The Effect Of Ubiquitin Ligase Nedd4l On Toll-like Receptor Signaling Transduction Of Macrophage

Innate immunity is the first barrier against the invasion of foreign pathogens.Through recognizing the conservative structure named pathogen associated molecular patterns?PAMPs?of microorganisms by pattern recognition receptors?PRRs?,innate immune cells can quickly be activated and elicit immune responses to clear pathogens effectively.Toll-like receptors?TLRs?are a critical family of membrane receptors of PRRs and different TLRs can recognize different PAMPs.After activation of TLRs,signals can be transmitted by two different signaling pathways,the MyD88-dependent pathway and the TRIF-dependent pathway.Besides TLR3,all TLRs can activate downstream signals through the MyD88-dependent pathway,activating MAPKs?mitogen-activated protein kinase?and NF-?B signaling pathway to produce a variety of inflammatory cytokines and chemokines.TLR3 and TLR4 can activate the TBK1-IRF3 signal through TRIF-dependent pathway to induce type I interferon production.As important signal transduction molecules for recognizing pathogen components,how TLR signaling pathways are activated and regulated is a hot topic of research.Ubiquitination,as a very important post-translational modification of proteins,plays a crucial role in the regulation of TLR signaling.Nedd4l?neural precursor cell expressed developmentally down-regulated 4-like?,also known as Nedd4-2,is an E3 ubiquitin ligase of the Nedd4 family.The most well-known and most widely studied function of Nedd4l is its ability to regulate sodium channels in epithelial cells.Itch and Nedd4 are two E3 ubiquitin ligase of Nedd4 family that can regulate T cell function in adaptive immunity.Itch mediates the ubiquitination and degradation of the transcription factor JunB,thereby reducing IL4 transcription and expression and inhibiting Th2 responses.Nedd4 promotes the ubiquitination and degradation of Cbl-b,a negative regulator of T cell activation,thereby promoting T cell activation and enhancing adaptive immune responses.Although it is highly homologous to Itch and Nedd4 which belong to the E3 ubiquitin ligase family,there is no report about the regulation of innate immune responses by Nedd4l.Therefore,we aim to investigate the role of Nedd4l in regulating innate immune responses,especially the TLR mediated signaling.We first studied the effects of Nedd4l on the production of type I interferon and pro-inflammatory factors.Primary peritoneal macrophages of control wild-type mice(Nedd4l^+/+)and Nedd4l deficient mice(Nedd4l^-/-)were isolated and stimulated with TLR4specific agonist LPS.The deficiency of Nedd4l significantly attenuated the mRNA and protein levels of type I interferon IFN-?and pro-inflammatory factors IL-6 and TNF-?induced by LPS,indicating that Nedd4l has a positive regulatory effect on TLR4-mediated innate immune function in macrophages.Determining Nedd4l has a regulatory effect on the TLR response,the next step is to detect changes of the associated signaling pathways.We used LPS to stimulate primary peritoneal macrophages of control wild-type mice(Nedd4l^+/+)and Nedd4l deficient mice(Nedd4l^-/-)and found that Nedd4l does not affect NF-?B p65 and ERK1/2,p38 and JNK of MAPK signaling pathway.However,the deficiency of Nedd4l inhibits the phosphorylation of TBK1 and IRF3,and Nedd4l promotes the formation of TRAF3/TBK1 complex,suggesting that Nedd4l promotes TRAF3-dependent TLR signaling.It was further found that Nedd4l can directly bind TRAF3,and the WW domain of Nedd4l is the essential domain for binding to TRAF3.In addition,the deficiency of Nedd4l also significantly reduced the level of LPS-induced c-Rel in macrophages.It has been reported that TRAF3is involved in the regulation of c-Rel expression in macrophages,which is consistent with our results that Nedd4l regulates TRAF3-mediated signaling.Since ubiquitin ligase Nedd4l can interact with TRAF3,we hypothesized that Nedd4l enhances TLR signaling transduction by promoting ubiquitination of TRAF3.Deficiency of Nedd4l significantly reduced LPS-induced ubiquitination of TRAF3 in macrophages,including ubiquitination at K48 and K63,indicating that Nedd4l enhances ubiquitination of TRAF3.This conclusion was further confirmed by in vitro ubiquitination experiments.Deletion of the