Anti-TLR5 (aa 517-634) polyclonal antibody

Cerebral pericytes are mural cells embedded in the basement membrane of capillaries. Increasing evidence suggests that they play important role in controlling neurovascular functions, i.e. cerebral blood flow, angiogenesis and permeability of the blood-brain barrier. These cells can also influence neuroinflammation which is highly regulated by the innate immune system. Therefore, we systematically tested the pattern recognition receptor expression of brain pericytes. We detected expression of NOD1, NOD2, NLRC5, NLRP1-3, NLRP5, NLRP9, NLRP10 and NLRX mRNA in non-treated cells. Among the ten known human TLRs, TLR2, TLR4, TLR5, TLR6 and TLR10 were found to be expressed. Inflammatory mediators induced the expression of NLRA, NLRC4 and TLR9 and increased the levels of NOD2, TLR2, inflammasome-forming caspases and inflammasome-cleaved interleukins. Oxidative stress, on the other hand, upregulated expression of TLR10 and NLRP9. Activation of selected pattern recognition receptors can lead to inflammasome assembly and caspase-dependent secretion of IL-1 beta. TNF-alpha and IFN-gamma increased the levels of pro-IL-1 beta and pro-caspase-1 proteins; however, no canonical activation of NLRP1, NLRP2, NLRP3 or NLRC4 inflammasomes could be observed in human brain vascular pericytes. On the other hand, we could demonstrate secretion of active IL-1 beta in response to non-canonical inflammasome activation, i.e. intracellular LPS or infection with E. coli bacteria. Our in vitro results indicate that pericytes might have an important regulatory role in neuroinflammation. (C) 2017 Elsevier Inc. All rights reserved.

Flagellin is a major component of the flagellar filament. Flagellin also functions as a specific ligand that stimulates innate immunity through direct interaction with Toll-like receptor 5 (TLR5) in the host. Because flagellin activates the immune response, it has been of interest to develop as a vaccine adjuvant in subunit vaccines or antigen fusion vaccines. Despite the widespread application of flagellin fusion in preventing infectious diseases, flagellin-antigen fusion designs have never been biophysically and structurally characterized. Moreover, flagellin from Salmonella species has been used extensively despite containing hypervariable regions not required for TLR5 that can cause an unexpected immune response. In this study, flagellin from Bacillus cereus (BcFlg) was identified as the smallest flagellin molecule containing only the conserved TLR5-activating D0 and D1 domains. The crystal structure of BcFlg was determined to provide a scheme for fusion designs. Through homology-based modeling and comparative structural analyses, diverse fusion strategies were proposed. Moreover, cellular and biophysical analysis of an array of fusion constructs indicated that insertion fusion at BcFlg residues 178-180 does not interfere with the protein stability or TLR5-stimulating capacity of flagellin, suggesting its usefulness in the development and optimization of flagellin fusion vaccines.