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Zinc salts inhibit in vitro Toll-like receptor 2 surface expression by keratinocytes |
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European Journal of Dermatology. Volume 17, Number 6, 492-6, November-December 2007, Investigative report |
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DOI : 10.1684/ejd.2007.0263 |
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Author(s) : Véronique Jarrousse, Nathalie Castex-Rizzi, Amir Khammari, Marie Charveron, Brigitte Dréno , INSERM U601, 9, quai Moncousu, 44093 Nantes cedex 01, FranceFax: (+33) 2400831173, Clinique dermatologique, CHU Hôtel-dieu, 1, place A. Ricordeau, 44035 Nantes, France, Institut de recherche Pierre Fabre, Laboratoire de biologie cellulaire cutanée, Toulouse, France. | |||||
Summary : Propionibacterium acnes (P. acnes) plays an important role in the induction and maintenance of the inflammatory phase of acne. At the therapeutic level, it has been shown that zinc salts could have a beneficial effect on mild and moderate inflammatory acne lesions. However, their mechanisms of action are still only partially known. Immediate early immune response is a crucial route in the development of inflammatory reaction and, specifically, activation of Toll-like Receptors (TLRs) leading to nuclear factor (NF)-κB translocation and production of inflammatory cytokines such as interleukin-8 (IL-8). The aim of this work was to determine if cytokine secretion and innate immunity could be targets of zinc salts.Normal Human Epidermal Keratinocytes (NHEK) and skin explants were stimulated by P. acnes extracts and incubated (3 h) with zinc salts (1 μg/mL). Then we successively studied TLR2 expression by immunohistochemistry and IL-8 production by ELISA. After incubation with zinc salts, the increase of TLR2 surface expression in skin upon membrane fraction (FM) of P. acnes challenge was decreased as compared to that in control samples. However, this inhibition does not modify IL-8 secretion by keratinocytes.In conclusion the inhibition of TLR2 surface expression by keratinocytes could be one of the anti-inflammatory mechanisms of zinc salts in acne. | |||||
Keywords : keratinocytes, interleukin-8, Propionibacterium acnes, TLR2, zinc | |||||
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ARTICLE
Auteur(s) : Véronique Jarrousse1, Nathalie Castex-Rizzi3, Amir Khammari1,2, Marie Charveron3, Brigitte Dréno1,2 1INSERM U601, 9, quai Moncousu, 44093 Nantes cedex
01, FranceFax: (+33) 2400831173 accepté le 30 Mai 2007 Acne vulgaris is a skin disorder of the pilosebaceous follicles that commonly occurs in adolescence and in young adulthood. Although the precise mechanisms that induce the development and progression of acne still remain unclear, three main factors have been identified: an androgen-stimulated increase in the production of sebum, obstruction of the pilo-sebaceous follicles resulting from abnormal proliferation and differentiation of follicular epithelium, and finally a bacterial factor with Propionibacterium acnes which plays a central role in the initiation and the maintenance of inflammatory reactions in and around the pilo-sebaceous follicles.Zinc was recognized to be essential for human health in 1963 [1]. Several studies [1, 2] have shown a beneficial effect of zinc salts on inflammatory lesions in mild and moderate acne. However, the mechanisms by which zinc salts act on skin inflammation are still only partially known [3]. Indeed, zinc inhibits polymorphonuclear cell chemotaxis [4], inhibits the growth of P. acnes [5], activates natural killer (NK) cells and the phagocytosic capacity of granulocytes [6, 7]. Its anti-inflammatory activity in acne could also be related to a decrease in tumor necrosis factor (TNF)-α and IL-6 production [8] and modulation of the expression of integrins [9, 10], mainly intracellular adhesion molecule (ICAM)-1 and leucocyte function associated antigen (LFA)-3 [11]. Zinc salts also have a specific action