Human TLR 8 senses UR / URR motifs in bacterial and mitochondrial RNA
2015; Springer Nature; Volume: 16; Issue: 12 Linguagem: Inglês
10.15252/embr.201540861
ISSN1469-3178
AutoresAnne Krüger, Marina Oldenburg, Chiranjeevi Chebrolu, Daniela Beißer, Julia Kolter, Anna M. Sigmund, Jörg Steinmann, Simon Schäfer, Hubertus Hochrein, Sven Rahmann, Hermann Wagner, Philipp Henneke, Veit Hornung, Jan Buer, Carsten J. Kirschning,
Tópico(s)NF-κB Signaling Pathways
ResumoScientific Report6 November 2015Open Access Human TLR8 senses UR/URR motifs in bacterial and mitochondrial RNA Anne Krüger Anne Krüger Institute of Medical Microbiology, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Marina Oldenburg Marina Oldenburg Institute of Medical Microbiology, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Chiranjeevi Chebrolu Chiranjeevi Chebrolu Institute of Medical Microbiology, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Daniela Beisser Daniela Beisser Genome Informatics, Institute of Human Genetics, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Julia Kolter Julia Kolter Centre of Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany Search for more papers by this author Anna M Sigmund Anna M Sigmund Institute of Medical Microbiology, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Jörg Steinmann Jörg Steinmann Institute of Medical Microbiology, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Simon Schäfer Simon Schäfer Clinic of Anesthesia, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Hubertus Hochrein Hubertus Hochrein Department of Research Immunology, Bavarian Nordic GmbH, Martinsried, Germany Search for more papers by this author Sven Rahmann Sven Rahmann Genome Informatics, Institute of Human Genetics, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Hermann Wagner Hermann Wagner Institute for Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Munich, Germany Search for more papers by this author Philipp Henneke Philipp Henneke Centre of Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany Search for more papers by this author Veit Hornung Veit Hornung Institute of Clinical Chemistry and Clinical Pharmacology, University of Bonn, Bonn, Germany Search for more papers by this author Jan Buer Jan Buer Institute of Medical Microbiology, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Carsten J Kirschning Corresponding Author Carsten J Kirschning Institute of Medical Microbiology, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Anne Krüger Anne Krüger Institute of Medical Microbiology, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Marina Oldenburg Marina Oldenburg Institute of Medical Microbiology, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Chiranjeevi Chebrolu Chiranjeevi Chebrolu Institute of Medical Microbiology, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Daniela Beisser Daniela Beisser Genome Informatics, Institute of Human Genetics, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Julia Kolter Julia Kolter Centre of Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany Search for more papers by this author Anna M Sigmund Anna M Sigmund Institute of Medical Microbiology, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Jörg Steinmann Jörg Steinmann Institute of Medical Microbiology, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Simon Schäfer Simon Schäfer Clinic of Anesthesia, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Hubertus Hochrein Hubertus Hochrein Department of Research Immunology, Bavarian Nordic GmbH, Martinsried, Germany Search for more papers by this author Sven Rahmann Sven Rahmann Genome Informatics, Institute of Human Genetics, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Hermann Wagner Hermann Wagner Institute for Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Munich, Germany Search for more papers by this author Philipp Henneke Philipp Henneke Centre of Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany Search for more papers by this author Veit Hornung Veit Hornung Institute of Clinical Chemistry and Clinical Pharmacology, University of Bonn, Bonn, Germany