Targeting Ly6G increases fibrosis after MI by inducing neutrophil phenotype switch and modulating their communication with post MI heart cells

Abstract

Introduction

Myocardial infarction (MI) generates a complex immune response involved in tissue repair and remodeling. Neutrophils infiltrating the heart present a heterogeneous profile, appear dynamically in the tissue and show multifaceted beneficial and deleterious roles after MI. However, studies assessing the role of neutrophils after MI rely on partially efficient depletion strategy targeting Ly6G in mice, rendering them possibly biased.

Objective

We aimed to decipher the role of neutrophil subsets in cardiac repair and in shaping cardiac immune cell phenotypes in the heart after MI.

Method

We used a mouse model of experimental MI induced by permanent coronary artery ligation, and depleted neutrophils using a combination of antibodies (Rat IgG2aκ anti-Ly6G + anti-rat IgG2aκ). Five days after MI, we analyzed cardiac CD45+ cells by scRNA-seq in combination with CITE-seq for surface marker analysis. Fourteen days after MI, we evaluated survival rate and cardiac remodeling by histology.

Results

Despite 70% decrease in neutrophil number after depletion in the heart 3 days after MI, a specific neutrophil population appears in response to α-Ly6G antibody, that arbors a unique transcriptomic signature 5 days after MI. We show that targeting Ly6G increases cardiac fibrosis and affects immune cell infiltration composition in the heart. Depletion of neutrophils strongly increases γδ T cells, monocytes and interferon response-associated macrophages accumulation in the heart. Pseudo-bulk analysis reveal differential gene expression in these populations after neutrophil depletion, indicating neutrophil ability to modulate immune cells recruitment and phenotype. We further show that neutrophils and γδ T cells can communicate directly with fibroblasts: α-Ly6G-induced neutrophil population is more likely to drive fibrosis and γδ T cells lead to increase proliferation and inflammatory activation of fibroblasts in vitro.

Conclusion

Overall, our data indicate that neutrophils shape local cardiac immune responses after MI and suggest a multifaceted role of neutrophils subpopulations. Results observed after α-Ly6G based depletion might rather be due to specific neutrophil population recruitment than absence of neutrophils. γδ T cells recruitment is highly affected by α-Ly6G antibody treatment and can participate to increased fibrosis.