Hyperinsulinemia promotes atherosclerosis by inducing metabolic reprogramming of autoreactive CD4+ T cells

Background: Accumulating evidence suggests that pro-inflammatory autoreactive CD4⁺ T cells are causally involved in atherogenesis. CD4⁺ T cells reactive to Apolipoprotein B (ApoB), the core protein of most lipoproteins, predominantly exhibit regulatory T cell(T_reg)-like signatures in healthy mice and humans, but acquire proinflammatory phenotypes that contribute to atherosclerosis progression. The mechanisms driving this phenotype switching are largely unknown. In other diseases, metabolic reprogramming has been shown to impact CD4⁺ T cells.

Results: We demonstrate that atherosclerosis-prone Apoe-/- mice exposed to a western diet (WD) exhibit an enhanced induction of a dysfunctional, pro-inflammatory CD4⁺ T cell phenotype compared to chow diet(CD)-fed Apoe-/- mice. This phenotype was characterized by increased proliferation, activation, and inflammatory conversion of CD4⁺ T cells in lymph nodes and atherosclerotic lesions, resulting in an accumulation of interferon γ (Ifnγ)-producing effector memory (T_EM) and, more specifically, ApoB reactive (ApoB⁺) CD4⁺ T cells. By utilizing RNA sequencing, flow cytometry, seahorse metabolic flux assay, and metabolomics we revealed that antigen-experienced T_EM and ApoB⁺ CD4⁺ T cells in WD-fed mice were rendered glycolytic. To test whether glucose transport into CD4⁺ T cells was involved in this process, we constructed Apoe-/- mice harboring a tamoxifen-inducible CD4⁺ T cell specific depletion of the glucose transporter Glut1 (CD4^Cre-ERT2 Glut1^fl/fl Apoe-/- mice). These mice were protected from the pro-inflammatory effector CD4⁺ T cell phenotype and displayed reduced atherosclerosis. Through conditional Glut1 knockout in Foxp3 lineage tracker (Foxp3^{eGFP-Cre-ERT2} Glut1^fl/flROSA26^{fl-stop-fl-RFP}) Apoe-/- mice, we furthermore demonstrated that enhanced glycolytic metabolism directly induces the conversion of T_reg CD4⁺ T cells to Foxp3^- exT_reg cells with cytotoxic and effector-like phenotypes. By utilizing mouse models of insulin deficiency and CD4⁺ T cell-specific Insulin receptor (InsR) deficiency (CD4^Cre-ERT2 InsR^fl/fl Apoe-/- mice), we mechanistically identified hyperinsulinemia as a critical driver of the diet-related metabolic reprogramming and proinflammatory conversion of CD4⁺ T cells during atherogenesis. Combined single-cell RNA sequencing, surface protein analysis by CITE-Seq, and T cell receptor (TCR) sequencing demonstrated that blood CD4⁺ T cells in humans with advanced coronary artery disease (CAD) were similarly enriched for highly glycolytic, proliferative and inflammatory T_EM and ApoB⁺ CD4⁺ T cells.

Conclusions: Exposure of Apoe-/- mice to a WD induces expansion, activation and pro-inflammatory conversion of autoreactive CD4⁺ T cells which associates with glycolytic metabolic reprogramming. CD4⁺ T cells in humans with advanced CAD are similarly biased, which substantiates the translational relevance of these findings. Conditional knockout and lineage tracing experiments in mice revealed that diet-related hyperinsulinemia is causally involved in the generation of the observed, pathogenic CD4⁺ T cell phenotype. Our findings may pave the way for targeted metabolic interventions to prevent or treat the proatherogenic conversion of CD4⁺ T cells.