Abstract
Abstract Background Inflammatory Bowel Disease (IBD) is a gastrointestinal disorder comprising Ulcerative Colitis (UC) and Crohn´s Disease (CD) characterized by an exacerbated and chronic immune response. Although GPR84 has emerged as a proinflammatory receptor in some pathologies such as atherosclerosis or reflux esophagitis, its role in IBD remains unclear. We aim to analyze the protein expression of GPR84 in IBD patients and its role in the cytokine secretion in macrophages. Methods Surgical resections from UC (n=5) and CD patients (n=5) were obtained. Healthy colon and ileum from colon-carcinoma patients (n=10) were used as controls. Protein expression of GPR84 and CD68 was analyzed by WesternBlot and immunohistochemistry. U937 monocytes were differentiated into macrophages with PMA (0,01μM) during 48 hours and treated with the GPR84 agonist decanoic acid (50-250μM) and a proinflammatory cocktail (0,1μM LPS + 20ng/mL IFNγ) during 24 hours. Some of them were treated with the GPR84 antagonist (10μM) or transiently silenced with a specific siRNA-GPR84. RNA was isolated and gene expression of pro- and anti-inflammatory cytokines was quantified by qPCR. Cytokine levels in supernatant were also quantified by Luminex. Data were expressed as fold induction vs control (mean±SEM) and compared by t-test. A p-value<0.05 was considered statistically significant. Results Protein levels of GPR84 were significantly increased in UC patients (150.3±11.75 vs 100.0±11.85), while no differences were observed in CD patients vs healthy ileums. Immunohistochemistry revealed a slight staining of this receptor in intestinal epithelial cells, whereas several cells of the lamina propria were strongly stained. Double immunohistochemistry confirmed that intestinal CD68 macrophages expressed GPR84 in both UC and CD patients. U937-derived macrophages treated with decanoic acid showed a significant increased expression of IL1b (2.68±0.86) and IL8 (2.44±0.48) vs vehicle-treated macrophages (0.998±0.23 and 1.05±0.17, respectively). In addition, IL1b levels in supernatant from macrophages treated with decanoic acid were significantly increased compared with the supernatant from vehicle-treated macrophages (4.50±1.05 vs 1.00±0.19). Moreover, the cotreatment with decanoic acid and GPR84 antagonist significantly reduced the expression of IL1b (0.99±0.29) vs vehicle-treated macrophages (1.94±0.26) and the transiently silencing of this receptor significantly reduced the expression of IL8 (0.89±0.25) vs siCtrl macrophages (4.02±1.59). Conclusion GPR84 is increased in UC patients and expressed in intestinal CD68+ macrophages. It regulates the expression of several cytokines pointing GPR84 as a promising pharmacological target for UC patients.
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call