EXPRESSION AND CHARACTERIZATION OF TOLL-LIKE RECEPTOR 10
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Toll-like receptors (TLRs), named after toll proteins identified in Drosophila melanogaster, are the pattern recognition receptors in the innate immune system that detect microbes. TLRs are mono, membrane-spanning, as well as non-catalytic receptors, which are mainly expressed in sentinel cells, such as the dendritic cells, neutrophils and macrophages. While humans have ten TLRs (TLR 1 to 10), the mouse has another three (TLRs 11, 12, 13). TLRs are made up of glycoproteins, which have luminal ligand-binding sites consisting of leucine-rich repeat (LRR) for detection of pathogens leading to activation of immune cells. TLR1, 2, 4, and 6 are responsible for recognition of lipids (such as triacetylated lipopeptide), peptidoglycan, and lipopolysaccharide (LPS). However, the TLR3, 7, 8, and 9 mainly recognize nucleic acids, such as double-stranded RNA (dsRNA) and CpG DNA, while the TLR13 detects ribosomal RNA sequences. So far, there are no data on the localization and immunological functions of TLR10. I studied the expression, localization and role of TLR10 in S. pneumoniae infection. First, I examined the expression of TLR10 in lungs of pig, cattle, dog, rat, and chickens. The light and electron microscopic data show TLR10 expression in vascular endothelium and smooth muscles in lungs of control and inflamed animals. Further, we found altered basal level of expression and localization of TLR10 in bovine neutrophils treated with E. coli lipopolysaccharide. These data show the expression of TLR10 in the lungs of tested animal species, and its alteration by LPS in bovine neutrophils. The next study was designed to investigate the regulation of TLR10 expression and to address its role in neutrophil chemotaxis. E. coli LPS activated human neutrophils showed temporal and spatial change in TLR10 expression. Confocal microscopy showed cytosolic and nuclear distribution of TLR10 in normal and activated neutrophils. TLR10 in E. coli LPS-activated neutrophils colocalized with flotallin-1, a lipid raft marker, and EEA-1, an early endosomal marker, suggested its endocytosis. Live cell imaging of LPS activated neutrophils showed TLR10 translocation to the leading edge. Neutrophils upon TLR10 knockdown were unable for fMLP-induced migration. TLR10 knockdown reduced the number of membrane pseudopods in activated neutrophils without altering the expression of key proteins of actin nucleation process, ARP-3 and Diap1. These data show TLR4-mediated pathway for regulation of TLR10 expression, and that TLR10 may have a role in neutrophil chemotaxis. Next, I examined the role of TLR10 in innate immune response to S. pneumoniae infection in U937 human macrophage cell line. S. pneumoniae are major causative agents of pneumonia, meningitis and bacteremia. A significant increase in TLR10 mRNA expression was found in S. pneumoniae (107 cfu for 6hr) challenged macrophages. TLR10 knockdown significantly reduced production of IL-1β, IL-8, IL-17 and TNF-α and no significant change in IL-10 expression, and also significantly diminished nuclear translocation of NF-κB but without affecting the phagocytosis of S. pneumoniae. Altogether, I report the that TLR10 is expressed in the normal and inflamed lungs in cattle, pigs, dogs, rats, chickens and humans. The expression of TLR10 is altered in activated neutrophils, and it plays a role in neutrophils chemotaxis and production of pro-inflammatory cytokines in macrophages infected with S. pneumoniae.
DegreeDoctor of Philosophy (Ph.D.)
DepartmentVeterinary Biomedical Sciences
ProgramVeterinary Biomedical Sciences
CommitteeMisra, Vikram; Mutwiri, George; Simko, Elemir
Copyright DateMarch 2016