Pathogenic Mechanism Of Aeromonas Hydrophila-induced Intestinal Inflammation In Grass Carp

Part I An experimental model for intestinal inflammation induced by Aeromonas hydrophila in grass carp(Ctenopharyngodon idella)Objective: Aeromonas hydrophila is a major opportunistic pathogen that can cause intestinal inflammation in a variety of farmed fish, but the exact pathogenesis of A.hydrophila-induced enteritis in fish is not well understood. The aim of the present study was to develop a model of intestinal inflammation in the grass carp by challenging with a strain of A. hydrophila, and therefore to provide a valuable experimental resource for better understanding the molecular events for bacterial intestinal inflammation.Methods: Grass carp were infected by anal intubation with different doses of A.hydrophila. The appropriate dose for the induction of experimental intestinal inflammation models was determined based on the disease activity index(DAI) by scoring changes in gross signs and symptoms. For the induction of intestinal inflammation, fish were challenged with A. hydrophila at 2.7 × 106 CFU/fish. The histopathological changes in the intestine were examined by light and scanning electron microscopy. The intestinal myeloperoxidase(MPO) activities in infected fish were measured during the whole experimental period. Moreover, the expression levels of the pro-inflammatory cytokines interleukin 1β(IL-1β), IL-8, and tumor necrosis factor α(TNF-α) were monitored by quantitative real-time PCR.Results: Histopathological examination revealed that severe intestinal lesions,including the fusion or shedding of villi, accompanied by the inflammatory cell infiltration,developed 3 d after bacterial infection. Ultrastructural observations also showed that microvilli shed due to bacterial infection. The injured intestinal villi were gradually repaired during the subsequent 18 days of the experimental period. Results also indicated that the inflammatory responses induced by A. hydrophila infection were closely associated with the expression of pro-inflammatory cytokines IL-1β, IL-8, and TNF-α andMPO activity. The highest m RNA levels of these cytokines and highest MPO activity in the intestine were observed on day 3 after challenge.Conclusion: Our data suggest that the intestinal lesions caused by A. hydrophila infection and their repair were completely consistent with the time course of inflammation-related genes or biomarkers. Thus, we have developed an experimental model of intestinal inflammation in an intensively farmed fish. This model is expected to be useful in clarifying the pathogenesis of bacterial enteritis in the grass carp and other farmed fish, and in testing antimicrobial drugs for potential use in aquaculture.Part II Comparative analysis of the intestine transcriptome of grass carp with Aeromonas hydrophila-induced intestinal inflammationObjective: Grass carp is a non-model fish species with limited nucleotide and protein sequences available in public databases. Therefore, less is known about the molecular immune pathways, metabolism profiles, and fundamental developmental mechanisms in grass carp. By using transcriptome sequencing, this study aims to assess the gene expression profiles in the intestine and characterize the changes in intestinal gene expression patterns after infection with A. hydrophila, and to yield insight into the underlying mechanisms in A. hydrophila-induced intestinal inflammation.Methods: The thymus, head kidney, spleen, liver, skin, gill and intestine from healthy grass carp were collected to isolate total RNA, respectively. The RNA samples with high-quality properties were pooled in equal amount and used for next-generation sequencing and de novo transcriptome assembly. The intestinal tissues were also harvested from fish that developed obvious inflammatory signs 1 d after challenge with A.hydrophila and used to isolate total RNA. Control RNA sample was isolated from the intestine of healthy fish. These RNA samples were subjected to Illumina sequencing,respectively. The expression changes of intestinal genes after bacterial infection were examined by high-throughput techniques. The biological functions of differentially expressed genes were analyzed by GO and KEGG annotations. Moreover, the RNA samples were isolated from intestinal tissues of healthy fish or from those harvested 1 d,and/or 3 d after bacterial infection and reverse-transcripted into c DNA, and then the q PCR was performed to examine temporal expression patterns of differentially expressed genes detected by transcriptome sequencing, and to find the relevance of inflammation-related genes to A. hydrophila-induced intestinal inflammation.Results: Illumina sequencing generated 45,867,282 clean reads, all of which were assembled de novo into 120,964 contigs and 67,413 unigenes. By BLAST searching COG database, 23,275 unigenes, belonging to 25 specific groups, were correctly annotated. Total26,567 transcripts were identified by gene ontology(GO) annotation, and distributed in 53 functional groups, categorized into the biological process, cellular component, and molecular function clusters. 