Sensitizing function of eRNA on TLR2 activation by Pam2CSK4.

<p>Macrophages were treated for 2 h with different concentrations of Pam2CSK4 in the presence of eRNA (10 μg/ml) or buffer (A, C-F) or a fixed concentration of Pam2CSK4 (0.1 ng/ml) or buffer in the presence of eRNA, tRNA, or eDNA (each 10 μg/ml) (B). Prior to cell stimulation, nucleic acids and Pam2CSK4 were preincubated for 1 h at 37°C. Cell supernatants were analyzed for the release of TNF-α by ELISA. The mRNA expression of TNF-α (C), IL-6 (D), IL1β (E), and MCP-1 (F) was assessed from macrophage lysates by qRT-PCR. Values are expressed as mean ± SEM; N = 3–12; *P < 0.05 between groups.</p

Time-dependent cytokine expression from macrophages treated with eRNA/Pam2CSK4.

<p>Following preincubation of eRNA (10 μg/ml) and Pam2CSK4 (0.1 ng/ml) for 1 h at 37°C, macrophages were treated with these combined agonists (eRNA+Pam2CSK4), eRNA, Pam, or buffer for different time periods as indicated. Supernatants of cells were analyzed for the release of TNF-α (A) and IL-6 (B) by ELISA. mRNA expression of TNF-α (C), IL-6 (D), IL-1β (E), and MCP-1 (F) was assessed from cell lysates by qRT-PCR. Values are expressed as mean ± SEM; N = 3; *P < 0.05 versus buffer-treated group.</p

Sensitizing function of different concentrations of eRNA on TLR2 activation.

<p>Macrophages were treated for 2 h with different concentrations of eRNA in the presence of Pam2CSK4 (0.1 ng/ml) or buffer. Prior to cell stimulation, eRNA and Pam2CSK4 were preincubated for 1 h at 37°C. Supernatants of cells were analyzed for the release of TNF-α by ELISA (A). mRNA expression of TNF-α (B), IL-6 (C), IL1β (D), and MCP-1 (E), was assessed from cell lysates by qRT-PCR. Values are expressed as mean ± SEM; N = 3; *P < 0.05 between groups.</p

Intracellular signaling involved in the synergistic eRNA/Pam2CSK4-mediated TLR2 activation.

<p>Macrophages were pretreated with buffer, neutralizing antibodies against TLR2 (1 μg/ml), TAPI (10 μg/ml), Bay (100 μM), PD98059 (20 μM), or SB203580 (10 μM) for 30 min, and stimulated for 2 h with eRNA (10 μg/ml)/Pam2CSK4 (Pam, 0.1 ng/ml), which were preincubated for 1 h at 37°C. Supernatants were analyzed for the release of TNF-α by ELISA (A). Cell lysates were used for the quantification of mRNA expression of TNF-α (B), IL-6 (C), IL-1β (D), and MCP-1 (E) by qRT-PCR. TNF-α release and mRNA expression induced by eRNA/Pam in the absence of additives were set to 100%. Values are expressed as mean ± SEM; N = 3; *P < 0.05 versus values from eRNA/Pam-treated cells in the absence of additives.</p

Sensitizing function of eRNA on TLR2 activation by TLR2-agonists FSL-1 and Pam3CSK4.

<p>Macrophages were treated for 2 h with different concentrations of FSL-1 (A,C) or Pam3CSK4 (B,D) in the presence of eRNA (10 μg/ml) or buffer. Prior to cell stimulation, eRNA was preincubated with FSL-1 or Pam3CSK4 for 1 h at 37°C. Supernatants of cells were analyzed for the release of TNF-α by ELISA (A,B) and mRNA expression of TNF-α was determined from cell lysates by qRT-PCR (C,D). Values are expressed as mean ± SEM; N = 3–8; *P < 0.05 between groups.</p

Sham Surgery and Inter-Individual Heterogeneity Are Major Determinants of Monocyte Subset Kinetics in a Mouse Model of Myocardial Infarction

