Fig 1: Conserved and divergent responses to sustained atherogenic dyslipidemia.a, Plasma levels of cholesterol in dyslipidemic APOE cKO and D374Y mice with respective littermate controls, maintained on a high fat diet for 20 weeks. (APOE cKO n = 16 vs 11; and D374Y n = 7 vs 3). Plots are ± SEM, two-sided t-test b, Representative liver sections stained with ORO in APOE cKO and D374Y mice after 20 weeks on a high fat diet (scale bars = 100 μm, n = 2 vs 2 for D374Y and n = 3 vs 3 for APOE cKO, where n indicates a different mouse). c, Representative liver sections stained with H&E in APOE cKO and D374Y mice after 20 weeks on a high fat diet (scale bars = 100 μm, n = 2 vs 2 in each group where n indicates a different mouse).d, Unique and common upregulated genes in both strains at 8, 12, and 20 weeks on a high fat diet. Unique genes for each strain are indicated at the bottom. e, Flow cytometry of CLEC4F levels on Kupffer cells from female D374Y versus littermate control after 8 weeks of high fat diet. f, Heatmap of the 72 genes upregulated at all time points in both strains, expressed in 76 human single cell types. g, Flow cytometry plots showing percentages of blood and liver monocytes, and liver Kupffer cells in dyslipidemic D374Y mice given clodronate liposome or Dil liposomes control for 8 weeks while on a high fat diet. h, Immunohistochemistry of liver to detect CD5L production in control or clodronate treated D374Y mice after 8 weeks of high fat diet (scale bars = 100 μm). i, The 14 genes not affected by clodronate-induced Kupffer cells depletion, expressed in liver myeloid cell clusters. j, Correlation between plasma IL18BP and liver PCSK9 expression in humans.
Fig 2: The transcriptional response of the liver to sustained dyslipidemia.a, Heat maps of bulk mRNA-seq of whole liver showing the 72 significant differentially expressed genes common to both dyslipidemic strains versus respective littermate controls after 8 weeks, 12 weeks and 20 weeks on an HFD (n = 2–5). b, Expression of the 72 conserved genes in liver cells using clusters taken from ref. 19. c, Pie chart showing how expression of the 72 conserved genes correlates with PCSK9 transcript levels in human liver samples (n = 261). d, Correlations with human PCSK9 levels for 12 of the 34 positively correlating genes from c. e, Representative ORO liver sections (top) and confocal images of F4/80 immunofluorescence (bottom) in the livers of D374Y dyslipidemic mice treated with clodronate liposomes and Dil liposomes (control) for 8 weeks while on an HFD (scale bar, 100 μm; n = 2 D3747 control and n = 2 D3747 clodronate, where every n represents a different mouse). f, Effect of long-term administration of clodronate liposomes on the 72 differentially expressed genes (n = 2–3). g, Top, percentages of blood and liver monocytes (live CD45+CD19−CD3e−CD64−Ly6C+) and liver KCs (live CD45+CD19−CD3e−CD64+Ly6C−F4/80+TIM4+) in D374Y dyslipidemic mice after 8 weeks of treatment with clodronate liposomes (n = 2 D374Y clodronate and n = 3 D374Y control mice). Bottom, heat maps showing variations in the expression of signature genes for KCs, monocytes, LAMs and CD207+ macrophages in the liver of D374Y mice after long-term clodronate exposure (n = 2 D374Y clodronate and n = 3 D374Y control mice). h, Transcript per million values for a clodronate-sensitive gene (Cd5l) and a clodronate-insensitive gene (Ctsd) from D374Y and littermate control and D374Y treated with Dil liposomes or clodronate liposomes (n = 3 littermate control, n = 5 D374Y, n = 3 D374Y control and n = 2 D374Y clodronate mice). i, Total liver cholesterol (mg) in D374Y dyslipidemic mice treated with either clodronate liposomes or Dil liposomes for 8 weeks while on an HFD (n = 2 D374Y clodronate and n = 3 D374Y control mice). j, Plasma levels of IL18BP (ng ml−1) after 8 weeks on an HFD in dyslipidemic APOE cKO and D374Y mice versus respective littermate controls (n = 5 APOE cKO and n = 3 littermate control mice; n = 5 D374Y and n = 5 littermate control mice). All plots are ±s.e.m. Two-sided t-test (g,i,j). cDC, conventional dendritic cell; HSPC, hematopoietic stem and progenitor cell; ILC, innate lymphoid cell; Macs, macrophages; Mig., migratory; NK, natural killer; pDC, plasmacytoid dendritic cell.
Fig 3: Ablating KCs prevents the hepatic response to atherogenic dyslipidemia.a, Volcano plot of genes downregulated in female D374Y mice treated with clodronate liposome during 10-d post-tamoxifen administration while maintained on a chow diet (n = 3 versus n = 3; P value from DESeq2 two-sided Wald test). b,c, Effects of clodronate liposomes on the expression of core identity genes of KCs (b), LAMs, monocytes and CV and capsular macrophages (c) in the liver of D374Y mice. d, Effect of clodronate liposomes on the day 10 conserved gene expression in both APOE cKO and D374Y mice. e, IL18BP plasma levels in dyslipidemic APOE cKO and D374Y mice given clodronate liposomes or Dil liposomes controls (ng ml−1, APOE cKO n = 3 and D374Y n = 4). f, Volcano plot and heat map indicating minimal response of the liver to dyslipidemia when comparing littermate control versus D374Y with both treated with clodronate liposomes, as determined by mRNA-seq (n = 3 versus n = 3; P value from DESeq2 two-sided Wald test). g, Total plasma (mg dl−1) and liver (µg mg−1 of protein) cholesterol and triglyceride measurements in dyslipidemic APOE cKO and D374Y mice given clodronate liposomes or Dil liposomes controls with respective littermate controls also administered clodronate liposomes (APOE cKO n = 4 versus n = 3 versus n = 4 and D374Y n = 3 versus n = 4 versus n = 3). h, Plasma CD5l (µg ml−1) concentrations as determined by ELISA in D374Y and littermate control mice 10 d after tamoxifen administration (n = 9 versus n = 9). i, Plasma CD5l (µg ml−1) concentrations in dyslipidemic D374Y mice and littermate control mice given clodronate liposomes and dyslipidemic D374Y mice administered Dil liposomes controls (n = 3 versus n = 4 versus n = 3). All experiments were conducted on day 10 after tamoxifen treatment, and mice were maintained on a normal chow diet. All plots are ±s.e.m. Two-sided t-test (e,h) or one-way ANOVA (g,i).
Fig 4: APOB lipoproteins are required for the KC inflammatory response to an HFD.a, Venn diagram of unique and overlapping differentially expressed liver genes after 4 weeks of HFD after tamoxifen induction in female APOE cKO (n = 3) and D374Y (n = 2 versus n = 3) mice versus respective littermate controls, as determined by bulk mRNA-seq. b, Expression of conserved genes from the week 4 HFD analysis in the liver day 10 scRNA-seq dataset. c, ELISA for secreted CD5L in plasma after 4 weeks on an HFD (APOE cKO n = 5 versus n = 5 and D374Y n = 8 versus n = 8). d, Identification of conserved transcriptional response to 4 weeks of HFD from littermate controls only of both strains. e, Heat map for expression of conserved genes from APOE cKO and D374Y mice compared to littermate controls alone in response to 4 weeks of HFD. All plots are ±s.e.m. Two-sided t-test (c).
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