Single-nuclei transcriptome analysis of channel catfish spleen provides insight into the immunome of an aquaculture-relevant species.

The catfish industry is the largest sector of U.S. aquaculture production. Given its role in food production, the catfish immune response to industry-relevant pathogens has been extensively studied and has provided crucial information on innate and adaptive immune function during disease progression. To further examine the channel catfish immune system, we performed single-cell RNA sequencing on nuclei isolated from whole spleens, a major lymphoid organ in teleost fish. Libraries were prepared using the 10X Genomics Chromium X with the Next GEM Single Cell 3' reagents and sequenced on an Illumina sequencer. Each demultiplexed sample was aligned to the Coco_2.0 channel catfish reference assembly, filtered, and counted to generate feature-barcode matrices. From whole spleen samples, outputs were analyzed both individually and as an integrated dataset. The three splenic transcriptome libraries generated an average of 278,717,872 reads from a mean 8,157 cells. The integrated data included 19,613 cells, counts for 20,121 genes, with a median 665 genes/cell. Cluster analysis of all cells identified 17 clusters which were classified as erythroid, hematopoietic stem cells, B cells, T cells, myeloid cells, and endothelial cells. Subcluster analysis was carried out on the immune cell populations. Here, distinct subclusters such as immature B cells, mature B cells, plasma cells, γδ T cells, dendritic cells, and macrophages were further identified. Differential gene expression analyses allowed for the identification of the most highly expressed genes for each cluster and subcluster. This dataset is a rich cellular gene expression resource for investigation of the channel catfish and teleost splenic immunome.

Interleukin-26 in channel catfish (Ictalurus punctatus): Molecular characterization and immune functional activity.

IL-26 is a crucial inflammatory cytokine that participates in defending host cells against infections. We initially cloned and identified the cDNA sequences of interleukin (IL)-26 in channel catfish (Ictalurus punctatus). The open reading frame (ORF) of IpIL-26 was 537 bp in length, encoding 178 amino acids (aa). Constitutive expression of IpIL-26 was observed in tested tissues, with the highest level found in the gill and spleen. To explore the function of IpIL-26 in channel catfish, different stimuli were used to act on both channel catfish and channel catfish kidney cells (CCK). The expression of IpIL-26 could be up-regulated by bacteria and viruses in multiple tissues. In vitro, recombinant IpIL-26 (rIpIL-26) could induce the expression levels of inflammatory cytokines such as TNF-α, IL-1ß, IL-6, IL-20, and IL-22 playing vital roles in defending the host against infections. Our results demonstrated that IpIL-26 might be an essential cytokine, significantly affecting the immune defense of channel catfish against pathogen infections.

TLR7 in channel catfish (Ictalurus punctatus) is expressed in the endolysosome and is stimulated by synthetic ssRNA analogs, imiquimod, and resiquimod.

Toll-like receptors (TLRs) are pivotal pattern recognition receptors (PRRs) and key mediators of innate immunity. Despite the significance of channel catfish (Ictalurus punctatus) in comparative immunology and aquaculture, its 20 TLR genes remain largely functionally uncharacterized. In this study, our aim was to determine the catfish TLR7 agonists, signaling potential, and cellular localization. Using a mammalian reporter system, we identified imiquimod and resiquimod, typical ssRNA analogs, as potent catfish TLR7 agonists. Notably, unlike grass carp TLR7, catfish TLR7 lacks the ability to respond to poly (I:C). Confocal microscopy revealed predominant catfish TLR7 expression in lysosomes, co-localizing with the endosomal chaperone protein, UNC93B1. Furthermore, imiquimod stimulation elicited robust IFNb transcription in peripheral blood leukocytes isolated from adult catfish. These findings underscore the conservation of TLR7 signaling in catfish, reminiscent of mammalian TLR7 responses. Our study sheds light on the functional aspects of catfish TLR7 and contributes to a better understanding of its role in immune defense mechanisms.

A Comprehensive Annotation of the Channel Catfish (Ictalurus punctatus) T Cell Receptor Alpha/Delta, Beta, and Gamma Loci.

