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.

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.

Seasonal changes in the lymphoid organs of wild brown trout, Salmo trutta L: a morphometrical study.

A morphometrical evaluation was made of the seasonal changes affecting the numbers of lymphocytes in the thymus, spleen and pronephros of wild brown trout, Salmo trutta, while the size of the thymus and the three thymic zones were also determined. Results reveal statistically significant changes throughout the year in the number of lymphocytes in the lymphoid organs studied. The spleen and pronephros have similar annual patterns of lymphocyte distribution with high numbers in two seasons, spring and autumn, and two periods of lymphoid involution in winter and summer. The highest numbers of thymocytes occur in trout caught in May and August, and the lowest in winter. In addition to normal lymphocytes, degenerated lymphoid cells that show pale cytoplasm devoid of cell organelles, also occurred in all the lymphoid organs. A negative correlation exists between the numbers of normal lymphocytes and that of degenerated lymphoid cells. The thymic size, as well as that of the subcapsular, inner and outer thymic zones, undergo very significant changes over the year. We discuss the relevance of cell proliferation, cell migration and in situ cell death for the circannual variations observed in the cell content of trout lymphoid organs, together with the possible causes.

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.

Seasonal intrathymic erythropoietic activity in trout.

Thymus glands of two salmonid species, Salmo trutta and Oncorhynchus mykiss, caught monthly throughout the year, were examined by light and transmission electron microscopy. Erythropoietic foci, consisting of both developing and mature erythroid cells, occurred in the subcapsular, inner, and outer thymic zones from April to November. We discuss the possible physiological significance of this seasonal erythropoietic activity, together with the role played by the thymic cell microenvironments and endocrine factors.

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.