TLR4al senses heme as a key damage/danger-associated molecular pattern to activate immune responses in lower vertebrates.

Toll-like receptor 4 (TLR4), a critical pattern recognition receptor, detects microbe- and damage/danger-associated molecular patterns to trigger immune responses in mammals. However, the functions and mechanisms remain largely unclear in lower vertebrates. This study systematically investigates the evolutionary divergence, subcellular localization and ligand of TLR4 in lower vertebrates by grass carp (Ctenopharyngodon idella) as a model species. TLR4 emerges and expands in a few teleosts but is absent in most fishes. We standardized the nomenclature of multiple TLR4 variants (3 or 4) in cyprinids. Tetrapods generally contain 1 TLR4. Astonishingly, CiTLR4ba, CiTLR4al, and CiTLR4bb localize to lysosomes but not cytomembrane like their mammalian counterparts, in which they recognize ligands that are engulfed into lysosomes. However, CiTLR4bc is soluble in cytoplasm due to the absence of signal peptide and transmembrane domain. In ligand recognition, CiTLR4 variants exhibit obvious heterogeneity. CiTLR4bc exhibits no binding activity to any of the tested ligands [lipopolysaccharide, peptidoglycan, bacterial dsDNA, polyinosinic-polycytidylic acid, heme]. Interestingly, both CiTLR4ba and CiTLR4bb bind polyinosinic-polycytidylic acid. Excitedly, CiTLR4al conservatively recognizes heme, a central damage/danger-associated molecular pattern (DAMP) released from damaged erythrocytes, in teleost and human. Further, 5 key high-affinity heme-binding sites (C113, H185, C295, H342, and C458) were identified in CiTLR4al. Each single key site mutation attenuates the heme-binding ability and downstream immune responses of CiTLR4al, supporting a multivalent heme-CiTLR4al interaction mechanism. The present study systematically elucidates the evolution of TLR4, as well as its complexity and conservation in vertebrates, offering direct evidence for the adaptive evolution of TLR4 in vertebrates. The results also contribute to the immune mechanism of hemolytic diseases.