Abstract
BackgroundBacterial DNA is well-known for its potent immunostimulatory properties which have been attributed to the abundance of CpG dinucleotides within the genomes of prokaryotes. Research has found that mammalian TLR9 is a receptor which mediates the immune response to CpG DNA; however, its functional properties in non-mammalian vertebrates are still poorly characterized. Leukocytes isolated from lower vertebrates, including teleosts, respond to CpG DNA and TLR9 has been identified in many fish species; however, the ligand-binding properties of fish TLR9 have, so far, not been studied. The fact that some vertebrates, like chicken, lack TLR9 and use an alternative molecule (TLR21) as a receptor for CpGs has questioned the functional conservation of TLR9 within vertebrates.ResultsIn the current study, TLR9 from Atlantic salmon (SsTLR9) has been found to interact with synthetic oligonucleotides via a CpG-independent but a pH-dependent mechanism. The endogenous receptor, expressed by primary mononuclear phagocytes colocalizes with CpG oligonucleotides (ODNs) in vesicles that appear to be endosomes. When overexpressed in salmonid cell lines, SsTLR9 spontaneously activates ISRE-containing promoters of genes involved in the IFN response; however, the transgenic receptor fails to translocate to CpG-containing endosomes. This indicates that only specific immune cell types have the ability to relocate the receptor to the appropriate cellular compartments where it may become activated by its ligand. In addition, through co-precipitation and mass spectrometry, two salmon proteins - hnRNPA0 and NCOA5, which both contain RNA-binding domains (RRM), were found to bind CpG ODNs, suggesting they may be involved in the CpG response in salmon leukocytes.ConclusionThe presented data are the first to demonstrate that the DNA-binding properties of TLR9 are conserved between teleosts and mammals. The current study also identifies additional molecules which may function as mediators of the immunostimulatory properties of foreign DNA.
Highlights
Bacterial DNA is well-known for its potent immunostimulatory properties which have been attributed to the abundance of CpG dinucleotides within the genomes of prokaryotes
Adherent mononuclear phagocytes isolated from head kidney were first stimulated for 48 hours with IFN-γ [20] in order to upregulate the SsTLR9 protein [15] and stimulated for 2 hours with biotinylated CpG-B prior to lysis and coprecipitation with streptavidin-conjugated magnetic beads
Addition of a 5-fold higher dose of non-labelled CpG-B (10 μM) to the biotinylated CpGs prior to stimulation significantly reduced the amount of the coprecipitated SsTLR9, which further confirmed the specificity of the binding
Summary
Bacterial DNA is well-known for its potent immunostimulatory properties which have been attributed to the abundance of CpG dinucleotides within the genomes of prokaryotes. Research has found that mammalian TLR9 is a receptor which mediates the immune response to CpG DNA; its functional properties in non-mammalian vertebrates are still poorly characterized. Toll-like receptor 9 (TLR9) has been identified as a major player in the innate immune response to bacterial DNA and synthetic oligodeoxynucleotides (ODNs) that contain unmethylated CpG motifs [3]. Acting as innate immune receptors, members of this family have been found to mediate the response to different bacteria- and virus-derived molecules, including lipopolysaccharide, bacterial lipopeptides [5], double-stranded RNA [6] and CpGs [3]. The receptor signals through Myeloid differentiation primary response gene 88 (MyD88) to activate transcription factors including nuclear factor kappa-B (NFkB) and interferon-regulatory factor 7 (IRF7) involved in the upregulation of proinflammatory genes and type I interferon (IFN), respectively [7]
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