Regulatory functions of Fpr2 in microbial sepsis: a new pharmacological target?

Thomas Gobbetti1, Sina Coldewey1, Simon McArthur1, Pauline le Faouder2, Nicolas Cenac2, Christoph Thiemermann1, Mauro Perretti1. 1William Harvey Research Institute, Queen Mary University, London, UK, 2INSERM, UMR1043 - Université Paul Sabatier - UMR n°5281, Toulouse, France

Introduction: Sepsis is medical condition difficult to treat in view of the paradoxical concomitance of excessive inflammation (SIRS) temporally aligned with an immunosuppressed state (CARS). Fpr2 transduces the pro-resolving properties of two effectors of resolution, Lipoxin A4 and Annexin A1. In view of the complexity of processes that characterize resolution, we questioned whether Fpr2 could exert regulatory functions in sepsis.

Methods and Results: Male 8 month C57Bl/6 WT and Fpr2 KO (bearing knocked in GFP) mice (n=6) were subjected to sepsis by coecal ligature and puncture (CLP) or sham operation (ketamine 100 mg/mL/xylazine 20mg/ml). Mice were killed 24h later, where hypothermia was observed: 26.7±0.6°C vs. 33±1.9°C for Fpr2 KO and WT, respectively. Echocardiography revealed compromised cardiac function in the Fpr2 KO with higher reduction in ejection fraction and fractional shortening. Focusing on organ injury, whilst there were differences in lung and liver damage, only the Fpr2 KO mice displayed kidney injury, measured as plasma creatinine and urea and tissue myeloperoxidase activity. FACS analysis of peritoneal lavages revealed a reduction in monocyte numbers in the Fpr2 KO, reflected by a significant increase in the ratio of granulocytes (PMNs) to monocytes (P<0.01). Analysis of soluble mediators in lavages by cytokine bead array (CBA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed increased levels of CXCL1 and CCL2, TNFa, IL-1a/6/10, and the lipids PGE2, LTB4 and LXA4 in Fpr2 KO vs. WT mice. Intriguingly, IFNg and IL-17a were only detectable in Fpr2 KO. Quantification of bacterial CFUs in peritoneal lavages revealed compromised bacterial clearance by Fpr2 KO mice. Finally, in vitro treatment of murine PMNs with AnnexinA1 (10nM for 4hours) was able to increase the phagocytic activity in WT but not in Fpr2 KO cells displaying a prophagocytic Fpr2-mediated effect of AnnexinA1.

Evidence for an engagement of the receptor in sepsis was obtained by monitoring Fpr2 gene promoter activity using the GFP proxy marker and flow cytometry: >3-fold increase in GFP signal was quantified in PMNs and monocytes following 24 h post-CLP (n=6, P<0.01). This response seems mediated by TNFa as replicated with mouse and human primary PMNs and monocytes.

Conclusion: Major alterations in the response to sepsis were detected in Fpr2 KO mice, with increased cardiac and kidney dysfunction, impaired monocyte recruitment and augmented inflammatory soluble mediator generation. These effects may be consequent to inadequate clearance of bacteria by phagocytosis and over-shooting of the host response to the infection. Annexin A1 displayed a prophagocytic Fpr2-mediated effect. Fpr2 expression is modulated during sepsis and this response seems to be mediated by TNFa as replicated with mouse macrophages and human primary monocytes. Together our data strongly suggest Fpr2 system as a novel therapeutic target for regulating the host response in sepsis without affecting the host immune response

Funding: Welcome Trust Programme Grant (086867/Z/08/Z).