Roles And Mechanisms Of CUL4B In Regulating Toll-like Receptor-triggered Proinflammatory Responses

Innate immunity is the first line to defense against invading pathogens Macrophages are responsible for the rapid recognition and elimination of pathogen Pathogen recognition by the innate immune system is actually specific,relying on germline-encoded pattern-recognition receptors(PRRs)that have evolved to detect components of foreign pathogens referred to as pathogen-associated molecular patterns(PAMPs).Upon PAMP recognition,PRRs initiate a serious of signaling programs for killing infectious microbes.Toll like receptor(TLR),which is an evolutionarily conservative PRR,plays an important role in the recognition and elimination of microbial pathogens.Different TLRs recognize PAMPs from different microbial pathogens.After recognition of PAMPs,TLRs trigger of downstream signaling cascades leading to the activation of transcription factor nuclear factor-kappaB(NF-?B),which controls induction of chemokines and proinflammatory cytokines to initiate innate immune responses Therefore,TLRs have a critical role in innate immune responses.However,aberrant TLR signal can also lead to aberrant inflammatory response and many immune related diseases.One of them is sepsis,which is a pathological syndrome characterized by persistent overinflammation and immunosuppression.It has a high incidence rate and mortality.Although the current understanding about the pathogenic process of sepsis has greatly increased,it is still lacking effective treatment for sepsis.Therefore,it is important to explore the underlying mechanism of TLR signaling pathways to avoid aberrant regulation of TLR signaling.Cullin 4B(CUL4B)functions as a scaffold protein in the Cullin-4B Ring E3 ligase complex(CRL4B)and participates in regulating diverse physiologically and developmentally controlled processes by targeting specific substrates for ubiquitin-dependent degradation or modification.Mutations in human CUL4B are a common cause of X-linked mental retardation syndrome.In addition to being mentally retarded,patients with CUL4B mutations also have an elevated monocyte count.It is suggested that CUL4B may play a role in regulating the function of monocyte or macrophages.Recently,we showed that CRL4B complex functions a transcriptional coreprssor by catalyzing H2AK119 monoubiquitination and coordinating with PRC2 to epigenetically inactivate the transcription of many tumor suppressors and thus promote tumorigenesis.In contrast to its oncogenic role in cancer cells,CUL4B in the hematopoietic system functions to impede tumor progression by restricting the accumulation of myeloid-derived suppressive cells(MDSCs).These results indicate that CUL4B is involved in epigenetic regulation.At present,epigenetic modification has been shown to play an important role in the infections and innate immune response caused by pathogens.At the same time,with the improved understanding of the TLR signal regulation network,the role of epigenetic modification for TLR-induced genes has gradually emerged.However,the role of CUL4B as a transcription repressor in TLR-mediated innate immune responses is unclear.Based on the above facts,the purpose of this study is to investigate the role of CUL4B in TLR-mediated innate immune responses using a variety of mouse infection modelsPart I CUL4B deficiency potentiates TLR-mediated immune responsesIn this part,we use pathogen induced mouse disease model to explore the roles of CUL4B in the TLR mediated innate immune response.We have achieved the following results1.We generated myeloid-specific Cul4b knockout mice(referred to as MKO mice)by crossing Cul4b-floxed mice with LysM-driven transgenic mice.Analysis by flow cytometry showed that the percentage of the macrophages,neutrophils,and dendritic cells in bone marrow and spleens of the MKO mice were comparable to those in wild-type mice.2.WT and Cul4b-deficient BMDMs were administrated with ligands of TLR and the transcription of proinflammatory cytokines were examined.We found that the mRNA expression levels of Tnf?,Ill?,116 and Ifn? were significantly increased in Cul4b-deficient macrophages treated with TLR2/3/4 ligands.Consistent with their mRNA expression,ELISA showed that deletion of CUL4B significantly increased the TLR2/3/4 ligand-induced secretion of TNF-?,IL-1?,and IL-6.These results suggest that CUL4B negatively regulates the TLR2/3/4-mediated production of proinflammatory3.To determine the role of CUL4B in the TLR4-mediated immune response in vivo.WT and MKO mice were intraperitoneally injected with LPS or S.typhimurium,and the serum levels of proinflammatory cytokines were measured.We found that the serum levels of TNF-?,IL-6,and IL-1? were higher in Cul4b MKO mice than in control mice.Consistent with increase in cytokine production in the sera,Cul4b MKO mice died earlier and had lower survival rates than control mice when challenged with LPS or S.typhimurium4.We then challenged MKO mice with poly(I:C)in vivo.Compared to WT mice,Cul4b deficient mice produced significantly higher levels of IL-6,TNF-? and IFN? in the serum and