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.

PIAS Factors from Rainbow Trout Control NF-κB- and STAT-Dependent Gene Expression.

Four 'protein inhibitors of activated STAT' (PIAS) control STAT-dependent and NF-κB-dependent immune signalling in humans. The genome of rainbow trout (Oncorhynchus mykiss) contains eight pias genes, which encode at least 14 different pias transcripts that are differentially expressed in a tissue- and cell-specific manner. Pias1a2 was the most strongly expressed variant among the analysed pias genes in most tissues, while pias4a2 was commonly low or absent. Since the knock-out of Pias factors in salmonid CHSE cells using CRISPR/Cas9 technology failed, three structurally different Pias protein variants were selected for overexpression studies in CHSE-214 cells. All three factors quenched the basal activity of an NF-κB promoter in a dose-dependent fashion, while the activity of an Mx promoter remained unaffected. Nevertheless, all three overexpressed Pias variants from trout strongly reduced the transcript level of the antiviral Stat-dependent mx gene in ifnγ-expressing CHSE-214 cells. Unlike mx, the overexpressed Pias factors modulated the transcript levels of NF-κB-dependent immune genes (mainly il6, il10, ifna3, and stat4) in ifnγ-expressing CHSE-214 cells in different ways. This dissimilar modulation of expression may result from the physical cooperation of the Pias proteins from trout with differential sets of interacting factors bound to distinct nuclear structures, as reflected by the differential nuclear localisation of trout Pias factors. In conclusion, this study provides evidence for the multiplication of pias genes and their sub-functionalisation during salmonid evolution.

Functionalization of Photosensitized Silica Nanoparticles for Advanced Photodynamic Therapy of Cancer.

BODIPY dyes have recently attracted attention as potential photosensitizers. In this work, commercial and novel photosensitizers (PSs) based on BODIPY chromophores (haloBODIPYs and orthogonal dimers strategically designed with intense bands in the blue, green or red region of the visible spectra and high singlet oxygen production) were covalently linked to mesoporous silica nanoparticles (MSNs) further functionalized with PEG and folic acid (FA). MSNs approximately 50 nm in size with different functional groups were synthesized to allow multiple alternatives of PS-PEG-FA decoration of their external surface. Different combinations varying the type of PS (commercial Rose Bengal, Thionine and Chlorine e6 or custom-made BODIPY-based), the linkage design, and the length of PEG are detailed. All the nanosystems were physicochemically characterized (morphology, diameter, size distribution and PS loaded amount) and photophysically studied (absorption capacity, fluorescence efficiency, and singlet oxygen production) in suspension. For the most promising PS-PEG-FA silica nanoplatforms, the biocompatibility in dark conditions and the phototoxicity under suitable irradiation wavelengths (blue, green, or red) at regulated light doses (10-15 J/cm2) were compared with PSs free in solution in HeLa cells in vitro.

Manipulating Charge-Transfer States in BODIPYs: A Model Strategy to Rapidly Develop Photodynamic Theragnostic Agents.

On the basis of a family of BINOL (1,1'-bi-2-naphthol)-based O-BODIPY (dioxyboron dipyrromethene) dyes, it is demonstrated that chemical manipulation of the chromophoric push-pull character, by playing with the electron-donating capability of the BINOL moiety (BINOL versus 3,3'-dibromoBINOL) and with the electron-acceptor ability of the BODIPY core (alkyl substitution degree), is a workable strategy to finely balance fluorescence (singlet-state emitting action) versus the capability to photogenerate cytotoxic reactive oxygen species (triplet-state photosensitizing action). It is also shown that the promotion of a suitable charge-transfer character in the involved chromophore upon excitation enhances the probability of an intersystem crossing phenomenon, which is required to populate the triple state enabling singlet oxygen production. The reported strategy opens up new perspectives for rapid development of smarter agents for photodynamic theragnosis, including heavy-atom-free agents, from a selected organic fluorophore precursor.

The Atlantic salmon interleukin 4/13 receptor family: Structure, tissue distribution and modulation of gene expression.

