ABSTRACT
Exposure to high environmental ammonia (HEA) levels increases the vulnerability of fishes to parasitic, viral and bacterial diseases. We tested the hypothesis that elevated plasma cortisol levels play a role in the HEA-mediated immunosuppression in fishes. To this end, we tested the effect of exogenous cortisol treatment on the lipopolysaccharide (LPS)-induced immune response in zebrafish (Danio rerio). Also, to test whether glucocorticoid receptor (GR) signaling is involved in HEA-mediated immunosuppression, zebrafish were treated with mifepristone, a GR antagonist, and the LPS-induced immune response assessed after HEA exposure. We evaluated a panel of important immunity-related genes including interleukin 1ß (il1b) and suppressor of cytokine signaling (socs-1a, 2, 3) and acute phase response genes [serum amyloid A (saa), transferrin (tfa), leukocyte cell-derived chemotaxin 2-like (lect2l), haptoglobin (hp), hepcidin (=hepatic anti-microbial peptide hamp), and complement component 3b (c3b)] by real-time quantitative PCR. Our results demonstrate that exogenous cortisol administration as well as elevated cortisol levels in response to HEA exposure modulate mRNA transcript levels of key mediators of the innate immune response in zebrafish. Mifepristone treatment reduced whole body cortisol levels and eliminated the HEA-mediated changes in transcript abundance of socs1a, il1b, as well as APR genes. Together, these results suggest that the HEA effect on the innate immune response is in part mediated by cortisol signaling, while the mode of action, including the receptors involved remains to be elucidated.
Subject(s)
Altitude , Ammonia/adverse effects , Environment , Hydrocortisone/metabolism , Immunity , Zebrafish/immunology , Acute-Phase Proteins/metabolism , Animals , Gene Expression Regulation/drug effects , Immunity/drug effects , Immunity/genetics , Lipopolysaccharides/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Zebrafish/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Ammonia is a toxic by-product of amino acid catabolism and a common environmental pollutant that has been associated with increased disease susceptibility in fish although the mechanism is not well understood. We addressed the hypothesis that elevated environmental ammonia acts by impairing the acute phase response (APR). Specifically, we determined the impact of sub-lethal acute (24 h) and chronic (14 d) ammonia exposure on acute phase protein gene expression in zebrafish (Danio rerio) in response to a challenge with bacterial lipopolysaccharide (LPS: i.p. 10 µg/g after 24h). A panel of LPS-responsive genes (SAA, HAMP, LECT2, Hp and IL1ß) were identified and evaluated by real-time quantitative PCR. Ammonia was found to impair induction of SAA, HAMP and LECT2 by 50-90%. Both short (15 min, 1h and 24h) and long-term (14 days) exposure to high environmental ammonia concentrations significantly elevated whole-body cortisol levels compared with control fish. Our results reveal for the first time that exposure to high environmental levels of ammonia suppresses the innate immune response in fish. We hypothesize that high environmental ammonia-mediated elevation of cortisol levels in zebrafish may be playing a key role in this immunosuppression, while the mechanisms involved remains to be elucidated.
Subject(s)
Acute-Phase Reaction/immunology , Ammonia/poisoning , Environmental Exposure/adverse effects , Lipopolysaccharides/pharmacology , Zebrafish/immunology , Animals , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/immunology , C-Reactive Protein/genetics , C-Reactive Protein/immunology , Hepcidins , Hydrocortisone/analysis , Immunity, Innate/drug effects , Immunity, Innate/immunology , Interleukin-10/genetics , Interleukin-10/immunology , Interleukin-1beta/genetics , Interleukin-1beta/immunology , RNA/chemistry , RNA/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Serum Amyloid A Protein/genetics , Serum Amyloid A Protein/immunology , Statistics, Nonparametric , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/immunology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , Zebrafish/genetics , Zebrafish Proteins/genetics , Zebrafish Proteins/immunologyABSTRACT
Hepcidin, an antimicrobial peptide described as a key regulator of iron metabolism, is known to respond in mammals to several stimuli, including iron overload, anemia, hypoxia and inflammation, through a number of molecular pathways. In order to understand the molecular pathways involved in the regulation of hepcidin expression in teleost fish, we have isolated for European sea bass (Dicentrarchus labrax) several coding sequences of known molecules involved on these pathways in mammals, namely jak3, stat3, tmprss6, bmp6, bmpr2, hjv, smad4, smad5, tfr1 and tfr2. The transcription levels of the isolated genes were evaluated by real-time PCR on fish subjected to experimental iron modulation (overload/deficiency) or infection with Photobacterium damsela. Results show that genes associated with the major pathway of the inflammatory response (IL6/JAK/STAT pathway) in mammals are also modulated in sea bass, being up-regulated during infection. Similarly, genes of the pathways classically associated with the response to variations in iron status (the HJV/BMP/SMAD and HFE/TfR pathways) are also modulated, mostly through down-regulation in iron deficiency and up-regulation during iron overload. Interestingly, many of these genes are also found to be up-regulated during infection, which may indicate a crosstalk between the known pathways of hepcidin regulation. These observations suggest the evolutionary conservation of the mechanisms of hepcidin regulation in teleost fish.
