Abstract
Background: Following myocardial infarction (MI), cardiac fibroblasts (CFs) adopt distinct phenotypes to ensure scar formation and healing. Although leukocytes are a critical driver of post-MI healing, the role of neutrophils in modulating CF phenotype remains insufficiently explored. We therefore investigated the impact of soluble mediators released by neutrophil subtypes found post-MI-pro-inflammatory (N1) and anti-inflammatory (N2)-on shaping CFs phenotype. Methods: In vitro, human 3D grown CFs were indirectly co-cultured with N1 or N2 neutrophil-like cells using a two-chamber Transwell system. After 24 h, expression of inflammatory, remodeling, and pro-fibrotic markers was evaluated in fibroblasts and conditioned media. In vivo, soluble mediators derived from polarized mouse neutrophils (SN1 or SN2) were injected into the infarcted myocardium of C57BL/6J after MI surgery. The effects on the healing process were investigated at 1 and 7 days post-MI. Results: In vitro, CFs were found to exhibit a pro-inflammatory and matrix-degrading phenotype following indirect co-culture with N1 cells, characterized by overexpression of IL-1β, IL-6, MCP-1, and metalloproteases MMP-3/MMP-9. In vivo, both SN1 and SN2 treatments significantly reduced pro-inflammatory markers IL-1β and IL-6 gene expression at day 1 post-MI (inflammatory phase). At day 7 post-MI (resolution phase), SN1/SN2 treatments continued to limit local inflammation, while mitigating fibrotic remodeling by reducing CCN2, α-SMA, and key extracellular matrix proteins. Conclusions: Together, these findings suggest that while N1-derived mediators promote a pro-inflammatory fibroblast phenotype in vitro, factors secreted by both N1 and N2 support a more balanced reparative response in vivo, by limiting local inflammation and potentially mitigating adverse remodeling post-MI.