Sfxn5 Regulation of Actin Polymerization for Neutrophil Spreading Depends on a Citrate–Cholesterol–PI(4,5)P2 Pathway

Abstract Cell spreading is an initial and critical step in neutrophil adhesion and migration, leading to neutrophil recruitment to inflammatory tissues. Sideroflexin (Sfxn) family proteins are metabolite transporters located in the mitochondrial membrane. Recombinant SFXN5 protein is a citrate transporter in vitro; however, whether Sfxn5 regulates any cellular behavior or function remains unknown. In this study, we found that small interfering RNA transfection or morpholino injection achieving Sfxn5 deficiency in neutrophils significantly decreased neutrophil recruitment in mice and zebrafish, respectively. Sfxn5 deficiency impaired neutrophil spreading and spreading-associated cellular phenotypes, such as cell adhesion, chemotaxis, and ROS production. Actin polymerization is critical for neutrophil spreading, and we found that actin polymerization in spreading neutrophils was partially inhibited by Sfxn5 deficiency. Mechanistically, we observed that the levels of cytosolic citrate and its downstream metabolic products, acetyl-CoA and cholesterol, were decreased in Sfxn5-deficient neutrophils. The levels of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a mediator for the regulation of actin polymerization by cholesterol, were reduced in the plasma membrane of Sfxn5-deficient neutrophils. Exogenous supplementation with citrate or cholesterol partially reversed the reduction in PI(4,5)P2 levels, defective neutrophil actin polymerization, and cell spreading. Altogether, we demonstrated that Sfxn5 maintains cytosolic citrate levels and ensures the synthesis of sufficient cholesterol to promote actin polymerization in a PI(4,5)P2-dependent manner during neutrophil spreading, which is essential for the eventual inflammatory recruitment of neutrophils. Our study revealed the importance of Sfxn5 in neutrophil spreading and migration, thus identifying, to our knowledge, for the first time, the physiological cellular functions of the Sfxn5 gene.