P058 Phosphodiesterase 8A domains at the site of leucocyte–endothelial cell adhesion in ulcerative colitis submucosa

Abstract

Development of anti-adhesion therapies like Etrolizumab and Alicaforsen highlights the potential for inhibiting recruitment pathways in ulcerative colitis (UC). Recent in vitro mouse work from our group shows that disruption of Phosphodiesterase (PDE) 8A domains inhibits T cell adhesion to ICAM-1 and endothelial cells.1 PDE inhibitors are known immunosuppressive agents, yet no PDE inhibitors are approved in UC due to dose-limiting side effects. As PDE8A is the most efficient in vivo hydrolyser of cAMP, PDE8A inhibitors are good candidates for overcoming the challenges presented mainly by PDE4 inhibitors. PDE8A expression in colorectal cancer (CRC) is well documented and PDE8A is reported in UC intestinal crypts by gene and protein array. However, the in situ localisation of PDE8A in UC submucosa during recruitment of leukocytes is unknown. Therefore, we developed an image analysis method to highlight PDE8A domains and investigated whether PDE8A domains were present at the site of leucocyte-endothelial cell adhesion in the submucosa of inflamed UC resections. PDE8A protein expression in five involved and two uninvolved UC colon resections was determined by immunohistochemistry using anti-PDE8A (ab61815) or IgG isotype (ab125938). Six paired biopsies from CRC (involved and uninvolved) were used as controls. Antibodies were titrated to 1:250 to reveal subcellular domains masked by diffuse staining at 1:100. Images were analysed using the saturation feature (0-255) in Image J software to assign PDE8A staining intensity as a representation of subcellular domains (Figure A). This in situ study performed in human UC tissue reveals PDE8A domains in the plasma membrane of submucosal leucocytes and endothelial cells participating in adhesion and transmigration (see Figure B). The PDE8A domains and high intensity staining were seen in both involved and uninvolved UC. In contrast, the control CRC dysplasia showed increased PDE8A staining compared with adjacent non-dysplastic mucosa. A) PDE8A domain visualisation using Image J saturation analysis B) PDE8A domains are present at the site of leukocyte-endothelial cell adhesion (black arrows). Images from involved UC resections of one (A) and three (B) patients. The in situ localisation of PDE8A in UC is consistent with its role in regulating inflammation and adhesion. Together with a favourable therapeutic index, the presence of PDE8A domains at the plasma membrane in involved and uninvolved UC suggests PDE8A is a promising target for anti-adhesion therapy in UC and other immune mediated diseases, perhaps in early disease. Future studies are planned to determine the ability of a PDE8A disruptor to inhibit adhesion molecules in whole blood cultures and intestinal explants. 1. Basole CP, Nguyen RK, Lamothe K, et al. PDE8 controls CD4+ T cell motility through the PDE8A-Raf-1 kinase signaling complex. 2017. http://www.sciencedirect.com/science/article/pii/S0898656817302231