The complete germline repertoires of the channel catfish, Ictalurus punctatus, T cell receptor (TR) loci, TRAD, TRB, and TRG were obtained by analyzing genomic data from PacBio sequencing. The catfish TRB locus spans 214 kb, and contains 112 TRBV genes, a single TRBD gene, 31 TRBJ genes and two TRBC genes. In contrast, the TRAD locus is very large, at 1,285 kb. It consists of four TRDD genes, one TRDJ gene followed by the exons for TRDC, 125 TRAJ genes and the exons encoding the TRAC. Downstream of the TRAC, are 140 TRADV genes, and all of them are in the opposite transcriptional orientation. The catfish TRGC locus spans 151 kb and consists of four diverse V-J-C cassettes. Altogether, this locus contains 15 TRGV genes and 10 TRGJ genes. To place our data into context, we also analyzed the zebrafish TR germline gene repertoires. Overall, our findings demonstrated that catfish possesses a more restricted repertoire compared to the zebrafish. For example, the 140 TRADV genes in catfish form eight subgroups based on members sharing 75% nucleotide identity. However, the 149 TRAD genes in zebrafish form 53 subgroups. This difference in subgroup numbers between catfish and zebrafish is best explained by expansions of catfish TRADV subgroups, which likely occurred through multiple, relatively recent gene duplications. Similarly, 112 catfish TRBV genes form 30 subgroups, while the 51 zebrafish TRBV genes are placed into 36 subgroups. Notably, several catfish and zebrafish TRB subgroups share ancestor nodes. In addition, the complete catfish TR gene annotation was used to compile a TR gene segment database, which was applied in clonotype analysis of an available gynogenetic channel catfish transcriptome. Combined, the TR annotation and clonotype analysis suggested that the expressed TRA, TRB, and TRD repertoires were generated by different mechanisms. The diversity of the TRB repertoire depends on the number of TRBV subgroups and TRBJ genes, while TRA diversity relies on the many different TRAJ genes, which appear to be only minimally trimmed. In contrast, TRD diversity relies on nucleotide additions and the utilization of up to four TRDD segments.

Virulence and live vaccine potential of Edwardsiella piscicida phoP and phoQ mutants in catfish against edwardsiellosis.

Edwardsiella piscicida is a Gram-negative facultative intracellular bacterium causing edwardsiellosis in catfish, the largest aquaculture industry in the United States. A safe and effective vaccine is an urgent need to avoid economic losses associated with E. piscicida outbreaks. PhoP/PhoQ is a two-component signal transduction system (TCS) that plays an important role in bacterial pathogenesis through sense and response to environmental and host stress signals. This study aimed to explore the contribution of PhoQ/PhoP in E. piscicida virulence and develop live attenuated vaccines against E. piscicida infection in channel catfish (Ictalurus punctatus) and hybrid catfish (channel catfish ♀ × blue catfish (I. furcatus) ♂). In the current study, two in-frame deletion mutants were constructed by deleting phoP (ETAC_09785) and phoQ (ETAC_09790) genes in E. piscicida strain C07-087, and the virulence and protection efficacy of the constructed strains were evaluated in catfish following intraperitoneal injection. Both EpΔphoP and EpΔphoQ strains had a delayed adaptation to oxidative stress (0.2% H2 O2 ) compared to E. piscicida wild type. The EpΔphoP and EpΔphoQ mutants produced significantly less biofilm compared to wild-type E. piscicida. Notably, EpΔphoP and EpΔphoQ mutants were significantly attenuated in channel catfish compared with wild-type E. piscicida (6.63% and 4.17% versus 49.16% mortalities), and channel catfish vaccinated with EpΔphoP and EpΔphoQ were significantly protected (95.65% and 97.92% survival) against E. piscicida infection at 21 days post-vaccination. In hybrid catfish, EpΔphoP was significantly more attenuated than EpΔphoQ, but EpΔphoQ provided significantly better protection than EpΔphoP. EpΔphoP and EpΔphoQ strains both induced specific antibodies in channel catfish against E. piscicida at 14 and 21 days post-vaccination. This result indicated that EpΔphoP and EpΔphoQ mutants were safe and protective in channel catfish fingerlings, while EpΔphoP was safe in hybrid catfish. Our findings show that PhoP and PhoQ are required for adaptation to oxidative stress and biofilm formation and may help E. piscicida face tough environmental challenges; thus, functional PhoP and PhoQ are critical for a successful infection.

Direct and pleiotropic effects of the Masou Salmon Delta-5 Desaturase transgene in F1 channel catfish (Ictalurus punctatus).

