PACAP binds conserved receptors and modulates cytokine gene expression and protein secretion in trout cell lines.

Antimicrobial peptides (AMPs) are an alternative to antibiotics for treatment and prevention of infections with a lower risk of bacterial resistance. Pituitary adenylate cyclase activating polypeptide (PACAP) is an outstanding AMP with versatile effects including antimicrobial activity and modulation of immune responses. The objective of this research was to study PACAP immunomodulatory effect on rainbow trout cell lines infected with Aeromonas salmonicida. PACAP from Clarias gariepinus (PACAP1) and a modified PACAP (PACAP5) were tested. RT-qPCR results showed that il1b and il8 expression in RTgutGC was significantly downregulated while tgfb expression was upregulated after PACAP treatment. Importantly, the concentration of IL-1ß and IFN-γ increased in the conditioned media of RTS11 cells incubated with PACAP1 and exposed to A. salmonicida. There was a poor correlation between gene expression and protein concentration, suggesting a stimulation of the translation of IL-1ß protein from previously accumulated transcripts or the cleavage of accumulated IL-1ß precursor. In-silico studies of PACAP-receptor interactions showed a turn of the peptide characteristic of PACAP-PAC1 interaction, correlated with the higher number of interactions observed with this specific receptor, which is also in agreement with the higher PACAP specificity described for PAC1 compared to VPAC1 and VPACA2. Finally, the in silico analysis revealed nine amino acids related to the PACAP receptor-associated functionality.

Effects of artificial photoperiods on antigen-dependent immune responses in rainbow trout (Oncorhynchus mykiss).

In this study, we investigated the effects of the artificial photoperiods that mimic summer (16L:8D; 16 h Light: 8 h Dark) and winter (8L:16D) solstices, equinoxes (12L:12D), and the artificial 24-h light regimen (24L:0D) on the leukocyte populations and the T helper and regulatory type responses on rainbow trout (Oncorhynchus mykiss). Using flow cytometry analysis, we found that photoperiod induces changes in head kidney leukocyte subsets. The lymphoid subset increased in the 16L:8D summer solstice regime. The analysis using antibodies against B and T cells showed the increase of CD4-1+ T lymphocytes and other unidentified lymphoid cells, with no changes in the B cells. To investigate the modulatory influence of the photoperiod on the fish T cell response, we quantified in the head kidney the transcript levels of genes involved in the Th1 type response (t-bet, ifn-Æ´, il-12p35, il-12p40c), Th2 type response (gata3, il-4/13a), Th17 response (ror-Æ´t, il-17a/f), T regulatory response (foxp3α, il-10a, tgf-ß1), and the T cell growth factor il-2. The results showed that the seasonal photoperiod alone has a limited influence on the expression of these genes, as the only difference was observed in il-14/13a and il-10a transcripts of fish kept on the 16L:8D regimen. In addition, the 24L:0D treatment used in aquaculture produces a reduction of il-14/13a and il-17a/f. We also evaluated the effect of photoperiod in the presence of an antigenic stimulus. Thus, in fish immunized with the recombinant viral protein 1 (rVP1) of infectious pancreatic necrosis virus (IPNV), the photoperiod had a striking influence on the type of adaptive immune response. Each photoperiod fosters a unique immune signature of antigenic response. A classical type 1 response is observed in fish subjected to the 16L:8D photoperiod. In contrast, fish in the 12L:12D photoperiod showed only the upregulation of il-12p40c. Furthermore, none of the cytokines were increased in fish maintained on the artificial 24L:0D regimen, and a decrease in the master transcription factors (t-bet, ror-Æ´t, and foxp3α) was observed. Thus, fish on the 12L:12D and 24L:0D photoperiod appear hyporesponsive regarding the T cell response. Altogether, this study showed that photoperiods modify the magnitude and quality of the T-helper response in rainbow trout and thus impact essential mechanisms for the generation of immune memory and protection against microorganisms.

Infectious pancreatic necrosis virus (IPNV) recombinant viral protein 1 (VP1) and VP2-Flagellin fusion protein elicit distinct expression profiles of cytokines involved in type 1, type 2, and regulatory T cell response in rainbow trout (Oncorhynchus mykiss).