Search for more papers by this author Jan Buer Jan Buer Institute of Medical Microbiology, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Carsten J Kirschning Corresponding Author Carsten J Kirschning Institute of Medical Microbiology, University of Duisburg-Essen, Essen, Germany Search for more papers by this author Author Information Anne Krüger1,‡, Marina Oldenburg1,‡, Chiranjeevi Chebrolu1,‡, Daniela Beisser2, Julia Kolter3, Anna M Sigmund1, Jörg Steinmann1, Simon Schäfer4, Hubertus Hochrein5, Sven Rahmann2, Hermann Wagner6, Philipp Henneke3, Veit Hornung7, Jan Buer1 and Carsten J Kirschning 1 1Institute of Medical Microbiology, University of Duisburg-Essen, Essen, Germany 2Genome Informatics, Institute of Human Genetics, University of Duisburg-Essen, Essen, Germany 3Centre of Chronic Immunodeficiency, University Medical Center Freiburg, Freiburg, Germany 4Clinic of Anesthesia, University of Duisburg-Essen, Essen, Germany 5Department of Research Immunology, Bavarian Nordic GmbH, Martinsried, Germany 6Institute for Medical Microbiology, Immunology and Hygiene, Technical University of Munich, Munich, Germany 7Institute of Clinical Chemistry and Clinical Pharmacology, University of Bonn, Bonn, Germany ‡These authors contributed equally to this work *Corresponding author. Tel: +49 201 723 1824; E-mail: [email protected] EMBO Reports (2015)16:1656-1663https://doi.org/10.15252/embr.201540861 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Toll-like receptor (TLR) 13 and TLR2 are the major sensors of Gram-positive bacteria in mice. TLR13 recognizes Sa19, a specific 23S ribosomal (r) RNA-derived fragment and bacterial modification of Sa19 ablates binding to TLR13, and to antibiotics such as erythromycin. Similarly, RNase A-treated Staphylococcus aureus activate human peripheral blood mononuclear cells (PBMCs) only via TLR2, implying single-stranded (ss) RNA as major stimulant. Here, we identify human TLR8 as functional TLR13 equivalent that promiscuously senses ssRNA. Accordingly, Sa19 and mitochondrial (mt) 16S rRNA sequence-derived oligoribonucleotides (ORNs) stimulate PBMCs in a MyD88-dependent manner. These ORNs, as well as S. aureus-, Escherichia coli-, and mt-RNA, also activate differentiated human monocytoid THP-1 cells, provided they express TLR8. Moreover, Unc93b1−/−- and Tlr8−/−-THP-1 cells are refractory, while endogenous and ectopically expressed TLR8 confers responsiveness in a UR/URR RNA ligand consensus motif-dependent manner. If TLR8 function is inhibited by suppression of lysosomal function, antibiotic treatment efficiently blocks bacteria-driven inflammatory responses in infected human whole blood cultures. Sepsis therapy might thus benefit from interfering with TLR8 function. Synopsis This study shows that human monocyte TLR8 senses bacterial ribosomal and transfer RNA, as well as mitochondrial RNA. TLR8 recognizes a UR/URR RNA-ligand consensus motif in contrast to the bacteria-borne 10-mer motif detected by its murine TLR13 counterpart. Various molecular subpopulations of both Gram-positive and Gram-negative bacterial RNAs activate endosomal human TLR8. Large (16S) ribosomal mitochondrial RNA sequence-derived oligoribonucleotides and total mitochondrial RNA activate human TLR8. TLR8 mediates human monocyte activation upon S. aureus and E. coli infection besides TLR2. Lysosomal inhibition by chloroquine largely impairs bacterial infection-driven immune activation ex vivo. Introduction Toll-like receptors (TLRs) mediate recognition of pathogen- and host-derived “danger associated molecular patterns” (P-/DAMPs) driving severe inflammation such as in sepsis, which is mostly induced by Gram-positive and Gram-negative bacterial infection 1. Anti-inflammatory interventions in the acute sepsis phase are thus promising therapeutic approaches 2. Indeed, blockade of TLR2 and TLR4 at the start of antibiotic therapy inhibited human peripheral blood mononuclear cell (PBMC) activation and also saved mice upon Gram-negative but not Gram-positive bacterial infection 3. Accordingly, the Sa19 segment of bacterial 23S ribosomal (r) RNA is a murine (m) TLR13 ligand strongly