Alternating high-fat diet enhances atherosclerosis by neutrophil reprogramming

Abstract

Systemic immune responses caused by chronic hypercholesterolaemia contribute to atherosclerosis initiation, progression and complications¹. However, individuals often change their dietary habits over time², and the effects of an alternating high-fat diet (HFD) on atherosclerosis remain unclear. Here, to address this relevant issue, we developed a protocol using atherosclerosis-prone mice to compare an alternating versus continuous HFD while maintaining similar overall exposure periods. We found that an alternating HFD accelerated atherosclerosis in Ldlr^-/- and Apoe^-/- mice compared with a continuous HFD. This pro-atherogenic effect of the alternating HFD was also observed in Apoe^-/-Rag2^-/- mice lacking T, B and natural killer T cells, ruling out the role of the adaptive immune system in the observed phenotype. Discontinuing the HFD in the alternating HFD group downregulated RUNX1³, promoting inflammatory signalling in bone marrow myeloid progenitors. After re-exposure to an HFD, these cells produced IL-1β, leading to emergency myelopoiesis and increased neutrophil levels in blood. Neutrophils infiltrated plaques and released neutrophil extracellular traps, exacerbating atherosclerosis. Specific depletion of neutrophils or inhibition of IL-1β pathways abolished emergency myelopoiesis and reversed the pro-atherogenic effects of the alternating HFD. This study highlights the role of IL-1β-dependent neutrophil progenitor reprogramming in accelerated atherosclerosis induced by alternating HFD.

Extended Data figure 1.. Characteristics of mice and plaque composition in continuous vs alternate HFD.

a, cholesterol burden evaluated using the area under the curve of repetitive plasma cholesterol levels measured during the experimental protocol (n=6/group). b, body weight of mice at sacrifice (female Ldlr^−/− n=10/group; male Ldlr^−/− n=5/group; male Apoe^−/− n=12/group; male Apoe^−/−Rag2^−/− n=7 Cont. n=9 Alt.). c, representative photomicrographs and quantitative analysis of macrophage accumulation (MOMA staining, red) in atherosclerotic lesions of Apoe^−/− after 8 weeks of continuous or alternate HFD regimen (2 experiments pooled, n=12 Cont. n=13 Alt.); Scale bar 100 μm. d, representative photomicrographs and quantitative analysis of acellular area (Masson’s Trichrome) of Apoe^−/− after 8 weeks of continuous or alternate HFD regimen (2 experiments pooled, n=11 Cont. n=13 Alt.); Scale bar 100 μm. Data are presented as mean values +/− SD. P values were calculated using two-tailed Mann-Whitney test.

Extended Data Figure 2.. Microbiota and immune characterization.

a, Ldlr^−/− mice were exposed or not transiently to HF diet during 4 weeks and four weeks later gut microbiota was analyzed. b, bacterial diversity on the basis of Shannon and Chao in the fecal samples (n=4/group). c, bacterial-taxon-based analysis at the phylum level in the fecal microbiota. d, blood cell counts at sacrifice of Ldlr^−/− mice fed either a continuous or an alternate HFD (2 experiments pooled, n=12 Cont. n=13 Alt.). e, spleen cell count at sacrifice of Ldlr^−/− mice fed either a continuous or an alternate HFD (2 experiments pooled, n=12 Cont. n=13 Alt.). f, gating strategy by flow cytometry in the blood. Classical monocytes were defined as NK1.1-CD11b+Ly6G-Ly6Chigh cells; Non-classical monocytes were defined as NK1.1-CD11b+Ly6G-Ly6Clow cells; neutrophils were defined as NK1.1-CD11b+Ly6G+ cells. CD4+ T Lymphocytes were selected as NK1.1-B220-CD11b-CD3+CD4+ cells, CD8+ T Lymphocytes were selected as NK1.1-B220-CD11b-CD3+CD8+ cells. B cells were defined as NK1.1-CD11b-B220+ cells. g, cholesterolemia at sacrifice of splenectomized Apoe^−/− mice fed either a continuous or an alternate HFD (2 experiments pooled, n=7/group). Data are presented as mean values +/− SD (d, e, g). Data are presented as box and whiskers (1^st quartile, 3^rd quartile, median) in b. P values were calculated using two-tailed Mann-Whitney test.

