Adaptive Evolution Of Non - Viral TLRs In Mammals And Validation Of TLR4 Function

Unlike the adaptive immunity only found in vertebrates, innate immunity, the first line of defense against pathogens invasion, is widely exist in animals, plants and other multicellular organisms. Toll-like receptors (TLRs) are a cornerstone of innate immune system, which can be activated by specific pathogen-associated molecular patterns (PAMPs). It also plays a regulatory role in triggering the adaptive immunity in vertebrates. So it is a bridge between innate immunity and adaptive immunity. TLRs can be classified into non-viral TLRs and viral TLRs with respect to their location and ligand recognition. Non-viral TLRs are located on the cell membrane and mainly recognized for bacteria. In response to the diversity living environments and feeding habits of mammals, the virulence, species, and prevalence of microbial pathogens that mammals faced are different. Our research selected 6 non-viral TLRs gene family members of 27 mammals represent species to analyze selective pressure and carry on TLR4 function verification experiments to discuss the following scientific questions:1) whether adaptive evolution occurs on non-viral TLRs genes across mammals and cetacean phylogeny? 2) Which lineages of mammals are under positive selected pressure? 3) whether TLR4 have a difference function between cetaceans and terrestrial mammals and explore the reason.The selective pressure analysis implemented in PAML showed significant evidence of positive selection acting on six non-viral TLRs gene across mammals lineage and detected 93 positive selected sites which located on extracellular domain. It suggested that the non-viral TLRs has undergone adaptive evolution in mammals. The analysis also presented that the different groups of mammals under different intensity of selection pressure. We speculated that microbial pathogens, animal diseases and feeding habits are important factors that promote mammals undergone adaptive changes. Futhermore, we conducted a selective pressure analysis on cetaceans and found four non-viral TLRs gene (TLR1, TLR2, TLR4, TLR5, TLR6) under positive selection. It revealed that cetacean innate immunity gene has undergone adaptive evolution durning the secondary aquatic adaptation.More importantly, our research found bovine TLR4 showed a stronger lipopolysaccharide (LPS) response compared with dolphin across detected the relative expression of IL8 and NF-κB. We speculate that it because of the number and stucture of bacterial pathogens in marine is quite different from that of the land. In further study, we verified that the difference sensitivity to LPS response between bovine and dolphin TLR4 mainly due to the variance of extracellular domain. During the transition from terrestrial to full aquatic habitat, cetacean faced the challenge of infectious pathogens in changing habitats. So TLR4 must have undergone adaptive evolution to keep in accordance with pathogens of the co-evolution arms race model.