Modulating inflammation through FPR2/ALX in experimental stroke

HK Smith, FNE Gavins. Imperial College, London, UK

Ischaemia/reperfusion (I/R) injury following stroke can impair recovery. Members of the formyl peptide receptor family (FPR; Fpr in mouse) can act as endogenous anti-inflammatory/pro-resolving agents. We investigated the pharmacological potential of FPR ligands in modulating inflammation in a model of global cerebral I/R.

Male C57BL/6 mice (23-28g) underwent bilateral common carotid artery occlusion (BCCAO; 5min) and reperfusion (40min or 2h) under deep anaesthesia (pentobarbital sodium; 100mg/kg). Resulting cerebral leukocyte-endothelial (L-E) interactions were viewed through a cranial window using intravital microscopy (IVM). Rhodamine 6G (100μl; 0.02%) was administered 5min prior to IVM for visualisation of leukocytes under fluorescence.

At the start of reperfusion, mice were administered 100μl i.v.: 15-epi-lipoxin A4 (15-epi-LXA4 (selective FPR2/ALX agonist; 0.5μg or 4μg; vehicle=saline+ethanol); Annexin A1 mimetic peptide, Ac2-26 (AnxA1Ac2-26; FPR1 agonist; 100μg; vehicle=saline), Boc2 (pan-FPR antagonist; 10µg; vehicle=saline), WRW4 (FPR2/ALX-selective antagonist; 55μg; vehicle=saline) or Ac2-26+Boc2. L-E interactions per 100μm vessel length were quantified as rolling leukocyte flux (mm-2min-1), rolling velocity (μmsec-1), and leukocyte adhesion (mm-2min-1) and expressed as mean ±SEM by unpaired t test; n=4-7 mice/group.

At 40 min, both doses of 15-epi-LXA4 reduced inflammation from levels seen in vehicle treatment groups (56.1±9.0 (0.5μg) or 61.2±13.4 (4.0 μg) vs. 236.9±26.7 rolling cells mm-2min-1; 19.9±19.9 (0.5μg) or 53.6±14.7 (4.0μg) vs. 267.2±34.8 adherent cells mm-2min-1), although this reduction was not sustained to the same extent at 2h reperfusion at the lower dose.

AnxA1Ac2-26 treatment decreased adhesion (p<0.05) at 40min vs. vehicle (80.0±44.7 vs. 315.4±37.25 adherent cells mm-2min-1) and all L-E interactions by 80-90% at 2h (58.2±21.22 vs. 112.6±74.47 rolling cells mm-2min-1; 77.3±15.2 vs. 610.7±102.3 adherent cells mm-2min-1). Combined treatment with AnxA1Ac2-26 and FPR antagonists Boc2 or WRW4 abrogated the effects of AnxA1Ac2-26 at 40min (adhesion=454.2±78.43 and 326.2±50.2 cells mm-2min-1; p<0.05), suggesting an Fpr2/3 mechanism. Abrogation was not sustained at to the same extent at 2h.

The data presented here demonstrate that 15-epi-LXA4 and AnxA1Ac2-26 both reduce L-E interactions in an experimental model of global I/R, and that this activity is dose and time-dependent, and likely to occur through Fpr2/3 in our model. These data indicate the significance of Fpr2/3 in a model of cerebral I/R, and that modulation of the receptor could produce significant protective effects in clinical stroke.