Extended Data Figure 3.. Single-cell RNA-seq analysis of aortic leukocytes.

a, UMAP plot of total CD45+ leukocytes and annotation of cell identities based on b) expression of marker transcripts and c) expression of cell surface markers; d) reclustering of leukocytes annotated as monocytes and macrophages identifies aortic monocyte/macrophage subpopulations; e) selected marker genes of monocyte/macrophage clusters used for their annotation; f) percentage of monocyte/macrophage subsets among total monocytes/macrophages across experimental conditions. In f, each datapoint represents a pool of two aortas, all aortas were processed for scRNA-seq together via cell hashing (see methods) (n=3 healthy, n=5 Cont. n=5 Alt.). Data are presented as mean values +/− SD (f).

Extended Data Figure 4.. Modulation of HFD induced neutrophils.

a, neutrophils were defined as CD45+CD11b+Ly6G+ cells ; immature neutrophils were defined as CD62L+ cells. FMO, Fluorescent Minus One. b, quantitative analysis and representative photomicrographs of NET content (citrullinated histone 3 staining, yellow) in atherosclerotic lesions of male Ldlr^−/− mice fed either continuous (Black) or alternate (Red) HFD (n=5/group) at week 16. Scale bar 100 μm. c, neutrophils were defined as CD45+NK1.1-CD11b+CD115-Ly6C-CXCR2+ cells. d, experimental protocol to validate murine anti-Ly6G mAb. e, kinetic of blood neutrophils in the blood at baseline and just before mAb re-injection at H48. n=5 mice/timepoint. P values were calculated using two-tailed Mann-Whitney test. Data are presented as mean values +/− SD (b) or mean values +/− SEM (e).

Extended Data Figure 5.. Characterization of murine models.

a, plasma cholesterol levels of male Ldlr^−/− mice fed either continuous (Black) or alternate (Red) HFD and treated by isotype control or anti-Ly6G mAb depleting antibody (isotype, n=7/group; anti-Ly6G, n=8/group). b, male Ldlr^−/− mice, called exposed (n=7), were put under a HFD during 4 weeks from 6 to 10 weeks of age and next subsequently subjected to regular chow diet for 8 weeks. Non-exposed mice were put only under a regular chow diet (n=5). Blood monocyte (NK1.1-CD11b+Ly6C+Ly6G-) and neutrophil (NK1.1-CD11b+Ly6G+) counts at sacrifice. Data are presented as mean values +/− SD.

Extended Data Figure 6.. Flow cytometry gating strategy to identify progenitors in the bone marrow.

long-term (LT) and short-term (ST) hematopoietic stem cells (HSC). LT-HSC were defined as Lin-CD45+MHCII-ckithighScahighCD48-CD150+ and ST-HSC were defined as Lin-CD45+MHCII-ckithighScahighCD48-CD150-. BM progenitors : granulocyte/macrophage progenitors (GMP, Lin-CD45+MHCII-ckithighSca-CD16+CD34+), Megakaryocyte/erythroid progenitors (MEP, Lin-CD45+MHCII-ckithighSca-CD16-CD34-), Common Lymphoid progenitors (CLP, Lin-CD45+MHCII-ckitlowSca+CD135+).

Extended Data Figure 7.. Characterization of neutrophilic progenitors in the bone marrow using ScRNA seq.

a, UMAP plot of bone marrow Lin- population isolated from HFD non-exposed and transiently HFD exposed mice. b, feature plots of marker genes commonly used in the literature to define BM progenitor cell subsets. c, bubble chart showing the expression of marker genes in each cell cluster. d, UMAP clustering of BM Lin- cells from non-exposed and HFD exposed mice (n=4). Lin- BM cells captured from individual mice color coded separately. e, total numbers and percentages of scRNA-Seq based annotated Lin- cell subsets. Each bone marrow scRNA-Seq dataset resulted within the range of 4561–10207 annotated cells after various quality filtering methods (n=5/group). f, heatmap of all DEGs in neutrophilic progenitors of HFD non-exposed and exposed mice. Data are presented as mean values +/− SD (e).

Extended Data Figure 8.. Impact of re-exposure on myeloid cells.

a, red blood cells and platelets counts in the blood of Ldlr^−/− mice one week after HFD re-exposure (Week 13), (n=10/group). b, kinetic of plasma cholesterol levels (n=5 C57Bl6, Cd36^−/− , Tlr4^−/− n=3 Myd88^−/− ). P values were calculated using paired non-parametric test for each group at 2 timepoints. c, C57Bl6 mice were put under a high fat diet (HFD) during 4 weeks, then under a chow diet (CD) during 5 weeks and finally under a HFD during 2 weeks. BM cells were isolated after 2 weeks of exposure or 2 weeks of re-exposure and il1b mRNA levels were quantified by qPCR (n=5 expo and n=4 re-expo). P values were calculated using two-tailed Mann-Whitney test. Data are presented as mean values +/− SD (a,c) or mean values +/− SEM (b).

