Introduction
Reducing the use of antibiotics in aquaculture is one of the biggest challenges of this sector. Antimicrobial peptides (AMPs) are considered promising alternatives to these drugs, but studies evaluating the potential use of AMPs in the production of sea bass are still missing, with only one report addressing the effects of synthetic hepcidins in infected sea bass (Álvarez et al, 2016). Most mammals present a single hepcidin gene, with limited antimicrobial activity and a major function in the regulation of iron metabolism, by inhibiting the iron exporter ferroportin (Nemeth et al, 2004). However, many fish, including the European sea bass (Dicentrarchus labrax), present two hepcidin types that have subfunctionalized, with the single type 1 hepcidin regulating systemic body iron, and the various type 2 hepcidins having an almost exclusively antimicrobial role (Neves et al, 2015). As such, in this study we evaluated the potential of bass derived hepcidins to treat or prevent iron disorders and infectious diseases, by administering them to animals subjected to various experimental models of infection or iron modulation.
Material and Methods
Several experimental models were performed to test if sea bass hepcidins can be effectively used to treat/prevent iron disorders or bacterial infections: 1) peptide administration - intraperitoneally (i.p) administered 100 µl of a 50 μM solution of hamp1 (QSHLSLCRWCCNCCRGNKGCGFCCKF) or hamp2 (HSSPGGCRFCCNCCPN-MSGCGVCCRF) to healthy sea bass; 2) iron overload – i.p. administered 100 µl (5 mg) of iron dextran; 3) iron overload+peptide – iron overload followed 24 hours later by peptide administration, as before; 4) infection – i.p. infected with 105 CFU of Photobacterium damselae spp damselae PP3; 5) infection+peptide – infection followed 24 hours later by peptide administration, as before; 6) peptide+infection – peptide administration followed 24 hours later by infection, as before. At pre-determined time points, animals were terminally sampled, and blood, serum and tissues collected, for determination of haematological and serological parameters, tissue iron content, CFU counts and gene expression analysis.
Results
We observed that administration of hamp1 can have a significant impact on several iron related parameters, when administered to healthy animals. This leads to a condition of anemia, which might be connected to the slight increase in mortality observed when hamp1 is also administered to infected animals, making them more susceptible. However, when administered to iron overloaded animals, it seems to limit the severity of its effects. Hamp2 on the other hand has shown no significant impact on iron metabolism, and had no discernible effects during iron overload. But when administered to infected animals, either before or after infection, reduced mortality very significantly (from around 55% to between 6-17%), decreased bacterial loads and reduced the impact on several iron related parameters, in particular limiting the development of anemia of inflammation.
Discussion and conclusions
Our findings show that when hamp1 is administered to healthy animals interferes with the iron metabolism, leading to significant anemia. Administration during infection leads to an even more severe anemia of inflammation, likely contributing to the increased mortality. However, hamp1 seems to attenuate the effects of iron overload, and as such might have applications in the treatment of iron disorders. Hamp2 on the other hand presents itself as a viable alternative to the use of other prophylactic or therapeutic substances, in particular antibiotics, since it is a bioactive molecule that does not interfere with iron metabolism and is very effective in controlling bacterial infections. Additionally, the effects of hamp2 are likely not limited to a direct antimicrobial effect, but also by stimulation of the overall inflammatory response, but further studies are needed to address this. Nevertheless, costs of production of antimicrobial peptides still represent a significant barrier to their more generalized use.
Acknowledgments
This work was funded by the structured program of R&D&I ATLANTIDA - Platform for the monitoring of the North Atlantic Ocean and tools for the sustainable exploitation of the marine resources (NORTE-01-0145-FEDER-000040), and by individual funding from the Portuguese Foundation for Science and Technology (FCT) through SFRH/BD/114899/2016 (CB).
References
Álvarez, C.A., Acosta, F., Montero, D., Guzmán, F., Torres, E., Vega, B., Mercado, L., 2016. Synthetic hepcidin from fish: Uptake and protection against Vibrio anguillarum in sea bass (Dicentrarchus labrax). Fish Shellfish Immunol. 55, 662–670.
Neves, J. V, Caldas, C., Vieira, I., Ramos, M.F., S Rodrigues, P.N., 2015. Multiple Hepcidins in a Teleost Fish, Dicentrarchus labrax: Different Hepcidins for Different Roles. J. Immunol. 195, 2696–2709.
Nemeth, E., Tuttle, M.S., Powelson, J., Vaughn, M.B., Donovan, A., Ward, D.M., Kaplan, J., Ganz, T., 2004. Hepcidin Regulates Cellular Iron Efflux by Binding to Ferroportin and Inducing Its Internalization. Science 306, 2090–2093.