operative primarily in Gram-positive bacterial infection 4567. Sa19 sequence modification, as is frequently found in methicillin-resistant Staphylococcus aureus (MRSA) strains that are also macrolide, lincosamide and streptogramin antibiotic (MLS) resistant, ablates recognition by TLR13 (termed immune escape) 58. On the other hand, bacterial transfer (t) RNA is recognized via TLR7, and Gram-negative bacterial RNA activates human (h) TLR8 91011. Whether Gram-positive bacterial RNA drives a strong immune activity immediately upon acute infection also in humans as it had been observed in mice is largely unknown. Previous studies searching for a human pattern recognition receptor (PRR) equivalent to mTLR13 have concluded that mainly TLR2 is operative in response to Gram-positive bacteria 512. Here, we demonstrate that not only Sa19, but also variants of it including mitochondrial (mt) 16S rRNA-borne derivatives strongly stimulate human immune cells via TLR8 exclusively. Ligand recognition of TLR8 was found to be promiscuous, in that its ligand consensus motif was broader than “UGG” 13, since “UAA” and “UGA” motifs were also activating. Human TLR8 thus senses both bacterial pathogen-derived rRNAs and self-mitoribosomal (mtr) RNA wherein the latter descends from bacteria according to the endosymbiotic theory. Specifically, we observed that TLR8-mediated RNA sensing is critically involved in recognition of viable Escherichia coli and S. aureus. For example, the inhibition of lysosomal function, which affects bacteria-driven TLR8 and also TLR7 activation, allowed antibiotics to inhibit consequent inflammation. Our results identify TLR8 as major bacteria and mitochondria sensor and implicate a novel therapeutic interference in acute inflammation pathology. Results and Discussion Specific bacterial and mitochondrial RNAs activate human immune cells dependent on MyD88 and Unc93B1 but largely independent on TLR7 In quest for a human equivalent of TLR13, we analyzed whether the 23S rRNA segment Sa19—with 19 bases of appropriate length 14—or heat-inactivated S. aureus (hiSa) stimulate PBMCs. Both compounds were active. Thus, blockade of TLR2 with a neutralizing and human/mouse cross-reactive monoclonal antibody (mAb, named T2.5) was ineffective unless the hiSa solution was incubated with single-stranded (ss) RNA-specific RNase A (Fig 1A) 515. We thus concluded that both Sa19 “like” ssRNA segments as well as TLR2 ligands are candidates of S. aureus-derived immune stimulatory PAMPs. Figure 1. Specific bacterial and mitochondrial ribosomal RNA segments and fractions induce proinflammatory but not type I IFN cytokine production through an endosomal TLR A. Cytokine release by PBMCs challenged with heat-inactivated S. aureus (hiSa), and/or RNase A (RA)-treated solution, or Sa19 (TLR13 activating ORN) upon TLR2 blockade (T2.5; n.p., not performed; **P ≤ 0.01; n = 3; unpaired t-test). B. Alignment of Sa19 and mitochondrial 16S rRNA Sa19 “like” segment (mt, mitochondrial; PTL, peptidyl transferase loop; D, domain; _, transition region) sequence with common core motif (blue) and uracils (red U); *conserved in human (Hs), cattle (Bt), mouse and rat; G/A underlined, mutated core motif; ma, 6N methylation of adenosine 7. C. Cytokine release of PBMCs transfected with ORNs including TLR7/8 ligand RNA40 (n = 3). D, E. Activity of PBMCs upon bacterial RNA challenge, pretreatment with chloroquine in (D) only (S, Svedberg; r, ribosomal; tot, total; n = 3). F. Activity of PBMCs upon mitochondrial (mt) RNA challenge (Hs, human; triangle, increasing doses; LFA, Lipofectamine 2000; n = 3). G. Activity of PBMCs challenged with ORNs and total S. aureus (Sa) RNA (Myd88d/d, mutant MyD88 expression not impairing LPS-driven IL-8 production; n = 2). H. Responsiveness of undifferentiated (undiff) and 3- or 8-day PMA-differentiated (ddi) THP-1 cells to Sa19 challenge (n = 3). I. Activity of parental and Unc93b1−/−-3ddiTHP-1 cells challenged with ORNs (n = 3). Data information: Graphs show mean ± SD; –, unchallenged; n.d., not detected; P3C, Pam3CSK4; Loxo, loxoribine. Download figure Download PowerPoint Also, self-RNA–antimicrobial