Extended Data Figure 9.. Impact of re-exposure on BM progenitors and myeloid cells.

a, proportion of GMP expressing Runx1 in the BM at week 4, 12 and 16 in Ldlr^−/− mice receiving either continuous or Alternate HFD (Flow cytometry). n=7/group 4W-16W and n=8/group W12. P values were calculated using two-tailed Mann-Whitney test. b, flow cytometry characterization of BM subsets producing pro-IL-1β or expressing IL1R receptor. Neutrophils were defined as gated CD45+Cd11b+Ly6G+, monocytes were defined ad CD45+CD11b+Ly6G-Ly6C+ and GMP as Lin-CD45+cKit+Sca1-CD34+CD16/32+. c, strategy for cell sorting of BM monocytes and neutrophils. d, quantification of Il-1β mRNA levels on sorted monocytes and neutrophils at week 13 (n=5/group). P values were calculated using two-tailed Mann-Whitney test. Data are presented as mean values +/− SD.

Extended Data Figure 10.. Modulation of IL1β pathways.

a, cytokine production by stimulated splenocytes at sacrifice from continuous or alternate HFD fed chimeric Il1β^−/−Ldlr^−/− mice (n=9/group). b, cytokine production by stimulated BM cells at sacrifice from continuous or alternate LysMCre^+/−Il1r^lox/loxLdlr^−/− groups (n=9 Cont. N=10 Alt.). c, cytokine production by stimulated splenocytes at sacrifice from anti-IL1β-treated Ldlr^−/− mice fed either continuous or alternate HFD (n=7 Cont. n=8 Alt.). d, experimental protocol, 6-week-old Apoe^−/− mice were fed either alternate (Red) or continuous (Grey) HFD and treated by NLRP3 inflammasome pharmacological inhibitor (MCC950, 10 mg/kg every 2 days IP) during the last 4 weeks of the protocol. e, comparison in MCC950-treated alternate group of the variations of blood neutrophil count induced by exposition versus re-exposition to HFD (n=5/group). f, plasma cholesterol levels at sacrifice. g, quantitative analysis of atherosclerotic lesions in the aortic sinus of MCC950-treated Apoe^−/− mice (n=5/group). Scale bar 200 um. h, cytokine production by stimulated splenocytes isolated at sacrifice from MCC950-treated Apoe^−/− mice (n=5/group). P values were calculated using two-tailed Mann-Whitney test. Data are presented as mean values +/− SD.

Figure 1.. Alternate HFD accelerates experimental atherosclerosis.

a, experimental protocol comparing athero-prone mice fed either alternate (Red) or continuous (Black) HFD. b, kinetics of plasma cholesterol levels of Ldlr^−/− mice (n=6/group/timepoint). c, kinetics of body weight of Ldlr^−/− mice (n=6/group/timepoint). d, at sacrifice, cholesterolemia, quantitative analysis and representative photomicrographs of atherosclerotic lesions in the aortic sinus of female Ldlr^−/− mice mice (n=10/group). Scale bar 200 μm. e, quantitative analysis and representative photomicrographs of atherosclerotic lesions along the thoraco-abdominal aorta of female Ldlr^−/− mice mice (n=5/group). Scale bar 1 mm. f, at sacrifice, plasma cholesterol levels, quantitative analysis and representative photomicrographs of atherosclerotic lesions in the aortic sinus of male Ldlr^−/− mice (n=8/group). Scale bar 200 μm. g, at sacrifice, plasma cholesterol levels, quantitative analysis and representative photomicrographs of atherosclerotic lesions in the aortic sinus of male Apoe^−/− mice (n=12/group). Scale bar 200 μm. h, representative photomicrographs and quantitative analysis of macrophage accumulation (MOMA staining, red) in atherosclerotic lesions of male Ldlr^−/− mice (n=8/group). Scale bar 100 μm. i, representative photomicrographs and quantitative analysis of acellular area (Masson’s Trichrome) in atherosclerotic lesions of male Ldlr^−/− mice mice (n=10/group). Scale bar 100 μm. Data are presented as mean values +/− SD. P values were calculated using two-tailed Mann-Whitney test.

