Elucidating the changes in the heterogeneity and function of radiation-induced cardiac macrophages using single-cell RNA sequencing

Chunxiang Cao; Ran Wu; Shubei Wang; Lingfang Zhuang; Peizhan Chen; Shuyan Li; Qian Zhu; Huan Li; Yingying Lin; Min Li; Lu Cao; Jiayi Chen

doi:10.3389/fimmu.2024.1363278

Elucidating the changes in the heterogeneity and function of radiation-induced cardiac macrophages using single-cell RNA sequencing

Front Immunol. 2024 Mar 27:15:1363278. doi: 10.3389/fimmu.2024.1363278. eCollection 2024.

Authors

Chunxiang Cao^#^{1

2}, Ran Wu^#^{1

2}, Shubei Wang^#^{1

2}, Lingfang Zhuang³, Peizhan Chen⁴, Shuyan Li^{1

2}, Qian Zhu^{1

2}, Huan Li^{1

2}, Yingying Lin^{1

2}, Min Li^{1

2}, Lu Cao^{1

2}, Jiayi Chen^{1

2}

Affiliations

¹ Department of Radiation Oncology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
² Shanghai Key Laboratory of Proton-therapy, Shanghai, China.
³ Department of Cardiovascular Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
⁴ Clinical Research Center, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

^# Contributed equally.

Abstract

Purpose: A mouse model of irradiation (IR)-induced heart injury was established to investigate the early changes in cardiac function after radiation and the role of cardiac macrophages in this process.

Methods: Cardiac function was evaluated by heart-to-tibia ratio, lung-to-heart ratio and echocardiography. Immunofluorescence staining and flow cytometry analysis were used to evaluate the changes of macrophages in the heart. Immune cells from heart tissues were sorted by magnetic beads for single-cell RNA sequencing, and the subsets of macrophages were identified and analyzed. Trajectory analysis was used to explore the differentiation relationship of each macrophage subset. The differentially expressed genes (DEGs) were compared, and the related enriched pathways were identified. Single-cell regulatory network inference and clustering (SCENIC) analysis was performed to identify the potential transcription factors (TFs) which participated in this process.

Results: Cardiac function temporarily decreased on Day 7 and returned to normal level on Day 35, accompanied by macrophages decreased and increased respectively. Then, we identified 7 clusters of macrophages by single-cell RNA sequencing and found two kinds of stage specific macrophages: senescence-associated macrophage (Cdkn1a^highC5ar1^high) on Day 7 and interferon-associated macrophage (Ccr2^highIsg15^high) on Day 35. Moreover, we observed cardiac macrophages polarized over these two-time points based on M1/M2 and CCR2/major histocompatibility complex II (MHCII) expression. Finally, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses suggested that macrophages on Day 7 were characterized by an inflammatory senescent phenotype with enhanced chemotaxis and inflammatory factors, while macrophages on Day 35 showed enhanced phagocytosis with reduced inflammation, which was associated with interferon-related pathways. SCENIC analysis showed AP-1 family members were associated with IR-induced macrophages changes.

Conclusion: We are the first study to characterize the diversity, features, and evolution of macrophages during the early stages in an IR-induced cardiac injury animal model.

Keywords: DNA damage; heart injury; interferon; irradiation; macrophage; mitochondrial dysfunction; senescence; single-cell RNA sequencing.

Publication types

Research Support, Non-U.S. Gov't
Comment

MeSH terms

Animals
Inflammation / metabolism
Interferons / metabolism
Macrophages*
Mice
Phagocytosis*
Sequence Analysis, RNA

Substances

Interferons

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study was supported in part by National Natural Science Foundation of China (grant number 82373514, 82373202, 81972963, 82102819, 82203457), the National Key Research and Development Program of China (grant number 2022YFC2404602), Scientific and Technological Innovation Action Plan of Shanghai Science and Technology Committee (grant number 22Y31900103), Beijing Science and Technology Innovation Medical Development Foundation (grant number KC2021-JX-0170-9), Shanghai Sailing Program (grant number 21YF1427700), Shanghai Science and Technology Innovation Action Plan (grant number 23Y41900100).