Study On NLRP3 Inflammasome Activation Regulated By Bile Acid Receptor Agonists

Bile acids(BAs)function both as detergent to help solubilization of lipids and as hormones through bile acid receptors,including the transmembrane guanosine-binding protein coupled receptor-5(TGR5),the farnesoid X receptor(FXR),the pregnane X receptor(PXR),the constitutive androstane receptor(CAR)and the vitamin D receptor(VDR).Recent studies on BAs show that activation of TGR5 in monocytes and macrophages reduce the phagocytic activity and pro-inflammatory cytokine production,suggesting an immunomodulatory action of BAs via TGR5.Inflammasomes are cytosolic multimeric protein complexes,including the nucleotide-binding domain and leucine-rich-repeat-cotaining protein(NLR)or absent in melanoma 2(AIM2),the apoptosis-associated speck-like protein(ASC)and caspase-1,that control the secretion of interleukin-1(IL-1)family members such as IL-1? and IL-18.The ubiquitination of the NLR family pyrin domain containing 3(NLRP3)acts as an autoinhibitory signal during the activation of NLRP3 inflammasome,while the ubiquitination of ASC is needed for the ASC oligomerization,which is an essential step of the assembly of NLRP3 inflammasome.In toxicology studies,the activation of NLRP3 inflammasome has been considered as a mechanism of being exposed to toxics.Studying into the mechanism of NLRP3 inflammasome activation may help us understand the mechanism of being exposed to toxics better.We found that the primary and the secondary BAs could block the activation of NLRP3 inflammasome.Next we confirmed that TGR5 is responsible for the inhibition of NLRP3 inflammasome activation,via INT777,which is an angonist of TGR5.In addition,H89,which is the inhibitor of the protein kinase A(PKA),could rescue the INT777-induced inhibition of NLRP3 inflammasome activation.Moreover,a remarkable evaluation of NLRP3 ubiquitination casued by INT777 could be observed in wild type mouse macrophages,while INT777 could not induce the ubiquitination of NLRP3 in the TGR5-deficient mouse macropahges.Next,we found that activation of PKA,which is downstream of TGR5,by forskolin induced the ubiquitination of NLRP3,suggesting a crosstalk between phosphorylation and ubiquitination.Then we confirm that NLRP3 could be directly phosphorylated by PKA at Ser 291 through Phos-tag and the in vitro kinase assay.In addition,the S291A mutation impaired the PKA-induced ubiquitination of NLRP3.Finally,we found the relationship between the mutations of Cryopyrin-Associated Periodic Syndromes(CAPS)and the phosphorylation of Ser 291.GW4064 is an agonist of FXR.To our surprise,GW4064 could inhibit the activation of NLRP3 inflammasome independent of FXR.Moreover,we found that GW4064 could inhibit the ubiquintination and the oligomerization of ASC,which is essential for NLRP3 inflammasome activation.In vivo,we found that GW4064 alleviated the NLRP3-related disease models,including peritonitis induced by aluminum salts(Alum)and sepsis induced by lipopolysaccharide(LPS).We have provided new evidence for the mechanism of the xenobiotics toxicity.