Theranostics 2021; 11(19):9358-9375. doi:10.7150/thno.62797

Research Paper

IRGM/Irgm1 facilitates macrophage apoptosis through ROS generation and MAPK signal transduction: Irgm1+/- mice display increases atherosclerotic plaque stability

Shaohong Fang1,2*, Song Sun2,3*, Hengxuan Cai1,2*, Xiaoyi Zou2,3, Shanjie Wang1,2, Xinran Hao2,3, Xin Wan2,3, Jiangtian Tian1,2, Zhaoying Li1,2, Zhongze He2,3, Wei Huang2,3, Chenchen Liang1,2, Zhenming Zhang1,2, Liming Yang4, Jinwei Tian1,2✉, Bo Yu1,2✉, Bo Sun2,3✉

1. Department of Cardiology, Second Affiliated Hospital of Harbin Medical University, Harbin 150086, China.
2. The Key Laboratory of Myocardial Ischemia, Chinese Ministry of Education, Harbin 150086, China.
3. Department of Neurobiology, Harbin Medical University; Neurobiology Key Laboratory, Education Department of Heilongjiang Province, Harbin 150086, China.
4. Department of Pathophysiology, Harbin Medical University-Daqing, Daqing 163319, China.
*These authors contributed equally to this work.

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Citation:
Fang S, Sun S, Cai H, Zou X, Wang S, Hao X, Wan X, Tian J, Li Z, He Z, Huang W, Liang C, Zhang Z, Yang L, Tian J, Yu B, Sun B. IRGM/Irgm1 facilitates macrophage apoptosis through ROS generation and MAPK signal transduction: Irgm1+/- mice display increases atherosclerotic plaque stability. Theranostics 2021; 11(19):9358-9375. doi:10.7150/thno.62797. Available from https://www.thno.org/v11p9358.htm

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Abstract

Rationale: Atherosclerosis plaque rupture (PR) is the pathological basis and chief culprit of most acute cardiovascular events and death. Given the complex and important role of macrophage apoptosis and autophagy in affecting plaque stability, an important unanswered question include is whether, and how, immunity-related GTPase family M protein (IRGM) and its mouse orthologue IRGM1 affect macrophage survival and atherosclerotic plaque stability.

Methods: To investigate whether serum IRGM of ST-segment elevation myocardial infarction (STEMI) patients is related to plaque morphology, we divided 85 STEMI patients into those with and without plaque rupture (PR and non-PR, respectively) based on OCT image analysis, and quantified the patients' serum IRGM levels. Next, we engineered Irgm1 deficient mice (Irgm1+/-) and chimera mice with Irgm1 deficiency in the bone marrow on an ApoE-/- background, which were then fed a high-fat diet for 16 weeks. Pathological staining was used to detect necrotic plaque cores, ratios of neutral lipids and cholesterol crystal, as well as collagen fiber contents in these mice to characterize plaque stability. In addition, immunofluorescence, immunohistochemical staining and western blot were used to detect the apoptosis of macrophages in the plaques. In vitro, THP-1 and RAW264.7 cells were stimulated with ox-LDL to mimic the in vivo environment, and IRGM/IRGM1 expression were modified by specific siRNA (knockdown) or IRGM plasmid (knocked-in). The effect of IRGM/Irgm1 on autophagy and apoptosis of macrophages induced by ox-LDL was then evaluated. In addition, we introduced inhibitors of the JNK/p38/ERK signaling pathway to verify the specific mechanism by which Irgm1 regulates RAW264.7 cell apoptosis.

Results: The serum IRGM levels of PR patients is significantly higher than that of non-PR patients and healthy volunteers, which may be an effective predictor of PR. On a high-fat diet, Irgm1-deficient mice exhibit reduced necrotic plaque cores, as well as neutral lipid and cholesterol crystal ratios, with increased collagen fiber content. Additionally, macrophage apoptosis is inhibited in the plaques of Irgm1-deficient mice. In vitro, IRGM/Irgm1 deficiency rapidly inhibits ox-LDL-induced macrophage autophagy while inhibiting ox-LDL-induced macrophage apoptosis in late stages. Additionally, IRGM/Irgm1 deficiency suppresses reactive oxygen species (ROS) production in macrophages, while removal of ROS effectively inhibits macrophage apoptosis induced by IRGM overexpression. We further show that Irgm1 can affect macrophage apoptosis by regulating JNK/p38/ERK phosphorylation in the MAPK signaling pathway.

Conclusions: Serum IRGM may be related to the process of PR in STEMI patients, and IRGM/Irgm1 deficiency increases plaque stability. In addition, IRGM/Irgm1 deficiency suppresses macrophage apoptosis by inhibiting ROS generation and MAPK signaling transduction. Cumulatively, these results suggest that targeting IRGM may represent a new treatment strategy for the prevention and treatment of acute cardiovascular deaths caused by PR.

Keywords: IRGM/Irgm1, macrophage, apoptosis, atherosclerosis, plaque stability