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Alternate high fat diet aggravates atherosclerosis through an IL1B- 2 dependent reprogramming of neutrophil progenitors

ABSTRACT: Systemic immune responses induced by chronic hypercholesterolemia contribute to the initiation, progression and complications of atherosclerosis. However, it is common for individuals to change dietary habits over time. The consequences of an alternating high fat diet and the associated variations in plasma cholesterol levels on atherosclerosis are unknown. To address this relevant issue, we developed a novel protocol in atherosclerosis-prone mice that allowed us to compare the effects of alternate versus continuous high fat diet (HFD) on atherosclerosis, while keeping the overall period of exposure to HFD similar between groups. We showed that an alternate HFD lead to accelerated atherosclerosis in Ldlr-/- and Apoe 55 -/- mice compared to continuous HFD. This pro-atherogenic effect of alternate HFD was also observed in Apoe-/-Rag2 57 -/- mice lacking T, B, and NKT cells, ruling out the role of the adaptive immune system in the observed phenotype. Employing various complementary in vivo approaches, we discovered that discontinuing HFD in the alternate group downregulated Runx1, a negative regulator of inflammatory signaling pathways in the myeloid lineage. Consequently, after re61 exposure to HFD, myeloid progenitors were primed to respond to HFD re-exposure by differentiating into IL-1-producing immature neutrophils, and inducing a state of emergency myelopoiesis. Subsequently, neutrophils markedly increased in the peripheral blood, infiltrated atherosclerotic lesions and locally released neutrophil extracellular traps. Anti-Ly6G neutrophil depletion abolished the pro-atherogenic effects of alternate HFD. Specific deletion of Il1b and Il1 receptor confirmed that the IL-1 signaling pathway was a major driver of the pro67 inflammatory and pro-atherogenic neutrophil signature. Finally, treatment with anti-IL-1 neutralizing antibody or an inflammasome inhibitor during re-exposure to HFD reversed the pro-atherogenic effects of alternate HFD. In summary, we showed that alternate HFD accelerates atherosclerosis through IL-1-dependent neutrophil progenitor reprogramming.

ORGANISM(S): Mus musculus

PROVIDER: GSE254105 | GEO | 2024/04/23

REPOSITORIES: GEO

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Project description:Systemic immune responses induced by chronic hypercholesterolemia contribute to the initiation, progression and complications of atherosclerosis. However, the consequences of an alternate high fat diet and the associated variations in plasma cholesterol levels on atherosclerosis are unknown. To address this relevant issue concerning common situations where dietary habits change over time, we developed a novel protocol in athero-prone mice that allowed us to compare the effects of alternate versus continuous high fat diet (HFD) on atherosclerosis, the overall period of time of exposure to HFD being similar between groups. We showed that alternate HFD lead to accelerated atherosclerosis in Ldlr-/- and Apoe-/- mice compared to continuous HFD. The pro-atherogenic effect of alternate HFD was also found in Apoe-/-Rag2-/- mice lacking T, B, and NKT cells, ruling out a role for the adaptive immune system in the observed phenotype. Using different complementary in vivo approaches, we found that lipid-rich diet discontinuation in alternate group downregulated Runx1, a negative regulator downstream of TLR-4 pathways. As a result, after re-exposure to HFD, myeloid progenitors were more sensitive to hypercholesterolemia leading to acute differentiation into IL-1b- producing immature neutrophils, which caused a state of emergency myelopoiesis. Consecutively, neutrophils markedly increased in the peripheral blood, infiltrated atherosclerotic lesions and locally released neutrophil extracellular traps. Anti-Ly6G neutrophil depletion abolished the pro-atherogenic effects of alternate HFD. Specific deletion of Il1b and Il1b receptor confirmed that the IL-1b signaling pathway was a major driver of the pro-inflammatory and pro-atherogenic neutrophil signature. Finally, treatment with anti-IL-1b neutralizing antibody or inflammasome inhibitor during re-exposure to HFD reversed the pro-atherogenic effects of alternate HFD. In summary, we showed that alternate HFD accelerates atherosclerosis through IL-1b-dependent neutrophil progenitor reprogramming.

