D182 Blocks TLR4-elicited Inflammatory Responses Via IRAK4

Inflammation is a host response to traumatic,infectious,post-ischaemic,toxic or autoimmune injury that leads to the release of a large amount of inflammatory mediators.Excess production of these inflammatory mediators is involved in many diseases including rheumatoid arthritis,asthma,pulmonary fibrosis,type 2 diabetes mellitus,atherosclerosis,and cancer.Toll-like receptors(TLRs)are a type of pattern recognition receptors implicated in the defense system against pathogens.They recognize pathogen-associated molecular patterns(PAMPs)molecules traumatic,infectious,post-ischaemic,toxic or autoimmune injury,which are broadly shared by pathogens but distinguishable from host molecules.All TLRs activate through different signal pathways,inducing the production of proinflammatory cytokines or eliciting a type I interferon response.Although full activation of TLR responses is necessary for the elimination of inflammtion to invade pathogens,excessive inflammatory activation through TLR signaling may promote the development of immuno-pathological conditions such as infectious disease and autoimmune disease.One of the TLRs,TLR4,engaged by LPS found on Gram-negative bacteria,orchestrates the immunity response on certain monocytes and macrophages.It is the only known TLR able to activate both MyD8 8-dependent induction of genes encoding inflammatory molecules and TRIF-dependent production of type I interferon.When in excess,LPS/TLR4 signaling leads to serious systemic disorders with a high mortality rate,for example,during sepsis,septic shock,or systemic inflammatory response syndrome.Sepsis remains a critical problem,with significant mortality and morbidity despite intense efforts to find effective therapies.The prevailing theory of sepsis is an unregulated inflammatory response or cytokine storm resulting in organ damage and death.Thus,suppression and/or inhibition of the above-mentioned signaling molecules may have great potential for anti-inflammatory or anticytokine therapies in sepsis.Our group recently found that D182 inhibited the expression of LPS-challenged NO production in murine macrophage cell line RAW264.7.The data has been suggested that D182 could be applied to inflammatory lesions to modulate the inflammatory response and alleviate spesis.In our present study,we demonstrated that D182 shows protective effects in two murine models that D182(ⅰ)efficiently protected the mice from spesis death(ⅱ)attenuated multi-organ inflammatory damage(ⅲ)modulated the production of such proinflammatory cytokines and chemokines as TNF-α,IL-6,MCP-land MIP-la and(ⅳ)decreased the generation of bacteria in peritoneal and blood.Next we selected murine macrophage cell line RAW264.7 and primary peritoneal macrophages to evaluate D182 pharmacological effects and revealed the underling mechanisms.On the production of inflammatory mediators in LPS-induced macrophages,CCK-8 and Elisa tests confirmed that 5-45 μM D182 significantly reduced the production of TNF-α,IL-6 and MCP-l,without any cytotoxic effect on these cells.Using flow cytometric technique,we confirmed that D182 didn’t affect LPS binding to macrophages.Based on TLR4 signal pathway theories,we analyzed TLR4,MyD88,IRAK4,TRAF6 proteins in this pathway by Docking,which is a method predicting the preferred orientation of one molecule to D182 when binds to each other to form a stable complex.we found that D182 could bind to the active center of IRAK4 and the interaction energy between D182 and IRAK4 was 31.07.Then the molecular dynamics simulations and interaction energy calculations were both performed to show theoretically the binding strength of IRAK4(the protein data bank code:2NRY)with the ligands of D182.The total interaction energies,van der Waals energies and electrostatics energies all confirmed that 2NRY…D182 complex interaction energies were so strong.Furthermore,D182 was located in the active loops formed by ILE22,MET29,VAL37,ALA48,VAL80,TYR96,TYR98,MET99,GLY102,and LEU 152.There were hydrophobic interactions between the side chains of these residues and the ligands.The D182…2NRY interaction involved hydrogen bonding interactions between the oxygen in the methoxy group of D182 and the hydrogen in the peptide group of MET99 of 2NRY.The average time evolution of the distances(A)between the oxygen in the methoxy group of D182 and the hydrogen in the peptide group of MET99 of 2NRY was 2.14 A.These results indicated that the interaction between 2NRY and D182 was strong.Sequentially,a direct binding assay using surface plasmon resonance ascertained the stoichiometry,binding affinity and kinetics for the interaction of our compound with the binding groove of IRAK4.In the experimental set-up used with the BIAcore T200,sensorgrams and steady state curves indicated that a compound binds to a target protein..In particular,the activities of D182 on IRAK4 in cells were assessed.Immuno blot showed that D182 significantly reduced IRAK4 phosphorylation,blocked the degredation of IRAKI and TRAF6.To further elucidate the molecular mechanisms of D182 inhibition on IRAK4,the activations of NF-κB and MAPKs ’down-stream’ the IRAK4 were investigated.Pre-exposure of RAW264.7 cells to D182 resulted in the rapid inhibitory effect of the LPS-mediated phosphorylation and degradation of IκBα at 15 min,inhibited P65 phosphorylation and translocation into the nucleus.NF-κB luciferase construct clearly inhibited luciferase activity by D182 on LPS-induced NF-κB activation.Although D182-treated macrophages showed diminished activation of P38 compared with LPS-only macrophages,activation of JNK and ERK1/2 was minimally affected.Accordingly,AP-1 luciferase reporter gene assay appeared to be differentially expressed in D182-pertreated cells,and it confirmed that only in high dose treated group(45 κM),D182 significantly impaired AP-1 activation in LPS-stimulated RAW264.7 cells.Last to determine D182 inhibiting