Knock-in of <i>Ikkα^AA/AA</i> in haematopoietic cells does not influence apoptosis in atherosclerotic lesions.

<p>Analysis of aortic root lesions of <i>Apoe^−/−</i> mice transplanted with <i>Ikkα^AA/AAApoe^−/−</i> or <i>Ikkα^+/+Apoe^−/−</i> BM and receiving a high-fat diet for 13 weeks. (<b>A</b>) Quantification of necrotic cores as percentage of plaque area. (B–C) Quantification of apoptotic cells (Tunel⁺, B) and apoptotic macrophages (Tunel⁺Mac2⁺, C) as percentage of all plaque cells. Graphs represent the mean ± SEM (n = 10–12).</p

<i>Ikkα^AA/AAApoe^−/−</i> BM-chimeras have less B-cells, T_reg and effector memory T-cells, and more naive T-cells.

<p>Shown is flow cytometric analysis of peripheral blood, thymus and secondary lymphoid organs of <i>Apoe^−/−</i> mice transplanted with <i>Ikkα^AA/AAApoe^−/−</i> or <i>Ikkα^+/+Apoe^−/−</i> BM and receiving a high-cholesterol diet for 13 weeks. (<b>A</b>) Cd19⁺ B-cell and Cd3⁺ T-cell populations as percentage of Cd45⁺ leukocytes, and Cd4⁺ and Cd8a⁺ T-cell subsets as percentage of Cd3⁺ T-cells in peripheral blood. (<b>B</b>) Cd3⁺Cd4⁺Cd25⁺Foxp3⁺ regulatory T-cell (T_reg) levels as percentage of Cd3⁺ T-cells and Cd45⁺ leukocytes. (<b>C</b>) Cd3⁺Cd44^lowCd62L^high naive T-cells and Cd3⁺Cd44^highCd62L^low effector memory T-cells as percentage of Cd3⁺ T-cells and Cd45⁺ leukocytes. Left Y-axes belong to naive T-cells, right Y-axes to effector memory T-cells. (<b>D</b>) Cd11c⁺MhcII⁺ conventional dendritic cells (cDCs) and Cd11c⁺Cd11b⁻440c⁺ plasmacytoid DCs (pDCs) as percentage of Cd45⁺ leukocytes <i>(left)</i>. Surface expression of MhcII on splenic cDCs and pDCs <i>(right)</i>. (<b>A–D</b>) All graphs represent the mean ± SEM (n = 18–19); 2-tailed t-test, *P<0.05, **P<0.01, ***P<0.001.</p

<i>Ikkα^AA/AA</i> knock-in does not enhance or prolong NF-κB p65 activity, or majorly influence cytokine expression in <i>Apoe^−/−</i> macrophages <i>in vitro</i>.

<p>(A) BM-derived macrophages from <i>Ikkα^AA/AAApoe^−/−</i> and <i>Ikkα^+/+Apoe^−/−</i> mice were stimulated <i>in vitro</i> with 10 ng/ml Tnf-α, 50 µg/ml of mildly oxidized LDL or 100 ng/ml LPS for the indicated time. Activation of p65 was quantified in nuclear extracts using a TransAm p65 assay. Graphs represent the mean ± SEM (n = 2); 2-way ANOVA with Bonferroni post-test, ***P<0.001. (B) BM-derived macrophages from <i>Ikkα^AA/AAApoe^−/−</i> and <i>Ikkα^+/+Apoe^−/−</i> mice were stimulated <i>in vitro</i> for 24 h with 10 ng/ml Tnf-α or 50 µg/ml heavily oxidized LDL. Cytokine concentrations in the supernatants are displayed for Tnf-α, Il-6 and Mcp1. Graphs represent mean ± SEM (n = 9 from 3 independent experiments); 2-way ANOVA with Bonferroni post-test, ***P<0.001. (C) Concentrations of Tnf-α, Mcp1 and Il-6 in serum of <i>Apoe^−/−</i> mice transplanted with <i>Ikkα^AA/AAApoe^−/−</i> or <i>Ikkα^+/+Apoe^−/−</i> BM and receiving a high-fat diet for 8 weeks. Graphs represent the mean ± SEM (n = 7–9).</p

Leukocyte numbers and frequencies of leukocyte subsets in <i>Apoe^−/−</i> mice transplanted with <i>Ikkα^AA/AAApoe^−/−</i> or <i>Ikkα^+/+Apoe^−/−</i> BM.

<p>Peripheral blood leukocyte counts were determined after 8 weeks of high-fat diet (n = 8). Leukocyte subset frequencies were quantified by flow cytometry after 13 weeks of high-fat diet (n = 18–19).</p

<i>Ikkα^AA/AA</i> knock-in does not influence macrophage lipid uptake.

