Bacterial tRNA induces cytokine production in human and mouse immune cells. (A) Bacterial total RNA and purified tRNA were analyzed on a 1% agarose gel and detected with ethidium bromide. One representative gel is shown (n > 10). (B and C) Human PBMCs (black bars; B) and WT mouse FLT3L induced DCs (gray bars; C) were stimulated with various concentrations of purified tRNA from S. aureus and A. lwoffii (10, 2, 0.4, and 0.1 µg/ml) or with 10 µg/ml tRNA digested with P1 nuclease (B). For each stimulation, tRNA was complexed to DOTAP and incubated with immune cells for 20 h with subsequent IFN-α detection by ELISA. (D) TLR2/4 double-deficient DCs (gray bars) or enriched human monocytes (black bars) were stimulated with tRNA at 2 µg/ml (±nuclease P1 treatment) and IL-6, IL12p40, and TNF production was analyzed. For B–D, one representative experiment of at least two independent experiments consisting of two PBMCs donors or two individual mice is shown (n = 2 ± SD).