WW domain of Nedd4l,and deletion or mutation of the HECT domain disrupt the enzymatic activity of Nedd4l and so eliminate Nedd4l-mediated ubiquitination of TRAF3.The deletion of the C2 domain increases Nedd4l-mediated ubiquitination of TRAF3,consistent with the previous report that the C2 domain can inhibit its enzymatic activity.By using ubiquitin mutants with different lysine residue mutated,we found that the direct ubiquitination modification of TRAF3 by Nedd4l is K29-linked ubiquitination,rather than K48 or K63-linked ubiquitination.Taken together,we hypothesized that Nedd4l catalyzes the K29-linked ubiquitination to promote ubiquitination of TRAF3 at K48 and K63.Next,we discovered the ubiquitination of several sites of TRAF3 including lysine residues K273,K315 and cysteine residues C56 and C124 were affected by Nedd4l through LC-MS/MS.It was verified by experiments that the C56 and C124 residues are the ubiquitination sites of the K29 linked ubiquitin chain catalyzed by Nedd4l.In addition,mutations in C56 and C124 promoted ubiquitination of TRAF3 and increased expression of the NF-?B and IRF3 luciferase reporter genes.Taken together,the above results demonstrate that Nedd4l promotes TRAF3-mediated signal transduction by catalyzing K29-linked C56 and C124 ubiquitination of TRAF3.In summary,our study found for the first time that the E3 ubiquitin ligase Nedd4l has a regulatory role in the innate immune responses.Nedd4l promotes the production of type I interferons and pro-inflammatory cytokines via TLR signaling in innate immunity by catalyzing K29-linked cysteine residues ubiquitination of TRAF3.This study not only confirmed that Nedd4l is a key regulator of innate immunity,but also revealed a more complex regulatory mode of TRAF3 ubiquitination and the significance of cysteine ubiquitination in innate immunity.To investigate whether ubiquitination of cysteine is also involved in other molecules of pattern recognition receptor signaling will bring new insights into the regulatory mechanisms of innate immunity and will also provide new targets and methods for anti-infective immunotherapy.Nedd4l is closely related to the development of many tumors.In some studies,Nedd4l was found as a tumor suppressor,but some studies have also reported that Nedd4l can promote tumor progression.Therefore,the role and mechanism of Nedd4l in tumors needs further study.In tumor tissues,tumor-associated macrophage?TAM?accounts for the largest number of innate immune cell in the tumor microenvironment.It can differentiate into different types and acquire different functions when stimulated by different signals in the microenvironment.The M1 type exerts an anti-tumor effect,while the M2 type has a tumor-promoting effect.The dual role of macrophages in tumors suggests significant phenotypic heterogeneity,plasticity and a diversity of functions.Therefore,to explore the mechanism of macrophage polarization,and inducing the polarization of M2 TAM to M1TAM may provide new targets for tumor immunotherapy.Previous studies have shown that ubiquitination is involved in the regulation of immune cell differentiation,and ubiquitin ligase can regulate the polarization of macrophages.Therefore,we explored whether the E3 ubiquitin ligase Nedd4l also plays a role in macrophage polarization and tumor development.We stimulated immortalized bone marrow-derived macrophages?BMDM?originally separated from Nedd4l deficient mice(Nedd4l^-/-)and their littermate control wild-type mice(Nedd4l^+/+)to M1 or M2 macrophage.And the mRNA levels of their associated marker molecules were detected.The mRNA expression of iNOS and IL12?M1 related genes?decreased with Nedd4l deficiency,while the mRNA expression of Arg-1,IL10,Fizz1 and Ym1?M2 related genes?increased,indicating that Nedd4l can promote the polarization of M1 macrophages.Then,we inoculated melanoma cells to the myeloid cell-specific Nedd4l conditional knockout mice(Nedd4l^fp/fpLyz2-Cre^+/-)and its control mice(Nedd4l^fp/fpLyz2-Cre^-/-),and observed the effect of Nedd4l in macrophages on tumor growth.The tumor in control mice grew slower than that in Nedd4l conditional knockout mice,however,there was no statistical differences between the two groups.How Nedd4l affects macrophage polarization and tumor development needs to be further explored in the future.