Fish cell cultures as in vitro models of inflammatory responses elicited by immunostimulants. Expression of regulatory genes of the innate immune response.

We report the differential expression of various genes related to the regulation of the innate immune responses, including pro-inflammatory (IL-1ß1, IL-8, TNF-α1, TNF-α2) and immune-suppressing (IL-10) cytokines, interferon-induced Mx-1 protein, enzymes regulating nitric oxide (inducible nitric oxide synthase, arginase-2) and eicosanoid (COX-2) production, and Toll-like pathogen pattern-recognition receptors TLR-3, TLR-5 and TLR-9, in two lympho-haematopoietic stromal cell lines derived from the spleen (trout splenic stroma, TSS) and the pronephros (trout pronephric stroma-2, TPS-2) of rainbow trout (Oncorhynchus mykiss), as well as in primary cultures of rainbow trout head kidney macrophages, after their exposure to the well-known immunostimulants LPS, levamisole and poly I:C. Although there were differences in the responses between the two stromal cell lines, using reverse transcription followed by real time polymerase chain reaction (RT-qPCR) we demonstrated that exposure to the immunostimulants, particularly poly I:C and LPS, resulted in significant changes in the expression of the immunoregulatory genes in the two stromal cell lines in many cases their responses resembling in fold change magnitudes and in response profiles to those observed in the primary macrophage cultures. Exposure to poly I:C and, with lower fold change values, to LPS produced upregulation of the pro- (IL-1ß, IL-8, TNF-α) and anti-inflammatory (IL-10) cytokine genes, as well as of the Mx-1 gene. Furthermore, the immunostimulation elicited the upregulation of COX-2, iNOS and arginase-2 genes in the cell lines. Likewise, the TSS and TPS-2 cell lines significantly upregulated the expression of TLR-3, TLR-5 and TLR-9 genes after exposure to the immunostimulants, thus explaining the ability of the stromal cells to recognise and respond to the immunostimulants. Such results give support to an important role of lympho-haematopoietic stromal cells in the development and control of pro-inflammatory responses in fish. The upregulation of genes of pro-inflammatory cytokines and of mediators of the innate immune responses correlates well with the previously demonstrated functional capacities, including phagocytosis, microbicidal activity and NO production, exhibited by the TSS and TPS-2 stromal cell lines when exposed to the same immunostimulants. On the other hand, the expression of immunosuppressing genes (IL-10, COX-2 and arginase-2) demonstrate that the lympho-haematopoietic stromal cells are also able to contribute to the control of inflammatory responses. This study reinforce the possibility of using histotypic cell cultures, as those formed by the TSS and TPS-2 cell lines, formed by heterogeneous cell populations that partially replicates the cell-cell and cell-extracellular matrix interactions, to develop cost-effective and repetitive in vitro systems for the screening of immunostimulant candidates for aquaculture, as they are able to replicate in vitro immune regulatory networks occurring in vivo.

Fish cell cultures as in vitro models of pro-inflammatory responses elicited by immunostimulants.

We have tested the elicitation of innate defence-related responses in two stromal cell lines derived from the spleen (trout splenic stroma, TSS) and the pronephros (trout pronephric stroma-2, TPS-2) of rainbow trout (Oncorhynchus mykiss) after they were exposed to different concentrations of lipopolysaccharide (LPS), levamisole, or polyinosinic polycytidylic acid (poly-I:C). For comparison, cultures of rainbow trout head kidney macrophages were also included in the study, and the effect of the immunostimulants on the phagocytic activity, the intracellular and extracellular reactive oxygen species and nitric oxide production were assayed. Although the responses varied depending upon the concentration of the immunostimulants and the particular cell line, our results demonstrate that those activities were enhanced in the TSS and TPS-2 cell lines after exposure to any of the immunostimulants. These results indicate that the stromal cells of the main lympho-haemopoietic organs of O. mykiss develop innate defence responses, which are enhanced by well-known immunostimulants. In addition, such enhancement of the defence responses in the TSS and TPS-2 cell lines could be also elicited when they were exposed to conditioned supernatants from levamisole- or poly I:C-stimulated HK macrophage cultures, thus demonstrating that the haemopoietic stromal cells respond to macrophage-derived factors. Moreover, we demonstrate that the stromal cell lines constitutively expressed the Toll-like receptors TLR3, TLR5 and TLR9 genes. The results are discussed considering the role of the lympho-haemopoietic stromal cells in the innate immune responses, and the possibility of using histiotypic cell cultures of non-leucocyte cells of the haemopoietic organs to develop in vitro methods to select new immunostimulant candidates for aquaculture.

