Myeloid CD40 deficiency reduces atherosclerosis by impairing macrophages' transition into a pro-inflammatory state

Abstract

Aims: CD40 and its ligand, CD40L, play a critical role in driving atherosclerotic plaque development. Disrupted CD40-signalling reduces experimental atherosclerosis and induces a favourable stable plaque phenotype. We recently showed that small molecule-based inhibition of CD40-tumour necrosis factor receptor associated factor-6 interactions attenuates atherosclerosis in hyperlipidaemic mice via macrophage-driven mechanisms. The present study aims to detail the function of myeloid CD40 in atherosclerosis using myeloid-specific CD40-deficient mice.

Method and results: Cd40flox/flox and LysM-cre Cd40flox/flox mice on an Apoe-/- background were generated (CD40wt and CD40mac-/-, respectively). Atherosclerotic lesion size, as well as plaque macrophage content, was reduced in CD40mac-/- compared to CD40wt mice, and their plaques displayed a reduction in necrotic core size. Transcriptomics analysis of the CD40mac-/- atherosclerotic aorta revealed downregulated pathways of immune pathways and inflammatory responses. Loss of CD40 in macrophages changed the representation of aortic macrophage subsets. Mass cytometry analysis revealed a higher content of a subset of alternative or resident-like CD206+CD209b- macrophages in the atherosclerotic aorta of CD40mac-/- compared to CD40wt mice. RNA-sequencing of bone marrow-derived macrophages of CD40mac-/- mice demonstrated upregulation of genes associated with alternatively activated macrophages (including Folr2, Thbs1, Sdc1, and Tns1).

Conclusions: We here show that absence of CD40 signalling in myeloid cells reduces atherosclerosis and limits systemic inflammation by preventing a shift in macrophage polarization towards pro-inflammatory states. Our study confirms the merit of macrophage-targeted inhibition of CD40 as a valuable therapeutic strategy to combat atherosclerosis.

Keywords: Atherosclerosis; CD40; Inflammation; Macrophage.

Graphical Abstract

Figure 1

CD40 deficiency and inflammation in CD40^mac−/− mice. CD40 expression in LPS or control stimulated BMDMs isolated from CD40^mac−/− compared to CD40^wt mice (A; n = 3 mice; pooled cells, n = 6 technical replicates). Western blot showing CD40-expression after stimulation with LPS in CD40^wt and CD40^mac−/− BMDMs (B; n = 3 mice, pooled cells, representative blot shown with an α-tubulin loading control ∼50 kDa.). Flow cytometric analysis of 22 weeks old CD40^wt and CD40^mac−/− showing ratios of circulating CD45⁺CD11b⁺Ly6G^- monocytes, CD45⁺CD11b⁺Ly6G⁺SiglecF⁺ neutrophils, CD45⁺CD11b⁺Ly6G^-SiglecF⁺ eosinophils, CD45⁺CD11b⁺CD11c⁺MHCII⁺ dendritic cells in (C), ratios of circulating CD45⁺CD19⁺ B-cells and CD45 ⁺ CD3⁺ T-cells in (D) and ratios of CD45 ⁺ CD3 ⁺ CD4/8 ⁺ CD62L^+/-CD44^-/+ effector, naïve and memory T-cells in CD40^wt and CD40^mac−/− lymph nodes (E). Gene expression in CD40^wt and CD40^mac−/− lymph nodes of chemokines and cytokines analysed by qPCR (mRNA gene expression presented as fold-change against the respective housekeeping gene) is shown in (F), and by Luminex of plasma in (G). N = 8/6 for CD40^wt and CD40^mac−/− mice, respectively, in C; n = 7 in (D–E); n = 10/12 mice, respectively, in (F), and n = 14/18 mice, respectively, in (G). Data is shown as mean ± SD. Statistical analyses were performed using the unpaired t-test in (A–B) and the Mann–Whitney U test (with multiple comparisons adjusted for using the Holm-Šídák test) in (C–G).

