Background: 5-aminosalicylate (5-ASA) drugs constitute a major therapeutic option for ulcerative colitis (UC), but their mechanism of action remains incompletely understood. We employed an in vivo model of acute inflammation in humans both with and without UC to elucidate the functional impacts of 5-ASAs on the acute inflammatory response.
Methods: Acute inflammation was provoked by intradermal injection of killed E. coli (EC) in 26 UC patients and 23 healthy controls (HC), with individuals in both cohorts either on no immunomodulatory treatment or on 5-ASAs. Inflammatory exudates were sampled at 4h and 48h by raising suction blisters over the inoculation sites. Cells were characterised by polychromatic flow cytometry; and lipid mediators by mass spectrometry. The impact of lipid mediators on macrophage pro-inflammatory cytokine secretion was determined by ELISA following in vitro stimulation of peripheral-blood monocyte-derived macrophages with killed EC.
Results: An excessive inflammatory reaction clinically observed in UC was normalised in patients taking 5-ASA therapy. This enhanced resolution was associated with increased concentrations of the hydroxy fatty acids 9-oxo-octadecadienoic acid (OxoODE) and 13-OxoODE. To characterise the effect of these novel mediators, cultured macrophages were co-incubated with EC, in the presence of increasing concentrations of either 9-OxoODE or 13-OxoODE. 9-OxoODE led to a dose-dependent suppression of both TNF-α (p=0.0001) and MIP-1β secretion (p=0.002), and 13-OxoODE inhibited TNF-α secretion (p=0.01), at concentrations reflective of those detected in the in vivo model.
9-OxoODE was more potent than 13-OxoODE for TNF-α inhibition, with an IC50 of 100nM. These effects were completely reversed by GW9662, an antagonist at the PPAR-γ receptor (p=0.006). Notably, the in vivo profile of lipid mediators in HC treated for 5 days with 5-ASAs differed from that seen in UC, associated with subtle differences in the resolving cellular inflammatory infiltrates and cytokine milieu. This has important implications for understanding the generation of hydroxy fatty acids in vivo in patients.
Conclusions: We have uncovered an important novel pathway through which 5-ASAs normalise the overly exuberant acute inflammatory response in UC, by generation of hydroxy fatty acids that harness a previously unexplored pro-resolution pathway. These exert anti-inflammatory actions through the PPAR-γ receptor, providing potential new drug targets in this disease.