peptide complexes stimulate human dendritic cells (DCs) via TLRs and the endosymbiotic theory implies that self-mitochondria originate from bacteria 1617. Since 23S bacterial rRNA comprises Sa19, we analyzed the sequence of its 16S mtrRNA human, cattle, mouse and rat orthologs and designed specific 19-mer Sa19 “like” oligoribonucleotides (ORNs, Figs 1B and EV1A and B). The 16S mtrRNA Sa19 orthologous segment termed *mtPTL, like Sa19 located within domain V of the large rRNA is conserved yet mutated toward “GAGAAGA”, while integer Sa19 “GGAAAGA” motifs are contained in other regions of 16S mtrRNAs 18. We also used two Sa19 adenosine (A) 7 variants that lack murine immune cell stimulatory capacity (Figs 1B and EV1C and D) 57. None of the Sa19 “like” RNA segments applied activated mTLR13, and only *mtPTL carrying a TLR7-prone “UAU” motif activated the murine immune system at all and through TLR7 (Fig EV1C–H) 1419. Total mtRNA was refractory (Fig EV1D), as if *mtPTL was rendered inactive in the context of total mtRNA. This finding was reminiscent of TLR13 “muteness” toward the E. coli Sa19 within total RNA 5. Click here to expand this figure. Figure EV1. Scheme of bacterial and mitochondrial (mt) largest rRNAs, Sa19 “like” segments (ORNs) within the latter, and mostly non-responsiveness of the murine immune system to ORNs and mtRNA A. Schematic of both, bacterial 23S and mitochondrial 16S rRNAs (I to VI, domains; red rectangles, Sa19 like segments within 16S mtrRNA; D, domain; PTL, peptidyl transferase loop within V). B. Sa19 “like” 16S rRNA segment information (abbr., abbreviation; term., terminal; *interspecies sequence conservation). C. Luciferase activity (Rel. luc. act.) of transfected and challenged HEK293 cells (n = 2). D. Cytokine release by bone marrow-derived macrophages (BM; LyoVec, transfection reagent; Hs, human; Rn, rat; n = 2). E. Serum cytokine concentrations of mice challenged i.v. with phosphorothioate-stabilized ORNs (n = 3). F. Cytokine release of splenocytes transfected with ORNs with various transfectants (pLA, poly-L-arginine; LFA, Lipofectamine 2000; TR, transfection reagent only; n = 2). G, H. Cytokine release of challenged wild-type (Wt) and k.o. BMs (n = 3). Data information: (C–H) Graphs show mean ± SD; –, unchallenged; n.d., not detected. Download figure Download PowerPoint We next analyzed human PBMCs for their responsiveness to different bacteria and self-derived RNAs. While all Sa19 “like” ORNs applied including TLR13-silent, point-mutated, and methylated ones induced robust TNF production in PBMCs, surprisingly none triggered substantial type I interferon (IFN) release (Fig 1C). This result indicated a lack of TLR7 involvement because TLR7 ligands such as RNA40 and bacterial tRNA induce PBMC type I interferon (IFN) production by activating plasmacytoid DC TLR7 1419. Next, we analyzed bacterial rRNA activities (Fig EV2A) 5910. 23S, 16S, and 5S rRNA of both Gram-positive and Gram-negative bacteria induced substantial TNF release from PBMCs, provided their lysosomes were functional (Fig 1D). While total bacterial RNA encompassing tRNA triggered substantial IFNα release, neither bacterial rRNAs, nor total mtRNA hardly induced IFNα release, while the latter RNA elicited strong IL-6 production (Figs 1E and F, and EV2B). Click here to expand this figure. Figure EV2. RNA subpopulation isolation, candidate mRNAs, mTLR13-hTLR8 alignment, TLR8 mRNA knockdown, and responsiveness as well as TLR7/8 expression of differentiated k.o. THP-1 cells A, B. Electrophoresis gels of total S. aureus RNA with tRNA and 5S rRNA (left panel) or isolated 5S rRNA (right panel; PAGE) and total or purified mammalian mtRNA (agarose), respectively (kb, kilobases; M, RNA size marker; tot, total; S, Svedberg; r, ribosomal; hmw, high molecular weight; t, transfer; mt, mitochondrial RNA; sup, supernatant at initial mitochondria separation step; Rn, rat; Hs, human; *S not applicable). C. Receptor mRNA amounts increased ≥ 2-fold in 3-day differentiated (ddi) as compared to undifferentiated (undiff) and 8ddiTHP-1 cells, except for the data marked by a frame (depicted for