<p>(<b>A</b>) Flow cytometric analysis of Dil-oxLDL uptake by BM-derived macrophages. Cells were incubated without or with Dil-oxLDL (1 µg/ml or 10 µg/ml) for 3 h or 24 h, as indicated. To test for actin-dependent uptake, additional controls were simultaneously treated with cytochalasin D (cytD), as indicated. Representative flow cytometric histograms are shown. Graphs represent the mean ± SEM (n = 3); 2-way ANOVA with Bonferroni post-test. (<b>B–D</b>) Intracellular lipid depositions in aortic root lesions were stained with Nile Red and co-stained with Mac2 in order to quantify lipid-laden macrophages. (B) Nile Red staining was quantified relative to the plaque area (left graph), and Nile Red⁺ cells were quantified as percentage of total plaque cells (right graph). (C) Lipid uptake by macrophages was quantified as Nile Red⁺Mac2⁺ area as % of Mac2⁺ area (left graph), and as Nile Red⁺Mac2⁺ cells as % of Mac2⁺-cells. (D) Shown are representative pictures from Nile Red staining (red fluorescence; left image). The middle and right image demonstrate Image J analyses. Displayed are the plaque area (thin red line), macrophage area (green), plaque cell nuclei (blue), Nile Red⁺ area (yellow) and Mac2⁺ Nile Red⁺ area (white, lipid deposits in macrophages). Graphs represent the mean ± SEM (n = 5–6).</p

Knock-in of <i>Ikkα^AA/AA</i> in haematopoietic cells does not influence advanced atherosclerosis in <i>Apoe^−/−</i> mice.

<p><i>Apoe^−/−</i> mice were transplanted with <i>Ikkα^AA/AAApoe^−/−</i> or <i>Ikkα^+/+Apoe^−/−</i> BM and received a high-cholesterol diet for 13 weeks before analysis. (<b>A,B</b>) Body weight (A) and lipid analysis in blood serum (B) (n = 18). (<b>C</b>) Cholesterol levels after HPLC-based size fractionation of pooled blood serum samples. (<b>D,E</b>) Atherosclerotic lesion sizes in the aorta (D) and aortic root (E) (n = 16–19). Representative pictures of Oil-Red-O⁺ lipid depositions in aorta and aortic root are shown. Scale bar = 500 µm. (<b>F</b>) Classification of aortic root lesions according to their severity. Shown is the plaque distribution as percentage of the total number of lesions examined (n = 18–27). (A,B,D,E) Graphs represent the mean ± SEM; 2-tailed t-test, ***P<0.001.</p

<i>Ikkα^AA/AA</i> knock-in in haematopoietic cells does not influence earlier stages of atherogenesis in <i>Apoe^−/−</i> mice.

<p><i>Apoe^−/−</i> mice were transplanted with <i>Ikkα^AA/AAApoe^−/−</i> or <i>Ikkα^+/+Apoe^−/−</i> BM and received a high-cholesterol diet for 8 weeks before analysis. (<b>A,B</b>) Body weight (A) and lipid analysis in blood serum (B) (n = 7–9). (<b>C</b>) Cholesterol levels after HPLC-based size fractionation of pooled blood serum samples. (<b>D,E</b>) Atherosclerotic lesion sizes in the aorta (D) and aortic root (E). Representative pictures of Oil-Red-O⁺ lipid depositions in aorta and aortic root are shown. Scale bar = 500 µm. (<b>F</b>) Classification of aortic root lesions according to their severity. Shown is the plaque distribution as percentage of the total number of lesions examined (n = 22–26). (A,B,D,E) Graphs represent the mean ± SEM.</p

Knock-in of <i>Ikkα^AA/AA</i> in haematopoietic cells does not influence the cellular composition of atherosclerotic lesions.

<p>Immunofluorescent stainings of aortic roots from <i>Apoe^−/−</i> mice reconstituted with <i>Ikkα^AA/AAApoe</i>^−/− or <i>Ikkα^+/+Apoe</i>^−/− BM and receiving a high-fat diet for 13 weeks. (<b>A–C</b>) Quantification of Mac2⁺ macrophages (A, n = 17–18), Sma⁺ SMCs (B, n = 16–17) and Cd3⁺ T-cells (C, n = 7–8) as percentage of all plaque cells <i>(left)</i> and relative to the plaque area <i>(right)</i>. Graphs represent the mean ± SEM. Representative pictures are shown. Scale bar = 100 µm.</p