<div><p>Aims</p><p>Mouse models of myocardial infarction (MI) are commonly used to explore the pathophysiological role of the monocytic response in myocardial injury and to develop translational strategies. However, no study thus far has examined the potential impact of inter-individual variability and sham surgical procedures on monocyte subset kinetics after experimental MI in mice. Our goal was to investigate determinants of systemic myeloid cell subset shifts in C57BL/6 mice following MI by developing a protocol for sequential extensive flow cytometry (FCM).</p><p>Methods and Results</p><p>Following cross-sectional multiplex FCM analysis we provide for the first time a detailed description of absolute quantities, relative subset composition, and biological variability of circulating classical, intermediate, and non-classical monocyte subsets in C57BL/6 mice. By using intra-individual longitudinal measurements after MI induction, a time course of classical and non-classical monocytosis was recorded. This approach disclosed a significant reduction of monocyte subset dispersion across all investigated time points following MI. We found that in the current invasive model of chronic MI the global pattern of systemic monocyte kinetics is mainly determined by a nonspecific inflammatory response to sham surgery and not by the extent of myocardial injury.</p><p>Conclusions</p><p>Application of sequential multiplexed FCM may help to reduce the impact of biological variability in C57BL/6 mice. Furthermore, the confounding influence of sham surgical procedures should always be considered when measuring monocyte subset kinetics in a murine model of MI.</p></div

Inter-individual variability of circulating leukocyte subsets in C57BL/6 mice.

<p>(<b>A</b>) Cross-sectional cell frequency analysis of 180 gender- and age-matched wild-type (WT) mice. Box-and-whiskers plots of the absolute cell numbers. The box shows the 25^th to 75^th percentiles, and the line in the box indicates the median value. Horizontal bars outside the box indicate 10^th to 90^th percentiles and the circles indicate 1^st to 99^th percentiles. (CV – standard deviation/mean). (<b>B</b>) Frequencies of main monocyte subsets in WT mice as based on Ly6C/CD43 classification are displayed. (<b>C</b>) Occurrence of monocytosis in WT animals depends on shifts towards classical (Ly6C^hiCD43^low) phenotype. (<b>D</b>) Analysis of MHC-II-pos compartment reveals predominance of “non-classical” (Ly6C^lowCD43^high) and “intermediate” phenotype. (<b>D</b>) Mean fluorescence intensity (MFI) for F4/80 in the major monocyte subsets in WT mice.</p

Polychromatic FCM is adaptable for low-volume measurements in mice.

<p>(<b>A</b>) Basic FCM gating strategy and phenotypic definitions for granulocyte and monocyte subsets as used in the current study. Monocytes (Lin<b>⁻</b>CD11b<b>⁻</b>Iab<b>⁻</b>) and MHC(Iab)⁺ cells were separated into main subsets based on the Ly6C/CD43 expression properties. (<b>B</b>) FCM immunobead-based assay for rapid leukocyte quantification in mice. (<b>C</b>) Comparison of assay-standardized (50 µl) and volume-reduced (20 µl) blood samples (n = 10 mice) show high correlation across the entire cell frequency range for all measured myeloid subsets.</p

Sham surgery determines monocyte subset kinetics in a mouse model of MI.

<p>(<b>A</b>) Flow chart of the time-course study. (<b>B</b>) Mean ejection fraction (EF) and end-diastolic volume (EDV) obtained by sequential magnetic resonance imaging (MRI) in C57BL/6 mice (n = 15). Significance between time points was calculated by one-way ANOVA with Tukeys' post-hoc test: * p<0.05, ** p<0.01, *** p<0.001. (<b>C</b>) Sham-operated (n = 7) and MI mice displayed a similar intra-individual time course of circulating monocyte subsets. Calculated individual cell-delta data were used to display the intra-individual leukocyte subset kinetics and differences between MI, sham-operated and control (n = 4) groups. Significance between groups calculated by two-way ANOVA with Benferroni post-hoc test (MI vs. SHAM, ns - not significant). (<b>D</b>) Subset composition changes within the circulating MHCII^neg monocyte compartment following MI. (<b>E</b>) Example of MRI analysis with a short axis of hearts with mild and severe MI. EDV: end-diastolic volume, ESV: end-systolic volume. (<b>F</b>) Selection of mild versus severe MI based on the chronic impairment of the left ventricle ejection fraction (LVEF<35%) and increased LV dilatation (EDV) at day 21. (<b>G</b>) Monocyte time-course kinetics for both MI groups shows no correlation between development of blood monocytosis and the extent of myocardial injury.</p

Sequential FCM may reduce the impact of inter-individual variability.

<p>Time course of the coefficient of variation for monocyte subset absolute cell numbers following MI within (<b>A</b>) inter-group (independently operated mice, n = 18–23/group) and (<b>B</b>) intra-group (sequential analysis, n = 8) experimental setups.</p