induced a significant decrease of colitis associated to a decreases and an increase of CAM and AAM markers respectively
Aims
The aim of this study was to investigate the role of PTS in colitis using a pre-clinical and clinical approaches
Methods
Serum level of PTS and mRNA expression of CgA were quantified in active UC and healthy individuals & mice using ELISA & RT-qPCR. Colitis was induced in CgA-/- and wild type (WT) mice by administrating dextran sulfate sodium in drinking water (DSS 5%, 5 days), and PTS treatment (2.5mg/kg/day, 6 days) or vehicle started 1 day before induction. Disease activity index (DAI) was daily evaluated, and macroscopic scores at sacrifice day. The serum level of CRP was quantified using ELISA. Colonic MPO, IL-1β, IL-6, TNF-α, MIP-1α, MIP-1β, ARG-1, YM1 & TGFβ were assessed using ELISA or RT-qPCR. Naïve peritoneal macrophages were isolated from CgA-/- and WT mice and treated with PTS (200ng/ml; 2, 24h) then exposed for 6 h to LPS (100 ng/ml) or to IL-4/IL-13 (20ng/ml) to promote CAMs or AAMs. CAMs markers (IL-6, IL-1β, TNF-α, MIP-1α, MIP-β) and AAMs markers (ARG-1, YM1, TGFβ) were quantified by using ELISA and/or RT-qPCR. In vitro chemotaxis activity of PTS (200 ng/ml) on naïve Mo was assessed by migration assay using MCP-1 (30ng/ml)
Results
CgA & PTS levels significantly increased in human active UC and WT colitic mice. In colitic CgA-/- mice, we confirmed a significant increase of colitis through the regulation of all the pro and anti-inflammatory markers. PTS treatment significantly increased the onset and severity of colitis in CgA-/- and in WT mice. Macroscopic score, CRP, colonic MPO, IL-1β, IL-6, TNF-α, MIP-1α and MIP-1β increased significantly, while ARG-1, YM1, TGFβ decreased in colitic WT & CgA-/- mice treated with PTS. IL-6, IL-1β, TNF-α, MIP-1α & MIP-β were significantly increased in PTS-conditioned CgA-/- CAMs but not in WT, however, a significant decrease of ARG-1, YM1, & TGFβ was demonstrated in both groups. Meanwhile, PTS-conditioned CgA-/- & WT AMS expressed significantly less ARG-1, YM1, & TGFβ when compared to IL-4/IL-13 control condition. In undifferentiated macrophages, PTS significantly increased macrophages migration
Conclusions
These findings suggest that PTS contributes to the pathogenesis of colitis and inflammatory process via the modulation of Mo phenotype and function. Targeting PTS may lead to novel therapeutic strategies in IBD
Funding Agencies
CCC, CIHR, NRCResearch Manitoba, Children’s Hospital Research Institute, NSERC