Elsevier

Molecular Immunology

Volume 87, July 2017, Pages 267-283
Molecular Immunology

Research paper
Chronic stress impairs the local immune response during cutaneous repair in gilthead sea bream (Sparus aurata, L.)

https://doi.org/10.1016/j.molimm.2017.04.008Get rights and content

Highlights

  • A cutaneous stress axis exists in teleost fish skin.

  • Stress associated transcripts are down-regulated by chronic stress in teleost fish skin.

  • Chronic stress impairs teleost skin regeneration and the reestablishment of the physical barrier.

  • The inflammatory response during cutaneous repair is suppressed by chronic stress.

Abstract

Scale removal in fish triggers a damage-repair program to re-establish the lost epidermis and scale and an associated local immune response. In mammals, chronic stress is known to delay wound healing and to modulate the cutaneous stress axis, but this is unstudied in teleost fish the most successful extant vertebrates. The present study was designed to test the hypothesis that chronic stress impairs cutaneous repair in teleost fish as a consequence of suppression of the immune response. The hypothesis was tested by removing the scales and damaging the skin on one side of the body of fish previously exposed for 4 weeks to a chronic crowding stress and then evaluating cutaneous repair for 1 week. Scale removal caused the loss of the epidermis although at 3 days it was re-established. At this stage the basement membrane was significantly thicker (p = 0.038) and the hypodermis was significantly thinner (p = 0.016) in the regenerating skin of stressed fish relative to the control fish. At 3 days, stressed fish also had a significantly lower plasma osmolality (p = 0.015) than control fish indicative of reduced barrier function. Chronic stress caused a significant down-regulation of the glucocorticoid receptor (gr) in skin before damage (time 0, p = 0.005) and of star at 3 and 7 days (p < 0.05) after regeneration relative to control fish. In regenerating skin key transcripts of cutaneous repair, pcna, colivα1 and mmp9, and the inflammatory response, tgfβ1, csf-1r, mpo and crtac2, were down-regulated (p < 0.05) by chronic stress. Irrespective of chronic stress and in contrast to intact skin many hyper pigmented masses, putative melanomacrophages, infiltrated the epidermis of regenerating skin. This study reveals that chronic stress suppresses the local immune response to scale removal and impairs the expression of key transcripts of wound healing. Elements of the stress axis were identified and modulated by chronic stress during cutaneous repair in gilthead seabream skin.

Introduction

The skin, together with its appendages, serves as a barrier and has an essential role in maintaining whole organism homeostasis and for this reason damage is rapidly repaired. In vertebrates the skin is composed of an outer epidermis, the dermis composed of connective tissue and is separated from underlying structures such as the muscle by a subcutaneous layer of adipose tissue (hypodermis) (Bacha and Bacha, 2000, Kuehnel, 2003). In general vertebrate skin has a relatively well conserved organization, although habitat specific specializations have emerged and fish skin is protected by scales and composed of living non-keratinized epidermal cells covered with a mucous layer (Elliott, 2011, Hawkes, 1974, Spearman, 1973). Despite the obvious differences that exist between piscine and mammalian skin, this tissue is proposed to arise through common developmental pathways and has a similar role in innate immunity (Rakers et al., 2010, Wu et al., 2004).

Recent studies of human skin reveal that in addition to being an endocrine responsive tissue it also produces hormones, neurotransmitters and neuropeptides (Slominski, 2005, Slominski and Wortsman, 2000, Slominski et al., 2007). In particular, the key elements of the hypothalamic-pituitary-adrenal (HPA) axis are present in skin indicating that in addition to the classical central stress axis a second cutaneous stress axis also exists. The cutaneous stress axis in human skin maintains local homeostasis and has a regulatory role in skin immunity and the pigmentary system, although its involvement in cutaneous wound healing is poorly studied (Slominski et al., 2007). The emergence of a cutaneous stress axis in vertebrates is largely unstudied, particularly in the most successful group of extant vertebrates, the teleost fish. Although the results of a recent microarray study of the cutaneous response to an ectoparasite infestation in Atlantic salmon revealed the up-regulation of genes involved in steroid metabolism (Krasnov et al., 2012) and hinted at the existence of a cutaneous stress axis.

The skin is the largest neuroendocrine-immune tissue and is the main barrier between an organism and its external environment and an integrated response of the endocrine, immune and central nervous system is essential for maintenance of internal homeostasis (Brazzini et al., 2003, Slominski et al., 2012). For this reason damage to this barrier in mammals leads to a rapid multi-phase repair process which relies on the innate immune response (Barrientos et al., 2008). An emerging and widely studied concept in mammals is the cross-talk that occurs between the stress axis and the immune system (Dhabhar, 2008). The release of glucocorticoids as a result of prolonged stress down-regulates pro-inflammatory cytokines (Schleimer et al., 1990) and this delays repair of the barrier in mammals (Glaser et al., 1999, Godbout and Glaser, 2006, Vileikyte, 2007, Walburn et al., 2009). In common with other vertebrates, acute stress in fish is reported to act as an immunoenhancer (Fast et al., 2008, Tort, 2011, Weyts et al., 1999), although chronic stress (prolonged stress) suppresses the immune response (Fast et al., 2008, Tort, 2011, Weyts et al., 1999).