IRAK4 activity in MyD88-dependent TLR signaling,murine macrophage cell line RAW264.7 was treated with the TLR4 agonist LPS.mRNA expression for MyD88-dependent genes TNF-α,IL-6 and MCP-1 in LPS-treated macrophages was significantly high compared with the D182 treated cells.Then we analysed whole-genome-widely for the genes that account for the effect of D182 in LPS-stimulated RAW264.7 cells,using a Agilent 4 × 44 K Mouse Whole-Genome oligonucleotide microarray.D182 significantly inhibited LPS-induced NO production and iNOS expression in RAW264.7 cells.The results of the microarray analysis showed that 4,570 genes were differentially expressed between "C"(Control),"L"(LPS-only)and "D"(D 182 pretreated+LPS)groups.To further understand the biological relevance of these gene expression profiles,we carried out bioinformatic analyses to determine the Gene Ontology(GO)terms and pathways most significantly associated with these genes.The main GO categories in biological progress(BP)were related to innate immune response,inflammatory response,immune response,defense response to virus,cell cycle,cell surface receptor signaling pathway,cellular response to interferon-beta,defense response to protozoan,response to virus,positive regulation of cell proliferation,negative regulation of viral genome replication,cellular response to lipopolysaccharide,elevation of cytosolic calcium ion concentration,and defense response to bacterium.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis revealed the enrichment of a variety of processes of D182 physiology represented mostly by immunity pathways,such as TLR signaling pathway,NF-κB signaling pathway,JAK-STAT signaling pathway,cytokine-cytokine receptor interaction,cell adhesion molecules,herpes simplex infection,herpes simplex infection,etc.Such GO and KEGG pathway analyses results were in accordance with our purpose,and then we focused on genes belonging to categories of macrophage-mediated immunity.The system allowed us to pin down the genes related to immune response.Once again,hierarchical clustering was used to focuse on the gene differentially expressed in BP categories.As a result,87 genes could be truly the representatives accounting for the intrinsic susceptibility to LPS and its induced reversal in the RAW cells.This simple dichotomy classification further allowed us to analyze the differentially expressed genes for their gene ontology and coexpression with ease and confidence.Focusing on the gene categories,we identified the major cellular processes involved the reversed susceptibility in the LPS-induced cells.Prominently,these include 13 genes as "key regulatory" genes:Mtor,B4galtl,Jmjd6,Dock2,Fzd4,Mdm4,IRAK4,Ngf,Defa-rsl,Vegfa,Rps6ka4,Ajapl,Clrb.We then mapped all the "key regulatory"genes on KEGG pathways from the database,and a clear signal pathway picture emerged.With D182 pre-treatment,up-reuglated genes,down-reuglated genes and genes highly related to key regulatory genes are all presented in this picture,giving us strong suggestions to find novel regulatory genes and possible signal transduction pathway.So more deep-going work should be done to confirm such hypothesis.Lastly,in an application of bimolecular fluorescence complementation(BiFC)to visualize protein-protein interactions(PPIs)during drug-elicited processes,we compared V150L,V150A,I152L,L201V and L207V mutations in Venus using the interaction between the basic region-leucine zipper(bZIP)domains of Jun(bJun)and Fos(bFos)in the nucleus as a model system.I152L,V150L and V150A mutations were introduced to Venus N-terminal(VN)respectively.And V150A and I152L double mutation in VN155 at the same time.To Venus C-terminal(VC)fragment,L201V,L207V and the double mutations were introduced repectively.Each mutation fragment was coexpressed with wild-type VN or VC fragment in COS-1 cells to examine their self-assembly.The fluorescence intensity derived from the self-assembly was signifcantly reduced with all these mutations,while L207V mutation in VC fragment had the higher intensity of fluorescence than I152L in VN.It suggested that these substitutions could decrease the affinity between the N-and C-terminal fragments,which may lead to increasing the S/N ratio between proteins fused to the fragments.We also examined the BiFC signals of the Venus variants.We used transcription factors,bJun and bFos,known to form a hetero dimer,as positive control,and bJun and AbFos,lacking the carboxy-terminal half of bFos bZIP,as negative control for the PPIs in BiFC assay.The results suggested that point mutations of I152L and L207V effeciently reduced the non-specific fluorescent intensity and the S/N ratios of these two variants were much higher than wild type.The third we compared the two pairs of N-and C-terminal fragments VN155/VC155 and VN210/VC210,using the same model system.And VN210/VC210 fragment pair could not produce better S/N ratio improvement in BiFC assay.In summary,D182 rescued mice from lethal sepsis.Its mechanism might involve a down-regulation of inflammatory mediators production via IRAK4.Thus,D182 could be a useful therapeutic candidate for the treatment of sepsis and other inflammatory disorders.Microarray assay revealed that D182 possessing anti-inflammatory properties was involved in the regulation of immunity and defense related genes that act as key mediators in various inflammatory processes.Moreover in the same cell model,we first compared reported mutations that could specifically reduce the spontaneous self-assembly of the two nonfluorescent fragments in the Venus-based BiFC assay.Here we identified that I152L and L207V effeciently reduced the non-specific fluorescent intensity and the S/N ratios of these two variants were much higher than wild-type.Besides,VN210/VC210 fragment pair could not produce better S/N ratio improvement in BiFC assay.It might help to clarify our understanding of drug action and enhance the productivity of drug-discovery research.