Figure 1.

Methanosphaera stadtmanae and its RNA inducing an immune response with antiviral characteristics. (A,B) The expression of genes encoding for IFN-α14, IFN-β, and IFN-λ1 in moDCs (A) and PBMCs (B) after stimulation with M. stadtmanae for 3, 6, and 12 h was measured by qRT-PCR. The log2 ratios of all three genes to the reference gene HPRT are presented. The data from at least three different donors are shown as the mean ± SEM (n = 3–4). ns, not significant; *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (all compared with unstimulated control group; repeated measures one-way ANOVA with Dunnett’s post hoc test). (C) Confocal microscopy of cellular location of NF-κB p65, IRF1, and IRF5 (green) in moDCs after stimulation with M. stadtmanae for 4 h by immunolabelling. Nuclei were counterstained with Hoechst 33342 (blue). Scale bars: 10 µM. The images shown are representative examples from one of three independent experiments (n = 3). (D,E) ELISA of TNF-α and IL-1β in the supernatants of stimulated moDCs (D) or PBMCs (E) after 18 h. Cells were either untreated, treated with 10⁷ cells of M. stadtmanae, or with 5 µg/mL of total RNA from M. stadtmanae. RNA was complexed to DOTAP and pre-treated for 30 min at 37°C with RNase A where indicated. (F,G) ELISA of TNF-α and IL-1β in the supernatants of moDCs (F) or PBMCs (G) stimulated for 18 h with 10⁷ cells of M. stadtmanae or 2.5 µg/mL of purified rRNAs (complexed to DOTAP). In (D–G), the data shown are the mean ± SEM of at least four different donors (n = 4–7). ns, not significant; *P ≤ 0.05 and **P ≤ 0.01 (one-way ANOVA with Tukey post hoc test; in (D,E) all compared with unstimulated control group and in (F,G) the rRNA fractions are compared with each other). moDCs, monocyte-derived dendritic cells; PBMCs, peripheral blood mononuclear cells; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; ANOVA, analysis of variance; TNF-α, tumor necrosis factor-alpha; IL-1β, interleukin-1 beta.