on type 5α-reductase, shown in vitro [12].Among the different actors of immediate immunity, Toll-like receptors have been described, these are receptors specifically activated by microbial components [13, 14]. This activation of TLR by microbial antigens induces a strong production of different inflammatory cytokines by the cell, and among them IL-8 which strongly attracts and activates neutrophils and T lymphocytes. Kim et al., [15] demonstrated that P. acnes induces monocyte cytokine (IL-12 and IL-8) production through a TLR2-dependent pathway and that the expression of TLR2 in acne lesions indicates that activation of TLR2 can contribute to inflammation at the site of disease activity. Furthermore Nagy et al. [16] have shown that distinct strains of P. acnes upregulate human beta-defensins (hBD2) and IL-8 mRNA and protein expression in cultured keratinocytes.The aim of this work was to determine if the anti-inflammatory activity of zinc salts in acne could be linked to a modulation of TLR2 surface expression and interleukin (IL)-8 production by keratinocytes.Materials and methodsMaterialsBacterial extractsWe used three extracts of Propionibacterium acnes (IP 53113) provided by Pierre Fabre Laboratories (Toulouse, France). This reference strain was first described in 1968.Supernatant A (SA) and pellet C were obtained after bacterial culture centrifugation, several freeze/thaw cycles and a last centrifugation at 4000 rpm for 15 min. Supernatant B (SB) was obtained after a second centrifugation (4000 rpm, 15 min) of pellet C. The membrane fraction (FM) extract was obtained after reconstitution of pellets re-suspended in keratinocyte basal medium (KBM) without HC (hydrocortisone) (Promocell, Heidelberg, Allemagne). The distribution of cellular compartments in the three extracts (FM, SA and SB) was partially known: the wall and cytosolic membrane (peptidoglycane and lipoteichoic acid) was present in the FM extract and a low proportion of clever wall elements were present in the SB extract. The SA extract contained cytosolic proteins. Preliminary experiments were carried out in our laboratory to determine the effect of different dilutions of P. acnes extracts on keratinocyte viability (MTT test). The following extract dilutions: FM diluted at 1/2, SA and SB diluted at 1/5, were considered to be the most appropriate [17]. Trace elements15 mg zinc salts (Labcatal, Montrouge, France), were re-suspended in KBM without HC and incubated with NHEK or skin explants.The optimal amount of zinc salts was determined in preliminary experiments (dose-response studies, figure 1). Keratinocyte cultureNHEK from healthy prepuces were obtained from the pediatric surgery department of Nantes Hospital and were grown at 37 °C in a humid 5% CO2 atmosphere, in serum free keratinocyte growth medium (KSFM) (Invitrogen, Cergy-Pontoise, France) supplemented with 25 μg/mL bovine pituitary extract (BPE), 0.5 ng/mL of recombinant epidermal growth factor (EGF), 2.5 μg/mL fungizone (Bristol-Myers Squibb, Paris, France) and penicillin 100 UI/mL-streptomycine 100 μg/mL. Cells were used after a limited number (n = 2) of subcultures. NHEK were seeded at the density of 20000 per well in 4-wells chamber slides (Dutcher, Brumath, France) in low calcium (CaCL2 0.09 mM) defined medium KBM with HC. The medium was replaced by KBM without HC 24 h before starting the experiment.Skin explantsExplants were prepared from skin abdominal plasties received after plastic surgery. After fat removing, explants (4 mm diameter) were incubated at 37 °C in a moist atmosphere in the presence of 5% CO2 in solution in order to cause stimulation, or in control medium (KBM without HC). After incubation, explants were removed from the culture medium and frozen in liquid nitrogen before storage at – 80 °C.NHEK and skin explants stimulation by LPS or by P. acnes extractsNHEK and explants were stimulated for six hours at 37 °C with 1 μg/mL lipopolysaccharide (LPS) purified from Echerichia coli, 0111:B4 clone (Sigma) and