A neutrophil-intrinsic CKLF1-PKM2 axis drives glycolytic flux for de novo DAG synthesis and pro-inflammatory ROS...

Reactive oxygen species (ROS) burst and subsequent recruitment to the ischemic brain characterize neutrophil activation in the hyperacute phase of ischemic stroke, yet the underlying metabolic drivers remain elusive. Here, we report that neutrophil-intrinsic chemokine-like factor 1 (CKLF1) acts as a key immunometabolic regulator following stroke. CKLF1 was rapidly upregulated in neutrophils within 6 h post-ischemia in both humans and mice. Mechanistically, CKLF1 bound directly to the tetrameric form of pyruvate kinase M2 (PKM2), enhancing glycolytic flux and diverting intermediates toward de novo diacylglycerol (DAG) synthesis. This rewiring promoted protein kinase C-dependent phosphorylation of p47‑phox and assembly of NADPH oxidase, driving ROS production that reinforced neutrophil recruitment and adhesion via αMβ2 integrin. Genetic deletion of neutrophil CKLF1 attenuated neutrophil recruitment and improved functional recovery. Leveraging this mechanism, we developed a neutrophil-targeted nanotherapeutic using engineered outer membrane vesicles from E. coli W3110 to deliver a stapled CKLF1-derived peptide (C19) that disrupts CKLF1-PKM2 binding. This intervention suppressed glycolysis-driven ROS, reduced neutrophil recruitment and αMβ2 expression, and provided robust neuroprotection. Our findings reveal a CKLF1-PKM2-DAG axis that directs metabolic reprogramming and neutrophil pathogenicity in stroke, offering a novel target for immunometabolic therapy.