Figure 2.. Alternate HFD accelerates atherosclerosis independently of gut microbiota or adaptive immunity.

a, left, Principal Component Analysis of the top 500 most variable genes (n=6/group). b, experimental protocol comparing male Ldlr^−/− mice fed either alternate (Red) or continuous (Black) HFD and treated with antibiotics. c, at sacrifice, plasma cholesterol levels, quantitative analysis and representative photomicrographs of atherosclerotic lesions in the aortic sinus of male Ldlr^−/− mice fed either continuous or alternate HFD and treated with antibiotics (n=5 Cont. and n=7 Alt.). Scale bar 200 μm. d, mRNA levels of spleen cytokines at sacrifice (n=12 Cont. n=13 Alt.). e, cytokine production by stimulated splenocytes from continuous or alternate ldlr^−/−groups (n=12 Cont. n=13 Alt.). f, blood leukocyte subsets counts at sacrifice from continuous or alternate HFD ldlr^−/−groups (n=12 Cont. n=13 Alt). g, h, spleen leukocyte subsets counts and flow cytometry picture from continuous or alternate HFD ldlr^−/−groups (n=12 Cont. n=13 Alt.). i, representative photomicrographs and quantitative analysis of T cell content (CD3 staining, red) in atherosclerotic lesions of male Ldlr^−/− mice fed either continuous or alternate HFD (n=11 Cont. n=12 Alt.). Scale bar 100 μm. j, protocol with surgical splenectomy (Sx). k, quantitative analysis and representative photomicrographs of atherosclerotic lesions of splenectomized male Apoe^−/− mice mice fed either continuous or alternate HFD (n=6/group). Scale bar 200 μm. l, 6-week old male Apoe^−/−Rag2^−/− mice were fed either alternate or continuous HFD. m, plasma cholesterol levels, quantitative analysis and representative photomicrographs of atherosclerotic lesions of male Apoe^−/− Rag2^−/− mice fed either continuous or alternate HFD (n=7 Cont. n=9 Alt.); Scale bar 200 μm. n, cytokine production by stimulated splenocytes from continuous or alternate HFD Apoe^−/−Rag2^−/− groups (n=7 Cont. n=9 Alt.). Data are presented as mean values +/− SD. P values were calculated using two-tailed Mann-Whitney test.

Figure 3.. HFD re-exposure induces acute neutrophilia and NETosis responsible for accelerated atherosclerosis.

a, kinetics of plaque size in the aortic sinus (n=6/group/timepoint). b, left, Principal Component Analysis of the top 500 most variable genes. Right Top 10 enriched pathways of the analysis of 175 differentially expressed genes between the two diets (n=4 Cont. N=5 Alt.). c, kinetics of monocyte counts in the blood (n=6/group/timepoint). d, comparison in alternate group of the variations in blood monocyte counts induced by exposure versus re-exposure to HFD (n=11 Cont. n=10 Alt.). e, macrophage content (MOMA staining, red) in atherosclerotic lesions at Week 13 (n=5/group). Scale bar 100 μm. f, kinetics of neutrophil counts in the blood (n=6/group/timepoint). g, comparison in alternate group of the variations of blood neutrophil counts induced by exposure versus re-exposure to HFD (n=10/group). h, proportion of immature CD62L+ neutrophils in the blood (n=5/group). i, comparison of plasma levels of Cxcl1, Cxcl2 and Ccl3 during exposure versus re-exposure to HFD. Variations are expressed relative to baseline value (n=5/group). j, top left: UMAP plot centered on the two aortic neutrophil subpopulations; top right: percentage of Neutro1 and Neutro2 populations among total aortic neutrophils; bottom: expression of selected marker genes in aortic neutrophil subpopulations. k, NET content (yellow) in atherosclerotic lesions (n=5/group) at Week 13. Scale bar 100 μm. l, male Ldlr^−/− mice treated IP by isotype or anti-Ly6G depleting monoclonal antibody. m, examples of neutrophil depletion in the blood 48 hours after anti-Ly6G mAb injection. n, neutrophil kinetic in the blood of Ldlr^−/− mice treated with isotype control (n=6) or anti-Ly6G mAb (n=7/group). o, quantitative analysis and representative photomicrographs of MPO+ cells (Green) in atherosclerotic lesions of male Ldlr^−/− mice (isotype control n=7; anti-Ly6G mAb n=8); Scale bar 50 μm. p, atherosclerotic lesions in the aortic sinus of male Ldlr^−/− mice treated by isotype control (n=7/group) or anti-Ly6G mAb (n=8/group); Scale bar 200 μm. All data are presented as mean values +/− SD except for a,c,f presented as mean values +/− SEM. P values were calculated using two-tailed Mann-Whitney test or Kruskal-Wallis test (n,o,p).

