NF-κB Mediates Intestinal Inflammation In Artemia Sinica And Function Of CK2 In Immune Response Of Artemia

Host defense is known to mainly rely on innate immunity in invertebrates.The intestinal epithelium and the body surface epidermis are the first line of defense in the organism’s physiological barrier.After breaking through the first line of defense,the body’s innate immune response will be triggered.The nuclear factor-κB(NF-κB)family is an important transcription factor involved in the innate immune responses,regulating the transcription of cytokines and antimicrobial effectors as well as cellular differentiation,survival and proliferation.Recent work has highlighted the importance of NF-κB in the maintenance of epithelium barriers.NF-κB activation actively contributes to intestinal inflammation,while inhibition of NF-κB activation specifically in the intestinal epithelium causes severe intestinal inflammation.However,the molecular mechanisms of NF-κB regulation remain largely unknown.CK2,a highly conserved protein serine/threonine kinase,is ubiquitously distributed in eukaryotes.Some studies suggest that protein kinase CK2 is involved in the activation of NF-κB signaling pathway.It has structural activity and has been implicated in multiple cellular functions,such as cell cycle,apoptosis,and inflammation.To explore the mechanisms of NF-κB and CK2 in the innate immune system of crustaceans,the role of NF-κB in intestinal inflammation from the high-salt environment crustacean Artemia sinica was explored and the expression pattern and function of CK2 in two brine shrimps from different reproductive modes was analyzed.The main findings are as follows:1.As NF-κB sequence was identified from the transcriptome of A.sinica,with a typical N-terminal Rel homology region(RHD)to binding DNA and homo/heterodimerization.It contained an open reading frame(ORF)of 3066 bp,encoding 1021 amino acids.The expression of As NF-κB during embryonic development and bacterial challenge were studied by real-time quantitative PCR(q RT-PCR).As NF-κB levels in different developmental stages decreased from nauplius to adulthood.The expression level of As NF-κB was increased in the bacterial infection of Micrococcus lysodeikticu,Vibrio harveyi,and Vibrio anguillarum but decreased in the stimulation of Vibrio parahaemolyticus.When SN50 was employed to suppress NF-κB activation,cytokine interleukin 17(IL17)and mannose receptor C type(MRC)were inhibited.These data suggest that the NF-κB-regulated host defenses against pathogen infections and facilitation of IL17 and MRC expression during stimulation with different bacteria.2.Intestinal inflammation was induced by dextran sulfate sodium,and As NF-κB activity was inhibited in an intestinal injury model.In intestinal injury,IL17,MRC,ABF1/2,serpin B13,and β-arrestin significantly decreased with As NF-κB inhibition,whereas Lysozyme increased.In intestinal homeostasis,blocking of As NF-κB expression led to extensive damage to the intestinal epithelium and extensive blood cell infiltration in the lower layer.In the DSS-induced intestinal inflammation,high levels of As NF-κB activation resulted in less pervasive intestinal damage and the maintenance of intestinal barrier integrity.Intestinal injury robustly increased the expression of IL17.As NF-κB activation was enhanced by IL17 deficiency in the intestinal injury model.IL17 inhibition aggravated intestinal inflammation,leading to loss of epithelial architecture and the infiltration of inflammatory cells.These data indicate that NF-κB and IL17 play key mediator roles in the maintenance of gut epithelial integrity and immune homeostasis.3.The characteristics of the intestinal flora were analyzed through 16 s r DNA technology in the next-generation sequenc-ing(NGS).Proteobacteria and Firmicutes accounted for the major abundance of the intestinal microecology in both controls group and SN50 treatment group,whereas the abundance of Proteobacteria was increased and the abundance of Firmicutes was decreased in the SN50 treatment group.At the genus level,the abundance of Vibrio increased and the abundance of Exiguobacterium decreased in the SN50 treatment group.In the DSS-induced intestinal inflammation,the abundance of Proteobacteria was reduced and the abundance of Firmicutes was improved in high expression of As NF-κB group.At the genus level,the abundance of Vibrio and Bacillaceae declined in high expression of As NF-κB group.Vibrio alginolyticus and Exiguobacterium aurantiacum accounted for the major abundance of the intestinal microecology in intestinal inflammation.Vibrio alginolyticus was significantly decreased in high expression of As NF-κB group,while Exiguobacterium aurantiacum was increased.These results imply that NF-κB activity is critical for intestinal flora.4.High-throughput technology was used to detect the transcriptomes of geographical environments A.sinica and Qinghai Gahai Artemia parthenogenetica under bacterial stress.A.sinica differential genes associated with bacterial induction were mainly enriched in KEGG pathways such as Staphylococcus aureus infection,Marlaria and Systemic lupus erythematosus.A.parthenogenetica differential genes associated with bacterial induction were mainly enriched in Tuberculosis,Phagosome and ABC transporters.Based on the GO database,A.sinica differential genes involved in bacterial infection clustered in heme metabolic processne,gative regulation of response to stimulus and regulation of reactive oxygen species biosynthetic process.A.parthenogenetica differential genes involved in bacterial infection were described the functional properties,including chitin binding,Ikappa B kinase activity and protein tyrosine kinase activity.CK2α was identified from the transcriptomes of A.sinica and A.parthenogenetica,named As CK2α and Ap CK2α,respectively.CK2α is catalytic subunits of protein kinase CK2.The q RT-PCR results showed that As CK2α was higher than Ap CK2α in different developmental stages,while Ap CK2αhigher than As CK2α under M.lysodeikticus and V.harveyi challenge.Tissue location of As CK2α and Ap CK2α were detected in the head and chest.The results suggest that Artemia living in different geographical environments had different immune responses to bacterial infection.5.CK2 catalytic subunits As CK2α and regulatory subunit As CK2β were identified from transcriptomes of A.sinica.The ORF of As CK2α was obtained,a 1047 bp sequence encoding a predicted protein of 348 amino acids.As CK2β contained an ORF 666 bp,encoding 221 amino acids.As CK2α was highly expressed in nauplius and metanauplius.As CK2β had a decreasing trend from nauplius to adult.In response to stress with V.parahaemolyticus,As CK2α and As CK2β expression increased.In response to stress with V.anguillarum,As CK2α expression enhanced,while As CK2β expression reduced.The works indicate that CK2 two subunits have independent regulation in bacterial infection and play a vital role in the growth,development and bacterial stimulation of A.sinica.The findings of this study imply that the transcription factor NF-κB and the cytokine IL17 are pivotal mediators in the regulation of epithelial barrier protection and immune homeostasis in A.sinica,and CK2α is different expression in in the growth,development and immune responses of Artemia.