prepared with fetal calf serum (10%) as described [18].The different P. acnes extracts (FM 1/2, SA 1/5 and SB 1/5) were deposited on skin explants for 3 h (immunohistochemistry) or 24 h (cytokine assays). Incubation with zinc saltsAfter stimulation and two washes with PBS, zinc salts were added to NHEK and skin explants for 3 h at 1 μg/mL. Then, culture supernatants were collected and frozen at – 80 °C for subsequent IL-8 secretion assay. NHEK and explants were washed twice by PBS, then NHEK were fixed in acetone for 10 minutes and frozen at – 20 °C. Explants were frozen in liquid nitrogen before storage at – 80 °C.Five independent experimental series were performed for each condition. MethodsLabelling of smears and skin explants (immunohistochemistry)Sections of deep-frozen skin explants were fixed in acetone for 10 min. The non-specific sites were saturated for 30 min with TBS (Tris-Buffered Saline) 0.05% Tween20 w/v (Sigma), 0.1% BSA w/v (Bovine Serum Albumin) (Sigma). The polyclonal rabbit antibody anti-TLR-2 H-175 (TEBU, Le Perray-en-Yvelines, France) was deposited on the slides for 30 min in a humid environment at room temperature. Rinses of 10 min were made between each stage using TBS, 0.1% BSA, 0.05% Tween20. The slides were incubated successively with a secondary biotinylated antibody (DAKO ChemTek detection kit peroxidase/AEC, Rabbit/Mouse) (30 min) and streptavidin coupled with peroxidase (30 min) before deposing AEC (a peroxidase substrate) for 5 min. The reaction was stopped with distilled water (10 min) and counter-staining was done with Mayer haemalun (VWR International) for about 1 min. The slides were rinsed with distilled water, mounted in an aqueous medium and observed with a Leitz microscope (X20 objective). The control section included omission of the primary antibody [16]. Two different examiners read the slides. The intensity of the labelling was quantified according to semi-quantitative scale: none (0), weak (1), moderate (2) and strong (3).Production of IL-8 (cytokine assays)Explants and NHEK were stimulated in the presence of P. acnes extracts (FM 1/2, SA 1/5, SB 1/5). To determine the IL-8 concentration, the culture media were harvested after 24h of culture and stored at – 80 °C until assay. The concentration of IL-8 in the explant supernatants was measured by a commercially available enzyme-linked immunoabsorbent assay kit (BD Biosciences, le Pont de Claix, France), used in accordance with the manufacters instructions. All samples were assayed in duplicate.Statistical analysisResults were expressed as the mean ± standard deviation (SD). Data were compared to controls, which consisted of keratinocytes or explants treated in the same manner as the test samples but without trace elements or LPS or P. acnes extracts. The statistical significance of the data was determined by T test. A P < 0.05 was taken as significant.ResultsTLR2 surface expression on keratinocytesZinc salts decreased TLR2 expression by NHEK and skin explants previously stimulated by LPS (1 μg/mL).TLR2 expression on NHEK stimulated by LPS (figure 1A) was significantly (p = 0.047) stronger (2.67 ± 0.58) compared to control (1.33 ± 0.58). With zinc salts, TLR2 expression was significantly decreased in a dose-dependent manner from 2.33 (± 0.58) with 0.1 μg/ml of zinc salts to 1 (± 0.87) with 1.5 μg/mL. TLR2 expression on skin explants stimulated by LPS (figure 1B) was increased (2.40 ± 0.65) (not significantly) compared to control (1.80 ± 0.65). In the presence of the three concentrations of zinc salts (0.5 μg/mL, 1μg/mL and 1.5 μg/mL), the level of TLR2 expression was decreased (not significantly) and was similar to that observed in control. According to the results, the concentration of 1 μg/mL of zinc salts with 3h of incubation was used in further experiments. Zinc salts decreased TLR2 expression by keratinocytes in skin explants previously stimulated by FM P. acnes extracts. TLR2 protein was express through the epidermis except by basal layer. When skin