A splenic reservoir of pro-angiogenic neutrophils supports blood flow recovery after peripheral tissue ischemia

Abstract

Background: Neutrophils accumulate at sites of ischemic injury, where a distinct subset characterized by CD49d+CXCR4hiVEGFR1hi expression promotes angiogenesis and contributes to blood flow recovery. This study investigated the function, origin, and recruitment mechanisms of these pro-angiogenic neutrophils (PANs) in a murine model of hindlimb ischemia and explored whether a corresponding population responds to acute ischemia in humans. Methods: Using spectral flow cytometry and immunohistochemistry, PANs and classical neutrophils were characterized in ischemic gastrocnemius muscle following hindlimb ischemia in mice. Blood flow recovery was assessed by laser speckle flowmetry. To determine the origin and mobilization mechanisms of PANs, splenectomy, chemical sympathectomy with 6-hydroxydopamine (6-OHDA), and surgical splenic denervation were performed. Translational studies examined blood samples collected during elective abdominal aortic aneurysm repair to assess PAN mobilization following acute lower limb ischemia induced by aortic cross-clamping. Results: PANs rapidly accumulated in ischemic muscle through a CD49d-dependent mechanism, and blockade of CD49d impaired blood flow recovery. During homeostasis, PANs were maintained within a splenic reservoir and were rapidly released into circulation following ischemia. Splenectomy and disruption of splenic sympathetic signaling significantly reduced PAN recruitment to ischemic tissue and impaired functional recovery. Mobilization was driven by ischemia-induced sympathetic activation, which decreased CXCL12 levels in the splenic red pulp, disrupting CXCR4-mediated PAN retention. In humans, a phenotypically similar PAN population was identified in peripheral blood, which increased rapidly following lower limb ischemia during aortic cross-clamping. The speed of this response is consistent with release from an extramedullary reservoir, although its anatomical source remains unknown. Conclusion: These findings identify the spleen as a reservoir of pro-angiogenic neutrophils in mice and reveal a neuroimmune mechanism regulating their mobilization and recruitment after ischemic injury. The rapid appearance of PAN in human circulation after acute ischemia suggests that comparable extramedullary neutrophil reservoirs may also exist in humans.