Effects Of IL-6 On Immune Regulation After Aeromonas Hydrophil Infection In Zebrafish

In recent years,aquaculture industry is developing rapidly.However,due to the occurrence of germplasm decline,excessive breeding density and other problems,fish diseases occur frequently,among which,fish diseases caused by Aeromonas hydrophila have incurred serious economic losses to farmers.Fish’s resistance to disease is more dependent on non-specific immunity,thus it is particularly essential to explore the disease resistance mechanism of fish at the gene level.Interleukin-6（IL-6）is a pleiotropic cytokine that plays a critical role in acute stress and lipid metabolism and is considered to be the primary regulator of liver acute phase reaction.Zebrafish（Danio rerio）has been widely used to study disease resistance as a model organism,stress resistance and biological development.In order to explore the role of IL-6 in acute liver immune response in fish,and lay the foundation for subsequent research on the specific molecular pathways and regulatory mechanisms of IL-6 in bacterial immune response,zebrafish was selected as the research object.The sequence characteristics of IL-6 were analyzed by bioinformatics methods（phylogenetic analysis,amino acid sequence alignment,protein tertiary structure prediction,etc.）,and the expression changes of il-6 in different tissues of zebrafish after A.hydrophila injection were studied.In addition,IL-6^-/-zebrafish was constructed by using CRISPR/Cas9 technology,and the effects of IL-6 in response to A.hydrophila stress was analyzed by HE staining,A.hydrophila challenge test,histology and transcriptomics.The main outcomes are as follows:（1）Based on phylogenetic analysis,amino acid sequence alignment and protein tertiary structure prediction of IL-6 in different vertebrate species,it was found that the amino acid sequence identity of zebrafish and cyprinidae IL-6 was more than 70%,and the protein structure was highly conserved,which means that the function of zebrafish and cyprinidae IL-6 is relatively conserved.In response to A.hydrophila stress,the relative expression of il-6 in the liver,spleen,brain and gills of zebrafish was significantly up-regulated during inflammation.（2）In order to explore the role of IL-6 in response to A.hydrophila stress in fish,IL-6^-/-zebrafish was constructed by CRISPR/Cas9 technology.The results showed that there was no significant difference in body length and body weight between the3-month-old IL-6^-/-and WT zebrafish.However,at the age of 12 months,the body weight and length of IL-6^-/-zebrafish were significantly higher than that of WT zebrafish,which might be caused by the differences in lipid metabolism and food intake.IL-6 deficiency reduced the mortality of A.hydrophila infection in IL-6^-/-zebrafish.The results of HE staining showed that the inflammation in the liver of WT zebrafish was more severe.The bacterial load in liver,spleen,heart and brain of WT was higher than that of IL-6^-/-zebrafish.Through transcriptome analysis,it was found that IL-6^-/-zebrafish may compensate for the immune deficiency caused by IL-6 deficiency by up-regulating the expression of antimicrobial proteins such as LEAP-2 and LYZ,thereby improving the survival rate.（3）In this study,the liver transcriptomes of WT and IL-6^-/-before and after challenge with A.hydrophila were sequenced and analyzed.Compared with the healthy wild-type group（WT）,there were 3,201 up-regulated genes and 3,573 down-regulated genes in the wild-type group injected with A.hydrophila（WT-Ah）.Compared with the WT group,there were 1,498 up-regulated genes and 1,234 down-regulated genes in the healthy IL-6^-/-group（IL6-KO）.Compared with WT-Ah group,there were 2,432up-regulated genes and 1,706 down-regulated genes in IL-6^-/-after injection of A.hydrophila group（IL6-KO-Ah）.There were 3,450 up-regulated genes and 3,645down-regulated genes in the IL6-KO-Ah group compared with the IL6-KO group.Through GO and KEGG enrichment analysis,the results showed that DEGs in WT and IL-6^-/-after A.hydrophila injection were significantly enriched in immune-related aspects such as oxidation-reduction process and exogenous drug catabolic process compared with the corresponding healthy group.Compared with the WT group,DEGs in the IL6-KO group were mainly enriched in tricarboxylic acid cycle,regulation of humoral immune response,NADP metabolic process,regulation of complement activation and other metabolism and immune-related aspects.Compared with the WT-Ah group,DEGs in the IL6-KO-Ah group were mainly enriched in binding of sperm to zona pellucida,positive regulation of acrosome reaction,exogenous drug catabolic process,xenobiotic metabolic process,oxidation-reduction process and complement activation.