developed a more severe innate inflammatory response after being challenged with poly(I:C)Taken together,these data indicated that the deletion of Cul4b aggravated TLR3/4-induced or bacteria-induced septic shock.Therefore,CUL4B negatively regulates innate inflammatory responsesPart II CUL4B regulates TLR2/3/4-mediated immune response by inhibiting GSK3? activityIn the first part,we showed that the deletion of CUL4B aggravated TLR2/3/4-triggered immune responses.The underlying mechanisms remain to be determined.GSK3? has been shown as a key molecule in the production of proinflammatory cytokines in various TLR signaling pathways,and inhibitors of GSK3? can effectively inhibit the production of proinflammatory cytokines and promote the production of anti-inflammatory factor such as IL-10.Although these studies clearly demonstrate the importance of GSK3? in TLRs-mediated cytokine production,little is known about the regulatory mechanisms of GSK3? activity in TLR-mediated immune responses.Does CUL4B directly regulate GSK3? in TLR mediated inflammatory response?We did the following analyses1.We first examined the effects of CUL4B deletion on GSK3 ? activity,and found that the levels of GSK3? phosphorylated at Ser9,reflecting GSK3? inactivation,were significantly reduced in LPS stimulated Cul4b-deficient BMDMs.These results indicate that increased GSK3? activity may response for the enhanced production of TLR4-triggered proinflammatory cytokines in Cul4b MKO mice2.Similar to the LPS-triggered response,the level of GSK3? phosphorylated at serine 9 was decreased in poly(I:C)-triggered or PGN-triggered MKO BMDMs.These results indicate that increased GSK3? kinase activity may mediated the enhanced t TLR2/3-mediated immune response in Cul4b MKO mice3.Pretreatment with the GSK3? inhibitor SB216763 efficiently blocked increased TLR3/4-mediated cytokine production and increased the survival rate of LPS and poly(I:C)-challenged MKO mice.Together,these results suggest that CUL4B negatively regulates TLR3/4-mediated inflammatory responses via regulating GSK3?activity4.GSK3? has been shown to positively regulate TLR-triggered NF-?B activity,and suppress the activity of CREB.We next examined the transcription activity and the key molecules in these signaling pathways.We observed increased transcription activity of NF-?B and the expression level of related signal proteins in TLR2/3/4 ligands-treated Cul4b-deficient macrophages.In contrast,the levels of phosphorylated CREB and CREB activity were reduced in TLR2/3/4 ligands-treated Cul4b-deficient macrophages.Correspondently,the levels anti-inflammatory factor IL-10 were reduced in TLR2/3/4 ligands-treated Cul4b-deficient macrophages.These results indicate that CUL4B play a regulatory role in TLR2/3/4-mediated inflammatory response by suppressing the production of proinflammatory cytokines and promoting anti-inflammatory cytokine production.In conclusion,CUL4B regulates TLR2/3/4-mediated innate immune response by inhibiting GSK3? activity.Part III CUL4B negatively regulates TLR2/3/4-mediated immune response by repressing Pten transcriptionIn the second part we showed that CUL4B regulates TLR2/3/4-mediated immune response by inhibiting GSK3? activity.We next determined the mechanism by which CUL4B regulate GSK3? activity.1.As serine 9 of GSK3? is phosphory lated by AKT,we next determined the level of AKT phosphorylated at Thr308 and Ser473,which indicates maximal AKT activity.We found that the levels of phosphorylated AKT were significantly decreased in LPS stimulated,poly(I:C)-stimulated,or PGN-stimulated Cul4b-deficient macrophages,indicating that the decreased GSK3? phosphorylation at ser9 is due to decreased AKT kinase activity.2.The decrease in the level of AKT phosphorylation could be attributed to the decreased activities of kinases upstream of AKT or increased activities of phosphatases that dephosphorylate AKT.While no significant difference in the levels of AKT kinases and phosphatases was detected,PTEN,a negative regulator of AKT,was found to be significantly elevated in Cul4b deficient macrophages.Further analysis indicated that CUL4B regulated Pten at transcriptional level.3.The chromatin immunoprecipitation(ChIP)assay indicated that CUL4B binds the Pten promoter region.Importantly,we detected decrased binding of CUL4B to the Pten promoter during TLR-triggered responses,which is consistent with increased Pten expression during TLR agonist stimulation.Furtheremore,deletion of CUL4B significantly decreased the occupancy of CUL4B,EZH2,H2AK119ub1 and H3K27me3,but increased the level of H3K4me3 on the Pten promoter region.These results indncate that CUL4B complex epigenetically represses Pten transcription by coordinating with PRC2 complex.Taken together,these results indicate that the CUL4B complex represses the transcription of Pten,which inhibits AKT and thus restrains GSK3 ? activity.Our results reveal a CRL4B-based epigenetic mechanism by which TLR-mediated immune responses are modulated and may have implications for the development of novel anti-inflammatory strategies to manage infections and other diseases.