Scale loss caused by aggressive interactions between fish, abrasion and other environmental factors causes loss of the coating mucous layer, the epidermis and the superficial dermis attached to the scale and causes a cutaneous wound that reduces the efficiency of the skins barrier function. In teleost fish skin repair is much quicker than in mammals and damaged skin is rapidly covered by an epithelial cell layer and mucous. A second and much slower event during cutaneous repair in teleost fish is the development of the new scale with all the characteristics of the ontogenic scale and the associated reorganization of the connective tissue in the dermis (Bereiter-Hahn, 1986, Quilhac and Sire, 1999, Rakers et al., 2010, Vieira et al., 2011). The regulation of cutaneous wound repair involves the up-regulation of chemokines that attract macrophage to the site of inflammation and the release of growth factors by these cells causes cell cycle activation and up-regulation of extracellular matrix proteins and associated tissue proliferation and remodeling (Ibarz et al., 2013, Vieira et al., 2011). The repair of skin in fish differs from that in mammals as it does not result in scarring (Gurtner et al., 2008, Poss et al., 2003, Robson et al., 2001) making the fish an interesting model for comparative studies. The effect of prolonged exposure to stress on repair of skin after scale removal in teleost fish has never been explored.

The present study was designed therefore to test the hypothesis that chronic stress impairs teleost fish cutaneous repair as a consequence of the suppression of the immune response. We tested the hypothesis by damaging the skin on one side of the body by removing scales from fish pre-exposed to chronic stress for 4 weeks and then observed cutaneous regeneration during a further week of stress. Undamaged (control) and damaged skin was collected and analyzed from the same fish. Histology and histomorphometrics were used to assess changes in skin organization and to establish how the barrier changed. Cutaneous repair over time was studied by histology allied to qPCR to, i) evaluate changes in gene transcripts associated with wound healing and the cutaneous immune response and ii) to measure the infiltration of the damaged skin by dark pigmented masses.

Section snippets

Material and methods

Manipulation of animals was performed in compliance with international and national ethics guidelines for animal care and experimentation, under a “Group-I” license from the Portuguese Government Central Veterinary service to CCMAR and conducted by a certified investigator (DMP).

Biometric parameters

The general physiological status of the fish after 4 weeks of chronic stress (0 h) and during cutaneous wound healing (12 h, 3 and 7 days after scale removal) was assessed by measuring body weight, total length, K and HSI (Table 1). Two-way ANOVA showed that total length, K and HSI were not affected by time after scale removal or stress, or by the interaction between these effects (p > 0.05). At 0 h, no significant differences were detected between control and the stress group, for any of the

Discussion

The present study demonstrates for the first time that chronic stress impairs cutaneous repair in gilthead seabream. The impairment in repair is likely due to the immunosuppression elicited by chronic stress and also modulation of other transcripts associated with cutaneous repair. This study also reveals for the first time that elements of a cutaneous stress axis exists in gilthead seabream and that modulation of gr transcripts in the skin seems to be linked to the down-regulation of immune

Conclusion

The results of this study report for the first time in a teleost fish that chronic stress changes the molecular mechanisms of cutaneous repair after scale removal. Specifically, chronic stress suppresses the local inflammatory response and was associated with down-regulation of key elements of the repair process. This study also suggests that chronic stress modulates the expression of elements of the stress axis identified in skin and that this may impair cutaneous repair. Surprisingly, no

Conflicts of interest

The authors declare no conflict of interest.

Authors' contributions

DMP conceived and planned the project. APM performed the practical work including histology, plasma analysis and molecular biology. APM, LA and JCRC performed the bioinformatics analyses. DMP and APM analyzed and interpreted the data. DMP and APM drafted the manuscript and LA and JCRC revised it critically for important intellectual content. All authors have given their final approval of the version to be published.

Acknowledgments

The authors would like to thank Dr. Pedro Pousão (IPMA, Olhão) for kindly providing the gilthead seabream used in this study and also Mr. João Reis (Ramalhete Station) for animal care during the experiment. This work was supported by the European Regional Development Fund through COMPETE and the Portuguese Foundation for Science and Technology (FCT) (CCMAR/Multi/04326/2013), (PTDC/MAR/122296/2010). JCRC is funded by the project UID/Multi/04326/2013 and LA by a post-doctoral fellowship

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