Figure 2

Reduced atherosclerosis in the aortic arch of CD40^mac−/− mice. Lesion size (A) and mac3⁺ macrophage content (B) in CD40^wt and CD40^mac−/− aortic arches. Mac3⁺ macrophages (C) and CD206⁺ cell (D) content was further quantified separately in initial and advanced plaques. Necrotic regions were quantified in CD40^wt and CD40^mac−/− aortic arches (E; representative haematoxylin & eosin stain with necrotic areas marked by dashed lines). In vitro collagen production by primary murine VSMCs isolated from CD40-deficient and wild-type mice (F) and in immortalized murine VSMCs after stimulation with conditioned media from naïve, LPS- or IL4-activated primary CD40^wt and CD40^mac−/− BMDMs (G). Flow cytometric analysis of efferocytosis in primary bone marrow-derived macrophages isolated from CD40^wt and CD40^mac−/− mice (H). N = 19/17 mice, respectively, in (A), n = 17 mice in (B), n = 110 CD40^wt/142 CD40^mac−/− plaques, from n = 17 mice in (C), n = 90 CD40^wt/102 CD40^mac−/− plaques from n = 16/19 mice, respectively, in (D), n = 16/17 mice, respectively, in (E), n = 4 replicates in (F), n = 3 replicates in (G), and n = 6 replicates in (H). Scale bars represent 500 µm in (A) and 200 µm in (B) and (E). Data is shown as mean ± SD. Statistical tests were performed using the Mann-Whitney U test in (A–E) and the unpaired student’s t test in (F–H).

Figure 3

CD45⁺ cell populations in the atherosclerotic aorta of CD40^wt and CD40^mac−/− mice assessed by CyTOF analysis. Seven CD45⁺ leukocyte populations were identified by viSNE analysis: CD11b⁺ myeloid cells, Ly6G/C⁺ neutrophils, SiglecF⁺ eosinophils, CD103⁺ type 1 conventional dendritic cells (cDC1s), CD19⁺ B-cells, CD90.2⁺ T-cells and CD161⁺ natural killer (NK) cells (A; composite of all cells measured), with cell composition in CD40^mac−/− compared to CD40^wt mice shown in (B). N = 12/13, respectively for CD40^wt and CD40^mac−/− mice, with each sample representing two pooled aortas resulting in a final n = 6 samples. Statistical analyses were performed using a multiple t-test with P-values adjusted according to discoveries determined using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (Q = 1%).

Figure 4

Increased M2 macrophage ratio in CD40-deficient atherosclerotic aortas. Within the CD45⁺CD11b⁺ myeloid cell population in the atherosclerotic aorta eight sub-populations were identified (after neutrophil and eosin populations were subtracted): CD206⁺CD209b⁻ and CD206⁺CD209b⁺ macrophages, CCR2⁺ macrophages, CD11c⁺ macrophages, CD26^- and CD26⁺ type 2 conventional dendritic cells (cDC2) and Ly6C⁻ and Ly6C⁺ monocytes (A; composite of all cells measured) with cell composition in CD40^mac−/− compared to CD40^wt mice shown in (B). N = 12/13, respectively, for CD40^wt and CD40^mac−/− mice, with each sample representing two pooled aortas resulting in a final n = 6 samples. Statistical analyses were performed using a multiple t-test with P-values adjusted according to discoveries determined using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (Q = 1%).

Figure 5

Macrophage genes and pathways affected by CD40-depletion identified by RNA-sequencing. Volcano plot showing CD40^wt versus CD40^mac−/− BMDMs (stimulated with FGK45 and IFNγ (A). Top affected functions in CD40^mac−/− BMDMs were identified by IPA (B). Levels of IFNγ, IL-17A, IL-18, CXCL2 and CXCL10 were decreased in CD40^mac−/− compared to CD40^wt BMDMs (following a stimulation pulse by lipopolysaccharide/IFNγ) as analysed by Luminex (C). Black dashed lines represent cut-off values (adjusted P-value = 0.05, Log₂ fold change = 1). Red dashed lines represent an adjusted P-value = 0.05. N = 3 technical replicates of cells isolated from n = 3 mice. Statistical tests were performed using the Mann–Whitney U test in (C), with data shown as mean ± SD.

Figure 6

Gene expression resulting from CD40-triggering in native and oxLDL-loaded BMDMs. Gene expression levels relating to classical and alternative macrophage activation are shown in (A), and the main up-regulated pathways identified by IPA pathways analysis are shown in (B). N = 3 technical replicates. Grey boxes represent genes with non-significant z-scores or genes lacking sufficient information to produce z-scores.

Figure 7

Genes and pathways of the aorta affected by myeloid-specific CD40-depletion identified by RNA-sequencing. Volcano plot showing CD40^mac−/− versus CD40^wt aortas (A). Differentially regulated pathways in CD40^mac−/− and CD40^wt aortas were identified by GSEA (B). The top 25 main upstream activated/inhibited regulators were identified by IPA pathways analysis (C; red boxes represent –Log₁₀P-values and grey boxes represent activation z-scores). Black dashed lines represent cut-off values (P-value = 0.05, Log₂ fold change = 1). Red dashed lines represent an adjusted P-value = 0.05 and green dashed lines represent an unadjusted P-value = 0.05. N = 5 mice.