Channel catfish (Ictalurus punctatus) is the primary culture species in the US along with its hybrid made with male blue catfish, I. furcatus. In an effort to improve the nutritional value of channel catfish, the masou salmon Δ5-desaturase like gene (D5D) driven by the common carp beta-actin promoter (ßactin) was inserted into channel catfish. The objectives of this study were to determine the effectiveness of ßactin-D5D for improving n-3 fatty acid production in F1 transgenic channel catfish, as well as examine pleiotropic effects on growth, proximate analysis, disease resistance, and other performance traits. Transgenic F1 channel catfish showed a 33% increase in the relative proportion of n-3 fatty acids coupled with a 15% decrease in n-6 fatty acids and a 17% decrease in n-9 fatty acids when compared to non-transgenic full-siblings (P < 0.01, P < 0.01, P < 0.01). However, while the relative proportion of n-3 fatty acids was achieved, the total amount of fatty acids in the transgenic fish decreased resulting in a reduction of all fatty acids. Insertion of the ßactin-D5D transgene into channel catfish also had large effects on the body composition, and growth of channel catfish. Transgenic channel catfish grew faster, were more disease resistant, had higher protein and moisture percentage, but lower fat percentage than full-sib controls. There were sex effects as performance changes were more dramatic and significant in males. The ßactin-D5D transgenic channel catfish were also more uniform in their fatty acid composition, growth and other traits.

Adaptive immune responses in channel catfish exposed to Edwardsiella ictaluri live attenuated vaccine and wild type strains through the specific gene expression profiles.

We extend the previous findings on the differential activity of immune-related genes in the lymphoid organs of channel catfish in the 7 days post-challenge (dpc) with E. ictaluri live attenuated vaccines (LAVs) and wild type (WT) strains by assessing the expression of these genes in the 21 dpc. The expression of T and B cell-specific genes were significantly elevated in the spleen at 14 dpc and in the AK at 21 dpc in catfish treated with E. ictaluri WT and LAV strains compared to a non-treated control group. The gene expression of IFN-γ correlated with adaptive immunity genes in the lymphoid tissues of catfish. These data indicate that two novel LAVs were able to trigger the activation of T helper1 polarization cytokine IFN-γ gene and specific lymphocyte genes in the spleen followed by their activation in the AK of catfish without causing inflammation, thus providing protective immunity in E. ictaluri infection.

Dose effects of a DNA vaccine encoding immobilization antigen on immune response of channel catfish against Ichthyophthirius multifiliis.

Channel catfish (Ictalurus punctatus) vaccinated with pcDNA3.1-IAg52b plasmid DNA vaccine encoding immobilization antigen genes of Ichthyophthirius multifiliis (Ich) produced anti-Ich antibodies and were partially protected (20% survival) in a previous study. Here we evaluated whether a higher dose or two doses of pcDNA3.1-IAg52b vaccine could provide better protection for catfish against Ich. Fish were distributed into 6 groups and vaccinated using following schemes: 1.10 µg pcDNA3.1-IAg52b fish-1, 2.20 µg pcDNA3.1-IAg52b fish-1, 3. two doses of 10 µg pcDNA3.1-IAg52b fish-1 with 7 days between doses, 4.20 µg pcDNA3.1 fish-1 (mock-vaccinated control), 5.15,000 live theronts fish-1 (positive control), and 6. non-vaccinated and non-challenge control. Parasite infection levels, serum anti-Ich antibody levels, fish mortality and immune-related gene expression were determined during the trial. Fish vaccinated with a single dose of 20 µg pcDNA3.1-IAg52b fish-1 or two doses of 10 µg fish-1 had higher anti-Ich antibody levels than fish receiving a single dose of 10 µg fish-1. Survival was significantly higher in fish receiving 20 µg vaccine fish-1 (35.6%) or 2 doses of 10 µg fish-1 (48.9%) than fish injected with a single dose of 10 µg fish-1 (15.6%) or mock-vaccinated control (0%). Fish vaccinated at the dose 20 µg fish-1 had higher expression of vaccine DNA in muscle than fish vaccinated with 10 µg fish-1. Fish vaccinated with the DNA vaccine showed higher up-regulation than mock-vaccinated control in the expression of IgM, CD4, MHC I and TcR-α genes during most of time points after vaccination. Further studies are needed to improve efficacy of DNA vaccines by using multiple antigens in the DNA vaccines.