Interleukin (IL)-4 and IL-13 play a central role in T helper 2 immune response in mammals. The cell signalling is mediated by the type I heterodimeric receptor containing the IL-4Rα and γC chains, and the type II receptors formed by IL-4Rα and IL-13Rα1. In salmonid species, three paralogues of the IL-4 and IL-13 cytokines have been reported, il-4/13a, il-4/13b1 and il-4/13b2. In regard to receptors, two paralogues of each IL-4/13 receptor chains have been identified in rainbow trout while five genes named γc1, il-4rα, il-13rα1a, il-13rα1b, and il-13rα2 have identified in Atlantic salmon. Since Atlantic salmon is an important farmed fish species, the aim of this work was to get new insights into distribution, structure and expression regulation of the IL-4/13 receptors in salmon. By using qRT-PCR, it was shown that all γc1, il-4rα, il-13rα1a, il-13rα1b, and il-13rα2 receptor chains were expressed in lymphoid and non-lymphoid tissues of healthy salmon, nonetheless γC expression was higher in lymphoid than non-lymphoid tissues. The in silico structural analysis and homology modelling of the predicted receptor proteins showed that domains and most motifs present in the superior vertebrate chains are conserved in salmon suggesting a conserved role for these receptor chains. Only IL-13Rα1B is a receptor chain with a unique structure that seem not to be present in higher vertebrates but in fish species. In order to determine the regulatory role of IL-4/13 on the expression of receptor chains, Atlantic salmon il-4/13A gene was synthetized and cloned in pET15b. The recombinant IL-4/13A was produced in E. coli and the activity of the purified cytokine was confirmed in vitro. The regulatory role of IL-4/13A on the expression of their potential receptors was tested in salmon receiving the recombinant cytokine and effects were compared with those of the control group. The results showed that IL-4/13A induced the expression of its own gene and GATA-3, in the head kidney of fish but not in the spleen, while IL-10 increased in both lymphoid organs indicating a regulatory role of this cytokine on the induction of Th2 responses in salmon. IFN-γ and MHC class II were also later induced in head kidney. In regard to the expression of the receptor chains, IL-4/13A upregulated the expression of γC, IL-13Rα1A and IL-13Rα2A in the spleen but not in the head kidney of salmon, indicating a role on the modulation of cell signalling for the Th2 response. Furthermore, Piscirickettsia salmonis infection of Atlantic salmon occurred with an increase of γC and IL-13Rα1A suggesting a potential role of the IL-4/13 system in bacterial immunity or pathogenesis. This study contributes to a better understanding of the IL-4/13A system in salmon, which as a key axis for Th2 response may be involved not only in pathogen elimination but also in adaptive immune repair that seems critical tolerance to infectious diseases.

Functionalized Fluorescent Silica Nanoparticles for Bioimaging of Cancer Cells.

Functionalized fluorescent silica nanoparticles were designed and synthesized to selectively target cancer cells for bioimaging analysis. The synthesis method and characterization of functionalized fluorescent silica nanoparticles (50-60 nm), as well as internalization and subcellular localization in HeLa cells is reported here. The dye, rhodamine 101 (R101) was physically embedded during the sol-gel synthesis. The dye loading was optimized by varying the synthesis conditions (temperature and dye concentration added to the gel) and by the use of different organotriethoxysilanes as a second silica precursor. Additionally, R101, was also covalently bound to the functionalized external surface of the silica nanoparticles. The quantum yields of the dye-doped silica nanoparticles range from 0.25 to 0.50 and demonstrated an enhanced brightness of 230-260 fold respect to the free dye in solution. The shell of the nanoparticles was further decorated with PEG of 2000 Da and folic acid (FA) to ensure good stability in water and to enhance selectivity to cancer cells, respectively. In vitro assays with HeLa cells showed that fluorescent nanoparticles were internalized by cells accumulating exclusively into lysosomes. Quantitative analysis showed a significantly higher accumulation of FA functionalized fluorescent silica nanoparticles compared to nanoparticles without FA, proving that the former may represent good candidates for targeting cancer cells.

Methylthio BODIPY as a standard triplet photosensitizer for singlet oxygen production: a photophysical study.