28,386 unigenes were mapped to 259 KEGG pathway identifiers. Comparative transcriptome analysis revealed that 18,170 genes expressed in both the intestines of healthy and infected fish. A total of 549 differentially expressed genes were identified from transcriptome profiles, of which 315 were up-regulated and 234 were down-regulated. 36.43% differentially expressed genes clustered into the cellular component, 38.25% fell into the molecular function group, and 38.99% were concentrated in the biological process. Amongst the affected genes, 165 pathways were involved,including rheumatoid arthritis, antigen processing and presentation, phagosome, oxidative phosphorylation, type I diabetes mellitus, lysosome, ribosome, and cytokine-cytokine receptor interaction. The results of q PCR showed that there were significant differences in the expression level changes of inflammation-related genes in intestinal tissues after infection with A. hydrophila. The expression levels of IL-10, IL-10Ra, IL-10Rb, IL-2RG,IL-17 R, IL-22, IL-23 genes peaked 1 d post bacterial infection, while those of IL-12p40 and IL12Rb2 genes achieved maximum values at the 3rd day. In contrast, IL-23 R and IL-6genes were significantly up-regulated 3 d post bacterial infection. Notably, IL-21 gene was found to be significantly down-regulated 3 d after bacterial infection.Conclusion: The bacterial infection with A. hydrophila caused a variety of changes in the intestine of grass carp, including cellular component, functional impairment in cytokine activity, oxygen transporter activity, and transmembrane transport, as well as abnormal biological process in host defense and immune response, and induced significant up-regulation of inflammation-related genes, thus provoked intestinal inflammation.Part III Molecular cloning and characterization of grass carp IL-23p19 and its expression patterns in relation to intestinal inflammationObjective: To clone and characterize the IL-23p19 gene of grass carp and to define its expression pattern related to intestinal inflammation and possible role in the inflammatory process.Methods: The 3′- and 5′-rapid amplification of c DNA ends(RACE) was performed to identify full length c DNA of grass carp IL-23p19 gene by using specific primers designed based on a partial sequence from transcriptome data, and then the IL-23p19 genomic DNA was isolated. The exon-intron organization, coding products, and its evolutionary relatives were analyzed by bioinformatics methods. The recombinant p EGFP-C1-gc IL-23p19 was constructed and transfected into HEK293 T cell, and the subcellular localization of gc IL-23p19 protein was examined. The q PCR was performed to detect the expression profile of IL-23p19 in various tissues of healthy grass carp and in those after infection with A. hydrophila. Furthermore, the differences in intestinal IL-23p19 expression levels between different infection routes at different time points were also assessed.Results: We determined the full-length c DNA sequence of grass carp IL-23p19 by RACE technique. The full-length c DNA sequence contains an open reading frame of 567 nucleotides, encoding 188 amino acids. The alignment of the c DNA sequence with the corresponding genomic DNA showed that IL-23p19 gene consists of four exons and three introns, sharing identical exon-intron organization to its counterparts in zebrafish, another member in Cyprinidae, and in human, mouse and other mammals. The phylogenetic tree generated from the amino acid sequences revealed that gc IL-23p19 exhibited more similarity to those from common carp, zebrafish, both belonging to the same family as grass carp, and grouped together as a single branch, and then clustered with other teleost into a larger group that was clearly distinct from another group of birds and mammals. The results from confocal visualization demonstrated that IL-23p19 protein mainly localized in the cytoplasm of HEK293 T cells. The q PCR assays showed that IL-23p19 was constitutively expressed at varying levels in different tissues of healthy grass carp. The lowest IL-23p19 expression level occurred in head kidney, muscle, and liver, whereas the highest level was detected in gill, followed by blood, skin, and trunk kidney. The IL-23p19 expression levels