A new study published in Engineering uses single-cell RNA sequencing (scRNA-seq) to unpack how phytosterol oxidation products (POPs), common dietary lipid derivatives, alter vascular cell heterogeneity and drive atherosclerosis (AS) through defined inflammatory signaling pathways. Atherosclerosis is a chronic inflammatory cardiovascular disorder where macrophage function shapes plaque formation and progression, yet prior research has not fully clarified how food-derived POPs reshape aortic cellular profiles or the molecular cascades linking POP exposure to vascular inflammation.

The research team built a single-cell transcriptomic atlas of aortic tissue from ApoE⁻/⁻ mice fed standard atherogenic diets with or without supplementary POPs, resolving five primary aortic cell lineages: fibroblasts, myeloid cells, lymphocytes, smooth muscle cells and endothelial cells. Cross-group comparison of cell-cell communication networks revealed POP treatment rewired intercellular signaling across plaque microenvironments, with myeloid cells emerging as central coordinators of inflammatory cross-talk between distinct vascular cell populations. Further subclustering of myeloid subsets identified four macrophage subpopulations, among which M1-like pro-inflammatory macrophages showed elevated representation in POP-fed mice, marked by enriched expression of canonical pro-inflammatory genes including Nlrp3, Tnf and Ccl3. Pseudotime trajectory and RNA velocity analyses mapped monocyte differentiation toward M1-like macrophage states under POP exposure, highlighting Il1r1 as a top dynamic driver gene governing this inflammatory cell fate shift.

Gene enrichment and single-cell regulatory network analysis pinpointed the TLR4 signaling pathway and transcription factor IRF5 as dominant regulators of M1-like macrophage transcriptional activity in POP-treated samples. To validate this axis, the team ran in vitro assays on bone marrow-derived macrophages (BMDMs) and RAW264.7 macrophage cell lines stimulated with oxidized low-density lipoprotein (ox-LDL), the primary lipid accumulated within atherosclerotic plaques. 7-ketositosterol (7-KS), the most abundant POP found in processed foods, dose-dependently amplified secretion of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 while upregulating M1 surface markers iNOS and CD86, with no measurable change in anti-inflammatory mediator IL-10 release. Western blot and immunofluorescence assays confirmed 7-KS elevated protein levels of TLR4, MyD88 and TRAF6, and boosted IRF5 nuclear translocation to activate downstream inflammatory gene transcription. When cells were pre-treated with TLR4 inhibitor TLR4-IN-C34 or IRF5 inhibitor YE6144, the pro-inflammatory and M1-polarizing effects of 7-KS were largely eliminated, confirming the TLR4–IRF5 axis as an indispensable mediator of 7-KS activity.

To translate mouse-derived findings to human vascular pathology, researchers integrated four public human arterial scRNA-seq datasets covering healthy aortic tissue and advanced atherosclerotic carotid lesions. Across over 73,000 human vascular cells, inflammatory macrophage populations carrying matching POP-associated molecular signatures were significantly expanded in patient plaque samples, with GSEA confirming consistent enrichment of TLR signaling, MyD88-dependent TLR cascades and inflammatory response gene sets in human lesion macrophages. SCENIC transcription factor profiling detected heightened IRF5 regulon activity in human inflammatory macrophages, mirroring observations in murine M1-like macrophage populations.

The work establishes a single-cell framework for evaluating dietary component bioactivity and provides a conserved molecular signature linking dietary POP intake to vascular inflammation across mouse and human atherosclerotic tissue, while identifying potential molecular targets for mitigating diet-driven atherosclerotic progression.

The paper “Single-Cell RNA Sequencing Reveals 7-Ketositosterol Exacerbates Aortic Inflammation Through TLR4 Signaling-Regulated IRF5 Mediated M1 Macrophage Polarization,” is authored by Qinjun Zhang, Weisu Huang, Cheng Chen, Jianfu Shen, Baiyi Lu, Peiwu Li. Full text of the open access paper: https://doi.org/10.1016/j.eng.2025.07.021. For more information about Engineering, visit the website at https://www.sciencedirect.com/journal/engineering.