Human TLR8 senses UR/URR motifs in bacterial and mitochondrial RNA.

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.

Species and mediator specific TLR4 antagonism in primary human and murine immune cells by ßGlcN(1↔1)αGlc based lipid A mimetics.

Immune stimulatory pathogen associated molecular patterns (PAMPs) are major drivers of infection pathology. Infections with Gram-negative bacteria or negatively polar and single stranded RNA influenza virus are prominent causes of morbidity and mortality. Toll-like receptor (TLR) 4 is a major host sensor for both of the two infections. In order to inhibit TLR4 driven immune activation we recently developed synthetic tetra-acylated lipid A mimetics based on a conformationally restricted ßGlcN(1↔1)αGlcN disaccharide scaffold (DA-compounds) that antagonized ectopically overexpressed human and murine TLR4/MD-2 complexes. Here we comparatively analyzed human peripheral blood mononuclear cell (hPBMC) and murine bone marrow derived macrophage (mBM) activation upon 30 min of preincubation in vitro with six variably acylated DA-compounds. 16 h subsequent to consequent LPS challenge, we sampled culture supernatants for cytokine and NO concentration analysis. Four compounds significantly inhibited release of both TNF and IL-6 by hPBMCs upon LPS challenge. In contrast, three compounds effectively inhibited mBM production of MIP-2 and KC, and even five of them inhibited IL-6 and NO production. LPS driven like other TLR ligand driven mBM TNF release was largely unimpaired. The inhibitory effect was specific in that Clo75 driven cytokine release by both hPBMCs and mBMs was unimpaired by the compounds analyzed. Our results indicate biological species specificity of LPS antagonism by variably tetraacylated lipid A mimetics and validate three out of six DA-antagonists as promising candidates for development of therapeutically applicable anti-inflammatory compounds.