Implication of mucus‐secreting cells, acidophilic granulocytes and monocytes/macrophages in the resolution of skin inflammation caused by subcutaneous injection of λ/κ‐carrageenin to gilthead seabream (Sparus aurata) specimens

Abstract

To date, the mechanisms of inflammation have been poorly studied in fish of commercial interest, due to the lack of development of appropriate experimental models. The current study evaluated a local inflammation triggered by a polymeric carrageenin mixture (a mucopolysaccharide derived from the red seaweed Chondrus crispus) in the skin of gilthead seabream (Sparus aurata). Fish were injected subcutaneously with phosphate-buffered saline (as control) or λ/κ-carrageenin (1%), and skin samples from the injection sites were collected 1.5, 3 and 6 hr post-injection, processed for inclusion in paraplast and stained with haematoxylin-eosin, Alcian blue or periodic acid-Schiff. Furthermore, immunohistochemistry and expression analyses of several cells' markers and proinflammatory genes were also analysed in samples of the injected sites. Microscopic results indicated an increased number of skin mucus-secreting cells and acidophilic granulocytes in the skin of fish studied at 1.5 hr and 3 hr postinjection with carrageenin, respectively, with respect to the data obtained in control fish. Otherwise, both the gene expression of the non-specific cytotoxic cell marker (granzyme B, grb) and the proinflammatory cytokine (interleukin-1β, il-1β) were upregulated at 1.5 hr in the skin of fish injected with carrageenin compared with the control fish, whilst the gene expression of acidophilic granulocyte markers (NADPH oxidase subunit Phox22 and Phox40, phox22 and phox40) was up-regulated at 3 and 6 hr in the carrageenin group, compared with the control group. In addition, the gene expression of myeloperoxidase (mpo) was also up-regulated at 6 hr in the skin of fish injected with carrageenin in comparison with control samples. The present results indicate the chronological participation of two important immune cells involved in the resolution of the inflammation in the skin of gilthead seabream.

Implication of adipocytes from subcutaneous adipose tissue and fatty acids in skin inflammation caused by λ-carrageenin in gilthead seabream (Sparus aurata)

“…was increased in the skin of fish in the λ-carrageenin group compared to the control group. These results are in agreement with previous data pointing to a secondary role of eicosanoids in the inflammatory pathway in seabream induced by carrageenin compared to mammals, at least at these experimental time points [40]. Thus, not only the timing of eicosanoid production and the concentrations of the different eicosanoids, but also the sensitivity of the target cells and tissues to the eicosanoids generated, could be some of the particularities to be taken into account to explain this major difference in gilthead seabream relative to mammals [13].…”

“…Interestingly, immune cell membranes are constituted by phospholipids with a high content of n-6 PUFAs and a low content of n-3 PUFAs [13]. These results could also support the recruitment of immune cells at the inflammation zone at this experimental time (6 h), previously evidenced by our research group by histology and gene expression analysis [36,40]. Besides this, the presence of n-3 PUFAs has been associated with the activation of PPAR-γ, the subsequent inhibition of NF-κB (the main molecular regulator of inflammation), and therefore, with the reduction of the inflammatory response [13].…”

“…According to our histological study, an increase in adipocyte area in SAT was detected in fish injected with λ-carrageenin at 3 h p.i. These results could be the morphological evidence of the response of adipocytes to the proinflammatory mediators that are released, in the area of inflammation, by immune cells recruited at this time point (3 h) [36,38,40]. This morphological change could be necessary to induce the polarization of adipocytes in the inflamed zone towards a proinflammatory phenotype.…”

“…6 h p.i., compared to the control group. In addition, although carrageenin injection had no impact on systemic immunity of gilthead seabream, it led to an increase in the number of skin mucus-secreting cells and acidophilic granulocytes, as well as up-regulation of proinflammatory genes in the skin near the injection site [38,40]. These events were associated with increased skin thickness, edema, and emphysema in the underlying muscle, which were the symptoms observed in human necrotizing fasciitis [39].…”

“…More recently, the inflammatory response of gilthead seabream (Sparus aurata) has been studied by our team, providing diverse information on gene expression [36,37], humoral and cellular immune parameters [38] and the use of non-invasive techniques such as real-time ultrasound or micro-CT to study this complex process [39]. In addition, previous work has evidenced the local effect of mucus-secreting cells of the skin in the inflamed area at 3 h after carrageenin injection by immunohistochemical assay [40]. All these previous results encouraged us to perform the present study, looking for the possible role of other non-immune resident cells in the area of inflammation.…”