Bacterial RNA sensing by TLR8 requires RNase 6 processing and is inhibited by RNA 2'O-methylation.

TLR8 senses single-stranded RNA (ssRNA) fragments, processed via cleavage by ribonuclease (RNase) T2 and RNase A family members. Processing by these RNases releases uridines and purine-terminated residues resulting in TLR8 activation. Monocytes show high expression of RNase 6, yet this RNase has not been analyzed for its physiological contribution to the recognition of bacterial RNA by TLR8. Here, we show a role for RNase 6 in TLR8 activation. BLaER1 cells, transdifferentiated into monocyte-like cells, as well as primary monocytes deficient for RNASE6 show a dampened TLR8-dependent response upon stimulation with isolated bacterial RNA (bRNA) and also upon infection with live bacteria. Pretreatment of bacterial RNA with recombinant RNase 6 generates fragments that induce TLR8 stimulation in RNase 6 knockout cells. 2'O-RNA methyl modification, when introduced at the first uridine in the UA dinucleotide, impairs processing by RNase 6 and dampens TLR8 stimulation. In summary, our data show that RNase 6 processes bacterial RNA and generates uridine-terminated breakdown products that activate TLR8.

Quantitative and time-resolved miRNA pattern of early human T cell activation.

T cells are central to the immune response against various pathogens and cancer cells. Complex networks of transcriptional and post-transcriptional regulators, including microRNAs (miRNAs), coordinate the T cell activation process. Available miRNA datasets, however, do not sufficiently dissolve the dynamic changes of miRNA controlled networks upon T cell activation. Here, we established a quantitative and time-resolved expression pattern for the entire miRNome over a period of 24 h upon human T-cell activation. Based on our time-resolved datasets, we identified central miRNAs and specified common miRNA expression profiles. We found the most prominent quantitative expression changes for miR-155-5p with a range from initially 40 molecules/cell to 1600 molecules/cell upon T-cell activation. We established a comprehensive dynamic regulatory network of both the up- and downstream regulation of miR-155. Upstream, we highlight IRF4 and its complexes with SPI1 and BATF as central for the transcriptional regulation of miR-155. Downstream of miR-155-5p, we verified 17 of its target genes by the time-resolved data recorded after T cell activation. Our data provide comprehensive insights into the range of stimulus induced miRNA abundance changes and lay the ground to identify efficient points of intervention for modifying the T cell response.