Mechanisms Of NIK-mediated Innate Immunity And Inflammation In Grass Carp(Ctenopharyngodon Idellus)

NIK(NF-κB-inducible kinase)belongs to the mitogen-activated protein kinase family,which activates NF-κB and plays an important role in immunity,inflammation,apoptosis and a range of pathological responses.Among NF-κB non-classical pathways,the interplay between NIK and IKKα has been frequently studied in mammals and zebrafish.However,few have explored the relationship between NIK and other subunits of IKK complex.The relationship between IKKβ and NIK,as a splice protein in the classical NF-κB pathway,has never been investigated in fish.There is still a gap in the research on whether NIK is linked to classical NF-κB pathway.In this study,we investigated for the first time how grass carp NIK connects with NF-κB classical pathway in response to viral stimulation,and then regulates the expression of type I IFN,participates in inflammatory response,andinduces apoptosis.the full-length c DNA sequence of grass carp(Ctenopharyngodon idella)NIK(CiNIK)was cloned and identifiedby homologous cloning and RACE technology.The structural domains of grass carp NIK was analyzed by using the Smart Prediction website,and a NIK phylogenetic tree was constructed to analyze the affinities of NIK in each species.The results showed that the full-length of grass carp NIK was 2436 bp,including 5’UTR 386 bp,3’UTR 457 bp,ORF of grass carp NIK was 1593 bp.Grass carp NIK contains an STKc domain,which belongs to serine-threonine kinase.CIK cells were stimulated with GCRV and the expression of NIK was elevated in response to the stimulation of GCRV.Also,after grass carp were stimulated with GCRV,poly(I:C)and LPS,brain,intestine,gill,skin,eye,spleen,kidney,and liver tissues were extracted to detect the m RNA expression level of NIK.The results showed that the expression of NIK was increased in liver,kidney,intestine and spleen of grass carp under all types of stimulation.This suggests that CiNIK can respond to virus,LPS and double-stranded RNA virus analog stimuli.We constructed a grass carp NIK-associated eukaryotic expression vector to further investigate the function of grass carp NIK in cells.To investigate the cellular localization of CiNIK and Ci IKKβ,we performed immunofluorescence localization experiments,which showed that both CiNIK and Ci IKKβ were localized in the cytoplasm.To investigate the relationship between CiNIK and Ci IKKβ,we co-transfected CiNIK-FLAG and Ci IKKB-GFP into grass carp cells and performed immunofluorescence co-localization experiments.The results showed that the cytoplasm of grass carp emitted yellow fluorescence,which making clear the interaction between CiNIK and Ci IKKβ.To further validate this conclusion,we performed immunoprecipitation experiments to verify that CiNIK does interact with Ci IKKβ.Serine threonine kinase has autophosphorylation activity.In this study,we found that the autophosphorylation of CiNIK was enhanced by poly(I:C)stimulation.Overexpression of CiNIK increased the phosphorylation of serine 180 of Ci IKKβ.CiNIK phosphorylation activated p65 protein activity,which in turn activated classical NF-κB pathway.p65 activity changes indicated that NF-κB heterodimer would move from the cytoplasm to the nucleus,and then activate the genes related to innate immunity and antiviral pathway and inflammatory mechanism.To investigate the effect of grass carp NIK on inflammatory factors,we overexpressed grass carp NIK and IKKβ separately,and then co-transfected them to detect the m RNA expression level of grass carp IL-8.We found that CiNIK and Ci IKKβ could synergistically up-regulate the expression of IL-8.When CiNIK and Ci IKKβcoexisted,the expression was higher.When grass carp NIK was knocked down,the m RNA expression level of IL-8 was reduced.Since grass carp NIK can respond to stimulation by viral stimulants,grass carp NIK is likely involved in innate immune antiviral pathway.To test this conjecture,we overexpressed NIK in grass carp cells and found that both the protein level and m RNA level of IFN I were upregulated.Similarly,we overexpressed NIK in grass carp cells and found an increase in the expression of the apoptosis-related gene Bcl-xl and an up-regulation of the m RNA ratio of Bax/Bcl-2.This demonstrated that grass carp NIK was also involved in the apoptotic process.In short,this study cloned and characterized grass carp NIK.We confirmed that grass carp NIK can respond to viral stimulation and discovered that grass carp NIK can interact with grass carp IKKβ.In this paper,we also found that poly(I:C)could enhance the autophosphorylation of grass carp NIK,and overexpression of grass carp NIK could phosphorylate and activate grass carp IKKβ and p65,which in turn activated the NF-κB classical pathway,and regulated the expression of inflammatory factor IL-8 and innate immunity IFN I.Grass carp NIK regulates the apoptotic mechanism,and plays an important roles in the three aspects of immunity,inflammation and apoptosis.