Functional Study Of Interleukin 16/Integrin β1 Axis Regulate Genes Transcription In Hemocytes

Interleukin(IL)refers to lymphocytes that interact with white blood cells or immune cells.Interleukin-16(IL-16)was the first characteristic cytokine found to exhibit chemotactic activity in human T cells,and was originally designated as lymphocyte chemotactic factor(LCF).In vertebrates,IL-16 is mainly derived from CD8~+T cells,which can bind to CD4~+molecules to mediate the proliferation and differentiation of CD4~+cells,and further participate in inflammatory reactions related to various immune diseases.By comparing the whole genome sequence,we found the presence of interleukin-16-like molecule in Eriocheir sinensis,which is similar in structure to IL-16 in higher animals.The molecule has conserved PDZ domain in the carbon end,and also has the caspase3 cleavage site and NLS region.It is known from previous studies that EsIL-16 and integrinβ1 in Eriocheir sinensis have potential binding activity,and their interaction to form axis regulation is a necessary condition to effectively protect host from bacterial infection.However,the transcriptional mechanism of this regulation axis on immune defense of Eriocheir sinensis remains to be further explored.The expression pattern and transcriptional regulation of EsIL-16/Esintegrinβ1regulatory axis in Eriocheir sinensis were studied in this study.Primarily,we conducted expression pattern analysis of the target gene to be studied,and the results showed that the expression of EsIL-16 and Esintegrinβ1 m RNA could be significantly induced by Vibrio parahaemolyticus stimulation,and the expression of EsIL-16 was the highest at24h after bacterial infection.The expression level of Esintegrinβ1 was the highest at12h after bacterial infection.Then,circulating blood cells of Eriocheir sinensis were selected as experimental materials,and four treatment groups were set up.(1)Eriocheir sinensis blood cells treated with PBS,(2)Eriocheir sinensis blood cells infected with Vibrio parahaemolyticus,(3)blood cells infected with Vibrio parahaemolyticus and supplemented with EsIL-16 recombinant protein,(4)after down-expressing Esintegrinβ1,Blood cells stimulated with Vibrio parahaemolyticus and supplemented with EsIL-16 recombinant protein.Three independent parallel cells were set in each treatment group.After obtaining blood cell samples,total RNA was extracted and c DNA was reversed for transcriptome sequencing and bioinformatics analysis and verification.According to the results of transcription studies,through the comparison of differential genes,FPKM distribution method was used to detect the gene expression changes of samples under different treatments,and the density distribution of differentially expressed genes was obtained.Cluster analysis showed the expression levels of genes of different functional clusters in each treatment group.Transcriptional results showed that the activation of the regulatory axis was inhibited after the knockdown of Esintegrinβ1.Gene annotation identified differentially expressed genes that play important roles in physiological processes such as signal transduction,immunity,development and cell proliferation and metabolism between different treatments.KEGG enrichment analysis of differentially expressed genes showed that C-type lectin,extracellular mechanism receptor(ECMR),STAT signaling pathway,MAPK pathway,RAS and RAP1 signaling pathway are very classical innate immune function vectors,suggesting that the IL-16/Integrinβ1 regulatory axis may also mediate immune response.It provides important foundation and support for further research.In this paper,we initially established the mechanism of regulation of hemocyte gene expression by EsIL-16/Esintegrinβ1 axis of Eriocheir sinensis,and identified the main hemocyte gene information regulated by EsIL-16/Esintegrinβ1 axis.The potential signal transduction pathway of EsIL-16 regulating downstream gene transcription through Esintegrinβ1 receptor was screened.The results of this study provide an important reference for further exploring the mechanism of EsIL-16/Esintegrinβ1 axis regulating innate immunity of Eriocheir sinensis and other crustaceans,and help to enrich the understanding of cellular immune signal transduction pathways in crustaceans.