The Mechanism Of Synergistic Effect Of WISP1and TLRs Agonist On Inflammation In Macrophage

Background:WISP1（WNT1inducible signaling pathway protein1, CCN4） gene is a targetof canonical Wnt/β-catenin pathway. WISP1is a secreted protein, a member of theCCN family. CCN proteins are involved in multiple physiological and pathologicalevents as an extracellular sensor via a cell signalng pathway by binding integrinsreceptor. Integrin signaling activation plays an important role in acute lung injury.Our previous study identified that WISP1was one of the significant single nucleotidepolymorphisms in lung injury and showed macrophages played a vital role in theprocess.Objective:This study is aimed to uncover the mechanism of WISP1in the inflammatoryassociated with TLRs signaling transduction in macrophage.Methods:Macrophage cell line RAW264.7and primary macrophage isolated fromwild-type or TLR4or CD14or TRIF mouse peritoneal cavity were used for all assaysin this study. Recombinant WISP1protein was used to stimulate macrophage. LPSwas used as TLR4agonist and Poly I C was used for TLR3agonist. RGDs peptidecan efficiently bind to integrins used as a competitive inhibitor for WISP1-Integrininteraction. Macrophages were treated with WISP1or LPS or Poly IC alone orcombination with/without RGDs inhibitor. Cell culture media were then analyzed forTNF-α production by ELISA assays. Cells were collected for signaling pathwayactivation assays by Western blot using corresponding antibodies. WISP1effect onTLRs assembling on the lipid raft was conducted by isolation of lipid raft byultracentrifugation followed by Western bloting assays on fractions. The associationof integrins expression and TLRs signaling activation was analyzed by Western blotusing macrophage from wild-type and gene knock-out mouse. Results:1).WISP1treatment triggered phosphorylation of p38MAPK and NF-κB(p65)and secretion of TNF-α on RAW cells(P<0.01). In contrast, the pretreatment of RAWcells with RGDs to block integrin ligation decreased the level of TNF-α (P<0.05).2).WISP1-Integrins ligation promoted MD2recruted to the lipid raft. TLRsagonist (LPS/PolyI:C) enhanced phosphorylation of AKT, p38MAPK, NF-κB（p65）and upregulated expressions of β3and β1integrin of peritoneal macrophages. Theseeffects were inhibited in macrophage derived from TLR4or CD14or TRIF knock-outmouse model.3).There is a synergistic mechanism between TLRs agonist and WISP1inincresing TNF-α production mediated by integrin signalng pathway. Blockingintegrin signalng pathway by incubating macrophage with RGDS, the synergisticeffect of WISP1and TLRs agonists was lost.Conclusion:By ligation with integrins on the macrophage, WIPS1activates p38MAPK andNF-kB signaling pathways which lead to inflammatory cytokines expression andsecretion. In a similar manner, WISP1indirectly enhances TLRs receptor complexassembling on the lipid raft via interaction with integrins and consequentlystrengthens TLRs signaling activation. On the other hand, TLRs signaling activationupregulate integrins expression which positively facilitates WISP1-Integrin signalingpathway. This coupled-signaling events forming a positive regulation loop regulatesWISP1synergistic effect on inflammation in macrophag...