were significantly up-regulated after infection with A. hydrophila via intraperitoneal injection, and the highest upregulation was observed in intestine, followedby skin, blood, muscle, spleen, and gill. Results also indicated that different infection routes differentially regulated the intestinal IL-23p19 expression levels. The levels were increased by about 700-fold compared with healthy control 24 h post infection via anal intubation, while only 70-fold increase was observed by intraperitoneal injection. This might suggest that different gc IL-23p19 exhibits different proinflammatory effects in local and systemic immune responses.Conclusion: We report here for the first time the cloning, characterization and localization of IL-23p19 from farmed fish. The expression profiles suggest that gc IL-23p19 plays a key regulating role in the A. hydrophila-induced intestinal inflammation Part IV Molecular cloning and characterization of grass carp IL-23 R and its role in intestinal inflammationObjective: To clone and characterize the interleukin-23 receptor(IL-23R) gene from grass carp and to explore its biological functions in intestinal inflammation.Methods: The 3′- and 5′-rapid amplification of c DNA ends(RACE) was performed to identify full length c DNA of grass carp IL-23R(gc IL-23R) gene by using specific primers designed based on a partial sequence from transcriptome data. The antigenic epitope of gc IL-23 R was predicted by bioinformatics method from the c DNA sequences of two isoforms isolated in this study and chemically synthesized. And then the synthesized antigenic epitope sequence was cloned into prokaryotic expressing vector p GEX5 T to generate the recombinant construct p GEX5T-IL-23 R and expressed in E. coli BL21(DE3).The polyclonal antibody rgc IL-23 R p Ab was raised against the purified expressed product,the specificity of which was confirmed Western blot analysis. Immunohistological detection of gc IL-23 R protein in inflamed grass carp intestine after infection with A.hydrophila was performed by using rgc IL-23 R p Ab. The q PCR assay was conducted to quantify the expression levels of two isoforms of IL-23 R in various tissues before and after infection with A. hydrophila and to evaluate the effects of bacterial infection on the expression levels of different isoforms. The expression levels of two isoforms in intestinal tissues after bacterial infection via different infection routes were also compared.Results: Two splicing isoforms of grass carp IL-23 R, gc IL-23RX1 and gc IL-23RX2 were isolated by means of the RACE technique. The full-length c DNA sequence of gc IL-23RX1 and gc IL-23RX2 are 2875 bp and 2730 bp long, encoding 763 and 717 amino acids, respectively. Bioinformatics prediction showed both isoforms contain a 22-amino acid signal peptide and a transmembrane domain. The multiple sequence alignment of amino acids showed that compared to the WSXWS signature motif characteristic of cytokine receptors, the amino acid residues at corresponding positions in gc IL-23 R were MSEWS, which was also different from CSMWS of zebrafish. The gc IL-23 R gene shared the highest identity with zebrafish gene, and relatively high identity with those genes from birds and reptiles, but differed greatly from mammals. SDS-PAGE analysis confirmed that the apparent size of the recombinant antigenic epitope of gc IL-23 R matched well the predicted size. The specificity of the polyclonal antibody rgc IL-23 R p Ab was demonstrated by Western blot analysis. Immunohistological analysis of intestine tissues revealed that gc IL-23 R was expressed in enterocytes, and eosinophils and macrophages that infiltrated into intestinal mucosa, which was similar to that observed in mammals where IL-23 R was expressed in T cells, monocytes and dendritic cells. q PCR analysis indicated that both isoforms of IL-23 R could express in various tissues, but the expression levels of gc IL-23RX1 were globally higher than those of gc IL-23RX2. Intraperitoneal injection with A. hydrophila induced changes of expression levels of both isoforms in various tissues at varying degrees. Anal intubation had greater effects on the expression levels of gc IL-23RX1 and gc IL-23RX2 than intraperitoneal injection did, and the expression levels of gc IL-23RX2 changed more than those of gc IL-23RX1.Conclusion: We describe the first isolation and characterization of two splicing isoforms of IL-23 R from farmed fish. The IL-23 R gene plays an important role in local immune response in intestinal mucosa of grass carp with A. hydrophila-induced intestinal inflammation. Our data implied that these two isoforms might participate in the intestinal inflammation signaling in different ways.