In this study, we examined the cytokine immune response against two proteins of infectious pancreatic necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss), the virion-associated RNA polymerase VP1 and VP2-Flagellin (VP2-Flg) fusion protein. Since VP1 is not a structural protein, we hypothesize it can induce cellular immunity, an essential mechanism of the antiviral response. At the same time, the fusion construction VP2-Flg could be highly immunogenic due to the presence of the flagellin used as an adjuvant. Fish were immunized with the corresponding antigen in Montanide™, and the gene expression of a set of marker genes of Th1, Th2, and the immune regulatory response was quantified in the head kidney of immunized and control fish. Results indicate that VP1 induced upregulation of ifn-γ, il-12p40c, il-4/13a, il-4/13b2, il-10a, and tgf-ß1 in immunized fish. Expression of il-2a did not change in treated fish at the times tested. The antigen-dependent response was analysed by in vitro restimulation of head kidney leukocytes. In this assay, the group of cytokines upregulated after VP1-restimulation was consistent with those upregulated in the head kidney in vivo. Interestingly, VP1 induced il-2a expression after in vitro restimulation. The analysis of sorted lymphocytes showed that the increase of cytokines occurred in CD4-1+ T cells suggesting that Th differentiation happens in response to VP1. This is also consistent with the expression of t-bet and gata3, the master regulators for Th1/Th2 differentiation in the kidneys of immunized animals. A different cytokine expression profile was found after VP2-Flg administration, i.e., upregulation occurs for ifn-γ, il-4/13a, il-10a, and tgf-ß1, while down-regulation was observed in il-4/13b2 and il-2a. The cytokine response was due to flagellin; only the il-2a effect was dependent upon VP2 in the fusion protein. To the best of our knowledge this study reports for the first-time characteristics of the adaptive immune response induced in response to IPNV VP1 and the fusion protein VP2-Flg in fish. VP1 induces cytokines able to trigger the humoral and cell-mediated immune response in rainbow trout. The analysis of the fish response against VP2-Flg revealed the immunogenic properties of Aeromonas salmonicida flagellin, which can be further tested for adjuvanticity. The novel immunogenic effects of VP1 in rainbow trout open new opportunities for further IPNV vaccine development using this viral protein.

Antigen presentation in vertebrates: Structural and functional aspects.

Antigen presentation is a key process of the immune system and is responsible for the activation of T cells. The main characters are the major histocompatibility complex class I (MHC-I) and class II (MHC-II) molecules, and accessory proteins that act as chaperones for these glycoproteins. Current knowledge of this process and also the elucidation of the structural features of these proteins, has been extensively reviewed in humans. Unfortunately, this is not the case for non-human species, wherein the function and structural characteristic of the antigen presentation proteins is far from being understood. The majority of previous studies in non-human species, especially in teleost fish and lower vertebrates, are limited to the transcriptomic level, which leads to gaps in the knowledge about the functional process of antigen presentation in these species. This review summarizes what is known so far about antigen presentation pathways in vertebrates from a structural and functional perspective. The focus is not only on the MHC receptors, but also, on the forgotten characters of these pathways such as the proteins of the peptide loading complex, and the MHC-II chaperone invariant chain.

Interferon Gamma Induces the Increase of Cell-Surface Markers (CD80/86, CD83 and MHC-II) in Splenocytes From Atlantic Salmon.

Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and adaptive immune responses which has been widely characterized in several teleost families. In fish, IFNγ stimulates the expression of cytokines and chemokines associated with the pro-inflammatory response and enhances the production of nitrogen and oxygen reactive species in phagocytic cells. This work studied the effect of IFNγ on the expression of cell-surface markers on splenocytes of Atlantic salmon (Salmo salar). In vitro results showed that subpopulations of mononuclear splenocytes cultured for 15 days were capable of increasing gene expression and protein availability of cell-surface markers such as CD80/86, CD83 and MHC II, after being stimulated with recombinant IFNγ. These results were observed for subpopulations with characteristics associated with monocytes (51%), and features that could be related to lymphocytes (46.3%). In addition, a decrease in the expression of zbtb46 was detected in IFNγ-stimulated splenocytes. Finally, the expression of IFNγ and cell-surface markers was assessed in Atlantic salmon under field conditions. In vivo results showed that the expression of ifnγ increased simultaneously with the up-regulation of cd80/86, cd83 and mhcii during a natural outbreak of Piscirickettsia salmonis. Overall, the results obtained in this study allow us to propose IFNγ as a candidate molecule to stimulate the phenotypic progression of a small population of immune cells, which will increase antigen presenting cells markers. Thereby, modulatory strategies using IFNγ may generate a robust and coordinated immune response in fish against pathogens that affect aquaculture.

Revisiting the Teleost Thymus: Current Knowledge and Future Perspectives.

The thymus in vertebrates plays a critical role in producing functionally competent T-lymphocytes. Phylogenetically, the thymus emerges early during evolution in jawed cartilaginous fish, and it is usually a bilateral organ placed subcutaneously at the dorsal commissure of the operculum. In this review, we summarize the current understanding of the thymus localization, histology studies, cell composition, and function in teleost fishes. Furthermore, we consider environmental factors that affect thymus development, such as seasonal changes, photoperiod, water temperature fluctuations and hormones. Further analysis of the thymus cell distribution and function will help us understand how key stages for developing functional T cells occur in fish, and how thymus dynamics can be modulated by external factors like photoperiod. Overall, the information presented here helps identify the knowledge gaps and future steps needed for a better understanding of the immunobiology of fish thymus.