Table_1_Thoroughly Remold the Localization and Signaling Pathway of TLR22.docx

TLR22 exists in nearly all the poikilothermic vertebrates and plays a central role in the initiation of innate immunity and activation of adaptive immunity. TLR22 signaling pathway has been characterized in detail in fugu (Takifugu rubripes). Here, we thoroughly remold the localization and signaling pathways of TLR22. We characterized TLR22a and TLR22b in grass carp (Ctenopharyngodon idella), designated as CiTLR22a and CiTLR22b, and explored the ligand(s), adaptor(s), and signaling pathway(s). Results show that both CiTLR22a and CiTLR22b localize to lysosome, acidic compartment. Correspondingly, CiTLR22a and CiTLR22b directly bind and respond to dsRNA analog poly(I:C) at pH 5, but not at pH 7.4, the physiological pH. Moreover, CiTLR22a and CiTLR22b exhibit antagonistic function in signal transmission, wherein CiTLR22a facilitates the protein and phosphorylation levels of IRF7 and enhances the promoter activities of major IFNs and NF-κBs, while CiTLR22b downregulates IRF7 phosphorylation and IRF3 protein level and suppresses the IFN and NF-κB pathways. Further investigations revealed that CiTLR22a restrains grass carp reovirus (GCRV) replication and protects cells from GCRV infection, whereas CiTLR22b plays a negative role in response to GCRV infection. This is the first time to systematically clarify the signaling pathways of two isotype TLR22s; especially, subcellular localization and adaptor are different from previous TLR22 report, which results from technical limitations. The results will serve the antiviral immune mechanisms in poikilothermic vertebrates and evolutionary immunology.