A Fish Leukocyte Immune-Type Receptor Uses a Novel Intracytoplasmic Tail Networking Mechanism to Cross-Inhibit the Phagocytic Response.

Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are a family of immunoregulatory proteins shown to regulate several innate immune cell effector responses, including phagocytosis. The precise mechanisms of IpLITR-mediated regulation of the phagocytic process are not entirely understood, but we have previously shown that different IpLITR-types use classical as well as novel pathways for controlling immune cell-mediated target engulfment. To date, all functional assessments of IpLITR-mediated regulatory actions have focused on the independent characterization of select IpLITR-types in transfected cells. As members of the immunoglobulin superfamily, many IpLITRs share similar extracellular Ig-like domains, thus it is possible that various IpLITR actions are influenced by cross-talk mechanisms between different IpLITR-types; analogous to the paired innate receptor paradigm in mammals. Here, we describe in detail the co-expression of different IpLITR-types in the human embryonic AD293 cell line and examination of their receptor cross-talk mechanisms during the regulation of the phagocytic response using imaging flow cytometry, confocal microscopy, and immunoprecipitation protocols. Overall, our data provides interesting new insights into the integrated control of phagocytosis via the antagonistic networking of independent IpLITR-types that requires the selective recruitment of inhibitory signaling molecules for the initiation and sustained cross-inhibition of phagocytosis.

The miR-155 regulates cytokines expression by SOSC1 signal pathways of fish in vitro and in vivo.

MiR-155 is reported as immune regulated miRNA in mammalian corresponding to immunity, antibacterial and antiviral effects regulation. However, the roles and mechanisms of the miRNA have remained largely undefined. We herein comprehensively investigated the functions of miR-155 in vitro and in vivo by miR-155 mimics, agomir and antagomir in Cyprinus carpio and Ictalurus punctatus, with the target genes in the SOSC1 pathway certified in I. punctatus via luciferase reporter assays. Results showed that the miR-155 regulated the expressions of cytokines, including TNF-α, IFN-γ, IL-1ß, IL-6 and IL-10. Further research confirmed SOSC1 as one of the targets of the miRNA, and the JAK1/STAT3/SOSC1 signal pathway involved in the miR-155 effects on the expression of immune cytokines as well. Additionally, the changes of TLR2 in fish may also be related to miR-155 along with its target SOCS1, and the TLR2/MyD88 pathway may partly participate in the effects of the miR-155 on the cytokines. The research here confirmed that the miR-155 can regulate cytokines expression by SOSC1 signal pathways of fish in vitro and in vivo, which would provide resources for understanding and studying about immune regulation in fish.

Liver transcriptome analysis and cortisol immune-response modulation in lipopolysaccharide-stimulated in channel catfish (Ictalurus punctatus).

Channel catfish (Ictalurus punctatus) is an important aquaculture species in China. In channel catfish, diseases such as haemorrhagic, sepsis and tail-rot disease are all caused by bacteria as general in China. Most of the pathogenic bacteria are Gram-negative bacteria. Liver transcriptome analysis of the co-injection of cortisol and lipopolysaccharide (LPS) was performed in this study. Preliminary evidence from the results suggest that after the emergence of immune stress, cortisol will up-regulate the complement cascade pathway, down-regulate the coagulation cascade pathway, down-regulate the platelet activation pathway, down-regulate antigen presentation pathway, and show complex regulation relationship to inflammatory factors. At 12 h, the number of differential genes regulated by cortisol was about half less than the number of differential genes regulated by LPS. At 24 h, there was no significant difference between the number of differential genes regulated by cortisol and LPS, but the types of differential genes vary widely. KEGG enrichment analysis found that cortisol regulated LPS-stimulated immune responses mainly focus on cytokines, complement and coagulation cascades pathways, antigen presentation pathways, haematopoiesis, and inflammation. It is suggested that there may be some strategic choice in the regulation of immune response by cortisol. These results will help understand the pathogenesis and host defence system in bacterial disease caused by Gram-negative bacteria.

Cloning and characterization of antiviral cytotoxic T lymphocytes in channel catfish, Ictalurus punctatus.

