Prevention of CASP induced aortic cytokine and iNOS production following TLR9 antagonist treatment.

<p><b>A-E</b> 24 h of exposure to the synthetic TLR9 antagonist H154-thioate did not induce mRNA expression of the investigated cytokines, but prevented the CASP-dependent rise in mRNA expression of inflammatory mediators. There was no observable influence of H154-thioate on iNOS expression. <b>F</b> H154-thioate treatment prior to CASP completely prevented the CASP-induced arterial hypocontractility observed in WT animals (F) (*p<0.05; **p<0.01; ***p<0.001; n≥5 animals in each group; mean ± SEM).</p

Influence of TLR2 agonism on aortic contractility.

<p>LTA application to WT mice increased contraction force at 10⁻⁷ M phenylephrine. Results were standardized to the maximum contraction force, which was not different between both groups (*p<0.05; n≥5 animals in each group; mean ± SEM).</p

CASP induced aortic cytokine and iNOS production.

<p><b>A</b> Pro-inflammatory TNF-α was significantly up-regulated in WT mice compared to sham animals. CASP-surgery in TLR2-D animals lead to even higher levels of TNF-α compared to all other groups. <b>B, C</b> IL-1β and IL-6 were significantly up-regulated in TLR2-D animals after CASP compared to the other strains. <b>D</b> CASP induced a non-significant up-regulation of the anti-inflammatory cytokine IL-10 in WT, TLR2-D and CD14-D mice. <b>E</b> CASP induced a significant up-regulation of iNOS in TLR2-D mice only. (*p<0.05; **p<0.01; ***p<0.001; n≥5 animals in each group; mean ± SEM).</p

Arterial contractility 18 hours after CASP.

<p><b>A</b> A significant hypocontractility of aortic rings in CASP- WT animals, but not in sham-operated WT animals was observed at phenylephrine concentrations of 10^−7,5 to 10⁻⁵M. <b>B</b> TLR2-D mice exhibited a significantly lower maximal contractile response compared to WT animals (TLR2-D 19.07±0.454 mN vs. WT 23.59±0.872 mN; p<0.05). <b>C</b> Significant reduction of vascular contractility following CASP was also observed in TLR4-D mice compared to controls (p<0.05), but it was less severe than in WT animals. <b>D, E</b> CASP did not influence vascular contractility in TLR9-D and CD14-D mice, however CD14-D mice had a significantly higher EC50 of phenylephrine (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044531#pone.0044531.s001" target="_blank">Table S1</a>). <b>F</b> Standardized maximal loss of vascular contractility. In WT mice sham surgery led to a 17% decrease of vascular contractility, compared to 45% after CASP-treatment. TLR2-D animals lost 70% of their maximum contractile response, whereas CASP-treated TLR4-D mice lost only 27% and CD14-D as well as TLR9-D mice were insensitive to CASP (*p<0.05; n≥5 animals in each group; mean ± SEM).</p

Effects of CASP and expression of PRR expression in the murine vessel wall.

<p>PRR mRNA expression during control conditions and following CASP-surgery (TLR2 (A), TLR4 (B), TLR9 (C), CD14 (D)). TLR2-D mice showed significantly higher baseline levels of TLR4 (B), TLR9 (C) and CD14 (D) compared to the other groups. All PRRs appeared to be regulated after CASP; however, only TLR2 and CD14 mRNA expression reached the level of significance (A, D; *p<0.05; **p<0.01; ***p<0.001; n≥5 animals in each group; mean ± SEM).</p

Analysis of adhesion molecules.

