Glucocorticoids Enhance Toll-Like Receptor 2 Expression in Human

ORIGINAL ARTICLE Glucocorticoids Enhance Toll-Like Receptor 2 Expression in Human Keratinocytes Stimulated with Propionibacterium acnes or Proinflammatory Cytokines Michio Shibata1, Masako Katsuyama1, Tomoko Onodera1, Ritsuko Ehama1, Junichi Hosoi1 and Hachiro Tagami2 Toll-like receptors (TLRs) on keratinocytes are important cell surface receptors involved in the innate and acquired immune response to invading microorganisms. In acne vulgaris, TLR2 activation by Propionibacterium acnes (P. acnes) may induce skin inflammation via induction of various proinflammatory molecules that stimulate the invasion of inflammatory cells. Although corticosteroids themselves exert immunosuppressive or anti-inflammatory effects, it is well known clinically that systemic or topical glucocorticoid treatment provokes an acneiform reaction. Nevertheless, the effect of steroids on TLR2 expression in human keratinocytes remains unknown. Here, we found that the addition of glucocorticoids, such as dexamethasone and cortisol, to cultured human keratinocytes increased their TLR2 gene expression. Moreover, these glucocorticoids markedly enhanced TLR2 gene expression, which was further stimulated by P. acnes, tumor necrosis factor-a, and IL-1a. Gene expression of mitogen-activated protein kinase (MAPK) phosphatase-1 was also increased by the addition of dexamethasone. By using several inhibitors and activators, we found that TLR2 gene induction by glucocorticoids was mediated by the suppression of p38 MAPK activity following induction of MAPK phosphatase-1. These findings strongly suggest that steroid-induced TLR2 together with P. acnes existing as normal resident flora plays an important role in the exacerbation of acne vulgaris as well as in possible induction of corticosteroid-induced acne or in that of rosacea-like dermatitis. Journal of Investigative Dermatology (2009) 129, 375–382; doi:10.1038/jid.2008.237; published online 14 August 2008 INTRODUCTION reactions mediated mainly by NF-kB and mitogen-activated The immunology of the innate immune system has been a protein kinase (MAPK) pathways. TLR2 is well known to act rapidly progressing research area in the last decade. This as a receptor for Gram-positive Propionibacterium acnes important host defense system for animals against external (P. acnes). P. acnes, which colonizes the upper pilosebaceous organisms includes various mechanisms such as the Toll-like unit, is primarily responsible for inflammatory acne vulgaris. receptor (TLR) family, the complement activation system, and It acts through the induction of cytokine secretion and release antibacterial peptides. TLR family comprises a group of cell of substances such as proteases and hyaluronidases (Kim surface receptors that are important for both innate and et al., 2002; Jugeau et al., 2005). TLR2 activation by P. acnes acquired immune responses against invading microorganisms induces skin inflammation in acne lesions by the activation of (Akira et al., 2006). Many constituents of these pathogens, various inflammatory genes that encode cytokines, chemo- collectively known as the pathogen-associated molecular kines, and cell adhesion molecules with a resulting invasion pattern, bind to TLRs to induce various inflammatory of immune cells (Kim et al., 2002). Several reports have shown that TLR2 expression is enhanced by glucocorticoids in bronchial or other epithelial 1Shiseido Research Center, Yokohama, Japan and 2Tohoku University School of Medicine, Sendai, Japan cells (Imasato et al., 2002; Shuto et al., 2002; Hermoso et al., Correspondence: Dr Michio Shibata, Shiseido Research Center, 2-12-1 2004; Homma et al., 2004). TLR2 may also be involved in Fukuura, Kanazawa-ku, Yokohama 236-8643, Japan. susceptibility to lower respiratory tract infections upon E-mail; [email protected] inhalation of corticosteroids (Homma et al., 2004). However, Abbreviations: MAPK, mitogen-activated protein kinase; MKP-1, MAPK there have been no reports on the effect of glucocorticoids on þþ phosphatase-1; P. acnes, Propionibacterium acnes; PBS(À), Ca - and TLR2 expression in epidermal keratinocytes. Mg þþ-free phosphate-buffered saline; TLR, Toll-like receptor; TNF-a, tumor necrosis factor-a Cortisol, the main glucocorticoid in humans, is considered Received 24 December 2007; revised 15 April 2008; accepted 20 June 2008; a stress hormone because the blood cortisol level increases published online 14 August 2008 under conditions of psychosocial and physical stress (Evolahti & 2009 The Society for Investigative Dermatology www.jidonline.org 375 M Shibata et al. Glucocorticoids Enhance TLR2 in Keratinocytes et al., 2006). Animal and human studies strongly suggest an not occur at a significantly higher level (Figure 1d). We used important