Human Macrophages Modulate Neutrophil Transendothelial Migration in an Inflammation-on-a-Chip Model 2255264

Abstract

Abstract Introduction Dysregulated neutrophil function contributes to chronic inflammatory diseases, including ANCA-associated vasculitis, yet it remains unclear how macrophages contribute to this dysfunction. We engineered a microfluidic Vasculature-on-a-Chip with primary human neutrophils, macrophages, and a model endothelial blood vessel to address this gap. Methods We adapted our “Inflammation-on-a-Chip” device to embed monocyte-derived macrophages in collagen ECM adjacent to an endothelial lumen. We confirmed macrophage viability and differentiation (M1: M-CSF, IFN-γ, LPS; M2: M-CSF, IL-4). We measured endothelial structure, adhesion markers (ICAM-1, VE-cadherin), and permeability with polarized macrophages. Neutrophil transendothelial migration (TEM) and migration in response to sterile inflammation (IL-8, LTB4) were quantified for 8 h in coculture with M1 or M2 macrophages. Results Macrophages had polarization-dependent effects on both the vessel and the neutrophil response. Without inflammation, M1significantly increased vessel permeability vs M2. With inflammation, M2 co-culture preserved barrier function vs M1. While inflammatory stimuli and co-culture with either M1 or M2 trended toward higher ICAM−1 expression, macrophages imposed a clear polarization-dependent TEM control. M1 resembled cell-free controls, while M2 significantly reduced transmigration. Single-cell tracking showed M2-restricted ECM migration. Conclusion Human macrophages regulate neutrophil access within a 3D vascular microenvironment. Pro-resolution cells strongly inhibit both TEM and post-TEM motility. Ongoing work will identify M2-derived mediators for targeted, pro-resolution therapies. Funding Source National Institutes of Health (NIH) Grant R35 GM146737A T32NIH Biophysics Training Program Topic Categories Cellular Adhesion, Migration, and Inflammation (CAM)