Toll-like receptors (TLRs) constitute an important class of pattern-recognition receptors, which recognize a multitude of pathogen-associated molecular patterns (PAMPs). We focused on TLR4 and TLR20 of common carp (Cyprinus carpio), their signaling pathway and their possible functions as activating receptors involved in innate immune responses to various pathogens or immunostimulants. So far the expression of TLR4 and TLR20 genes has been reported only for few fish species of the Cypriniformes (zebrafish, grass carp, common carp) and close relatives Siluriformes (channel catfish). Multiple fish TLR4 genes, created by a (recent) duplication rather than speciation event, seem to exist. It is possible that these paralogs have evolved to express different ligand specificities. In mammals TLR4 recognizes lipopolysaccharides from Gram-negative bacteria. The ligand(s) for fish TLR4 have not been confirmed. Multiple TLR20 genes also seem to exist in zebrafish, but not in carp. Our phylogenetic analysis indicates an ancestral relationship could exist between TLR20 and TLR11/TLR12, two TLRs found in mice but not in humans with ligand specificity for profilin from parasitic Toxoplasma gondii. At present, the ligand of TLR20 is unknown. We have cloned full-length coding sequences of carp TLR4a/b and carp TLR20 to study the function of these molecules. Expression analysis using real-time PCR show both TLR4 and TLR20 are mainly expressed in immune organs such as head kidney, gut and spleen with neutrophilic granulocytes as the primary source. We have also investigated the expression of TLR4 and of TLR20 after infection with protozoan parasites or after infection with spring vireamia of carp virus (SVCV). For TLR4, neither challenge with parasites nor with virus induced major changes in gene expression. However, we find high gene expression of TLR20 after infection with the protozoan parasites Trypanoplasma borreli or Trypanosoma carassii. We used the full-length sequences to create fluorescent protein-tagged TLRs for visualization of localization of these receptors by fluorescence microscopy. Our study suggests both receptors are located mainly in the cytoplasmic region. To define the unknown ligands of TLR4 and TLR20 we overexpressed these receptors in both human cell lines (HEK 293) and fish cell lines (EPC, CLC) stably transfected with a promoter of the transcription factor NF-¿B and a luciferase reporter gene. These studies may help to identify ligands for carp TLR4 a/b and TLR20.
Pietretti, D., Forlenza, M., Fink, I. R., & Wiegertjes, G. F. (2013). A closer look at Toll-like receptor 4 (TLR4) and toll-like receptor 20 (TLR20) of common carp (Cyprinus carpio). Fish and Shellfish Immunology, 34(6), 1673-1673. https://doi.org/10.1016/j.fsi.2013.03.121