RESUMO
Human toll-like receptor 8 (TLR8) activation induces a potent T helper-1 (Th1) cell response critical for defense against intracellular pathogens, including protozoa. The receptor harbors two distinct binding sites, uridine and di- and/or trinucleotides, but the RNases upstream of TLR8 remain poorly characterized. We identified two endolysosomal endoribonucleases, RNase T2 and RNase 2, that act synergistically to release uridine from oligoribonucleotides. RNase T2 cleaves preferentially before, and RNase 2 after, uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic oligoribonucleotides all required RNase 2 and T2 processing to activate TLR8. Uridine supplementation restored RNA recognition in RNASE2-/- or RNASET2-/- but not RNASE2-/-RNASET2-/- cells. Primary immune cells from RNase T2-hypomorphic patients lacked a response to bacterial RNA but responded robustly to small-molecule TLR8 ligands. Our data identify an essential function of RNase T2 and RNase 2 upstream of TLR8 and provide insight into TLR8 activation.
Assuntos
Endorribonucleases/metabolismo , Monócitos/imunologia , Neutrófilos/imunologia , RNA Bacteriano/metabolismo , RNA de Protozoário/metabolismo , Receptor 8 Toll-Like/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Endorribonucleases/imunologia , Eritrócitos/imunologia , Eritrócitos/parasitologia , Escherichia coli/química , Escherichia coli/imunologia , Edição de Genes/métodos , Humanos , Listeria monocytogenes/química , Listeria monocytogenes/imunologia , Monócitos/microbiologia , Monócitos/parasitologia , Neutrófilos/microbiologia , Neutrófilos/parasitologia , Plasmodium falciparum/química , Plasmodium falciparum/imunologia , Cultura Primária de Células , Estabilidade de RNA , RNA Bacteriano/imunologia , RNA de Protozoário/imunologia , Serratia marcescens/química , Serratia marcescens/imunologia , Staphylococcus aureus/química , Staphylococcus aureus/imunologia , Streptococcus/química , Streptococcus/imunologia , Células THP-1 , Receptor 8 Toll-Like/imunologiaRESUMO
Antigen-presenting myeloid cells like monocytes detect invading pathogens via pattern recognition receptors (PRRs) and initiate adaptive and innate immune responses. As analysis of PRR signaling in primary human monocytes is hampered by their restricted expandability, human monocyte models like THP-1 cells are commonly used for loss-of-function studies, such as with CRISPR-Cas9 editing. A recently developed transdifferentiation cell culture system, BLaER1, enables lineage conversion from malignant B cells to monocytes and was found superior to THP-1 in mimicking PRR signaling, thus being the first model allowing TLR4 and inflammasome pathway analysis. Here, we identified an important caveat when investigating TLR4-driven signaling in BLaER1 cells. We show that this model contains glycosylphosphatidylinositol (GPI) anchor-deficient cells, which lack CD14 surface expression when differentiated to monocytes, resulting in diminished LPS/TLR4 but not TLR7/TLR8 responsiveness. This GPI anchor defect is caused by epigenetic silencing of PIGH, leading to a random distribution of intact and PIGH-deficient clones after single-cell cloning. Overexpressing PIGH restored GPI-anchored protein (including CD14) expression and LPS responsiveness. When studying CD14- or other GPI-anchored protein-dependent pathways, researchers should consider this anomaly and ensure equal GPI-anchored protein expression when comparing cells that have undergone single-cell cloning, e. g. after CRISPR-Cas9 editing.