Elsevier

Life Sciences

Volume 155, 15 June 2016, Pages 189-194
Life Sciences

Rosmarinic acid inhibits poly(I:C)-induced inflammatory reaction of epidermal keratinocytes

https://doi.org/10.1016/j.lfs.2016.05.023Get rights and content

Abstract

Aims

Keratinocytes are the predominant cells in the epidermis, exerting their primary role of physical barrier through sophisticated differentiation process. In addition, keratinocytes contribute to the activation of innate immunity, providing the surveillant role against external pathogens. It has been known that chronic skin inflammatory disease such as psoriasis can be provoked by viral pathogens including double-stranded RNA. In this study, we demonstrated that rosmarinic acid (RA) has an inhibitory potential on inflammatory reaction induced by double-stranded RNA mimic poly(I:C) in epidermal keratinocytes.

Main methods

We cultured human epidermal keratinocytes and induced inflammatory reaction by poly(I:C) treatment. The effect of RA on inflammatory reaction of keratinocytes was determined by RT-PCR and Western blot.

Key findings

RA significantly inhibited poly(I:C)-induced expression of inflammatory cytokines including IL-1β, IL-6, IL-8, CCL20, and TNF-α, and downregulated NF-κB signaling pathway in human keratinocytes. In addition, RA significantly inhibited poly(I:C)-induced inflammasome activation, in terms of secretion of active form of IL-1β and caspase-1. Furthermore, RA markedly inhibited poly(I:C)-induced NLRP3 and ASC expression.

Significance

These results indicate that RA can inhibit poly(I:C)-induced inflammatory reaction of keratinocytes, and suggest that it may be a potential candidate for the treatment of psoriasis.

Introduction

Skin is the largest organ, encompassing the entire body and demarcating the organism and environment. Its main function is to protect the body against harmful external insults [1]. The outmost layer epidermis is comprised of several cell types including keratinocytes, melanocytes and Langerhans cells. Among those, keratinocytes are the predominant cells and exert their role for physical barrier against external stimuli. In addition, keratinocytes contribute to innate immune surveillance through expressing various Toll-like receptors (TLRs), kinds of important pattern recognition receptors (PRRs). The specific recognition of pathogen-associated molecular patterns (PAMPs) by keratinocytes results in activation of inflammation-related intracellular signaling and production of inflammatory cytokines [2], [3].

Psoriasis is an immune-mediated inflammatory skin disorder characterized by hyperplasia of epidermal keratinocytes and infiltration of inflammatory cells [4]. It can be provoked or exacerbated by risk factors affecting the skin barrier function such as infections and mild trauma (scratching, piercings), suggesting that keratinocytes may be the potential origin cells for psoriasis. The cross-talk between innate and adaptive immunity is the fundamental mechanistic basis for psoriasis, in which many cytokines are functionally involved [5]. Especially, stimulation of keratinocytes with various PAMPs results in activation of innate immunity of keratinocytes, leading to the production of inflammatory cytokines related with psoriasis, including IL-1β and TNF-α [6]. Considering the importance of this initial innate immune response, blocking the inflammatory reaction of keratinocytes can be a good target for treating psoriasis.

Polyinosinic:polycytidylic acid (poly(I:C)) is a synthetic analogue of viral double-stranded RNA (dsRNA) which can induce innate immune response in a TLR3-dependent manner [7]. Keratinocytes express high level of TLR3, and stimulation of this receptor with its synthetic ligand poly(I:C) provokes inflammatory reaction in epidermal keratinocytes [8], [9]. In an attempt to find the therapeutics for psoriasis, we found that rosmarinic acid (RA) can be a possible candidate for drug development. It has been reported that RA possesses antioxidant and anti-inflammatory properties in macrophages [10]. However, the effect of RA on epidermal keratinocytes remains to be elucidated. In this study, we demonstrated that RA has an inhibitory potential on poly(I:C)-induced inflammatory reaction of epidermal keratinocytes.

Section snippets

Cell culture and drug preparations

Human skin tissues were obtained under the written informed consent of donors, in accordance with the ethical committee approval process of the Institutional Review Board of Chungnam National University Hospital (IRB file number: 2015-05-013). Primary keratinocytes were cultured according to the method previously described. Primary keratinocytes were isolated from epidermis, and then immortalized using the recombinant retrovirus expressing simian virus 40 T antigen (SV40Tag) [11].

Cytotoxicity of rosmarinic acid (RA) on keratinocytes

We tried to seek potential therapeutics for psoriasis, and found that rosmarinic acid (RA) (Fig. 1A) has inhibitory potential on inflammatory reaction of human keratinocytes. At first, we determined the cytotoxicity of RA on SV-HEKs. The MTT assay showed that RA did not induce cell death up to the dose of 100 μM (Fig. 1B). In LDH assay, the doses over 50 μM resulted in increased secretion of LDH from SV-HEKs (Fig. 1C). These results implicated that RA caused some damage of plasma membrane.

Discussion

Epidermal keratinocytes are innate immune cells that respond to TLR3 stimulation with high production of pro-inflammatory cytokines and chemokines such as IL-1β, TNFα, IL-6, IL-8 and CCL20 [13]. Psoriasis is an immune-mediated inflammatory skin disorder, and inflammatory cytokines secreted by psoriatic keratinocytes have important roles to recruit and activate immune cells such as neutrophils and activated T cells [14]. It has been described that psoriatic keratinocytes show increased

Conclusion

This study demonstrated that RA inhibited poly(I:C)-induced inflammatory reaction of epidermal keratinocytes, suggesting that RA could hold promise for the therapeutics of chronic inflammatory skin diseases such as psoriasis.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Acknowledgments

This research was supported by Chungnam National University Hospital Research Fund 2015, and a grant of the Korean Health Technology R&D Project (grant number: HN12C0062, Ministry of Health & Welfare, Republic of Korea).

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