At present, molecular studies on the immune response to pathogens in fish models are mainly focused on infectious disease pathogenesis. RNA-seq and microarray-based transcriptome profiling studies have revealed that the teleosts are useful in vivo models for identifying host determinants of responses to bacterial infection [27–30]. Furthermore, the RNA-seq approach has already been successfully applied to several infectious disease models of zebrafish [25–27]. However, none have applied the RNA-seq technology to elucidate the immune-related pathways underlying the zebrafish response to vaccination for more effective vaccine evaluation. In this work, in order to gain comprehensive insight into the immunogenetics of zebrafish following immunization with the putative E. tarda live attenuated vaccine, a high-throughput deep sequencing-by-synthesis technology was used to investigate the immunization-related gene expression patterns. DESeq analysis identified 4565 significantly differentially expressed genes in the zebrafish liver following WED immunization. GO and KEGG analysis revealed that the genes involved in the ER protein processing as well as the phagosome and antigen processing and presentation pathways are regulated at the early stage following WED immunization (Table 1 and Figure 2). Significantly, two class MHC pathways were found to be reversely regulated upon immunization, and the MHC class I pathway was activated and the MHC class II pathway was inhibited (Figures 4
5 and 6). Both the RNA-seq results and qPCR data from our study of zebrafish liver during the early stage after WED immunization indicated that activation of the MHC-I processing pathway in teleosts could elicit cellular immune responses for protection.
Once bacterial vaccines are administrated into the animal host, they are often internalized by phagocytes via different entry mechanism. However, the subsequent issues involved in microbial sensing and antigen processing are not well defined. In the conventional paradigm, MHC class II molecules present antigenic fragments acquired by the endocytic route to the immune system for recognition and activation of CD4+ T cells . MHC class I molecules, on the other hand, are restricted to surveying the cytosol for endogenous antigen from intracellular pathogens (such as bacteria, parasites, and viruses), tumors, or self-proteins, which are degraded into proteasomal products and then presented on MHC class I molecules to CD8+ T cells, thus exersting an irreplaceable role on cellular-mediated immuno-protection toward intracellular pathogens [43, 44]. E. tarda is believed to be an intracellular pathogen that can survive and replicate within large phagosomes in macrophages . Since WED is an attenuated strain from wild type E. tarda, it could be assumed that WED bacteria possess the ability to survive in phagosomes of APC cells and the internalized bacteria are recognized as endogenous or exogenous antigen which would be presented or cross-presented by the MHC-I pathway, and finally evoking a CD8+ CTL-mediated response to achieve immune protection.
In MHC-I antigen processing pathway, antigenic peptides are degraded in the cytoplasm by proteasome, then translocated into the ER and loaded onto MHC-I molecules with the help of several protein components. PA28, as an important proteasome activator, is a heterohexameric ring that binds to one or both ends of the 20 S proteasome [43, 46]. Upon binding, it increases the catalytic activity of all three of the proteasome active sites, leads to changes in substrate cleavage, thereby generating more MHC class I-presented peptides [46, 47]. Khan et al. reported that constitutive proteasomes were replaced with immune-proteasomes in mice livers starting at two days after Listeria monocytogenes infection. Immuno-proteasomes support the generation of MHC class I epitopes and shape immune-dominance hierarchies of CD8+ T cells . In mice, this switch is marked by the up-regulation of proteasome activator PA28 subunits, which alter the fragmentation of polypeptides through the proteasome and are inducible by IFN-γ . The study of immune responses to E. ictaluri infection in blue catfish liver demonstrated that both the PA28α and PA28β were up-regulated . In the study described herein, the genes encoding PA28 subunit 1, PA28 subunit 2 and PA28 subunit 3 were all up-regulated in zebrafish liver, which suggested a shift toward MHC class I antigen processing occurred at the early stage after WED immunization.
Heat shock proteins (HSP) are a type of highly conserved and ubiquitously expressed proteins that play an essential role as molecular chaperones in protein folding and transport within the cell  and possess the ability to stimulate MHC class I antigen processing . HSP/peptide complexes are taken up by APC via specific receptors, whose signaling leads to MHC-I presentation of HSP-associated peptides and the induction of specific CD8+ cytotoxic T cells . The antigenic peptides chaperoned by HSPs are known to be more efficient, by orders of magnitude, than the free peptides for presentation by MHC-I [49, 50]. In our work, three heat shock proteins (heat shock cognate 70 kDa protein, heat shock protein 4a and heat shock protein 90 kDa alpha 2) were found to be up-regulated following WED immunization, and the activated HSPs suggested that the internalized WED bacteria were processed and loaded onto MHC class I molecules, ultimately initiating initiate the CTLs.
As cited above, MHC class I molecules present antigenic peptides on cell surface for recognition by CD8+ T cells . Like other glycoproteins, the folding and assembly of MHC class I molecules require interactions with a number of chaperone molecules in the ER, some of which are specific to MHC class I molecules . Among the known ER chaperones, endoplasmin (grp94) possesses the ability to bind peptides suitable for assembly on to MHC class I molecules together with calreticulin . Calreticulin and calnexin are specialized ER lectin-binding chaperones to bind transiently to newly-synthesized glycoproteins, but the calreticulin has been suggested as unique to interactions with the HSP/grp94 complex, which leads to recruitment of ER protein 57 . The interaction between calnexin and MHC class I molecules is believed to stabilize the class I heavy chain and help it to associate with the β2m component [51, 53]. In this work, the three ER chaperons, calreticulin, calnexin and endoplasmin (grp94), were all found to be induced in WED-immunized zebrafish liver, providing further evidence that an active MHC class I processing pathway was stimulated by WED immunization. In addition, TAP binding protein, another molecule involved in MHC class I antigen loading [44, 49, 51, 53], and MHC class I complex ZE protein were also up-regulated in WED-immunized zebrafish liver, strongly suggesting a vigorous activation of the MHC-I processing pathway.
The MHC antigen processing-associated genes from zebrafish have been extensively characterized. However, little is known about their expression patterns in zebrafish following vaccine immunization. Recently, the coordinated up-regulation of MHC class I-related components including MHC class I alpha chain, β2m, calreticulin, endoplasmin, PA28α and PA28β were reported in large yellow croaker following poly I:C injection  and in catfish following an intracellular bacterial infection . In this work, the RNA-seq data were given to show a coordinated down-regulation of several MHC class II antigen processing and presentation components, including the MHC-II DAB, MHC-II beta chain, MHC-II invariant chain (CD74), MHC class II transactivator (CIITA), cathepsin B and lysosomal membrane glycoprotein 2 (lamp2). This complex process is illustrated in Figure 4 and the differentially expressed genes are listed in Table 3. Furthermore, qPCR data confirmed the co-inhibition of lamp2, MHC-II dab, CD74, and CIITA in zebrafish liver and spleen (Figure 6). In previous researches, a remarkable inhibition of MHC-II expression and antigen presentation was ever reported in some pathogen infection models, including Brucella abortus, and Mycobacterium tuberculosis[56–58]. For pathogens, an ability to impair the antigen processing and presentation of host has been proposed to facilitate chronic infection by decreasing T cell responses to microbial antigens. For vaccines, however, the underlying significance of suppression of the MHC-II expression and antigen presentation remains unknown.