Toll-like receptor 18 in Labeo rohita: molecular cloning, characterization, and analysis of its response in RBCs, PBLs, and macrophages after PAMP stimulation and bacterial infections

Toll-like receptors (TLRs) are key pattern recognition receptors (PRRs) that detect pathogen-associated molecular patterns (PAMPs) and elicit broadly acting innate immune responses. This study accomplished the identification, cloning, and sequencing of Labeo rohita TLR18 (LrTLR18). Structurally, LrTLR18 possesses a signal peptide, six leucine-rich repeats (LRRs), an LRR-C-terminal (LRR-CT) domain, a transmembrane (TM) region, and a Toll/IL-1 receptor (TIR) domain. Phylogenetically, LrTLR18 is evolutionarily closely related to the Schizothorax prenanti and Cyprinus carpio TLR18. The quantitative real-time PCR analysis revealed that LrTLR18 gene was expressed in all tested tissues, and among the tissues, the highest expression was observed in the eye followed by spleen, intestine, and gill in the descending order. During the ontogenic developments, the highest expression of LrTLR18 gene has been detected in the late blastula and gastrula stages. In response to the Aeromonas hydrophila and Edwardsiella tarda infections, LrTLR18 gene was differentially expressed in the blood, kidney, liver, and gills. Labeo rohita gills (LRG) cells in vitro infected with A. hydrophila and E. tarda, and red blood cells (RBCs) and peripheral blood leucocytes (PBLs) isolated from the rohu fingerlings infected with these pathogens revealed significantly (p < 0.05) enhanced expression of LrTLR18 gene. Following in vivo and in vitro stimulations with lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I: C), LrTLR18 gene expression was also significantly (p < 0.05) enhanced in various tissues, RBCs, PBLs, and macrophages. Together, these results highlight the important roles of the TLR18 against pathogenic invasions in fish.