Formyl Peptide Receptor 2 (Fpr2) triggers pro-resolving responses during vascular inflammation

Vincenzo Brancaleone, Bartomeu Colom, Andrew D Leinster, Sussan Nourshargh, Roderick J Flower, Mauro Perretti. WHRI, Barts and The London SMD, London, UK

Formyl peptide receptor 2 (Fpr2) is a G-protein coupled receptor associated with inflammatory events, transducing the effects of Annexin A1 (AnxA1), a glucocorticoid-inducible protein, and Lipoxin A4 (LXA4), a short-lived lipid derivative [Gavins et al., 2003, Fiore et al, 1994]. These two agonists elicit exquisite tissue-protective effects. To investigate the mechanisms behind Fpr2-regulated resolution of inflammation, we performed intravital microscopy of the mesentery using ischaemia/reperfusion (I/R) as stimulus.

Male Fpr2-/- [Dufton et al. 2010] and littermate controls (WT), 20±5 g body weight, were used for the study. Mice were anesthetised and the superior mesenteric artery was occluded and re-opened (I = 30min; R = 90min); blood aliquots were collected by cardiac puncture at specific times (pre- and post-I and post-I/R). Pharmacological treatments with LXA4 (1-100ng/mouse i.v., Calbiochem, UK) and the pan-Fpr antagonist Boc-2 (10μg/mouse i.p., MP biomedicals, UK) were performed immediately before occlusion or before re-opening. In some cases, platelet/neutrophil aggregates were determined by flow cytometry, labelling cells with anti-CD41 (MWReg30, eBioscience, UK) for the platelet, and Ly6G (1A8, BD Pharmingen, UK) for the neutrophils. Data are mean ± SEM of 6-8 mice per group.

From initial experiments, we chose a 30+90 min I/R, which led to augmented vascular response in Fpr2-/-; as an example, emigrated cells raised from 9.6±1.2 to 14.5±2.2 per 50x100 µm2, in WT and Fpr2-/-, respectively (n = 8; P<0.05). Similar results were quantified for cell adhesion, whilst rolling and velocity were not dramatically different between the genotypes. Confocal images indicated the almost pure (>90%) neutrophil recruitment in this setting of I/R. The inflammatory response to I/R was inhibited by LXA4 (100ng/mouse, Calbiochem, US), with ∼70% inhibition of cell emigration in WT mice (n = 6; one-way ANOVA, p<0.01), whereas the lipid was totally inactive in Fpr2-/- mice. Next, we monitored endogenous LXA4 generation by ELISA (Neogen, US), detecting a peak (∼75 pg/ml) at the end of ischaemia (∼80% above basal, ∼30 pg/ml) selectively in WT mice, but not in Fpr2-/-, mice. This increase in LXA4 generation post-ischaemia likely derives from platelet/neutrophil aggregation, as demonstrated by flow cytometry. In Fpr2-/- there was a reduced generation of these aggregates (-45%; n = 3). Administration of the Fpr pan-antagonist Boc-2 to WT mice before I/R procedure – but not after ischemia– elicited a phenotype similar to that of Fpr2-/- mice, i.e. higher degree of vascular inflammation.

Collectively these data show that Fpr2-/- mice, compared to WT, have a more inflamed phenotype and this is associated with a reduced production of LXA4. Furthermore, fundamental role for Fpr2 appears in driving this endogenous pro-resolving response during inflammation (by promoting platelet/neutrophil aggregates), where the inflammation itself switches this resolution pathway on.

Funded by the Wellcome Trust (programme grant 086867/Z/08/Z)

Gavins FN, Yona S, Kamal AM, Flower RJ, Perretti M (2003). Leukocyte adhesive actions of Annexin 1: ALXR- and FPR-related anti-inflammatory mechanisms. Blood 101: 4140-4147

Fiore S, Maddox JF, Perez HD, Serhan CN (1994). Identification of a human cDNA encoding a functional high affinity lipoxin A4 receptor. J Exp Med 180: 253-260

Dufton N, Hannon R, Brancaleone V, Dalli J, Patel HB, Gray M et al. (2010). Anti-inflammatory role of murine formyl-peptide receptor 2 ligand-specific effects on leukocytes responses and inflammatory inflammation. J Immunol 184: 2611-2619