Extended Data Fig. 1: Atherosclerosis-associated axon neogenesis in ATLOs.
From: Neuroimmune cardiovascular interfaces control atherosclerosis
a, Schematic view of aorta segments. b, Axon density in distinct aorta segments with or without plaque in aged WT and Apoe−/− mice; n = thorax: 361 sections in 3 WT, 121 or 209 sections in 3 Apoe−/− without or with plaque; abdomen: 254 sections in 6 WT, 109 sections in 3 Apoe−/− without plaque, 260 sections in 9 Apoe−/− with plaque. c, Axon density in thoracic (T) and abdominal (A) aorta segments with or without artery branches in non-atherosclerotic aorta segments; n = 61 or 148 sections without or with branch in 3 WT, 30 or 58 sections without or with branch in 3 Apoe−/− without plaque. d, Axon density in WT versus Apoe−/− aorta segments with plaque; n = 12–116 sections in 3 WT, 17–71 sections in 3 Apoe−/−. Abbreviations: AR, aortic root; AA, aortic arch; ICA, intercostal artery; CA, coeliac artery; SMA, superior mesenteric artery; RRA, right renal artery; LRA, left renal artery. e, Pearson correlation coefficient of axon density with plaque size in thorax and abdomen. n = 3 Apoe−/− mice. f, Enumeration of tubulin-β3 (TUBB3)+ immature axons; n = 3 WT, 3 ATLO. g, Detection of synaptophysin (Synpt+) axon endings (arrow) in ATLOs, their accumulation (arrowheads) at ATLO-media border (dashed line) and quantification in WT adventitia versus ATLOs; n = 4 WT, 4 ATLO; video 4. h, High resolution 3D reconstruction showing colocalization of CD68+ macrophages/monocytes, CD11c+ dendritic cells and CD3e+ T cells with NF200+ axons in ATLOs and their distance from adjacent axons (n = 4 ATLOs per cell-type). i, Colocalization of CD3e+ T cells with TH+ axons in ATLOs (n = 3). Arrows indicate interaction sites that are <1 μm apart; video 1. j, Synapsin (Syn)+ axon endings in WT adventitia and ATLO and accumulation of Syn+ puncta at ATLO-media border (arrowheads); video 3. n = 3 WT, 3 Apoe−/−. k,l, Colocalization of Syn+ or Synpt+ axon endings with CD45+ leukocyte forming axon-leukocyte junctions in ATLOs (arrowheads); n = 2; video 2. m, ATLOs lack ChAT+NFM+ parasympathetic axons but harbor ChAT+ leukocytes (arrow); quantification of ChAT+ T- and B- cells in ATLOs versus WT adventitia (n = 3 WT and 3 Apoe−/−) (FOV, field of view). n, Flow cytometry contour plots and quantification of ChAT+ T/B cells in ATLOs, RLNs and spleen (Spl) (n = 3 independent experiments, 2-3 mice per experiment). o-r, ADRβ2 expression in ATLOs. o, Differential gene expression of Adrb subtypes in aged WT versus Apoe−/− adventitia or plaque (n = 6 WT, 4 Apoe−/− no plaque, 4 ATLO, 3 plaque). p, 3D surface rendering of ADRβ2 expression in CD3e+ T cell in ATLO (n = 3). q, Flow cytometry gating strategy for immune cells. r, Flow cytometry contour plots and quantification of ADRβ2-expressing CD4+ T cells, CD4+CD44+CD62L- TEM cells, CD8+ T cells and B220+ B cells in ATLOs versus RLNs and spleen (n = 5 independent experiments, 1-2 mice per experiment). DAPI stains DNA in blue. For g,j, scale bars, 5µm; for h,i,k,l,p, scale bars, 2 µm; for m, scale bar, 50 µm. For b-d, center lines are medians, box limits are upper and lower quartiles and whiskers are 1.5x interquartile range. For f-h,m-o,r, data are means ± s.e.m. n represents biologically independent animals. Generalized estimating equations (b,c,d); two-sided unpaired Student´s t-test (f,g,m); one-way ANOVA with Bonferroni post hoc test (h,n,r); Generalized linear model with Bonferroni post hoc test (o)
