High density lipoproteins down-regulate transcriptional expression of pro-inflammatory factors and oxidative burst in head kidney leukocytes from rainbow trout, Oncorhynchus mykiss.

Teleosts are the first group of vertebrates possessing an acquired immune system; however, it is less developed than in mammals and is highly influenced by environmental changes. Therefore, innate immunity effectors play a more critical role in survival of pathogen-challenged fish. In a previous study we showed that trout high density lipoprotein (HDL), and its major apolipoprotein (ApoA-I) are widely expressed in primary defense barriers and other immune-relevant tissues, displaying important antibacterial activity in vitro. Here we show that trout HDL inhibits both basal and LPS-induced transcript expression of pro-inflammatory cytokines such as TNF-α and IL-1ß, and the acute phase protein serum amyloid A (A-SAA), in head kidney leukocytes (HLK) from rainbow trout. In addition, trout HDL was able to block the respiratory burst of PMA-stimulated HKL, at physiological concentrations and in a dose dependent manner. Moreover, this effect was only partially mimicked by supra-physiologic concentrations of the HDL-transported carotenoid, astaxanthin. These results constitute the first data suggesting that in addition to its antimicrobial activity, HDL would have a relevant immunomodulatory role in salmonid fish.

Serum amyloid A: a typical acute-phase reactant in rainbow trout?

Acute serum amyloid A (A-SAA) has been considered a major acute-phase reactant and an effector of innate immunity in all vertebrates. The work presented here shows that the expression of A-SAA is strongly induced in a wide variety of immune-relevant tissues in rainbow trout, either naturally infected with Flavobacterium psychrophilum or challenged with lipopolysaccharide (LPS) or CpG oligonucleotides (CpG ODN). Nevertheless, A-SAA was undetectable by Western blot either in the plasma or in high-density lipoprotein (HDL) of infected or challenged fish, using either an anti-mouse SAA1 IgG or an anti-trout A-SAA peptide serum, which recognise both the intact recombinant trout A-SAA and fragments derived from it. However, the anti-peptide serum was the immunoreactive in all primary defence barriers and in mononuclear cells of head kidney, spleen and liver. These findings reveal that, unlike mammalian SAA, trout A-SAA does not increase significantly in the plasma of diseased fish, suggesting it is more likely to be involved in local defence.

Apolipoprotein A-I, an antimicrobial protein in Oncorhynchus mykiss: evaluation of its expression in primary defence barriers and plasma levels in sick and healthy fish.

Antimicrobial proteins and peptides play an important role in the primary defence barriers in vertebrates and invertebrates. In a previous study it was shown that high-density lipoprotein (HDL) and its major apolipoproteins, ApoA-I and ApoA-II display antimicrobial activity in the carp (Cyprinus carpio L.). The aim of this study was to evaluate if ApoA-I conserves this defensive function in a salmonid fish like the rainbow trout, in spite of the low level of primary sequence conservation between fish ApoA-I. Here it is shown that trout ApoA-I displays an antimicrobial activity in the micromolar range against Gram positive and Gram negative bacteria, including some fish pathogens. In addition, its expression was also demonstrated by immunohistochemistry and RT-PCR in epidermis, gills and intestinal mucosa, which constitute the main primary defence barriers in fish. Finally, no significant difference in the hepatic expression and plasma levels of this abundant apolipoprotein was found in groups of healthy and diseased fish, in clear contrast with mammals where ApoA-I have been considered a negative acute phase reactant. These findings suggest that ApoA-I could constitute an important innate immunity effector in trout and perhaps other teleost fish.

Experimental infection of Flavobacterium psychrophilum in fins of Atlantic salmon Salmo salar revealed by scanning electron microscopy.

Infections caused by Flavobacterium psychrophilum include 'bacterial coldwater disease' (BCWD) and 'rainbow trout fry syndrome' (RTFS), which are severe diseases that can cause high mortality and significant losses in hatchery-reared salmonids worldwide. Usually, these conditions start with necrosis along the edge of the fins. As the infection progresses, both the fish surface and the internal organs can be involved. The aetiological agent produces a Ca-dependent protease that can be responsible for some of the pathogenic responses, although the precise nature of the response remains to be elucidated. Atlantic salmon Salmo salar were experimentally infected by F. psychrophilum in order to investigate the bacterial invasion in the fin tissues by scanning electron microscopy. The images showed numerous bacteria embedded in the mucous layer when this remained on the tegument. In other zones without mucus, it was observed that bacteria were present on the axis of fin rays, but not on the epidermal surface. The material on these axes was largely eroded by tubular boreholes, and bacterial rods could be seen in these perforations. EDX (Energy Dispersive X-ray) microanalysis of the axis of the fin rays showed significant amounts of P and Ca, revealing the ossification of the ray axis. The protease activity could explain the formation of the tubular boreholes, allowing the bacteria the necessary Ca for the activation of the enzyme. The erosion pattern suggests that the gliding motility of F. psychrophilum could be involved in this burrowing ability.

Okadaic acid inhibits angiotensin II, adrenocorticotropin and potassium-dependent aldosterone secretion.

The present study was designed to assess the effect of okadaic acid (OA), a protein phosphatase inhibitor, on aldosterone secretion in response to angiotensin II (AII), adrenocorticotropin (ACTH) and rises in external potassium concentration (K+). AII (10nM) caused a 20-fold increase in aldosterone production and OA reduced this response by 45%. ACTH (10nM) caused an 8.6-fold increase in aldosterone secretion and OA reduced this by 83%. Increasing K+ concentration from 3 to 12mM caused a 13-fold increase in aldosterone production, which OA inhibited by 36%. These results suggest that protein phosphatases participate in the control of adrenal steroid production, even though ACTH, AII and K+ act via different intracellular messenger systems.