The 2'-O-methylation status of a single guanosine controls transfer RNA-mediated Toll-like receptor 7 activation or inhibition

Abstract

Foreign RNA serves as pathogen-associated molecular pattern (PAMP) and is a potent immune stimulator for innate immune receptors. However, the role of single bacterial RNA species in immune activation has not been characterized in detail. We analyzed the immunostimulatory potential of transfer RNA (tRNA) from different bacteria. Interestingly, bacterial tRNA induced type I interferon (IFN) and inflammatory cytokines in mouse dendritic cells (DCs) and human peripheral blood mononuclear cells (PBMCs). Cytokine production was TLR7 dependent because TLR7-deficient mouse DCs did not respond and TLR7 inhibitory oligonucleotides inhibited tRNA-mediated activation. However, not all bacterial tRNA induced IFN-α because tRNA from Escherichia coli Nissle 1917 and Thermus thermophilus were non-immunostimulatory. Of note, tRNA from an E. coli knockout strain for tRNA (Gm18)-2'-O-methyltransferase (trmH) regained immunostimulatory potential. Additionally, in vitro methylation of this immunostimulatory Gm18-negative tRNA with recombinant trmH from T. thermophilus abolished its IFN-α inducing potential. More importantly, Gm18-modified tRNA acted as TLR7 antagonist and blocked IFN-α induction of influenza A virus-infected PBMCs.

Figure 1.

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).

Figure 2.

TLR7 mediates tRNA-induced immunostimulation. (A) WT and TLR7-deficient FLT3L-induced DCs were stimulated with 1 µg/ml RNA40 and 2 µg/ml tRNA from A. lwoffii complexed to DOTAP. The TLR9 ligand CpG-ODN2216 (1 µM) served as positive control. (B) Mouse FLT3L-induced DCs (gray bars) or human PBMCs (black bars) were incubated with 1 µg/ml RNA40 or 2 µg/ml tRNA from A. lwoffii and increasing amounts of the TLR7 inhibitory oligodeoxynucleotide IRS661 (1, 0.2, and 0.04 µM). Cells were incubated for 20 h with subsequent IFN-α detection by ELISA. For all panels, one representative experiment of at least two independent experiments is shown (n = 2 ± SD).

Figure 3.

tRNA from different bacterial species varies in their IFN-α–inducing potential. (A) Purified tRNA from different bacterial species were visualized on a 12% polyacrylamide gel (lane 1: B. subtilis; lane 2: P. aeruginosa; lane 3: T. thermophilus; lane 4: E. coli Nissle 1917; lane 5: L. lactis; lane 6: M. marburgensis). One representative gel is shown (n > 3). (B) Human PBMCs were stimulated with 2 µg/ml of purified tRNA from different bacterial species as indicated and IFN-α production is depicted as percentage of activity of the TLR7 agonist RNA40. Experimental data were obtained from PBMCs preparations of 3–10 different donors per strain. Statistical analysis was performed using the paired Student’s t test (***, P < 0.0001; *, P = 0.02).

Figure 4.

2′-O-methylation at tRNA position G18 is responsible for non-immunostimulatory character of E. coli Nissle 1917. (A) Human PBMCs were stimulated with 2, 0.4 and 0.1 µg/ml of purified tRNA from E. coli BW25113– and E. coli BW25113–derived knockout mutants for trmA (uracil-5-methyltransferase, m5U54), trmB (guanine-7-methyltransferase, m7G46), and trmH (Gm18-2′-O-methyltransferase). PBMCs were incubated with RNA for 20 h with subsequent IFN-α detection by ELISA. Representative experimental data from one out of four PBMC preparations are shown (n = 2 ± SD). (B) HPLC analysis of P1 nuclease and phosphatase-treated tRNA from E. coli BW25113, E. coli ΔtrmH, and E. coli ΔtrmH methylated in vitro with T. thermophilus trmH. (C) SDS-PAGE of purified recombinant T. thermophilus trmH protein expressed in E. coli BL21. (D) Purified tRNA from E. coli ΔtrmH was incubated with ¹⁴C-labeled SAM and with or without T. thermophilus trmH. tRNA was further visualized after PAGE by ethydium bromide (EtBr) staining and autoradiography (AR). For B–D, one representative experiment (n ≥ 2) is shown. (E) Human PBMCs were stimulated with purified tRNA (2 µg/ml) from A. lwoffii, T. thermophilus, E. coli ΔtrmH, and E. coli ΔtrmH methylated in vitro with recombinant trmH and T. thermophilus. PBMCs were incubated with RNA for 20 h with subsequent IFN-α detection by ELISA. (F) Human enriched monocytes were stimulated with 100 ng/ml LPS, 1 µM CpG2216, 1 µg/ml RNA40, nuclease P1, and 2 µg/ml tRNA from T. thermophilus and A. lwoffii ± nuclease P1 digestion. After incubation for 20 h, IL-6 production was measured by ELISA. Representative experimental data from one out of four PBMCs (E) or four monocyte (F) preparations are shown (n = 2 ± SD).

Figure 5.

Gm18-modified tRNA acts as antagonist for TLR7-mediated IFN-α production upon tRNA or ssRNA stimulation and viral infection. (A) Mouse FLT3L-induced DCs were stimulated with 2 µg/ml tRNA from A. lwoffii and various concentrations of tRNA from E. coli Nissle or T. thermophilus (2, 1.5, 1, 0.5, 0.2, 0.1, 0.05, 0.025, and 0.0125 µg/ml). (B) Human PBMCs were stimulated with 1 µg/ml RNA40 and various concentrations of tRNA from T. thermophilus or A. lwoffii (2, 0.5, 0.125, and 0.0313 µg/ml). (C) Human PBMCs were infected with various MOI (10, 1, and 0.1) of influenza virus A/PR/8/34 (IAV) and 2 µg/ml purified tRNA from T. thermophilus. For each stimulation, tRNA was complexed to DOTAP and incubated for 20 h with subsequent IFN-α detection by ELISA. For A–C, representative experimental data from one out of four responsive PBMCs or two mouse cell preparations are shown (n = 2 ± SD).