A complete photophysical study on the iodinated-BODIPY, 3,5-dimethyl-2,6-diiodo-8-thiomethyl-pyrromethene (MeSBDP), demonstrated that it is an excellent triplet photosensitizer for singlet oxygen production in a broad range of apolar and polar solvents. Besides its absorption and fluorescence emission spectra, the dynamics of its excited states including its intersystem crossing rate was characterized by femtosecond transient experiments. The photophysical study of its triplet state by nanosecond transient absorption spectroscopy and phosphorescence emission concluded to a diffusion-controlled quenching of 3MeSBDP by O2 and to a fraction of triplet state quenching by O2 close to unity. The high (>0.87) and solvent-insensitive singlet oxygen quantum yield φΔ measured by singlet oxygen phosphorescence emission, together with the noticeable photostability of MeSBSP, as well as the absence of quenching of singlet oxygen by MeSBDP itself, allows claiming it as an alternative standard photosensitizer for singlet oxygen production, under excitation either in the UV or in the visible range.

Isolation and Characterization of Salmonid CD4+ T Cells.

This study reports the isolation and functional characterization of rainbow trout (Oncorhynchus mykiss) CD4-1(+) T cells and the establishment of an IL-15-dependent CD4-1(+) T cell line. By using Abs specific for CD4-1 and CD3ε it was possible to isolate the double-positive T cells in spleen and head kidney. The morphology and the presence of transcripts for T cell markers in the sorted CD4-1(+)CD3ε(+) cells were studied next. Cells were found to express TCRα, TCRß, CD152 (CTLA-4), CD154 (CD40L), T-bet, GATA-3, and STAT-1. The sorted CD4-1(+) T cells also had a distinctive functional attribute of mammalian T lymphocytes, namely they could undergo Ag-specific proliferation, using OVA as a model Ag. The OVA-stimulated cells showed increased expression of several cytokines, including IFN-γ1, IL-4/13A, IL-15, IL-17D, IL-10, and TGF-ß1, perhaps indicating that T cell proliferation led to differentiation into distinct effector phenotypes. Using IL-15 as a growth factor, we have selected a lymphoid cell line derived from rainbow trout head kidney cells. The morphology, cell surface expression of CD4-1, and the presence of transcripts of T cell cytokines and transcription factors indicated that this is a CD4-1(+) T cell line. To our knowledge, this is the first demonstration of the presence of CD4-1(+)CD3ε(+) T cells in salmonids. As in mammals, CD4-1(+) T cells may be the master regulators of immune responses in fish, and therefore these findings and the new model T cell line developed will contribute to a greater understanding of T cell function and immune responses in teleost fish.

Adapting BODIPYs to singlet oxygen production on silica nanoparticles.

A modified Stöber method is used to synthesize spherical core-shell silica nanoparticles (NPs) with an external surface functionalized by amino groups and with an average size around 50 nm. Fluorescent dyes and photosensitizers of singlet oxygen were fixed, either separately or conjointly, respectively in the core or in the shell. Rhodamines were encapsulated in the core with relatively high fluorescence quantum yields (Φfl ≥ 0.3), allowing fluorescence tracking of the particles. Various photosensitizers of singlet oxygen (PS) were covalenty coupled to the shell, allowing singlet oxygen production. The stability of NP suspensions strongly deteriorated upon grafting the PS, affecting their apparent singlet oxygen quantum yields. Agglomeration of NPs depends both on the type and on the amount of grafted photosensitizer. New, lab-made, halogenated 4,4-difluoro-4-bora-3a,4a-diaza-s-indacenes (BODIPY) grafted to the NPs achieved higher singlet oxygen quantum yields (ΦΔ âˆ¼ 0.35-0.40) than Rose Bengal (RB) grafted NPs (ΦΔ âˆ¼ 0.10-0.27). Finally, we combined both fluorescence and PS functions in the same NP, namely a rhodamine in the silica core and a BODIPY or RB grafted in the shell, achieving the performance Φfl ∼ 0.10-0.20, ΦΔ âˆ¼ 0.16-0.25 with a single excitation wavelength. Thus, proper choice of the dyes, of their concentrations inside and on the NPs and the grafting method enables fine-tuning of singlet oxygen production and fluorescence emission.

Induction of anti-inflammatory cytokine expression by IPNV in persistent infection.