To determine the role of piscine anti-viral cytotoxic cells, we analyzed the response of channel catfish to Ictalurid herpesvirus 1, commonly designated channel catfish virus (CCV). Peripheral blood leukocytes (PBL) from catfish immunized with MHC-matched, CCV-infected G14D cells (G14D-CCV) showed marked lysis of G14D-CCV but little to no lysis of uninfected allogenic (3B11) or syngeneic (G14D) cells. Expansion of effectors by in vitro culture in the presence of irradiated G14D-CCV cells generated cultures with enhanced cytotoxicity and often broader target range. Cytotoxic effectors expressed rearranged TCR genes, perforin, granzyme, and IFN-γ. Four clonal cytotoxic lines were developed and unique TCR gene rearrangements including γδ were detected. Furthermore, catfish CTL clones were either CD4+/CD8- or CD4-/CD8-. Two CTL lines showed markedly enhanced killing of G14D-CCV targets, while the other two lines displayed a broader target range. Collectively, catfish virus-specific CTL display unique features that illustrate the diversity of the ectothermic vertebrate immune response.

Selective recruitment of Nck and Syk contribute to distinct leukocyte immune-type receptor-initiated target interactions.

The ability of phagocytes to recognize, immobilize, and engulf extracellular targets are fundamental immune cell processes that allow for the destruction of a variety of microbial intruders. The phagocytic process depends onsignalling events that initiate dynamic changes in the plasma membrane architecture that are required to accommodate the internalization of large particulate targets. To better understand fundamental molecular mechanisms responsible for facilitating phagocytic receptor-mediated regulation of cytoskeletal networks, our research has focused on investigating representative immunoregulatory proteins from the channel catfish (Ictalurus punctatus) leukocyte immune-type receptor family (IpLITRs). Specifically, we have shown that a specific IpLITR-type can regulate the constitutive deployment of filopodial-like structures to actively capture and secure targets to the phagocyte surface, which is followed by F-actin mediated membrane dynamics that are associated with the formation of phagocytic cup-like structures that precede target engulfment. In the present study, we use confocal imaging to examine the recruitment of mediators of the F-actin cytoskeleton during IpLITR-mediated regulation of membrane dynamics. Our results provide novel details regarding the dynamic recruitment of the signaling effectors Nck and Syk during classical as well as atypical IpLITR-induced phagocytic processes.

Effects of Live Attenuated Vaccine and Wild Type Strains of Edwardsiella ictaluri on Phagocytosis, Bacterial Killing, and Survival of Catfish B Cells.

Edwardsiella ictaluri, a Gram-negative facultative intracellular pathogen, is the causative agent of enteric septicemia of catfish (ESC). The innate functions of B cells have been demonstrated in several teleost fish, including zebrafish, rainbow trout, and channel catfish. Recently, our group has developed several protective E. ictaluri live attenuated vaccines (LAVs). However, the innate role of catfish B cells to phagocytose and destroy E. ictaluri wild-type (WT) and live attenuated vaccine (LAV) strains has not been evaluated. In this study, we assessed the efficacy of E. ictaluri WT and two LAVs on phagocytosis, microbial killing, and survival of catfish anterior kidney (AK) B cells. Initially, we documented active uptake of E. ictaluri WT and two LAVs in B cells by flow cytometry and light microscopy. Then, we observed the E. ictaluri strains-induced phagosome and/or phagolysosome formation in the cytoplasm of catfish magnetically sorted IgM+ B cells. Furthermore, we demonstrated that AK B cells were able to destroy the internalized E. ictaluri WT and LAV strains efficiently. Finally, we documented early and late apoptotic/necrotic manifestations induced by E. ictaluri in catfish AK B cells. In conclusion, our results suggest that both LAVs and WT strain initiate similar innate immune responses such as active phagocytic uptake, induced bactericidal activity as well as promote early and late apoptotic changes in catfish B cells. Our data suggest that phagocytic and microbicidal B cells may serve as professional APCs in initiation of protective adaptive immune responses against ESC in channel catfish.

Long-Lived Plasma Cells Secrete High-Affinity Antibodies Responding to a T-Dependent Immunization in a Teleost Fish.