<p>(A–C) The mRNA-expression levels of adhesion molecules were quantified in the LV tissue of TAC- and sham- operated animals. The expression of the respective mRNA was normalized to healthy baseline controls. Mean ±SEM; n: 3d TAC  = 6 mice, 3d Sham  = 3 mice, 6d TAC  = 6 mice, 6d Sham  = 3 mice. (D–I) Flow cytometric delineation of the Mean Fluorescence Intensity (MFI) of CD11b (D, G), CX3CR1 (E, H) and CD31 (F, I) on the surface of cardiac Ly6C^low (E–G) and Ly6C^high (H–J) macrophages. Mean ±SEM; n: 3d TAC  = 5 mice, 3d Sham  = 3 mice, 6d TAC  = 8 mice, 6d Sham  = 3 mice. *P<0.05.</p

Cardiac phagocytes.

<p>(A) Gating strategy to determine the subpopulations of Ly6C^low (black ellipse) and Ly6C^high macrophages (black dashed ellipse) in the cardiac tissue via F4/80 and Ly6C staining, previously gating all immune cells; concatenated plots of 3 healthy baseline mice. (B) Pie graphs representing the relative proportion of Ly6C^low and Ly6C^high macrophages in relation to the whole population of cardiac macrophages. Graphs were drawn with Microsoft Excel 2010; results are shown as mean, n: Baseline  = 10 mice, 3d TAC  = 4 mice, 6d TAC  = 4 mice, 21d TAC  = 4 mice. (C, D) Flow cytometric quantification of Ly6C^high and Ly6C^low macrophages. (E) Flow cytometric quantification of the respective immune cell/macrophage ratio. (C, D, E) Mean ±SEM; n: Baseline  = 10 mice, 3d TAC  = 5 mice, 3d Sham  = 4 mice, 6d TAC  = 8 mice, 6d Sham  = 4 mice, 21d TAC  = 6 mice, 21d Sham  = 7 mice. *P<0.05, **P<0.01, ***P<0.001.</p

Macrophage localization in the heart determined by immunostaining.

<p>(A) Representative cardiac sections of CX3CR1^GFP/+ mice 6 d after TAC, exhibiting macrophages in green, CD31 visualized in red by antibody staining. (A–C) Overwiew; * indicates the epicardial, + the mid-myocardial and # the endocardial regions; LV marks the left ventricle and RV the right ventricle. The arrow/BV marks one exemplary cardiac blood vessel. Scale bar: 300 µm. (D–F) Zoom in on the mid-myocardial and endocardial regions of the LV at the border to the RV. Scale bar: 80 µm; (G–I) Zoom in on the endocardial border zone of the LV and RV. Scale bar: 40 µm. (J–L) Zoom in on cardiac blood vessels in the mid-myocardial region of the LV, arrows indicate neighbouring macrophages. Scale bar: 35 µm.</p

Macrophage adhesion in pressure overload.

<p>(A) Gating strategy to determine the endothelial cells among all single cells in the heart via CD45 and CD31 staining (black ellipse), previously gating all single cells; concatenated plots of 5 healthy baseline mice. (B, C) Flow cytometric delineation of CD31- and ICAM-1-MFI of cardiac endothelial cells. Mean ±SEM; n: 3d TAC  = 5 mice, 3d Sham  = 3 mice, 6d TAC  = 8 mice, 6d Sham  = 3 mice *P<0.05, **P<0.01. (D–L) Representative cardiac sections of CX3CR1GFP/+ mice 6 d after TAC. Monocytes/macrophages marked by GFP fluorescence in green, CD31 visualized in red by antibody staining. Arrows indicate monocytes/macrophages at different steps of recruitment: adhering to the intravascular endothelium (D–F), migrating through the endothelium (G–I) and directly neighboring the blood vessels (J–L). Scale bars: 30 µm (D–L).</p

LTA affected the hemodynamic function of TAC operated wild type mice.

<p>3 d of pressure overload increased the left ventricular systolic pressure (A) and mean arterial blood pressure (B) of wild type mice (p<0.001, mean ± SEM, n = 7-13/group). LTA did not cause cardiac depression 6 h after stimulation in sham mice. In TAC pre-treated mice, LTA significantly reduced left ventricular and mean arterial blood pressure (*p<0.05, ***p<0.01). (C) TAC and LTA had no prominent influence on heart rate.</p