Ninjurins 1 Regulate Hematopoietic Stem Cell Amplification Through Canonical WNT Signaling Independently of Plasma Membrane Rupture Activity

Ninjurin-1 (NINJ1), a protein recently linked to plasma membrane rupture (PMR) during inflammasome activation, plays a well-established role in cell death. However, its function in hematopoiesis has remained unexplored. In this study, we identify a novel, PMR-independent role for Ninj1 in regulating hematopoiesis using zebrafish models. Ninj1 deficiency resulted in significantly reduced neutrophil, erythrocyte, and hematopoietic stem/progenitor cell (HSPC) numbers, independently of its plasma membrane rupture (PMR) activity. Surprisingly, Ninj1 deficiency also alleviated neutrophilia and keratinocyte hyperproliferation in a chronic inflammation model, revealing a role beyond inflammasome-mediated cell death. To further investigate thisnoncanonical function, we identified PMR-deficient Ninj1 mutants (A56P and D50A). Expression of the A56P mutant rescued hematopoietic defects in Ninj1-deficient larvae, while the dominant-negative D50A mutant recapitulated the phenotypes of Ninj1 deficiency, confirming that Ninj1 regulates hematopoiesis independently of PMR. Furthermore, Ninj1 deficiency increased susceptibility to intracellular bacterial infection due to impaired neutrophil and macrophage recruitment, while overexpression of the A56P mutant enhanced immune cell recruitment and resistance to infection. Finally, we discovered that Ninj1 and Ninj2 cooperatively regulate the amplification of nascent hematopoietic stem cells (HSCs) from the hemogenic endothelium via canonical Wnt signaling. Importantly, this regulatory mechanism is conserved in human HSPCs, underscoring its evolutionary significance and translational potential. These findings position Ninjurins as critical regulators of HSC development and immune responses, with promising implications for enhancing HSC production in regenerative medicine and developing therapies for hematological and infectious diseases.