Fig. 7: CLEC4A2 protects from atherosclerosis by promoting homeostatic functions in macrophages.

a CD45⁺CD11b⁺ cells from whole aortas of ApoE^−/− and ApoE^−/− Clec4a2^−/− mice fed a HFD for 12 weeks were analysed by scRNA-seq using 10X genomics platform. Aortic cells were pooled from 9 mice per genotype group. ivis clustering of myeloid cells pooled from 18 mice showed 13 clusters. b Proportion of 12 myeloid clusters in each genotype. c Volcano plot comparing gene expression (P < 0.05) in ApoE^−/− Clec4a2^−/− Cluster 1 (resident macrophages) vs. ApoE^−/− Cluster 1. Differential expression was assessed using the two-sided Robinson and Smyth exact test with Bonferroni’s correction for multiple-hypothesis testing. d and e IL-6 production by CSF1-BMDMs from WT and Clec4a2^−/− mice in response to FSL-1 (a ligand for TLR2) or LPS (a ligand for TLR4) treatment for 24 h, n = 4 mice each; the data are representative of four independent experiments. Two-tailed Student’s t-test. *P = 0.0224. ***P = 0.0001. f and g Representative images of oxidised low-density lipoprotein (oxLDL) uptake in CSF1-BMDMs from WT and Clec4a2^−/− mice in 24 h and percentages of oxLDL-laden BMDMs (Scale bar: 10 µm), n = 3 mice each; the data are representative of three independent experiments. Two-tailed Student’s t-test. *P = 0.0331. h The efficiency of reverse cholesterol efflux in oxLDL-laden BMDMs from WT and Clec4a2^−/− mice. n = 5-6 mice each; the data are representative of three independent experiments. Two-tailed Student’s t-test. **P = 0.0055. i Gene expression of cholesterol efflux genes (Abca1 and Abga1) in WT and Clec4a2^−/− BMDMs treated with oxLDL for 24 h. The expression levels were normalised against Actb. n = 4 mice each; the data are representative of two independent experiments. Two-tailed Student’s t-test. Abca1: **P = 0.0061, Abga1: **P = 0.0065. j The effect of SHP1 inhibition on oxLDL uptake in CSF1-BMDMs from WT and Clec4a2^−/− mice, n = 4 mice each, one experiment. Ordinary one-way ANOVA with Holm–Sidak’s multiple-comparison test. UT (WT) vs. inhibitor (WT): *P = 0.0185; UT (WT) vs. UT (Clec4a2^−/−): *P = 0.0388. k Gene expression of abca1 and abga1 in WT and Clec4a2^−/− BMDMs treated with oxLDL in the absence or presence of SHP1 inhibitor for 48 h. The expression levels were normalised against Actb (n = 4 mice each; one experiment). Ordinary one-way ANOVA with Holm–Sidak’s multiple-comparison test. Abca1: UT (WT) vs. inhibitor (WT): *P = 0.0314, UT (WT) vs. UT (Clec4a2^−/−): *P = 0.0171, UT (WT) vs. inhibitor (Clec4a2^−/−): **P = 0.0038. Abga1: UT (WT) vs. inhibitor (WT): *P = 0.0421, UT (WT) vs. UT (Clec4a2^−/−): *P = 0.0421, UT (WT) vs. inhibitor (Clec4a2^−/−): *P = 0.0421. All data are presented as mean ± SEM. Source data are provided as a Source Data file.