The recent discovery of long-lived plasma cells (LLPCs) in mammals, which provide a constant expression of specific high-affinity antibodies that mediate humoral memory, has caused a dramatic paradigm shift in the study of immunity and vaccine development. In teleost fish, there are few studies regarding the association between LLPCs and antibody production, and the affinity of the antibodies secreted by the LLPCs is poorly understood. In the present study, channel catfish (Ictalurus punctatus) were immunized with trinitrophenylated-keyhole limpet hemocyanin (TNP-KLH) to examine TNP-specific antibody titers, affinities, antibody-secreting cell (ASC) dynamic changes, and especially the affinity of secreted antibodies by LLPCs post-immunization. We demonstrated that TNP-specific LLPCs were generated starting at week 4 post-immunization, achieved a maximal number at week 8, and maintained a comparable level throughout the 18-week post-immunization period, which was correlated with the dynamics of serum antibody titers and affinity maturation in the response. The LLPCs appeared to mostly reside within, or migrate to, the anterior kidney (bone marrow-like tissue in mammals), but a small portion was also located in the spleen and peripheral blood. The antibodies produced by the LLPCs possessed high affinities, indicating that the generation and development of LLPCs were driven by affinity selection in teleosts. Collectively, the results of this study provide insights toward the evolutionary understanding of the affinity-dependent mechanism of LLPC generation and development.

Assessment of the Live Attenuated and Wild-Type Edwardsiella ictaluri-Induced Immune Gene Expression and Langerhans-Like Cell Profiles in the Immune-Related Organs of Catfish.

Edwardsiella ictaluri is a Gram-negative intracellular pathogen that causes enteric septicemia of catfish (ESC). Successful vaccination against intracellular pathogens requires T cell priming by antigen presenting cells (APCs) that bridge innate and adaptive immunity. However, the evidence on immunological mechanisms that underscore E. ictaluri pathogenesis and the protective role of live attenuated vaccines (LAVs) is scarce. We assessed the expression of immune genes related to antigen presentation by real-time PCR and the distribution patterns of Langerhans-like (L/CD207+) cells by immunohistochemistry in the immune-related tissues of channel catfish challenged with two novel E. ictaluri LAVs, EiΔevpB, and ESC-NDKL1 and wild type (WT) strain. Our results indicated significantly elevated expression of IFN-γ gene in the anterior kidney (AK) and spleen of vaccinated catfish at the early stages of exposure, which correlated with increased numbers of L/CD207+ cells. In general, the ESC-NDKL1-induced IFN-γ gene expression patterns in the AK resembled that of the patterns induced by EiΔevpB. However the MHCII gene expression patterns differed between the strains with significant increases at 6 h post-challenge (pc) with the EiΔevpB and at 7 d pc with the ESC-NDKL1 strains, respectively. Significant increases in activity of T helper type polarization genes such as IFN-γ and T cell co-receptors after exposure to ESC-NDKL1, in combination with elevated numbers of L/CD207+ cells at 7 d pc with both LAVs compared to uninfected and the WT-exposed counterparts, were documented in the spleen. The dominant pro-inflammatory environment with dramatically overexpressed inflammatory genes in the AK and 7 d pc in the spleen in response to E. ictaluri was found in exposed catfish. In general, the pro-inflammatory gene expression profiles in the ESC-NDKL1 pc showed more similarities to the WT strain-induced gene profiles compared to the EiΔevpB counterpart. In addition, E. ictaluri WT significantly decreased the numbers of Langerhans-like L/CD207+ cells in the AK and spleen at 3 and 7 days pc. In conclusion, we report the differential framework of initiation of innate and adaptive immune responses between E. ictaluri strains with both LAVs having a potential of satisfying the stringent requirements for successful vaccines.

FOXO genes in channel catfish and their response after bacterial infection.

FOXO proteins are a subgroup of the forkhead family of transcription factors that play crucial roles in lifespan regulation. In addition, FOXO proteins are also involved in immune responses. After a systematic study of FOXO genes in channel catfish, Ictalurus punctatus, seven FOXO genes were identified and characterized, including FOXO1a, FOXO1b, FOXO3a, FOXO3b, FOXO4, FOXO6a and FOXO6b. Through phylogenetic and syntenic analyses, it was found that FOXO1, FOXO3 and FOXO6 were duplicated in the catfish genome, as in the zebrafish genome. Analysis of the relative rates of nonsynonymous (dN) and synonymous (dS) substitutions revealed that the FOXO genes were globally strongly constrained by negative selection. Differential expression patterns were observed in the majority of FOXO genes after Edwardsiella ictaluri and Flavobacterium columnare infections. After E. ictaluri infection, four FOXO genes with orthologs in mammal species were significantly upregulated, where FOXO6b was the most dramatically upregulated. However, after F. columnare infection, the expression levels of almost all FOXO genes were not significantly affected. These results suggested that either a pathogenesis-specific pattern or tissue-specific pattern existed in catfish after these two bacterial infections. Taken together, these findings indicated that FOXO genes may play important roles in immune responses to bacterial infections in catfish.

