Study On The Innate Immune Mechanism Of Fish Pattern Recognition Receptor TLR18

Innate immunity mainly recognizes dangerous signals through pattern recognition receptors(PRRs)encoded by germline genes.There are four main types of PRRs,including Toll-like Receptors(TLRs),RIG-I-like Receptors(RLRs),NOD-like Receptors(NLRs),and C-type lectin-like Receptors(CLRs).Among them,Toll-like Receptors(TLRs)are receptors that can recognize multiple pathogen-associated molecular patterns(PAMPs)from bacteria,fungi and viruses.TLR18 is a fish-specific pattern recognition receptor composed of three domains,including extracellular LRR domain,transmembrane region and intracellular TIR domain,which is in accordance with the typical structural characteristics of Toll-like Receptors.Carp TLR18 can participate in the anti-bacterial immune response,but its immune regulation mechanism is still unknown.In this article,we will conduct a preliminary study on ligand recognition,cell localization,pathway-related adaptor molecules and mechanism of action of carp Toll-like receptor 18(TLR18).1.Research on ligand recognition and cell localization of carp TLR18Luciferase reporter gene found that in carp TLR18 overexpressing 293 T cells,after flagellin stimulation,the relative activity of the transcription factor NF-?B luciferase was significantly higher than that of the unstimulated group,and other ligand stimulation did not change;Flagellin-TLR18 chimera can activate the relative activity of NF-?B,but after deleting the LRR domain of TLR18,the chimera has no effect on NF-?B activity,confirming that TLR18 can recognize flagellin through the LRR domain.Immunofluorescence results showed that TLR18 is synthesized on the ribosome of the endoplasmic reticulum;further studies showed that TLR18 colocalized with Rab7.Therefore,TLR18 is transported to late endosomes after endoplasmic reticulum ribosome synthesis.2.Study on the mechanism of carp TLR18To explore the adaptor molecules of TLR18,we constructed expression vectors for adaptor molecules,namely My D88-p EGFP-N1,TIRAP-p EGFP-N1 and TRIF-p EGFPN1.TLR18 and adaptor molecule expression vectors were co-transfected into He La cells respectively.Immunofluorescence results showed that TLR18 can co-localize with My D88;Immunoprecipitation experiments further proved that TLR18 and My D88 interacted,indicating that TLR18 recruited My D88 as adaptor molecule.We then studied the signaling pathways involved in TLR18.After flagellin stimulation,Western Blotting showed that TLR18 significantly enhanced TRAF6 expression and phosphorylation of IKK?/? and p65.The q PCR results showed that the activation effect of TLR18 on downstream cytokines(il-1? and tnf-?)was significantly up-regulated compared with the control group.After inhibiting IRAK4 and IKK complexes by inhibitors,TLR18 significantly reduced the activation of downstream signaling molecules.Therefore,TLR18 can activate IRAK4,IKK?/? and TRAF6 signaling molecules,activate NF-?B,and then induce the production of pro-inflammatory cytokines,resulting in immune response.Through the above experiments,we have conducted a preliminary study on the natural immune regulation mechanism of the teleost fish specific pattern recognition receptor TLR18.It is preliminarily speculated that TLR18 recognizes flagellin and synthesizes in the endoplasmic reticulum.When it plays a role,TLR18 is transported to the late endosome,activates NF-?B via IRAK4,IKK?/? and TRAF6 signaling molecules,and induces cytokines(il-1? and tnf-?).This study provides a better research basis for the investigation of the regulation mechanism of fish Toll-like receptors against pathogen infection in the aquatic environment.