comparison; dual TLR7 probe set; CLEC7A, C-type lectin domain family 7A; FPR3, formyl peptide receptor 3; DDX58, DEAD box polypeptide; CLEC4A, C-type lectin domain; FPR1, formyl peptide receptor; SIGLEC1, sialic acid binding Ig-like lectin; n = 1), according to the comparative gene array-based transcriptome profiling. D. Alignment of murine (m) TLR13 and human (h) TLR8 sequences (46 residue signal and leucine-rich repeat N-terminal domain, LRRNT, of the latter is not depicted for clarity; dark blue, signal peptide and LRRNT; light blue, LRRs; violet, transmembrane domains; green, LRR C-terminal and cytoplasmic Toll-IL1 receptor resistance gene domains; orange, z-loop; yellow and turquoise, residues known to contribute to hTLR8 ligand recognition site one and two, respectively). E. Relative (Rel.) mRNA accumulation (accum.) in 3ddiTHP-1 cells upon transfection of siRNAs (scram., scramble control; untreat., untreated; *P ≤ 0.01; unpaired t-test; n = 3). F. Activity of 3ddiTHP-1 cells challenged with different amounts of heat-inactivated (hi) S. aureus and E. coli (Sa and Ec, respectively; –, unchallenged; n.d., not detected; Loxo, loxoribine; P3C, Pam3CSK4; corresponding to Fig 3C; n = 3). G. Immunoblot analysis of R848 overnight-primed 3ddiTHP-1 cell lysates (genotypes indicated, TLR7+ or TLR8+ HEK293 cell lysates for positive and cytoplasmic protein detection as loading controls; n = 3). Data information: Graphs show mean ± SD. Download figure Download PowerPoint PBMCs of a human individual expressing a nonfunctional Glu53Δ MyD88 mutant 20 failed to respond to S. aureus RNA and to Sa19 “like” ORNs (Fig 1G). Given that 3-day differentiated (3ddi) THP-1 cells turned out to be responsive toward Sa19 (Fig 1H), we applied THP-1 cells lacking expression of Unc93B1 (a chaperon translocating TLRs from the endoplasmic reticulum to endosomes) 2122. Like undifferentiated and 8ddiTHP-1 cells, Unc93b1−/−-3ddiTHP-1 counterparts failed to respond toward Sa19 “like” ORNs (Fig 1I). Upregulation or overexpression of TLR8 confers uridine-dependent specific bacterial and mitochondrial RNA responsiveness while TLR8 knockout abrogates it Unc93b1−/−-3ddiTHP-1 cells were also insensitive to large bacterial rRNAs (Fig 2A) 12. However, they responded well to hiSa and E. coli (Ec) via TLR2 (Fig 2B and C). Next, we comparatively profiled transcriptomes of undifferentiated, 3ddi-, and 8ddiTHP-1 cells. Selection of non-interleukin receptors that were at least twofold upregulated on the mRNA level in 3ddiTHP-1 cells led to seven candidate molecules out of which we considered mTLR13-like TLR8 as most promising candidate (Figs 2D and EV2C). In contrast to mTLR13, hTLR8 carries a z-loop (Fig EV2D) providing a functionally important cleavage site that contributes to ligand binding, and is deleted in mTLR8 13. In order to substantiate our selection of hTLR8, we analyzed its “gain and loss of” function. All Sa19 “like” ORNs activated hTLR8 overexpressing HEK293 cells, and BtmtD3_4 triggered also mRAW264.7 cell activity through hTLR8 (Fig 2E and F). Furthermore, knockdown of TLR8 mRNA abrogated responsiveness to BtmtD3_4, and to R848 (Figs 2G and EV2E). Figure 2. Transcriptome as well as gain- and loss-of-function analyses implicate TLR8 as bacteria and mitochondria ribosomal RNA sensor A. Cytokine release of 3-day differentiated (ddi) THP-1 cells toward stimulation with TLR ligands and bacterial RNAs (S, Svedberg; r, ribosomal; tot, total; n = 3). B, C. Responsiveness of Unc93b1−/−-3ddiTHP-1 cells treated with T2.5 and challenged with heat-inactivated (hi) bacteria (triangle, decreasing doses; n = 3). D. Ratios of constitutive mRNA amounts in 3- versus 8ddi (gray bar) and of each of both versus undifferentiated THP-1 cells (black or white bar, respectively) according to a transcriptome profiling result (n = 1). E, F. NF-κB-driven relative luciferase activity (Rel. luc. act.) of hTLR8-overexpressing HEK293 and murine RAW264.7 cells, respectively, upon challenges (vector, empty plasmid; n = 3). G. Transfection of TLR8 siRNA impairs 3ddiTHP-1 cell responsiveness (n = 3). Data information: Graphs show mean ± SD; *P ≤ 