Regulation Mechanisms Of The Miichthys Miiuy EIF3k And Ackr4a-mediated Autophagy In NF-κB Signaling

The immune system is generally divided into innate and acquired immunity,with most organisms relying primarily on innate immune mechanisms for survival.The innate immune system is a defence mechanism that develops within an organism and protects the host from invading pathogens by recognising the molecular structure of microorganisms through pattern recognition receptors(PRRs)and responding rapidly to them.Under normal physiological conditions,the inactive form of NF-κB dimer is sequestered into the cytoplasm by interacting with IKB inhibitory proteins(IκBα,IκBβand IκBε),and bacterial stimulation activates Toll-like receptors(TLRs)to mediate the NF-κB signaling pathway.As NF-κB activates and enters the nucleus,it induces the synthesis of cytokines and chemokines downstream of innate immunity to exert its antimicrobial activity and maintain systemic homeostasis.The clearance of bacteria by innate immunity constitutes a selective evolutionary pressure on bacteria to acquire evasion of immune attack.Autophagy is considered to be one of the host’s strategies to combat bacterial invasion,assisting the body to eliminate pathogenic bacteria and playing an important role in the resistance of eukaryotes to bacterial invasion.Normally,cells aim to remove invading extracellular bacteria and colonised bacteria from the cytoplasm through autophagy,but some bacteria are able to ’hijack’ autophagy to complete their self-growth and reproduction.As evolution has progressed,almost all innate immune systems,such as the traditional PRRs and inflammatory vesicles,have been combined with autophagy.Research on immune autophagy is still in its infancy and there are still many interesting puzzles and important questions waiting to be addressed.By investigating the involvement of autophagy in the regulation of the NF-κB signalling pathway,this study aims to explore the way autophagy regulates innate immunity during bacterial invasion and provides new insights into the impact of autophagy on fish-bacteria interactions.1.e IF3 k target My D88 for autophagic degradation and inhibit NF-κB signalingOptimal activation of NF-κB signaling is crucial for the initiation of inflammatory responses and eliminating invading bacteria.Bacteria have likewise evolved the ability to evade immunity;however,mechanisms by which bacteria dysregulate host NF-κB signaling are unclear.In this study,we determined the Co-IP product of My D88 by label free proteomic sequencing technology and screened the eukaryotic translation initiation factor e IF3 k that negatively regulates My D88.As a member of the eukaryotic translation initiation factor e IF3 family,e IF3 k not only performs the function of initiating translation,but also inhibits NF-κB signaling.The results of Luciferase assay and RT-q PCR showed that e IF3 k effectively inhibited the Luciferase reporter gene activity and m RNA level of inflammatory factors(IL-6,IL-8 and TNFα)and nuclear transcription factors(NF-κB)triggered by V.harveyi infection.It was also found that e IF3 k enhanced the ubiquitination of My D88 by E3 ubiquitin ligase Nrdp1.In addition,we determined that e IF3 k mediated My D88 degradation through autophagy,V.harveyi infection induced autophagy key gene ATG5,and ATG5 formed a complex with e IF3 k to induce autophagy.Mechanistically,we show that e IF3 k expression induced by V.harveyi enhances E3 ligase Nrdp1-mediated K27-linked ubiquitination of My D88,an upstream regulator of NF-κB pathway activation.And e IF3 k acts as a bridge linking ubiquitin-tagged My D88 and ATG5.Then My D88-e IF3k-ATG5 complex is transported to the autophagosome for degradation,and that innate immune signaling is subsequently terminated and does not attack invading V.harveyi.Therefore,our study identifies e IF3 k as a specific inhibitor of the My D88-dependent NF-κB pathway and suggests that e IF3 k may act as a selective autophagic receptor that synergizes with ATG5 to promote the autophagic degradation of My D88,which help V.harveyi to evade innate immunity.We conclude that V.harveyi can manipulate a host’s autophagy process to evade immunity in fish,and also provide a new perspective on mammalian resistance to bacterial invasion.2.ackr4 a induced autophagy to inhibited NF-κB signaling and blocked apoptosis to facilitate Vibrio harveyi infection.In the face of bacterial infection,the host destroys the invading pathogen in many ways.Autophagy and apoptosis are two recognized mechanisms of resistance to bacterial invasion.However,in the process of coevolution with the host,bacteria have also evolved the ability to evade immunity and use the host defense mechanism to achieve self replication.In this study,we used transcriptome sequencing to screen the differential genes before and after V.harveyi infection,and identify ackr4 a,an atypical chemokine receptor 4 family member,involved in innate immunity.The results of Luciferase assay and real-time quantitative PCR showed that ackr4 a effectively inhibited the Luciferase reporter gene activity and transcription level of inflammatory factors(IL-1β and TNFα)and nuclear transcription factors(NF-κB)triggered by V.harveyi infection.ackr4 a upregulated Beclin-1 and induced autophagy targeted My D88 degradation in the form of ackr4a-beclin-1 complex,and V.harveyi infection enhanced the binding of ackr4 a to Beclin-1.It was also found that ackr4 a could effectively block apoptosis and promote V.harveyi infection.In the early stage of apoptosis,the autophagy pathway induced by ackr4 a was enhanced and the key genes of apoptosis(Caspase 7 and Caspase 8)were inhibited.In addition,ackr4 a acts as the transcription target of AP-1.When V.harveyi is infected,the up-regulated AP-1 promotes ackr4 a transcription and induces autophagy.Mechanistically,V.harveyi-induced Ap-1activates ackr4 a transcription and expression.On the one hand,ackr4 a forms a complex with Beclin-1 and My D88,respectively,inducing autophagy and transporting My D88 into the lysosome for degradation to suppress inflammatory cytokine production.On the other hand,ackr4a-induced autophagy blocks apoptosis by inhibiting Caspase8.This study shows that V.harveyi can manipulate host’s autophagy to evade immunity,suggesting that V.harveyi has evolved a novel ability against immunity in fish.