Tissue ischemia induces mobilization of pro-angiogenic neutrophils from the spleen

Abstract

Abstract Pro-angiogenic neutrophils (PAN) form a small subset of circulating neutrophils expressing CD49d+ VEGFR1hi CXCR4hi, which play a crucial role in the revascularization of transplanted islets. To determine the role of PANs in re-establishing tissue perfusion following a large ischemic event we used the mouse model for peripheral artery disease, the hindlimb ischemia (HLI) model. We found that numbers of PANs in the affected gastrocnemius muscle increased ≈4 times 3h following ischemia induction and reached its peak 2 days following ischemia onset. Similarly, in patients undergoing surgery where peripheral perfusion is transiently clamped, numbers of circulating PANs increase ≈3 times 30 min post-HLI. In healthy mice, PANs are found in high frequency in the spleen (spleen: 9,8% of total neutrophils, blood: 0,78%). Splenectomy prior to induction of HLI reduced recruitment of PANs to the ischemic muscle, which demonstrates that the spleen acts as a reservoir for PANs. Furthermore, HLI downregulated the CXCR4-ligand CXCL12α in the splenic red pulp. This was prevented by chemically induced sympathectomy, resulting in increased retention of PANs in spleen, reduced PAN accumulation in the ischemic muscle and reduced blood flow recovery. Therefore, we propose that the downregulation of CXCL12α controlled by sympathetic nerve signaling pathways drives retention of PANs in the spleen, thereby regulating their release in response to HLI. In summary, we identified that the spleen contains a peripheral pool of PANs and emphasize the pivotal role of the neuro-immune axis in the recruitment of PANs to the site of ischemia. These discoveries open new avenues for drug development to limit damage and accelerate healing following ischemic events. This study was supported by the Swedish Research Council, Knut and Alice Wallenberg foundation, The Swedish Cancer Society, and O. E. and Edla Johansson’s foundation.