Beschreibung: TLRs 7 and 8 are pattern recognition receptors controlling antiviral host defense or autoimmune diseases. Apart from foreign and host RNA, synthetic RNA oligoribonucleotides (ORN) or small molecules of the imidazoquinoline family activate TLR7 and 8 and are being developed as therapeutic agonists. The structure-function relationships for RNA ORN and imidazoquinoline sensing and consequent downstream signaling by human TLR7 and TLR8 are unknown. Proteome- and genome-wide analyses in primary human monocyte-derived dendritic cells here showed that TLR8 sensing of RNA ORN versus imidazoquinoline translates to ligand-specific differential phosphorylation and transcriptional events. In addition, TLR7 and 8 ectodomains were found to discriminate between RNA ORN and imidazoquinolines by overlapping and nonoverlapping recognition sites to which murine loss-of-function mutations and human naturally occurring hyporesponsive polymorphisms map. Our data suggest TLR7 and TLR8 can signal in two different "modes" depending on the class of ligand. Considering RNA ORN and imidazoquinolines have been regarded as functionally interchangeable, our study highlights important functional incongruities whose understanding will be important for developing TLR7 or 8 therapeutics with desirable effector and safety profiles for in vivo application.

Beschreibung: During neuroinflammation, cytokines such as TNF-α and IFN- secreted by activated leukocytes and/or CNS resident cells have been shown to alter the phenotype and function of brain endothelial cells (BECs) leading to blood–brain barrier breakdown. In this study, we show that the human BEC line hCMEC/D3 expresses the receptors for TNF-α, TNF receptor 1 and TNF receptor 2, and for IFN-. BEC activation with TNF-α alone or in combination with IFN- induced endothelial leakage of paracellular tracers. At high cytokine concentrations (10 and 100 ng/ml), this effect was associated with caspase-3/7 activation and apoptotic cell death as evidenced by annexin V staining and DNA fragmentation (TUNEL) assays. In addition, inhibition of JNK and protein kinase C activation at these doses partially prevented activation of caspase-3/7, although only JNK inhibition was partially able to prevent the increase in BEC paracellular permeability induced by cytokines. By contrast, lower cytokine concentrations (1 ng/ml) also led to effector caspase activation, increased paracellular flux, and redistribution of zonula occludens-1 and VE-cadherin but failed to induce apoptosis. Under these conditions, specific caspase-3 and caspase-9, but not caspase-8, inhibitors partially blocked cytokine-induced disruption of tight and adherens junctions and BEC paracellular permeability. Our results suggest that the concentration of cytokines in the CNS endothelial microenvironment determines the extent of caspase-mediated barrier permeability changes, which may be generalized as a result of apoptosis or more subtle as a result of alterations in the organization of junctional complex molecules.