Infectious Pancreatic Necrosis Virus (IPNV) is the agent of a well-characterized acute disease that produces a systemic infection and high mortality in farmed fish species but also persistent infection in surviving fish after outbreaks. Because viral persistence of susceptible mammal hosts appears to be associated with the modulation of anti-inflammatory cytokine expression, in this study we examined the expression levels of key pro- and anti-inflammatory cytokines in kidney and spleen of trout, as well as humoral immune response (IgM and IgT) during experimental persistent viral infection and in the acute phase of infection as a comparison. IPNV infection in rainbow trout resulted in a distinct profile of cytokine expression depending on the type of infection, acute or persistent. Levels of early pro-inflammatory cytokines, IL-1ß and IL-8, did not increase in the head kidney of the fish with persistent asymptomatic infection but increased in some of the symptomatic infected fish. The antiviral cytokine IFNα was not significantly induced in any of the infected fish groups. The level of expression of the Th1-related cytokine IL-12 was significantly higher in trout with persistent asymptomatic infection than in symptomatic fish. This was also accompanied by an increase in IFNγ. The anti-inflammatory cytokines IL-10 and TGF-ß1 had distinct expression profiles. While IL-10 expression increased in all infected fish, TGF-ß1 was only up-regulated in fish with persistent infection. All infected fish had significantly lower total IgM levels than the non-infected fish whereas IgT levels did not change. Specific and neutralizing antibodies against IPNV were not observed in acute and persistent infection except in the group of fish with the lowest degree of clinical signs. Interestingly, the lack of humoral immune response could be associated with the high expression of anti-inflammatory cytokines, which might inhibit antibody production. The balance between pro-inflammatory Th1 type cytokines and the regulatory cytokines could explain the high percentage of survival and the resolution of the inflammatory response in the IPNV-infected fish but also the establishment of viral persistence.

Photosensitizer-Mesoporous Silica Nanoparticles Combination for Enhanced Photodynamic Therapy†.

Mesoporous silica nanoparticles (MSNs) are widely known for their versatile applications. One of the most extended is as drug delivery systems for the treatment of cancer and other diseases. This review compiles the most representative examples in the last years of functionalized MSNs as photosensitizer carriers for photodynamic therapy (PDT) against cancer. Several commercially available photosensitizers (PSs) demonstrated poor solubility in an aqueous medium and insufficient selectivity for cancer tissues. The tumor specificity of PSs is a key factor for enhancing the PDT effect and at the same time reducing side effects. The use of nanoparticles and particularly MSNs, in which PS is covalently anchored or physically embedded, can overcome these limitations. For that, PS-MSNs can be externally decorated with compounds of interest in order to act as an active target for certain cancer cells, demonstrating enhanced phototoxicity in vitro and in vivo. The objective of this review is to collect and compare different nanosystems based on PS-MSNs pointing out their advantages in PDT against diverse types of cancers.

The Acute Immune Responses of the Common Carp Cyprinus carpio to PLGA Microparticles-The Interactions of a Teleost Fish with a Foreign Material.

Poly lactic-co-glycolic acid (PLGA) particles safely and effectively deliver pharmaceutical ingredients, with many applications approved for clinical use in humans. In fishes, PLGA particles are being considered as carriers of therapeutic drugs and vaccine antigens. However, existing studies focus mainly on vaccine antigens, the endpoint immune responses to these (e.g., improved antibody titres), without deeper understanding of whether fishes react to the carrier. To test whether or not PLGA are recognized by or interact at all with the immune system of a teleost fish, we prepared, characterized and injected PLGA microparticles intraperitoneally into common carp. The influx, phenotype of inflammatory leukocytes, and their capacity to produce reactive oxygen species and phagocytose PLGA microparticles were tested by flow cytometry, qPCR, and microscopy. PLGA microparticles were indeed recognized. However, they induced only transient recruitment of inflammatory leukocytes that was resolved 4 days later whereas only the smallest µm-sized particles were phagocytosed. The overall response resembled that described in mammals against foreign materials. Given the similarities between our findings and those described in mammals, PLGA particles can be adapted to play a dual role as both antigen and drug carriers in fishes, depending on the administered dose and their design.

Variations in Rainbow Trout Immune Responses against A. salmonicida: Evidence of an Internal Seasonal Clock in Oncorhynchus mykiss.

