Mechanisms involved in the inflammatory process of gilthead seabream (Sparus aurata)

Abstract

Inflammation is a non-specific response triggered by the innate immune system against a stimulus of any etiology (injuries, trauma, infections, irritating products, etc.) to defend the organism, in this case fish, and restore it to the initial homeostasis. It presents a series of very characteristic symptoms called "cardinal signs of inflammation" by which it can be easily identifiable. Two types of inflammation can be differentiated: i) acute inflammation, or immediate response, which can be developed briefly upon detection of the initial stimulus, and last for days or weeks until its final resolution; and ii) chronic inflammation, produced by the inefficiency of defensive mechanisms to resolve acute inflammation and whose effects are highly related to the appearance of related chronic diseases. In this sense, inflammation is a very common event that occurs in the aquaculture industry due to the intense production conditions (overcrowding, quality water, deterioration of environmental conditions) to which fish are subjected to meet market demands. Under these situations, fish become more susceptible to wounds, trauma, and pathogens, that could be related to inflammation and the appearance of associated diseases, and which often translate into economic losses for the sector itself. Inflammation is an essential process to return fish to their initial state of health, since it participates directly in the repair of damaged tissue. However, to date no model of fish inflammation has been developed that allows to delve into its intrinsic mechanisms in an adequate way. This Doctoral Thesis aimed to improve the knowledge of the inflammation process on gilthead seabream (Sparus aurata), one of the most important Mediterranean cultured fish species. To get our objective we divided this Doctoral Thesis in three chapters. In Chapter I, the systemic effects caused by an injection of carrageenin were evaluated. Thereby, the fish were injected intramuscularly with PBS (control) or carrageenin (1%), and 1.5, 3 and 6 h post-injection, samples of skin mucus, serum, head-kidney and liver were obtained to determine the immunological and antioxidant status of gilthead seabream. Results evidenced that one intramuscular carrageenin injection of 1% is enough to provoke inflammation in the fish specimens, causing an increase in humoral activities in the skin mucus at 3 h post-injection. However, the carrageenin injection had no impact on the gilthead seabream systemic immunity. In addition, although a decrease in the hepatic catalase activity was observed at 3 and 6 h post-injection, no significant variations were detected in the gene expression of none of the genes studied in the liver, evidencing that this organ was not affected by carrageenin. The Chapter II of this Doctoral Thesis consisted in the study of the local effects triggered by carrageenin at the injection place. Thus, the fish were injected intramuscularly with PBS (control) or carrageenin (1%), and skin samples were obtained at 1.5, 3, and 6 h post-injection and processed on one side for histology and immunohistochemistry procedures, and on the other hand, for gene expression analysis, revealing an increase in the number of skin mucus-secreting cells and acidophilic granulocytes in the skin near the injection place at 1.5 and 3 h post-injection, respectively. Considering the results obtained, another assay was designed in which the fish were injected intramuscularly with PBS (control) or carrageenin, and the injection area was evaluated by real-time ultrasound and micro-CT at 1.5, 3, 6, 12 and 24 h after injection. The results evidenced the typical symptoms of human necrotizing fasciitis (increased skin thickness, edema, and emphysema) in the underlying muscle of animals injected at 1.5, 3, and 6 h, which were reduced to disappearance at 12-24 h after carrageenan administration. This symptomatology seems to be due to the molecules released at the site of inflammation and the possible degradation of carrageenin at that site, which would point to the final stages of the inflammatory response. The Chapter III of this Doctoral Thesis included the results obtained from the gene analysis of the inflammatory response caused by carrageenan in sea bream. The expression of 40 inflammatory related-genes was evaluated in vivo in the gilthead seabream in two separate trials: In the first experiment, fish specimens were injected with PBS (control) or carrageenin intramuscularly, and 1.5, 3 and 6 h post-injection and skin samples were obtained from the injected area and processed to evaluate gene expression. In the second assay, the same experimental design was replicated, but increasing the sampling time to 12 and 24 hours. The up-regulation of cell markers and pro-inflammatory gene expression, as well as the down-regulation of anti-inflammatory-related genes in the interval of 1.5-6 h post-injection, indicated the duration of the induction of the inflammatory response caused by carrageenin. Nonetheless, results obtained at 12 and 24 h post-injection seemed to indicate the activation of mechanisms necessary to terminate the inflammation response to carrageenin and restore skin homeostasis. These results were supported by an in silico assay carried out to study the phylogenetic conservation of the 40 inflammation related-molecules previously studied from protein sequences of gilthead seabream in comparison to other teleost protein sequences, the proteins of large yellow croaker (Larimichthys crocea L.) presenting the highest percentages of identity with the protein sequences of gilthead seabream. Furthermore, the most conserved proteins of inflammation were those involved in its regulation. This fact also supports the hypothesis that regulatory gene mechanisms could be activated 24 h after carrageenin injection in order to control the inflammatory response. Finally, all the findings of this Doctoral Thesis evidenced that one single injection of 1% carrageenin can be used to trigger a local inflammation in gilthead seabream, which constitute a model to continue studying this important process, both in health and disease in this fish species.