Participation of Hepcidins in the Inflammatory Response Triggered by λ-Carrageenin in Gilthead Seabream (Sparus aurata).

The role of hepcidins, antimicrobial peptides involved in iron metabolism, immunity, and inflammation, is studied. First, gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs) were incubated with λ-carrageenin to study the expression of hepcidin and iron metabolism-related genes. While the expression of most of the genes studied was upregulated, the expression of ferroportin gene (slc40a) was downregulated. In the second part of the study, seabream specimens were injected intramuscularly with λ-carrageenin or buffer (control). The expression of the same genes was evaluated in the head kidney, liver, and skin at different time points after injection. The expression of Hamp1m, ferritin b, and ferroportin genes (hamp1, fthb, and slc40a) was upregulated in the head kidney of fish from the λ-carrageenin-injected group, while the expression of Hamp2C and Hamp2E genes (hamp2.3 and hamp2.7) was downregulated. In the liver, the expression of hamp1, ferritin a (ftha), slc40a, Hamp2J, and Hamp2D (hamp2.5/6) genes was downregulated in the λ-carrageenin-injected group. In the skin, the expression of hamp1 and (Hamp2A Hamp2C) hamp2.1/3/4 genes was upregulated in the λ-carrageenin-injected group. A bioinformatic analysis was performed to predict the presence of transcription factor binding sites in the promoter region of hepcidins. The primary sequence of hepcidin was conserved among the different mature peptides, although changes in specific amino acid residues were identified. These changes affected the charge, hydrophobicity, and probability of hepcidins being antimicrobial peptides. This study sheds light on the poorly understood roles of hepcidins in fish. The results provide insight into the regulatory mechanisms of inflammation in fish and could contribute to the development of new strategies for treat inflammation in farm animals.

Campos-Sánchez JC ，Serna-Duque JA ，Alburquerque C ，Guardiola FA ，Esteban MÁ ... -  《-》

In vitro effects of λ-carrageenin in the head-kidney leucocytes of gilthead seabream (Sparus aurata).

The λ-carrageenin is a sulphated mucopolysaccharide that has been used for decades to induce experimental inflammation in mammals. However, it has been little considered in fish. We studied the in vitro effects of λ-carrageenin on gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs). For this purpose, HKLs were incubated with serial concentrations (from 0 to 1,000 μg mL-1) of λ-carrageenin for 3, 6, 12 and 24 h to assess its influence on cell viability and morphology, cell activity and modulation of several selected inflammation-related genes. The viability results demonstrated that λ-carrageenin has no negative effects on HKLs. The respiratory burst activity and phagocytic ability of HKLs after being incubated with λ-carrageenin (100 and 1,000 μg mL-1) for 24 h were increased, whereas the phagocytic capacity was inhibited by the higher dose at the same experimental time compared with control samples. However, the peroxidase activity of HKLs was not changed by incubation with λ-carrageenin. According to transmission electron microscopy results, incubation of HKLs with the higher dose of λ-carrageenin appeared to activate the cells being evident different morphological changes without sign of cell death. Furthermore, up-regulation of three proinflammatory cytokines (il1b, tnfa, and il6) and down-regulation of anti-inflammatory genes (tgfb) were denoted in HKLs incubated with carrageenin. The present results provide a detailed approach to the effects of λ-carrageenin on fish leucocytes, which could have some impact on how we understand the response of these cells when inducing an inflammatory process in fish.

Campos-Sánchez JC ，Guardiola FA ，Esteban MÁ 《-》

Effects of subcutaneous injection of λ/κ-carrageenin on the immune and liver antioxidant status of gilthead seabream (Sparus aurata).

This study investigated the acute inflammatory response induced by subcutaneous injection of carrageenin (1%) or phosphate-buffered saline (control) in gilthead seabream (Sparus aurata). Skin mucus, serum, head kidney (HK) and liver were sampled at 1.5, 3 and 6 hr post-injection (p.i.) to determine the immune and antioxidant status of this fish species. The skin mucus of the carrageenin group showed increased superoxide dismutase and peroxidase activities, lysozyme abundance, bactericidal activity against Vibrio anguillarum and Photobacterium damselae, and total immunoglobulins compared with those of the control group. However, the carrageenin-injected fish sampled at 6 hr p.i. showed decreased protease activity in the skin mucus and peroxidase activity in the HK leucocytes compared with the control. Moreover, the carrageenin injection had no effects on the systemic immune system, but it reduced the liver catalase activities at both 3 and 6 hr in the carrageenin group relative to those in the control group. The expression levels of several proinflammatory and cell marker genes in the HK and liver were also determined. In the HK, the expression levels of interleukin-1β and prostaglandin D synthase 1 were upregulated at 1.5 and 3 hr, respectively, in the carrageenin group compared with those in the control group. Contrarily, the expression of the NADPH oxidase subunit phox40 (an acidophilic granulocyte marker) in the carrageenin group at 6 hr was downregulated compared with that in the control group. These results suggested that subcutaneous injection of κ/λ-carrageenin in gilthead seabream triggered an acute skin inflammation characterized by the rapid recruitment of acidophilic granulocytes and the release of humoral mediators into the skin mucus.

Campos-Sánchez JC ，Guardiola FA ，García Beltrán JM ，Ceballos-Francisco D ，Esteban MÁ ... -  《-》

Hamp1 but not Hamp2 regulates ferroportin in fish with two functionally distinct hepcidin types.

Neves JV ，Ramos MF ，Moreira AC ，Silva T ，Gomes MS ，Rodrigues PNS ... -  《Scientific Reports》

Head kidney, liver and skin histopathology and gene expression in gilthead seabream (Sparus aurata L.) exposed to highly polluted marine sediments from Portman Bay (Spain).

Biomarkers have become crucial tools in modern environmental assessment as they can help to predict magnitude of pollution. The head-kidney (HK) and liver (hematopoietic and xenobiotic metabolism organs, respectively) are the key organs in all fish toxicological studies, although the skin has received less attention in this respect. The impact of two different types of polluted sediment collected from Portman Bay (Spain) on HK, liver and skin gene expression in gilthead seabream (Sparus aurata L.) exposed for two weeks to the sediments was determined by real time-PCR. The expression levels of different antioxidant enzyme genes [superoxide dismutase (sod) glutathione reductase (gr) and catalase (cat)] and immune-related genes [interleukin -1β (il-1b), immunoglobulin M (igm), T-Cell receptor (tcr-b), cyclooxygenase-2 (cox-2), colony-stimulating factor 1-receptor (csf-1r) and hepcidin (hep)] was analysed. Expression varied depending on both the organ and gene studied: tcr-b, csf-1r and hep genes were down-regulated in HK, as were gr, tcr-b and il-1b in liver and gr and il-1b in skin, while cox-2 was up-regulated in skin after exposure to both sediments. Concomitantly, histopathological alterations were also studied in HK, liver and skin. While no significant changes were detected in skin cells of fish reared in aquaria with polluted sediments marked changes in the general morphology of HK and liver were observed, accompanied by a substantial degree of cell death and melano-macrophage centre disorganization. The present study suggests that the biomarkers studied in gilthead seabream could be useful for assessing the impact of pollution in coastal environments.

Ben Hamed S ，Guardiola F ，Cuesta A ，Martínez S ，Martínez-Sánchez MJ ，Pérez-Sirvent C ，Esteban MÁ ... -  《-》