Molecular characterization, phylogenetic analysis and adjuvant effect of channel catfish interleukin-1ßs against Streptococcus iniae.

Channel catfish is one of the most extensively cultured species worldwide, which is widely used as a classical model for comparative immunology. Interleukin-1ß (IL1ß) is an immunoregulatory cytokine with the potential to enhance the immune response induced by vaccines in many animals. To characterize the molecular characterization and identify the immunoadjuvant role of channel catfish IL1ß, molecular cloning, phylogenetic analysis, and expression of two IL1ß genes were performed, the bioactivity of their recombinant proteins (rIL1ß1 and rIL1ß2) were detected in vitro and their adjuvant effects on a subunit vaccine encoding C5a peptidase (pSCPI) of Streptococcus iniae were evaluated. The results indicated that two IL1ßs remained highly conserved possessing five conserved motifs compared with other fish IL1ßs, although there were 28 nucleotide differences and 16 amino acid differences between channel catfish IL1ß1 and IL1ß2. Analysis of the ratios of nonsynonymous (dN) and synonymous (dS) substitutions revealed that fish IL1ß genes were subjected to negative/purifying selection with global dN/dS ratios value 0.425. The results of adjuvant effect showed that compared with injection of pSCPI alone, co-injecting pSCPI with both rIL1ß1 and rIL1ß2 significantly enhanced antibody levels, serum bactericidal activity, lysozyme activity, alternative complement hemolytic activity, and the expression of endogenous IL1ß and TNF-α in head kidney and spleen. Although vaccination with rIL1ß1 or rIL1ß2 failed to offer immunoprotection against S. iniae infection, the RPS (relative percent survival) of pSCPI+rIL1ß1 and pSCPI+rIL1ß2 groups were both higher than pSCPI alone (RPS, 50%), with 64.26% and 60.71%, respectively. Moreover, pSCPI+rIL1ß1+rIL1ß2 offered significantly higher (P < 0.05) immunoprotection (RPS, 75%) against S. iniae infection than pSCPI alone. Our present results not only enrich the molecular structure study of fish IL1ßs but also signify that two recombinant channel catfish IL1ßs can be used as potential adjuvants in a subunit vaccine model against bacterial infection, which are of profound importance to prevent and control bacterial disease in channel catfish.

Imaging flow cytometry and confocal microscopy-based examination of F-actin and phosphoinositide dynamics during leukocyte immune-type receptor-mediated phagocytic events.

Cells of the innate immune system rapidly detect and eliminate invading microbes using surface-expressed immunoregulatory receptors that translate extracellular binding events into potent effector responses. Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are a family of immunoregulatory proteins that have been shown to regulate several innate immune cell effector responses including the phagocytic process. The mechanisms by which these receptors regulate phagocytosis are not entirely understood but we have previously shown that different IpLITR-types use ITAM-dependent as well as ITAM-independent pathways for controlling target engulfment. The main objective of this study was to develop and use imaging flow cytometry and confocal microscopy-based assays to further examine both F-actin and phosphoinositide dynamics that occur during the different IpLITR-mediated phagocytic pathways. Results show that the ITAM-dependent IpLITR-induced phagocytic response promotes canonical changes in F-actin polymerization and PI(4,5)P2 redistributions. However, the ITAM-independent IpLITR phagocytic response induced unique patterns of F-actin and PI(4,5)P2 redistributions, which are likely due to its ability to regulate alternative signaling pathways. Additionally, both IpLITR-induced phagocytic pathways induced target internalization into PI(3)P-enriched phagosomes indicative of a maturing phagosome compartment. Overall, this imaging-based platform can be further applied to monitor the recruitment and distribution of signaling molecules during IpLITR-mediated phagocytic processes and may serve as a useful strategy for functional examinations of other immunoregulatory receptor-types in fish.