0.05; **P ≤ 0.01; unpaired t-test; –, unchallenged; P3C, Pam3CSK4; n.d., not detected; Sa, S. aureus; Ec, E. coli. Download figure Download PowerPoint Moreover, responsiveness of Tlr8−/−-3ddiTHP-1 cells to specific ORNs and bacterial rRNAs was strongly impaired, while total bacteria RNA recognition was widely operative (Fig 3A and B) 22. Otherwise, their sensitivity for hiSa and hiEc was normal, but largely TLR2 driven (Figs 3C and EV2F). Sensing of imidazoquinoline R848 and the guanosine (G) nucleotide analogue loxoribine by TLR7 was weak in Tlr8−/− cells yet abrogated by TLR7 mRNA expression knockdown (Figs EV2F and G, and EV3A) 1923. Besides loxoribine and RNA40, only total bacterial RNA and to a low degree also S. aureus 5S rRNA triggered IFNα release by PBMCs, which emphasized TLR8 specificity and lack of TLR7 specificity of the ORNs, bacterial rRNAs, as well as mtRNA (Fig 1C, E, and F) 14. Of note, mtRNA-driven activation of 3ddiTHP-1 cells was absent from Tlr8−/− and Unc93b1−/−-3ddiTHP-1 cells (Fig 3D). Figure 3. TLR8 recognizes UR/URR motif containing RNA A, B. Cytokine release of 3-day differentiated (ddi) THP-1 cells challenged with (A) ORNs or (B) bacterial RNA fractions (tot, total; S, Svedberg; r, ribosomal). C. Responsiveness of Tlr8−/−-3ddiTHP-1 cells challenged with heat-inactivated (hi) bacteria (triangle, decreasing doses; *P ≤ 0.05; **P ≤ 0.01; unpaired t-test) upon TLR2 blockade (T2.5). D. Responsiveness of 3ddiTHP-1 cells upon challenge with human (Hs) mitochondrial (mt) RNA (triangle, increasing doses). E–H. NF-κB-driven relative luciferase activity (Rel. luc. act.) of hTLR8+ HEK293 cells or cytokine release by PBMCs all transfected with the RNAs indicated and additional uridine (U) if indicated (n.p., not performed; vector, empty plasmid). Data information: Graphs show mean ± SD; n = 3; −, unchallenged; P3C, Pam3CSK4; Sa, S. aureus; Ec, E. coli; n.d., not detected. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. TLR7 mRNA knockdown in k.o. THP-1 cells and consequent responsiveness, Sa12 derivative sensitivity, bacterial infection-driven type I IFN or IL-6 production and inhibition of PBMCs or whole blood, respectively, and further species PBMC responsiveness Knockdown upon transfection of control (scramble) or two different TLR7 mRNA-specific siRNAs (siTLR7-1/3) in and cytokine release of respective 3-day differentiated THP-1 cells upon challenge (Rel., relative; accum., accumulation; Loxo, loxoribine; R848, small molecule, 50 μg/ml; tRNA, 200 ng/well of 96-well plate E. coli transfer RNA; n = 3). Sequence alignments of Sa12 and Sa12s6U with another Sa12 variant carrying in addition, as compared to the latter, ORN and, reminiscent of the respective motif in HsmtD1, a UGA motif (blue, core sequence; red, A6U; underlined, A7G). Diagram depicts PBMC activities upon challenges with Sa12 and derivatives (n = 3). PBMC type I IFN production upon pretreatments (T2.5, TLR2-neutralizing mAb; chloroquine, lysososmal function inhibitor) and challenge with TLR ligands or infection (5 × 104 cfu/ml; n = 2; corresponding to Fig 4B). Cytokine release of whole blood culture upon lysosome inhibition and challenge with heat-inactivated (hi) and viable bacteria (triangle, decreasing dose of infection; n = 3; corresponding to Fig 4C). Cytokine release of Sus (S.) scrofa and Macaca (M.) mulatta PBMCs upon challenge with ORN variants (n = 3). Data information: Graphs show mean ± SD; *P ≤ 0.05; **P ≤ 0.01; unpaired t-test; –, unchallenged; n.d., not detected; P3C, Pam3CSK4. Download figure Download PowerPoint TLR7 and most likely also TLR8 preferentially bind U/G rich viral-, si-, and self-RNA 1419232425. Structural data imply that the RNA specificity of TLR8 might be guided by the U and G content of ssRNA 13. We thus transfected Sa19 “like” ORNs differing in their U content (Fig 1B) and admixed U nucleosides into TLR8+ cells. The stimulatory power of ORNs that contained sparse U (such as Sa19 and Sa19 point mutants) substantially became enhanced by co-application of U nucleoside. Notably, ORN HsmtD1 containing like Sa19 just one U strongly activated cells. U-less Sa12 containing