Lymphatic Endothelial Cells Regulate Neutrophil Phenotypes and Function in a Microphysiological Model of Infection

ABSTRACT Early skin inflammation requires coordinated immune regulation, with neutrophils acting as first-line responders. While the blood vasculature and its role in neutrophil recruitment during infection has been extensively studied, the lymphatic system remains comparatively understudied despite its known role in immune cell trafficking. Growing evidence suggests lymphatic vessels actively participate in regulating inflammatory responses, yet whether they coordinate neutrophil behavior during skin infection remains unclear. Staphylococcus aureus is particularly problematic in this context, employing multiple immune evasion strategies and representing a major driver of antibiotic-resistant skin and soft tissue infections worldwide. To address this gap, we developed a human-based 3D microphysiological system incorporating luminal lymphatic endothelial vessels, a collagen matrix and bacteria to model an infected microenvironment. We evaluated neutrophil migration, phagocytosis and NETosis in response to Escherichia coli and S. aureus. Lymphatic endothelium amplified neutrophil migration in a bacterial-dependent manner, with E. coli promoting directional migration toward the vessel while S. aureus suppressed migration and directionality despite increased phagocytic uptake. S. aureus also induced myeloperoxidase-positive NETs with nuclear morphology consistent with vital NETosis, rescued by DNase treatment. To our knowledge, this is the first demonstration that lymphatic endothelium directly drives neutrophil behavior during skin infection. Competing Interest Statement David J. Beebe holds equity in Bellbrook Labs LLC, Salus Discovery LLC, Lynx Biosciences Inc., Stacks to the Future LLC, Flambeau Diagnostics LLC, Eolas Diagnostics, Inc., Navitro Biosciences LLC, and Onexio Biosystems LLC.