Abstract Body (Do not enter title and authors here): Background
Atherosclerotic cardiovascular disease (ASCVD), a chronic inflammatory disease, is the leading cause of morbidity and mortality worldwide. Macrophages play a central role in the development of ASCVD by maintaining the local inflammatory response. Endocytic traffic of cell-surface proteins is a crucial process that regulates the inflammatory response. Eps15 homology domain-containing protein1 (EHD1) is a key regulator of endocytic traffic. However, the role of EHD1 in macrophage function during the progression of atherosclerosis remains poorly understood.
Hypothesis
EHD1 modulates macrophage function by orchestrating the endocytic trafficking of cell surface receptors, thereby regulating the progression of atherosclerosis.
Goals
To investigate the role of EHD1 in macrophage activation and atherosclerosis.
Methods
First, EHD1 expression levels in macrophages were accessed by immunofluorescence. Second, the effect of EHD1 on macrophage activation was determined in EHD1-silenced and knockout macrophages. Third, cell-surface proteins controlled by EHD1 were identified by biotin labeling and mass spectrometry in Ehd1^-/- macrophages. Fourth, LDLR^-/- mice transplanted with Ehd1^-/- or wild-type bone marrow cells were fed with Western Diet for 12 weeks, followed by the pathological assessment. Single-cell RNA-seq (ScRNA-seq) was performed on CD45⁺ aortic cells to investigate the effect of EHD1 on the immune microenvironment.
Results
Compared with early lesions in both human and mouse models, EHD1 protein expression in lesional macrophages is significantly elevated in advanced atherosclerotic lesions, as demonstrated by immunofluorescence analysis. In cultured macrophages, LPS-induced expression of pro-inflammatory mediators, including TNF-α, IL-1β, IL-6, and NLRP3, is attenuated in EHD1-deficient macrophages. In vivo, studies demonstrate that recipients of Ehd1^-/- bone marrow develop smaller atherosclerotic lesions and MAC2⁺ area compared to controls. Consistent with in vitro findings, ScRNA-seq data reveals that the expression of Tnf, Il1b, and Nlrp3 is decreased in Ehd1^-/- inflammatory macrophages within the lesions. Mechanistically, EHD1 facilitates the endocytic recycling of TNF receptor II (TNFRII), leading to enhanced NF-κB activation in macrophages.
Conclusion
EHD1 enhances NF-κB signaling in macrophages by promoting the recycling of cell surface TNFRII, thereby amplifying inflammatory responses and exacerbating the progression of atherosclerosis.