In poikilothermic vertebrates, seasonality influences different immunological parameters such as leukocyte numbers, phagocytic activity, and antibody titers. This phenomenon has been described in different teleost species, with immunological parameters peaking during warmer months and decreased levels during winter. In this study, the cellular immune responses of rainbow trout (Oncorhynchus mykiss) kept under constant photoperiod and water temperature against intraperitoneally injected Aeromonas salmonicida during the summer and winter were investigated. The kinetics of different leukocyte subpopulations from peritoneal cavity, spleen, and head kidney in response to the bacteria was measured by flow cytometry. Furthermore, the kinetics of induced A. salmonicida-specific antibodies was evaluated by ELISA. Despite maintaining the photoperiod and water temperature as constant, different cell baselines were detected in all organs analyzed. During the winter months, B- and T-cell responses were decreased, contrary to what was observed during summer months. However, the specific antibody titers were similar between the two seasons. Natural antibodies, however, were greatly increased 12 h post-injection only during the wintertime. Altogether, our results suggest a bias toward innate immune responses and potential lymphoid immunosuppression in the wintertime in trout. These seasonal differences, despite photoperiod and water temperature being kept constant, suggest an internal inter-seasonal or circannual clock controlling the immune system and physiology of this teleost fish.

BINOL blocks as accessible triplet state modulators in BODIPY dyes.

BINOL moieties of different electronic demand are useful blocks for enabling the photo-production and modulation of triplet excited states in readily-accesible BINOL-based O-BODIPY dyes from standard F-BODIPY precursors. The rapid and rational development of smarter triplet-enabling BODIPY dyes on the basis of this strategy (e.g., TADF biomarker 4a or room temperature phosphor 4g) paves the way for advancing photonic applications based on organic triplet photosensitizers.

Halogen-free photosensitizers based on meso-enamine-BODIPYs for bioimaging and photodynamic therapy.

The search for efficient heavy atom free photosensitizers (PSs) for photodynamic therapy (PDT) is a very active field. We describe herein a simple and easily accessible molecular design based on the attachment of an enamine group as an electron-donor moiety at the meso position of the BODIPY core with different alkylation patterns. The effect of the alkylation degree and solvent polarity on the photophysical properties in terms of splitting absorption bands, fluorescence efficiencies and singlet oxygen production is analyzed in depth experimentally using spectroscopic techniques, including femtosecond and nanosecond transient absorption (fs- and ns-TA) and using computational simulations based on time-dependent density functional theory. The correlation between the theoretical/experimental results permits the rationalization of the observed photophysical behavior exhibited by meso-enamine-BODIPY compounds and the determination of mechanistic details, which rule the population of the triplet state manifold. The potential applicability as a theragnostic agent for the most promising compound is demonstrated through in vitro assays in HeLa cells by analyzing the internalization, localization and phototoxic action.

Identification of CD3ε, CD4, CD8ß splice variants of Atlantic salmon.

In vertebrates, CD3 complex and CD4 and CD8 co-receptors are essential for signal transduction during T cell activation. In the present study, we report the mRNA spliced variants of the Atlantic salmon CD3ε, CD4 and CD8ß and the effect of pathogen encounter on the expression of these variants. CD3ε is alternatively spliced in thymus, head kidney, spleen and gills to give rise to the complete mRNA sequence and to an alternative product that lacks the transmembrane exon. CD4 is also alternatively spliced in the thymus, head kidney, spleen and gills to form two variants, although the alternative product is barely detectable. The alternative product lacks the exon 1B encoding the D1 domain, which is essential for binding to MHC class II proteins. Two amplicons were also found for the CD8ß gene; sequencing analysis revealed that the main PCR product corresponds to the previously reported CD8ß sequence, whereas the variant sequence encodes a potential protein that lacks the Ig-like domain. The expression of CD3, CD4, CD8ß genes also analyzed in head kidney of LPS-treated and IPNV infected salmon and different patterns of expression were observed. The presence and balance of the different variants of T cell co-receptors could be related to the ability of fish to induce a particular type of immune response, as well as, the ability of the pathogen to modify the fish immune response.

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.

Exploring BODIPY Derivatives as Singlet Oxygen Photosensitizers for PDT.

This minireview is devoted to honoring the memory of Dr. Thomas Dougherty, a pioneer of modern photodynamic therapy (PDT). It compiles the most important inputs made by our research group since 2012 in the development of new photosensitizers based on BODIPY chromophore which, thanks to the rich BODIPY chemistry, allows a finely tuned design of the photophysical properties of this family of dyes to serve as efficient photosensitizers for the generation of singlet oxygen. These two factors, photophysical tuning and workable chemistry, have turned BODIPY chromophore as one of the most promising dyes for the development of improved photosensitizers for PDT. In this line, this minireview is mainly related to the establishment of chemical methods and structural designs for enabling efficient singlet oxygen generation in BODIPYs. The approaches include the incorporation of heavy atoms, such as halogens (iodine or bromine) in different number and positions on the BODIPY scaffold, and also transition metal atoms, by their complexation with Ir(III) center, for instance. On the other hand, low-toxicity approaches, without involving heavy metals, have been developed by preparing several orthogonal BODIPY dimers with different substitution patterns. The advantages and drawbacks of all these diverse molecular designs based on BODIPY structural framework are described.