The in vitro infection of the hematopoietic stroma of trout kidney by hemorrhagic septicemia rhabdovirus.

Viral hemorrhagic septicaemia virus (VHSV) infected the hematopoietic stromal cells (7,8) derived from pronephritic tissue of the rainbow trout, Oncorhynchuss mykiss, W., at their ninth passage in vitro. Viral infection resulted in the development of lytic cytopathic effects on confluent in vitro tridimensional network stromal cell cultures. Replication of VHSV in the stromal cell cultures was demonstrated by the increase in infectivity by epithelioma papulosum cyprini (EPC) cell culture assays and by the increase of the nucleoprotein antigen of VHSV by ELISA. By using anti-VHSV monoclonal antibodies (MAbs), flow cytometry studies demonstrated that only the infected stromal cells contained cytoplasmic viral antigens. The lytic infection of trout hematopoietic stromal cells in vitro could be relevant to the hemorrhagic pathology seen in the kidney of fish infected with VHSV.

Correlation between production of acyl homoserine lactones and proteases in an Aeromonas hydrophila aroA live vaccine.

Aeromonas hydrophila is a pathogen that causes disease in a wide range of homeothermic and poikilothermic hosts due to its multifactorial virulence. We have previously described the characterisation and use of an auxotrophic aroA mutant of the A. hydrophila AG2 strain as a live attenuated vaccine against A. hydrophila infections in rainbow trout (Oncorhynchus mykiss). In this study we report the expression of extracellular proteolytic activities and of quorum-sensing molecules by this mutant grown under different culture conditions, and in vaccine inocula. The aroA strain expresses extracellular proteases efficiently during in vitro growth and this ability is retained in vaccine inocula that were prepared by washing the bacterial cultures and resuspending the cells in phosphate-buffered saline. Since proteases are considered to be major bacterial antigens, the expression of these enzymes in the live attenuated vaccine may contribute to the superior protection afforded by these kind of vaccines. On the other hand, the production of serine- and metalloprotease activities in A. hydrophila has been described as controlled in a cell density-dependent fashion, through a mechanism known as quorum sensing. A microtiter method was developed that allowed correlation of the production of quorum-sensing molecules and of proteases produced by the aroA strain during in vitro growth and in the vaccine inocula. The production of both products was related to the type of culture medium and conditions used to grow the aroA mutant, whereas there was no correlation between the concentration of acyl homoserine lactones and protease production.

Alterations in the peripheral lymphoid organs and differential leukocyte counts in Saprolegnia-infected brown trout, Salmo trutta fario.

The number of circulating leukocytes and the structure of splenic and renal lymphoid tissue were comparatively analysed in healthy and Saprolegnia-infected wild brown trout, Salmo trutta fario. Sick trout showed lymphopenia, mainly due to decreased numbers of circulating small lymphocytes, and heterophilia. The splenic and renal lymphoid tissue of infected trout exhibited similar changes, consisting of cellular depletion, lymphoid cell degeneration, and vascular alterations with blood vessel enlargement and hypertrophy of sinusoidal endothelial cells. Furthermore, the endothelial cells in the spleen and kidney of the infected trout contained cytoplasmic vesicles filled with material of possible fungal origin. The absence of a reticular sheath was also evident in the splenic ellipsoids. These results suggest some immunodepression in Saprolegnia-infected trout which might favour the course of the disease.

Modulation of the immune response to an Aeromonas hydrophila aroA live vaccine in rainbow trout: effect of culture media on the humoral immune response and complement consumption.

