Mesenchymal stromal cells modulate neutrophil phenotype via paracrine signals

Abstract Background Despite the recognized importance of neutrophils in cardiac repair following myocardial infarction (MI), their interaction with mesenchymal stromal cells (MSCs), particularly regarding polarization phenotypes and functional impacts, remains unclear. Here, we investigated these interactions across controlled in vitro systems and an in vivo MI model. Methods Human HL-60 cells were differentiated into neutrophil-like cells (dHL-60) and polarized toward N1/N2 states to test the MSC paracrine effects using indirect transwell coculture. Readouts included gene expression, cytokine profiling and functional assays. To increase translational relevance, indirect ex vivo cocultures of human MSCs with primary neutrophils isolated from MI mice or from MI patients were analysed for gene expression and cytokine profiles in conditioned media. In vivo, syngeneic mouse MSCs were transplanted subcutaneously immediately after MI, and early cardiac function was evaluated by echocardiography. Cardiac neutrophils where quantified by flow cytometry, and Ly6G + neutrophils from infarcted hearts and peripheral blood were purified by MACS for bulk RNA-seq with targeted RT-qPCR validation. Results In vitro, MSCs suppressed pro-inflammatory mediators in N1-like neutrophils and enhanced reparative factors in N2-like cells. In vivo, remote MSC transplantation improved early cardiac performance, and reduced neutrophil accumulation in the infarct. Paradoxically, cardiac neutrophils showed transcriptomic enrichment of inflammatory pathways, whereas blood neutrophils showed reduced interferon-related programs. Conclusion Our findings indicate that MSCs can modulate neutrophil responses, underscoring the nuanced effects of MSC-based approaches in ischemic heart disease. These results suggest that anti-inflammatory effects observed under controlled conditions may not fully translate in vivo, highlighting the importance of context when evaluating MSC-based therapies.