Kinetics of local and systemic immune cell responses in whirling disease infection and resistance in rainbow trout.

BACKGROUND: Whirling disease (WD), caused by the myxozoan parasite Myxobolus cerebralis, is responsible for high mortalities in rainbow trout hatcheries and natural populations. To elucidate how resistant and susceptible rainbow trout strains respond to early invasion, a well-established model of WD was used to demonstrate the kinetics of local and systemic immune responses in two rainbow trout strains, the susceptible American Trout Lodge (TL) and the more resistant German Hofer strain (HO). METHODS: Parasite load and cellular immune responses were compared across several time points after M. cerebralis exposure to elucidate the kinetics of immune cells in resistant and susceptible rainbow trout in response to early invasion. In the course of the 20 days following exposure, leukocyte kinetics was monitored by flow cytometry in the caudal fin (CF), head kidney (HK) and spleen (SP). For the analysis of the leukocyte composition, cells were stained using a set of monoclonal antibodies with known specificity for distinct subpopulations of rainbow trout leukocytes. RESULTS: Experiments indicated general increases of CF, HK and SP myeloid cells, while decreases of B cells and T cells in the SP and HK were observed at several time points in the TL strain. On the other hand, in the HO strain, increases of T cells were dominant in CF, HK and SP at multiple time points. The differences between HO and TL were most distinct at 2, 4, 12 and 48 hours post-exposure (hpe) as well as at 4 days post-exposure (dpe), with the vast majority of innate immune response cells having higher values in the susceptible TL strain. Alteration of the leukocyte populations with augmented local cellular responses and excessive immune reactions likely lead to subsequent host tissue damage and supports parasite invasion and development in TL. CONCLUSIONS: The findings of this study highlight the significance of effective local and systemic immune reaction and indicate proper activation of T lymphocytes critical for host resistance during M. cerebralis infection. The present study provides insights into the cellular basis of protective immune responses against M. cerebralis and can help us to elucidate the mechanisms underlying the variation in resistance to WD.

Dawn to Dusk: Diurnal Rhythm of the Immune Response in Rainbow Trout (Oncorhynchus Mykiss).

The daily change of light and dark periods influences different physiological processes including feeding, resting and locomotor activity. Previously, several studies on mammalian models revealed a strong link between day-night rhythms and key immunological parameters. Since teleost fishes possess innate and adaptive immune responses like those observed in higher vertebrates, we aimed to elucidate how changes in light-dark cycles shape the immune system of fish. Using the rainbow trout laboratory model, we investigated the link between diurnal rhythms and immune competence of fish. Initially, the cell composition and phagocytic activity of leukocytes was analyzed in the circulation as well as in the head kidney, the functional ortholog of mammalian bone marrow. Once the baseline was established, we evaluated the ability of fish to respond to a bacterial stimulus, as well as the changes in antimicrobial activity of the serum. Our results suggest increased immune competence during the day, manifested by the higher presence of myeloid cells in the circulation; increased overall phagocytic activity; and higher capacity of the sera to inhibit the growth of Aeromonas salmonicida. Notably, our flow cytometric analysis identified the myeloid cells as the major population influenced by the time of day, whereas IgM+ B cells and thrombocytes did not vary in a significant manner. Interestingly, the presence of myeloid cells in blood and head kidney followed complementary trends. Thus, while we observed the highest number of myeloid cells in the blood during early morning, we witnessed a reverse trend in the head kidney, suggesting a homing of myeloid cells to reservoir niches with the onset of the dark phase. Further, the presence of myeloid cells was mirrored in the expression of the proinflammatory marker tnfa as well as in the number of leukocytes recruited to the peritoneal cavity in the peritonitis model of inflammation. Overall, the data suggest a connection between diurnal rhythms and the immune response of rainbow trout and highlight the relevance of rhythmicity and its influence on experimental work in the field of fish chronoimmunology.