Temporal and Tissue-Specific Dynamics of CX3CR1-positive Immune Cells After Myocardial Infarction and Their Regulation by ADAM10

In response to myocardial infarction (MI), CX3CR1-positive leukocytes are recruited to the injured heart via the transmembrane chemokine CX3CL1, whose shedding is regulated by the metalloprotease ADAM10. This recruitment can contribute to scar formation, cardiac fibrosis and reduced heart function. Our study aimed to (i) comprehensively characterize the temporal and tissue-specific dynamics of CX3CR1 positive immune cells after MI, and (ii) explore how reduced numbers of CX3CR1-positive, achieved via ADAM10 inhibition, impact extracellular matrix (ECM) remodelling using a multi-omics approach.

Immune cells were isolated from bone marrow, blood, and heart tissue at 1, 3 and 7 days after permanent LAD ligation. A panel of 17 markers for spectral flow cytometry was used to assess the proportion and expression intensity (MFI) of CX3CR1 positive immune cells, including neutrophils, monocytes, macrophages, T cells, and B cells. Additionally, we applied a multi-omics approach combining: (i) transcriptome analysis of neutrophils at days 1, 3, and 7 post-MI, (ii) cardiac tissue transcriptomes at day 3 post-MI (with and without ADAM10 inhibition), and (iii) ECM proteomics at day 28 post-MI (with and without ADAM10 inhibition). FACS analysis revealed that neutrophils, monocytes and macrophages display highest CX3CR1 expression in the post-MI heart, especially on days 1 and 3. CX3CR1-positive neutrophils maintain high expression levels over all timepoints, despite decreasing cell numbers. The highest absolute numbers of CX3CR1-positive cells were observed in monocytes (days 1 and 3) and macrophages (day 7) in the heart. In the bone marrow, monocytes and macrophages were consistently the dominant CX3CR1-positive populations. Transcriptomic data showed that CX3CR1-positive neutrophils expressed pro-fibrotic ECM-related genes including Col1a1, Col3a1, Emilin1, Postn, Spp1, Fn1, and Agrn, which were upregulated at all time points (days 1, 3, and 7). Importantly, these genes were significantly downregulated in infarcted mice treated with an ADAM10 inhibitor. Immunohistochemistry confirmed co-localization of ECM proteins (e.g. Collagen I and Emilin-1) with Ly6G+ neutrophils in the infarcted area at 3 days post-infarction. ECM proteomics at day 28 revealed that ADAM10 inhibition led to reduced levels of several pro-fibrotic proteins which were upregulated in post-MI neutrophils, indicating attenuated ECM remodelling.

Our findings highlight a critical role of CX3CR1-positive neutrophils in the early immune response and in promoting ECM deposition after MI. Targeting the ADAM10/CX3CL1/CX3CR1 axis effectively reduces CX3CR1-positive neutrophil recruitment and pro-fibrotic ECM accumulation, offering a promising therapeutic strategy to improve post-MI cardiac healing.