Signaling Mechanism And Anti-infection Effect Of Toll Like Receptor 8(TLR 8) In Pigs

Innate immunity is mediated by a series of pattern recognition receptors(PRRs)in the associated cells that recognize pathogen-associated pattern molecules(PAMPs).These pattern recognition receptors include Toll-like receptors(TLRs),RIG-I-like receptors(RLRs),NOD-like receptors(NLRs),C-type lectin receptors(CLRs),and cytoplasmic DNA receptors(CDRs).These PRRs activate downstream signaling pathways that induce innate immune responses through the production of inflammatory cytokines,type I interferons,and other mediators.Toll-like receptor is the earliest discovered family of natural immune receptors,in the family,TLR1,TLR2,TLR4,TLR5,and TLR6 are located primarily on the cell surface plasma membrane and are responsible for recognizing bacterial products,such as Lipid,lipoprotein and other microbial membrane components,whereas TLR3,TLR7,TLR8,and TLR9 are localized in the cell endosome and are mainly involved in the recogniton of nucleic acids of extracellular microorganisms.TLR8 belongs to the TLR7/8/9 subfamily.The members of the subfamily have conserved structure,cell localization and activation mechanisms,but the individual specific functions are still quite different.Due to the relative lack of information about the TLR8 signaling pathway and the similarity between TLR8 and TLR7,in most cases,information about the TLR8 signaling pathway referred to that of TLR7.In particular,animal TLR8 information has been lacked and needs to be investigated.Therefore,this study focused on porcine TLR8 and made preliminary exploration in terms of agonist recognition,cellular localization of TLR8 extracellular domain(ECD),signaling pathway and modulation,and anti-infection function etc.,so as to enrich the information of porcine TLR8 signaling mechinery and its anti-infection function.The specific contents are as follows:1.Identification of the interaction sites of porcine TLR8 with small molecular agonists imidazolinoline derivatives and their species specificityHuman TLR8(hTLR8)crystal structure has been resolved and the interaction sites of small molecular agonists have been known,whereas porcine TLR8(pTLR8)structure and its interaction sites of small molecular agonist need to be studied.Using human TLR8(PDB ID:3W3J)as the template,the three-dimensional structure model of pTLR8 ECD was constructed and further optimized.Ramachandran diagram analysis was performed to evaluate the quality of the model.Then the model was used for molecular docking with imidazolinoline derivatives R837 and R848 to obtain 9 recognition sites,8 of which have been successfully mutated,respectively,being Y336A,Q340A,K341 A,K342A,Y343A,F395A,I560A and G562A.These sites are same or close to the pTLR8 sites which are corresponding to the conserved and essential hTLR8 sites for small agonists,indicating the effectiveness of the molecular docking.Considering the important role of Z-loop area in the TLR8,the previously reported Z-loop related sequences were deleted or swabbed in pTLR8 and hTLR8,obtaining the mutants:hTLR8-ΔRQSYA,hTLR8-sub-pGQKNG,pTLR8-ΔGQKNG,pTLR8-sub-hRQSYA.Additoinaly,point mutations of the Z-loop related sequence were also made:G428A,K430A,N431A and,G432A.The wild-type TLR8 and mutant plasmids were transfected into HEK-293T cells,either alone or together with cell endosome protein Rab5 expression plasmid(pDsRed-C1-pRab5).Western blotting showed that all the proteins were expressed as expected.Con-focal fluorescence microscopy showed that all TLR8 proteins could be co-localized with Rab5,indicating the cell endosomal localization.Next,TLR8 or various mutants were co-transfected with NF-κB-promoter firefly luciferase reporter(Flue)and β-actin-promoter Renilla luciferase(RLuc)reporter,and the transfected HEK-293T cells were stimulated with R837,R848 or CL075.Dual luciferase reporter assay results showed that Y336,K341,K342,F395 and G562 are critical to the pTLR8 signaling pathway activated by R837,R848 and CL075.In terms of Z-loop related sequences,the Z-loop related sequence of hTLR8 is critical for CL075 as previously reported,however,it is not critical for R848.In contrast,the Z-loop related sequence of pTLR8 is not important for CL075,R837 and R848 at all.These results demonstrated that pTLR8 and hTLR8 possess similarity in response to small molecular agonsits,but also exhibit subtle differences and thus species specificity.2.Intracellular localization and the determinants of the porcine TLR8 ectodomain(ECD)Previously,we found that the TLR8 ECD was localized intracellularly;here we further investigated the TLR8 ECD subcellular localization and determining mechanism.First,the wild-type pTLR8,pTLR8-ECD,pTLR3-ECD,pTLR5-ECD,bTLR8-ECD,hTLR8-ECD,bovine herpesvirus 1(BHV-1)gD