Fig 1.
Sensitizing function of eRNA on TLR2 activation by Pam2CSK4.
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.
Fig 2.
Sensitizing function of different concentrations of eRNA on TLR2 activation.
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.
Fig 3.
Sensitizing function of eRNA on TLR2 activation by TLR2-agonists FSL-1 and Pam3CSK4.
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.
Fig 4.
Time-dependent cytokine expression from macrophages treated with eRNA/Pam2CSK4.
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.
Fig 5.
Intracellular signaling involved in the synergistic eRNA/Pam2CSK4-mediated TLR2 activation.
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.
Fig 6.
Influence of eRNA on the activation of TLRs.
Macrophages were treated for 2 h with different concentrations of LPS (A), R848 (B), or poly IC (C) in the presence of eRNA (10 μg/ml) or buffer. Prior to cell stimulation, eRNA was preincubated with each agonist for 1 h at 37°C and supernatants of cells were analyzed for the release of TNF-α by ELISA (A). mRNA expression of TNF-α (B), IL-6 (C), IL-1β (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.