The Aeromonas hydrophila aroA is an attenuated strain that has been assessed as a live vaccine in rainbow trout, Oncorhynchus mykiss. In this study the effects of different culture media used to grow the strain on its survival after in vitro exposure to rainbow trout serum, and on its immunogenicity in rainbow trout were compared. Four culture media were tested: Luria broth (LB), Luria broth with 0.25% glucose, trypticase soy broth (TSB), and brain-heart infusion broth (BHIB). Bacteria grown in culture media with glucose (TSB, BHIB and LB with 0.25% glucose) showed reduced complement consumption and a lower serum susceptibility. O. mykiss vaccinated with inocula prepared with BHIB- and LB-grown aroA cells resuspended in phosphate-buffered saline (PBS) showed higher and longer-lasting serum agglutinating antibody titres than those vaccinated with TSB-grown bacteria. Thus, a direct relationship between serum resistance and immunogenicity could not be established, but BHIB and LB culture media were the most effective in increasing the immunogenicity of the A. hydrophila aroA vaccine.

Tissue distribution and structure of barrier cells in the hematopoietic and lymphoid organs of salmonids.

BACKGROUND: Barrier cells have been recognized as a discrete group of fibroblastic- or myofibroblastic-like cells located in the lymphoid and hematopoietic organs of mammals. This paper reports the results of a morphological study of the main lymphoid organs of three salmonid species, in which cells structurally similar to the mammalian barrier cells were observed in healthy animals. METHODS: The spleen, kidney, and thymus of fingerlings of rainbow trout, Oncorhynchus mykiss, and Coho salmon, Oncorhynchus kisutch, and of adult brown trout, Salmo trutta fario, were processed for electron microscopy study using various fixation methods. Semithin sections were used for the Periodic Acid-Schiff (PAS) staining technique, and for the demonstration of the endogenous peroxidase activity. RESULTS: The kidney and spleen of all the species contained a variable, but usually low, number of electron-dense, elongated, and branched cells, ultrastructurally similar to the mammalian barrier cells. They also occurred in the thymus of some brown trout and Coho salmon, but not of rainbow trout. The electron density of this cell type was present after the various types of fixation procedures. They show numerous ribosomes, well-developed secretory organelles, electron-clear vesicles, large granules, and microfilaments. In all the salmonid species, barriers cells were positive for PAS staining and for endogenous peroxidase activity. A small number of barrier cells were in mitosis. In the different organs barrier cells appeared as isolated cells, or forming syncytial networks. They were found lining the blood sinusoids of the splenic red pulp and of the renal hematopoietic tissue, in the periellipsoidal sheaths, and closely associated with erythropoietic and plasmacytopoietic foci. CONCLUSIONS: Our results demonstrate the presence of cells closely resembling mammalian barrier cells in the hematopoietic and lymphoid organs of salmonids. The structure and tissue distribution of the salmonid barrier cells are discussed in relation to the functional roles described for this cell type in mammals.

Post-hatching development of the thymic epithelial cells in the rainbow trout Salmo gairdneri: an ultrastructural study.

This study reports the ultrastructure of subpopulations of epithelial cells of the thymic parenchyma during the post-hatching development of the rainbow trout, Salmo gairdner, kept at 14 degrees C. At hatching, the thymus contained a small number of medium and large thymocytes interspersed among three different types of epithelial cells: (1) epithelial cells adjacent to the connective tissue capsule; (2) ramified dark epithelial cells with electron-dense cytoplasm; and (3) pale electron-lucent epithelial cells displaying secretory-like features. All these cells types were anchored to one another by desmosomes and had apparently differentiated from the pharyngeal epithelium. At 4 days after hatching, the thymus enlarged, and numerous gaps occurred between the cell processes of contiguous epithelial cells adjacent to the capsular connective tissue. In 21-day-old trout, thymic trabeculae developed carrying blood vessels, and a subcapsular zone became evident containing lymphoblasts and large subcapsular epithelial cells. In 30-day-old trout, an outer thymic zone developed consisting of spindle-shaped epithelial cells which formed a dense network. At this stage, scattered cystic cells, which apparently differentiated from the pale epithelial cells, were present.

Spontaneous in vitro angiogenesis in a trout pronephric stromal cell line (TPS), and in TPS-haemopoietic co-cultures.