and gD-ECD were transfected into HEK-293T cells,and the protein expressions in cell culture supernants and in cells were detected by Western blotting and fluorescence microscopy,respectively.The results showed that all proteins were expressed in cytoplasm except for gD-ECD,which,as a control,could be secreted into cell culture medium.These results suggested that cellular localization of all ECD proteins from different species and TLR types are the common phenotype.In addition,pTLR8-ECD,bovine bTLR8-ECD and hTLR8-ECD are located in endoplasmic reticulum and endosome,but not in Golgi body and lysosome.To explore the roles of signal peptide and various post-translational modifications in the subcellular localization of TLR8-ECD,online prediction tools were first used to predict the pTLR8-ECD signal peptide and posttranslational modification sites including phosphorylation,ubiquitination,glycosylation,acetylation and palmitylation.The corresponding mutants were constructed into expression vector pEGFP-C1 or pEGFP-N1.HEK-293T cells were transfected with wild-type pTLR8-ECD or various mutants plus pDsRed-Cl-pRab5.The fluorescence microscopy results showed that phosphorylation,ubiquitination,glycosylation,acetylation and palmitoylation not affected the pTLR8-ECD endosomal localization,but pTLR8-ECD signal peptide indeed affected the protein endosomal localization.To explore the role of chaperone transporter UNC93B1 in the TLR8-ECD endosomal location,the CRISPR-Cas9 method was used to prepare the UNC93B1 KO porcine alveolar macrophages(PAMs).The pTLR8-ECD,bTLR8-ECD and hTLR8-ECD expression plasmids were co-transfected with pDsRed-Cl-pRab5 into PAM cells and UNC93B1 KO PAM cells,respectively.The fluorescence microscopy results showed that all the proteins could be localized to the endosomes in PAM cells,but the endosomal localizations of different TLR8-ECD dissappered in the UNC93B1 KO PAM cells,suggesting the important role of host UNC93B1 in the transportation of ECD protein to the cell endosome.3.The pTLR8 signaling specific genes,negative feedback regulation and anti-infection functionTo investigate the species specificity between pTLR8 and hTLR8,the pTLR8-NF-κB reporter and hTLR8-NF-κB reporter cells were constructed,and the TLR8 agonist R848 was used to stimulate the TLR8 signaling in the two types of reporter cells,respectively.Total RNA was extracted from the R848 stimulated and mock stimulated reporter cells,and subjected to transcriptome sequencing analysis.According to the analyzed transcriptome results,R848 activated 502 differential expression genes(DEGs)in pTLR8 reporter cells,and 1157 DEGs in hTLR8 reporter cells.Among these DEGs,149 DEGs are pTLR8 specific,804 DEGs are hTLR8 specific,and 353 DEGs are shared between pTLR8 and hTLR8 signaling.About 30 upregulated DEGs were confirmed by RT-qPCR in two reporter cells,PAMs and human THP-1 cells,which is consistent with the transcriptome results.The analysis of GO functional enrichment pTLR8 specific DEGs identified the item cell surface recognition,which contains seven pTLR8 specific genes,with three upregulating DEGs CATSPERG,HAVCR2 and CNTN2.The three upregulating DEGs were confirmed by RT-qPCR in PAMs,and HAVCR2(TIM-3)as as the immune-suppression gene,probably regulates the pTLR8 signaling and its anti-infection function in a negative feedback manner.To examine the possibility,the pTIM-3 expression vector was constructed.The expression plasmids pTIM-3 and pTLR8 were co-transfected into HEK-293T cells.After 12 hours of R848 stimulation,the NF-κB activity and the protein expressions of Akt,p-Akt,p65 and p-p65 were detected by promoter assay and Western blotting,respectively.In addition,the pTLR8 transfected cells were treated with Akt inhibitor A-443654 before stimulation R848 and NF-κB activity was detected by promoter assay.The results showed that pTIM-3 suppressed the R848 triggered pTLR8-NF-κB signaling by inhibiting the Akt phosphorylation.PAM cells stimulated with different concentrations of R848(0,5,10,20,30 μg/mL)exhibited the inhibitory effect on Salmonella typhimurium,demonstrating the anti-infectioin function of pTLR8 signaling.Further,we expressed and silenced Tim-3 gene in PAM cells,respectively,before stimulation of R848(0,5,10,20,30 μg/mL)to examine the effect of pTim-3 on Salmonella typhimurium growth.The results showed that the expression of pTim-3 significantly weakened the inhibition effect of pTLR8 signaling against Salmonella typhimurium,while Tim-3 silence enhanced the anti-infection effect of pTLR8 signaling.These data suggested that immune suppression gene TIM-3 negatively feeds back pTLR8 signaling and damages the anti-infection function of pTLR8 signaling;the finding provided a novel target to enhance the anti-infection function of TLR8 signaling.