Mechanism Role Of TrIL-1β In Takifugu Rubripes Against Cryptocaryon Irritans Infection

T.rubripes is an important commercial fish in the Chinese aquaculture industry.The huge market demand for T.rubripes is due to its nutritious and high quality meat,and the annual production of cultured fish continues to increase each year.The aquaculture industry of T.rubripes has been developing rapidly,which plays an important role in promoting China’s fish economy.However,the rapid development of the breeding industry and industrialized production have made T.rubripes susceptible to many pathogens infection,and the occurrence of cryptocaryoniasis has resulted in huge economic losses to the industry at present.This seriously hinders the development of aquaculture industry of T.rubripes and one of the diseases that enterprises urgently need to solve.In this study,we studied the immune defense mechanism of T.rubripes,and explored the effective ways of T.rubripes against C.irritans infection.The gill tissue and spleen tissue are the main immune tissues of fish.We were analyzed the gill tissue by transcriptomics,and the spleen tissue by proteomics and phosphorylated proteomics to screen out the important immune gene interleukin-1β(IL-1β).Molecular and cell biology methods were used to analyze the immunomodulatory function of IL-1β,and we speculated that IL-1β play an important immunoprotective role in the T.rubripes against C.irritans infection.Transcriptome sequencing was performed that the gill tissues of T.rubripes from control group and infection group were screening immune-related differentially expressed genes and immune signaling pathways.By principal component analysis(PCA),GO analysis and KEGG analysis,Immune-related categories included Toll-like receptors,MAPK,C-type lectin receptor,and NOD-like receptor signaling pathways were significantly enriched pathways.In immune-related signaling pathways,we found that IL-1β,AP-1,P38,MAPK,and NF-κB were significantly up-regulated DEGs in T.rubripes.It is speculated that these significantly enriched immune signaling pathways and significantly differentially expressed genes play an important role in the T.rubripes against C.irritans infection.To gain more understand the defense mechanism of T.rubripes against C.irritans infection,the spleen tissues were quantitatively analyzed by enrichment of phosphorylated peptides technology,and MS-based proteomics.We identified 5,307 proteins and 6,644 phosphorylated sites on 2,815 phosphoproteins using high-throughput proteomics analysis of the spleen of T.rubripes based on 26,421 unique peptides and 5,013 modified peptides,respectively.Using the combination of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses,screening for significantly different phosphoproteins,motif analysis and protein-protein interaction analysis,we ultimately identified hepcidin,IL-1β,quinone oxidoreductase PIG3,G-protein-signaling modulator,serine/threonine-protein kinase,MAPK 5-like,PKCθ type,ZFP 850 as potential biomarkers for T.rubripes immune responses.These proteins were involved in MAPK signaling pathway,TGF-β signaling pathway,necroptosis,p53 signaling pathway.The combined analysis of transcriptome analysis of gill tissue and proteomics analysis of spleen tissue can more accurates reflect the importance of the gene in the process of T.rubripes against C.irritans infection.Combined with the transcriptomic and proteomic data results show that we comprehensively analyzed the significantly up-regulation of interleukin-1β(IL-1β)gene expression in gill and spleen tissues.In our study,GO and KEGG analysis showed that IL-1β was involved in the MAPK signaling pathway,Toll-like receptor and NF-κB signaling pathway and other immune signaling pathways.Protein interaction analysis showed that IL-1β was interacted with multiple proteins and was located in a key position.IL-1β,as a major immunomodulatory factor has been the focus of researchers.In previous reports,IL-1β was playing important immunomodulatory role in bacterial and viral diseases of fish.So,we speculate that IL-1β may play an important role in the immunomodulatory process of T.rubripes against C.irritans infection.In this study,we performed that the potential function of T.rubripes IL-1β(TrIL-1β)in T.rubripes against C.irritans infection by method of the molecular characterization of TrIL-1βgene,immunofluorescence,transmission electron microscopy,cell biological experiments.Through the analysis of conserved domains and motifs,multiple sequences alignment and phylogenetic analysis and protein structure analysis,the results show that even though the sequence divergences existed among fish IL-1β,the components and positions of conserved domains and motifs of fish IL-1β remained highly conserved.Phylogenetic analysis indicated that the TrIL-1β protein sequence was most closed of Salmo salar(67.2%),followed by Danio rerio IL-1β(31.7%).The RT-q PCR results showed that IL-1β was widely expressed in all tissues(gills,spleen,head kidney,liver,intestine and skin),but the expression level was different,In the IL-1β expression profile,the highest expression level was detected in spleen tissue followed by gill and head kidney tissues.Moderate levels of m RNA expressions were detected in skin,liver,intestine.The IL-1β was extensively distributed in tissues of healthy T.rubripes.The incubation experiments incubation with 100 ng/m L TrIL-1βand trophonts for 1h revealed that the trophonts membrane gradually ruptured contents flowing out and TrIL-1β owned potential killing trophonts activity by destroying membrane.Immunofluorescence experiments showed that the red fluorescence intensity of IL-1β group was significantly higher than PBS group,BSA group and INF group,and indicating that recombinant TrIL-1β promote the expression of endogenous IL-1β.In the immunofluorescence images of gill tissues,IL-1β red fluorescence was found mainly on the trophonts membrane with the 3 days and filled the trophozoite with the 6 days,which again verified that IL-1β could gradually penetrate and destroy the trophonts membrane and kill trophonts,blocking reproductive cycle of C.irritans.The transmission electron microscopy showed that the IL-1β group had less tissue damage compared with BSA and INF groups.IL-1β-si RNA and IL-1β overexpression were performed in Head kidney primary cells(HKPC)and challenge experiments in vitro.RT-q PCR results showed that TrIL-1βwas verified to regulate and activate My D88/NF-κB and My D88/MAPK/p38 signaling pathways against C.irritans infection.In addition,IL-1β could promoted the differential expression of Ig M gene,showed that TrIL-1β was involved in humoral immunity of fish.At same time,cumulative mortality level experiment show that TrIL-1β could protect fish against C.irritans infection.Based on these data analysis showed that recombinant TrIL-1β induced high expression of endogenous IL-1β at the early stage of infection,which it was activated the My D88/NF-κB and My D88/MAPK/p38 pathways and secretion of IL-1β,IL-27,AP-1 and other cytokines enhance the immunity of fish and participate resistance to C.irritans infection and blocks reproductive cycle of C.irritans,delay the death time and improve the survival rate of fish.In this study,we found that IL-1β has potential biological function against C.irritans,and IL-1β was participates in immunoregulation and promotes the expression of related immune genes.These results suggest that recombinant TrIL-1β can be used as a potential cytokine adjuvant for subunit vaccine,which is important implications for the prevention and control of cryptocaryoniasis.