immunoregulatory role for the hypothalamic- phorbol 12-myristate 13-acetate-treated THP-1 cells as a pituitary-adrenocortical axis and the sympathoadrenomedul- positive control for TLR2 expression (Haehnel et al., 2002). lary system (Buske-Kirschbaum et al., 2002). Patients suffering TLR2 was found to be constitutively expressed in THP-1 cells from allergic inflammation have significantly attenuated with a high copy number even in the absence of TNF-a or cortisol responses to psychosocial stress (Buske-Kirschbaum dexamethasone (data not shown). TNF-a had no additive et al., 2002). Immobilization stress influences cutaneous effect on TLR2 expression, but dexamethasone remarkably functions by changing the levels of neuropeptides and enhanced TLR2 expression (Figure 1f). hormones, and it suppresses the contact hypersensitivity Next, we used the anti-human TLR2 monoclonal antibody response in mice (Singh et al., 1999). It is known that cortisol clone 1030A5.138 to detect TLR2 protein expression in suppresses the immune system via inhibition of the production keratinocytes (Figure 2). TLR2 staining intensity was ob- of inflammatory cytokines and chemokines (Hosoi et al., viously increased in cells stimulated with TNF-a in the 2001). Although corticosteroids themselves exert immunosup- presence of dexamethasone, indicating that enhancement by pressive and anti-inflammatory effects, it is well known glucocorticoid treatment occurred at both the gene and clinically that systemic or topical glucocorticoid treatment protein levels. In contrast, there were no fluorescence signals provokes an acneiform reaction (Plewig and Kligman, 1973; in samples stained with isotype control antibody (Figure 2e). Fung and Berger, 2000). Moreover, the prolonged use of potent topical corticosteroids on the face often results in Effect of the steroid receptor antagonist RU486 papulopustular eruptions accompanied by erythema (steroid In order to elucidate the mechanism of the effect of rosacea; Leyden et al., 1974) or persistent erythematous glucocorticoids on TLR2 expression in human keratinocytes, eruptions consisting of tiny papulopustules with a distribution we used the glucocorticoid receptor antagonist RU486, also mainly around the mouth (perioral dermatitis; Cotterill, 1979). known as mifepristone, (Flores et al., 2006), to determine if In this study, we found that two glucocorticoids, dex- the enhancement of TLR2 expression was mediated via the amethasone and cortisol, increased TLR2 gene expression in glucocorticoid receptor (Figure 1e). The addition of 1 mM cultured human keratinocytes without additional stimulation. RU486 to the culture completely blocked the enhancement Moreover, these glucocorticoids markedly enhanced TLR2 of TLR2 expression induced by dexamethasone but did not gene expression in keratinocytes that had been stimulated affect TNF-a-induced TLR2 expression, indicating that the with P. acnes or the inflammatory cytokines, tumor necrosis effect of dexamethasone was mediated by the glucocorticoid factor-a (TNF-a) and IL-1a. receptor. RESULTS The effect of glucocorticoids on P. acnes-induced TLR2 The effects of glucocorticoids on TLR2 gene expression in expression of keratinocytes TNF-a- or IL-1a-stimulated human keratinocytes We assessed TLR2 gene expression in keratinocytes when We confirmed mRNA expression of TLR2 in cultured human viable P. acnes and dexamethasone were added to the keratinocytes by SYBR Green-based quantitative PCR and culture (Figure 3). For these studies, we used several different then determined the effect of dexamethasone on TLR2 strains of P. acnes, including JCM6425 (4.1 Â 107 colony- expression (Figure 1a). Relatively low but detectable levels forming units per well), JCM6473 (4.4 Â 108 colony-forming of TLR2 expression were observed in untreated keratinocytes. units per well), and ATCC11827 (4.0 Â 107 colony-forming In contrast, expression was increased in the presence of 0.1 units per well). These P. acnes strains slightly increased TLR2 and 1 mM dexamethasone by four- to fivefold after 6 hours. expression of keratinocytes in the absence of dexamethasone. TNF-a also increased TLR2 expression by threefold. Most However, all of them enhanced keratinocyte TLR2 expression strikingly, dexamethasone increased TLR2 expression by over in the presence of dexamethasone by approximately fourfold 20-fold in keratinocytes stimulated with TNF-a. After 1 hour when compared to induction by P. acnes strains alone. We of incubation, TLR2 expression was low, but stimulation with did not find any differences in the degree of enhancement of dexamethasone and TNF-a still produced significant effects. TLR2 expression among the three different strains, although On the other hand, TNF-a-induced IL-8 expression was

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