a CGG motif failed to activate TLR8, while its mutant Sa12s6U carrying an A6U mutation resulting in containment of a UAA motif, included in duplicate in BtmtD3_4, while HsmtD1 carries a UGA motif, strongly activated cells (Fig 3E–H). Our data imply UR/URR rather than mere UG/UGG as RNA ligand consensus motif (Figs 3F and H, and EV3B) 13. Bacterial infection-driven cell activation is TLR8 dependent and inhibited by chloroquine in whole blood Next, we explored the impact of hTLR8 on protective interventions during infection. To test this, THP-1 cells pretreated with TLR2 neutralizing mAb were seeded with viable S. aureus, or with E. coli. In Tlr8−/−-3ddiTHP-1 cells, inhibition of activation by TLR2 blockade was substantial (Fig 4A). Furthermore, a combination of chloroquine—a broadly established lysosomal function inhibitor—and T2.5 pretreatment significantly impacted PBMCs. Specifically, a contribution of TLR2 to both, TNF and IFNα production upon Gram-positive bacteria-driven activation, was barely detectable since chloroquine alone was efficient, which was true also for Gram-negative bacterial infection in respect to IFNα but not TNF release (Figs 4B and EV3C). The failure of the control stimulus (and TLR8 ligand) BtmtD3_4 to induce substantial IFNα production while triggering that of TNF to similar degrees as compared to both infections implicated involvement of further endosomal pattern recognition in bacterial infections such as of tRNA through TLR7 (Figs 4B and EV3C). Also within whole blood, chloroquine inhibited TNF release more strongly upon infection with Gram-positive bacteria as compared to infection with Gram-negative bacteria, while its impact on IL-6 production (strongly induced by lipopeptide/TLR2) was the opposite (Figs 4C and EV3D). Altogether, these data imply TLR8 as one major bacteria- and self-mitochondria sensor and hint at an anti-inflammatory potential of TLR8 blockade in sepsis. Whether this potential extends toward other clinical syndromes, such as trauma-induced sterile inflammation, or autoimmunity, remains to be evaluated. Figure 4. Lysosomal function inhibition affecting TLR8 activity is anti-inflammatory upon both, Gram-positive and Gram-negative bacterial infection ex vivo Cytokine release of 3-day differentiated THP-1 cells of the indicated genotypes upon TLR2 blockade (T2.5) and challenge with TLR ligands, heat-inactivated (hi) or viable bacteria. PBMC activity upon endosome function inhibition (chloroquine) and TLR2 blockade (T2.5) followed by TLR ligand challenge or bacterial infection. Activity of whole blood culture upon endosome function inhibition (chloroquine) and challenge with TLR ligands, heat-inactivated (hi), or viable bacteria. Data information: Graphs show mean ± SD; n = 3; *P ≤ 0.05; **P ≤ 0.01; unpaired t-test; –, unchallenged; P3C, Pam3CSK4; n.d., not detected; triangles, decreasing doses of exponentially growing bacteria gentamicin-treated 1 h post-infection. Download figure Download PowerPoint Perspective and concluding remarks Upon submission of this study, four reports on Gram-positive bacterial RNA sensing by hTLR8 or on mTLR13 structure have been published and largely summarized 2627282930. Thus, S. aureus as well as Streptococcus pyogenes and Streptococcus agalactiae and Listeria monocytogenes total RNAs activate hTLR8 but not TLR7 2628. Total RNAs of further Gram-positive and probiotic bacteria, as well as Enterococcus faecalis (EC-12)-derived 23S and 16S rRNA drive TLR8-dependent, yet TLR7-independent IL-12 production 27. Our results extended these and earlier findings by implicating 5S beyond 23S and 16S rRNA of S. aureus and also Gram-negative E. coli as well as mtrRNA as immune stimulatory P-/DAMPs that activate hTLR8 with their UR/URR motif segments 1113. Function restricting TLR8 mutation in mice might underlie their TLR13 expression and thus the A7 methylation mediated Sa19 camouflage, which is inoperative in other tetrapodes including human, macaque, and hog (Figs 1C, EV3E and EV4). Evolvement of TLR8 as a bacteria sensor might have resulted from perpetua
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