This study describes angiogenic processes taking place in the in vitro micro-environment of a trout pronephric stroma cell line (TPS) under specific culture conditions, in which fetal calf serum, horse serum and hydrocortisone-sodium-21-hemisuccinate were used as supplements to the culture medium. When TPS cultures were kept in the same flask, i.e. without passages, for longer than 7 months, epithelioid cells differentiated into endothelial cells. Early stages of such differentiation were characterised by the presence of intracellular tubular vacuoles in clusters of neighbouring epithelioid cells. Subsequently, the endothelial cells reorganised and gave rise to microvascular structures, which branched over and into the TPS multilayers. The lining cells of the microvasculature showed typical characteristics of endothelial cells, such as ovoid or cubical shape, bundles of microfilaments and microtubules, and particularly numerous small vesicles at the apical pole, some of them fused to the plasma membrane. Similar angiogenic processes were also observed in long-term haemopoietic co-cultures formed by the TPS cell line and trout pronephric cell suspensions. Developing haemopoietic cells were observed at the basal pole of the vessels, and in the vascular lumen, where some immature cells appeared in close contact with the endothelium. These results indicate that the TPS cell line contains endothelial cell precursors, which are able to differentiate under certain culture conditions.

In vitro and in situ characterization of fish thymic nurse cells.

We present an enzyme- and immuno-cytochemical, and ultrastructural characterization of trout thymic nurse cells (TNCs). Our data suggest that isolated trout thymic multicellular complexes are epithelial cells with acidic compartments that may be involved in the processing of antigens and in the generation of the MHC-II proteins that these cell express, and also that isolated TNCs are the in vitro equivalent of the pale and intermediate electronlucent epithelial cells located in the inner zone of the trout thymus, constituting indirect evidence of the phylogenetical relationships of the inner zone of the teleost thymus with the thymic cortex of higher vertebrates.

Enzyme- and immuno-histochemical study of the thymic stroma in the rainbow trout, Salmo gairdneri, Richardson.

Owing to the lack of data about thymic non-lymphoid cells in fish we decided to perform a histochemical characterization of these cells in order to ascertain their relationships to other thymic components. In the present study we analyze the enzyme-histochemical patterns for acid phosphatase, alkaline phosphatase, non-specific sigma-naphthyl acetate esterase and 5' nucleotidase activities, as well as the presence of keratin demonstrated by immunoperoxidase staining, in the non-lymphoid cell populations of the thymus of the rainbow trout, Salmo gairdneri. According to their location in the organ, morphology and histochemical reactivities, we were able to define seven different subpopulations of keratin-positive epithelial cells: 1) Epithelial cells limiting with the capsular and septal connective tissues; 2) Subcapsular epithelial cells; 3) Stellate epithelial cells of the inner thymic zone; 4) Large, ovoid epithelial cells of the inner thymic zone; 5) Acidophilic epithelial cells of the outer thymic zone; 6) Cystic cells; and 7) Goblet cells. The significance of the heterogeneity of the epithelial cell (EC) population, its specific distribution in the organ, which apparently conforms distinct cell microenvironments, as well as the possible phylogenetical relationships between these microenvironments and the classical cortex and medulla of the mammalian thymus, are discussed.

Molecular characterization of the Aeromonas hydrophila aroA gene and potential use of an auxotrophic aroA mutant as a live attenuated vaccine.

The aroA gene of Aeromonas hydrophila SO2/2, encoding 5-enolpyruvylshikimate 3-phosphate synthase, was cloned by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829, and the nucleotide sequence was determined. The nucleotide sequence of the A. hydrophila aroA gene encoded a protein of 440 amino acids which showed a high degree of homology to other bacterial AroA proteins. To obtain an effective attenuated live vaccine against A. hydrophila infections in fish, the aroA gene was inactivated by the insertion of a DNA fragment containing a kanamycin resistance determinant and reintroduced by allelic exchange into the chromosome of A. hydrophila AG2 by means of the suicide vector pSUP202. The A. hydrophila mutant AG2 aroA::Ka(r) was highly attenuated when inoculated intraperitoneally into a rainbow trout, with a 50% lethal dose of >2 x 10(8) CFU. The mutants were not recoverable from the internal organs after 48 h postinoculation. Immunohistochemical studies demonstrated that immunopositive materials, but not whole cells, reacting with a polyclonal antiserum against A. hydrophila were present in the kidney and spleen 9 days postinjection. Vaccination of rainbow trout with the AroA mutant as a live vaccine conferred significant protection against the wild-type strain of A. hydrophila.