Figure 4.. Alternate HFD induces TLR4-dependent IL-1β production in the bone marrow responsible for neutrophil progenitor reprogramming.

a, experimental protocol with male Ldlr^−/− mice. b, at 18 weeks, proportion of long-term (LT) and short-term (ST) hematopoietic stem cells (HSC) (n=5 non-expo, n=6 expo). c, proportion of BM Granulocyte/macrophage progenitors (GMP), Megakaryocyte/Erythroid progenitors (MEP), Common Lymphoid progenitors (CLP) (n=5 non-expo, n=6 expo). d, monocyte and neutrophil counts in the BM (n=5/group). e, mRNA levels of cytokines in the BM (n=5 non expo, n=7 expo). f, Volcano plot showing gene expression down regulated (blue) and up-regulated (red) in BM progenitors (n=5/group). g, experimental protocol of BM cell transplantation. h, atherosclerotic lesions in the aortic sinus (n=10/group). Scale bar 200 μm. i, plasma cholesterol levels (n=10/group). j, cytokine production by stimulated BM cells (n=10/group). k,l, proportion of hematopoietic stem cells and progenitors in the BM of male Ldlr^−/− mice (n=6/group), one week after HFD re-exposure. m, immature CD62L+ neutrophils in the BM at week 13 (n=5/group). n, quantification of immature CD62L+ neutrophils producing IL-1 β in the BM at week 13 (N=5/group). o, cytokine production by stimulated BM cells (n=5/group). p, protocol of HFD re-exposure in C57Bl6 mice. q, kinetics of plasma cholesterol levels (n=5/group). r, kinetics of blood neutrophils in C57Bl6 (n=8/group). s, comparison in C57Bl6 (n=8 expo and n=7 re-expo), Cd36^−/− (n=5), Myd88^−/− (n=4) and Tlr4^−/− (n=4 expo n=5 re-expo) mice of the variations of blood neutrophil counts. t, Il-1β mRNA levels in the BM of C57Bl6 (n=8), Cd36^−/− (n=5), Myd88^−/− (n=4) and Tlr4^−/− (n=5) mice at sacrifice. u, experimental protocol with Ldlr^−/− mice. v, proportion of GMP expressing Runx1 in the BM at week 13 (n=8/group). w,x, experimental protocol with Runx1+/+ ^GMP (Runx1^lox/lox) and Runx1−/−^GMP (Runx1^lox/lox;Cebpa-Cre) mice and neutrophil kinetics in the blood (n=5/group). y, proportion of neutrophils producing IL-1 β in the BM after 4 weeks of HFD (n=5/group). Data are presented as mean values +/− SD. P values were calculated using two-tailed Mann-Whitney test or Kruskal-Wallis test (t).

Figure 5.. IL-1β signaling pathway blockade abolishes alternate HFD-induced neutrophilia and accelerated atherosclerosis.

a, experimental protocol with chimeric Il1β ^−/−Ldlr^−/− mice. b, comparison in alternate group of the variations of blood neutrophil count induced by exposition versus re-exposition to HFD (n=10/group). c, plasma cholesterol levels at sacrifice (n=8/group). d, quantitative analysis and representative photomicrographs of atherosclerotic lesions in the aortic sinus of chimeric Il1β ^−/− Ldlr^−/− mice. Scale bar 200 μm. e, cytokine production by stimulated BM cells at sacrifice from continuous or alternate HFD fed chimeric Il1β^−/−Ldlr^−/− mice (n=8/group). f, experimental protocol with chimeric LysMCre^+/−Il1r^lox/lox Ldlr^−/− mice. g, comparison in alternate LysMCre^+/−Il1r^lox/lox Ldlr^−/− group of the variations of blood neutrophil counts induced by exposure versus re-exposure to HFD (N=9/group). h, plasma cholesterol levels at sacrifice (n=9/group). i, quantitative analysis and representative photomicrographs of atherosclerotic lesions in the aortic sinus of LysMCre^+/−Il1r^lox/loxLdlr^−/− (n=10 Cont. n=9 Alt.). Scale bar 200 μm. j, cytokine production by stimulated BM cells at sacrifice (n=9/group). k, Ldlr^−/− mice were treated by anti-IL-1β neutralizing antibody during the last 4 weeks of the protocol. l, comparison in anti-IL-1β-treated alternate group of the variations of blood neutrophil count induced by exposition versus re-exposition to HFD (n=8/group). m, plasma cholesterol levels at sacrifice of anti-IL-1β-treated Ldlr^−/− mice fed either continuous (n=7) or alternate HFD (n=8). n, quantitative analysis and representative photomicrographs of atherosclerotic lesions in the aortic sinus of anti-IL1β -treated Ldlr^−/− mice (n=8). Scale bar 200 μm. o, cytokine production by stimulated BM cells at sacrifice from anti-IL1β-treated Ldlr^−/− mice fed either continuous (n=7) or alternate HFD (n=8). Data are presented as mean values +/− SD. P values were calculated using two-tailed Mann-Whitney test.