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. 2018 Oct 24;201(11):3392–3400. doi: 10.4049/jimmunol.1801008

FIGURE 4.

FIGURE 4.

Anti-miRs but not nucleases partially reduce CLP EV–induced cytokine production. (A) Anti-miR treatment of EVs partially attenuated the CLP EV–induced cytokine production. Prior to applying to BMDM cultures, sham EVs were treated with PBS or control oligonucleotide (Oligo) at 300 nM. CLP EVs (20 μg/ml) were incubated with PBS, control Oligos (300 nM), single anti-miR (anti–miR-146a, anti–miR-34a, and anti–mir-122, at 100 nM), or anti-miR combination at 300 nM, including anti–miR-146a, anti–miR-34a, and anti–miR-122 for 1 h. Cell culture media were collected 16 h after treatment and assayed for MIP-2 production using ELISA. Each bar represents triplicate samples with each experiment repeated twice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001. (B) RNase or Benzonase had no impact on CLP EV–induced MIP-2 production. miR-146a mimic (50 nM), splenic RNA (50 μg/ml), sham EVs (20 μg/ml), and CLP EVs (20 μg/ml), were incubated with PBS, RNase (10 μg/ml), or Benzonase (50 U/ml) for 1 h at room temperature before being applied to BMDM cultures. Sixteen hours later, media were collected for MIP-2 ELISA. Each bar represents triplicate samples with each experiment repeated twice. ***p < 0.001, #p < 0.0001, versus sham EVs. (C) DNase or Benzonase had no impact on CLP EV–induced MIP-2 production. miRNA-146a mimics (50 nM), thymic DNA (10 μg/ml), sham EVs (20 μg/ml), and CLP EVs (20 μg/ml) were incubated with PBS, DNase (50 U/ml), or Benzonase (50 U/ml) for 1 h at room temperature before being applied to BMDM. Sixteen hours later, medium MIP-2 was analyzed by ELISA. Each bar represents triplicate samples with each experiment repeated twice. ***p < 0.001, #p < 0.0001, versus sham EVs.