Project description:Atherosclerosis is a chronic inflammatory condition of our arteries and the main underlying pathology of myocardial infarction and stroke. The pathogenesis is age-dependent, but the links between disease progression, age, and atherogenic cytokines and chemokines are incompletely understood. Here, we studied the chemokine-like inflammatory cytokine macrophage migration inhibitory factor (MIF) in atherogenic Apoe–/– mice across different stages of aging and cholesterol-rich high-fat diet (HFD). MIF promotes atherosclerosis by mediating leukocyte recruitment, lesional inflammation, and suppressing atheroprotective B- cells. However, links between MIF and advanced atherosclerosis across aging have not been systematically explored. We compared effects of global Mif-gene deficiency in 30-, 42-, and 48-week-old Apoe–/– mice on HFD for 24, 36, or 42 weeks, respectively, and in 52-week- old mice on a 6-week HFD. Mif-deficient mice exhibited reduced atherosclerotic lesions in the 30/24- and 42/36-week-old groups, but atheroprotection, which in the applied Apoe–/– model was limited to lesions in the brachiocephalic artery and abdominal aorta, was not detected in the 48/42- and 52/6-week-old groups. This suggested that atheroprotection afforded by global Mif-gene deletion differs across aging stages and atherogenic diet duration. To characterize this phenotype and study the underlying mechanisms, we determined immune cells in the periphery and vascular lesions, obtained a multiplex cytokine/chemokine profile, and compared the transcriptome between the age-related phenotypes. We found that Mif- deficiency promotes lesional macrophage and T cell counts in younger but not aged mice, with sub-group analysis pointing towards a role for Trem2+ macrophage. The transcriptomic analysis identified MIF- and aging-dependent changes in lipid, cell respiration, and inflammation pathways, and key enriched genes such as Cpne7, Cd6, Plin1, Cd274 hinting towards effects on lesional T cells, lipids, and foamy macrophages. Moreover, Mif-deficient aged mice exhibited a distinct plasma cytokine/chemokine signature consistent with the notion that mediators known to drive inflamm’aging are either not down-regulated or even up- regulated in Mif-deficient aged mice compared to the corresponding younger ones. Lastly, Mif-deficiency favored formation of lymphocyte-rich peri-adventitial leukocyte clusters. While the causative contributions of these mechanistic pillars and their interplay will be subject to future scrutiny, our study suggests that atheroprotection due to global Mif-gene deficiency in atherogenic Apoe–/– mice is reduced upon advanced aging and identifies previously unrecognized cellular and molecular targets that could explain this phenotype shift. These observations enhance our understanding of inflamm’aging and MIF pathways in atherosclerosis and may have implications for translational MIF-directed strategies.

Project description:Background: Atherosclerosis leads high mortality, highlighting an urgent need for new therapeutic strategies. Protein kinases orchestrate multiple cellular events in atherosclerosis and may provide new therapeutic targets for atherosclerosis. Here, we identified a protein kinase, WEE1 G2 checkpoint kinase (WEE1), promoting inflammatory response in atherosclerosis. Methods: The ApoE-/- mice without macrophage-specific WEE1 deletion (ApoE-/-WEE1f/f) and ApoE-/- mice with macrophage-specific WEE1 deletion (ApoE-/-WEE1MCKO) were generated using bone marrow transplantation. These mice and WEE1 inhibitor MK1775 were used in a high-fat diet (HFD)-induced atherosclerotic model. Human atherosclerotic tissues, mouse primary peritoneal macrophages (MPMs), 293T cells, and recombinant proteins were utilized to investigate the pathogenic role and underlying mechanisms of WEE1. Results: We identified up-regulated p-WEE1 in macrophages in atherosclerotic lesions of HFD-fed ApoE-/- mice. Transcriptome sequencing analysis indicated that WEE1 promotes oxLDL-induced inflammation in macrophages. We then demonstrated that macrophage-specific deletion or pharmacological inhibition of WEE1 attenuates atherosclerosis by reducing inflammation both in vivo and in vitro. The overexpression of wild-type WEE1 or activating-mutant WEE1, but not inactivating-mutant WEE1, exacerbates inflammation in macrophages. Mechanistically, transcriptome sequencing analysis and co-immunoprecipitation followed by quantitative proteomics analysis identified p65 as a binding protein of WEE1. We confirmed that WEE1 directly interacts with the RHD domain of p65 via kinase domain and phosphorylates p65 at S536, thereby facilitating subsequent NF-κB activation and inflammatory response in macrophages. Conclusions: Our findings demonstrated that macrophage WEE1 promotes inflammatory atherosclerosis by directly binding to p65 and phosphorylate it at S536. This study provides WEE1 as a new p65 regulator in inflammation and a potential therapeutic target for atherosclerosis.

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Alternate high fat diet aggravates atherosclerosis through an IL1B- 2 dependent reprogramming of neutrophil progenitors

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