explants were stimulated with P. acnes extracts for 3 h (figure 2), the expression of TLR2 was induced: 2.20 (± 0.76) on skin explants incubated with FM1/2 extract compared to control: 1.70 (± 0.55) but not with the SA1/5 (1.80 ± 0.67) and SB1/5 (1.90 ± 0.76) extracts. After incubation of skin explants for 3h with zinc salts prior to FM1/2 extract, the expression of TLR2 was lower (at the limit of significance: p = 0.08) 1.20 (± 0.65) than control expression. The expression of TLR2 on skin explants incubated with SA: 1.80 (± 0.71) or SB: 1.90 (± 0.82) and then with zinc salts was not modified. IL-8 secretion in NHEK or skin explant supernatantsZinc salts had no effects on IL-8 secretion on NHEK nor on skin explants stimulated by P. acnes extracts.IL-8 secretion was significantly (p < 0.001) increased by FM: 515 pg/ml (± 30) in NHEK (figure 3A)) compared to control medium: 20 pg/mL (± 9). However, interleukin-8 secretion of keratinocytes after incubation with zinc salts (1 μg/mL for 3 h) was not modified. Regarding skin explants (figure 3B), we observed that IL-8 secretion was not stimulated by P. acnes extracts compared to control. IL-8 secretion pattern obtained with P acnes extracts was not modulated by zinc salts (1 μg/mL, for 3 h). DiscussionOur study shown that the expression of TLR2 by keratinocytes induced either by LPS or by FM P. acnes extract is decreased by zinc salts. This modulation seems to be obtained both with NHEK and skin explants. Explants have the advantage of being a model close to in vivo conditions. Thus, it permits study of the role of differentiation on the expression of an antigen by the keratinocytes and, moreover, localization of this marker in the epidermis. The model of NHEK has the advantage of studying keratinocytes alone and thus of ensuring a direct modulation on these cells.Recently, we have shown that extracts of P. acnes are able to increase TLR2, TLR4 and matrix metaloproteinase (MMP9) expression by keratinocytes [17]. In particular, we have shown on acne biopsies that TLR2 was clearly over expressed in comparison to TLR4. These facts suggested that P. acnes plays a central role in the induction and maintenance of the inflammatory phase of acne, through TLR2 expression. This present study focused on P. acnes (GRAM+) extract effects on TLR2 expression by keratinocytes. Indeed, TLR2 specifically recognizes a GRAM+ pattern (e.g. peptidoglycan) whereas TLR4 recognize a GRAM- pattern (LPS). However, it has been shown that commercial LPS preparations could stimulate epidermal keratinocytes to produce β-defensins 2 and IL-8 and that the LPS response was inhibited with mAB specific for TLR2, but not for CD14 and TLR4 [18]. Our study confirms that P. acnes is indeed able to induce the expression of TLR2. Interestingly, only the membrane fraction (FM) which contains peptidoglycan (PGN) and lipoteichoic acid, but neither supernatant A (SA) which contains cytosolic proteins nor supernatant B (SB) which is rich in membrane proteins are able to activate TLR2. Therefore, FM extracts of P. acnes contain one or several patterns which specifically activate TLR2 expression by keratinocytes. Zinc salts act mainly on inflammatory lesions in acne. It has been previously shown that its anti-inflammatory activity has different targets. Zinc is known to participate in the activation of nearly 300 enzymes. Some of these zinc-dependent enzymes are involved in DNA synthesis, cell division, and protein synthesis. Zinc is also required to stabilize three-dimensional structures of nearly 1000 transcription factors, such as “the zinc-finger” proteins. Zinc also plays important roles in immune functions. Zinc regulates several functions of lymphocytes, such as mitogenesis, antibody synthesis, the activation of T-cells and natural killer cells, and more specifically cellular immunity [19, 20]. Zinc deficiency has been reported to impair cellular immune functions. Our results add another anti-inflammatory target of zinc salts with TLR2. Indeed, we have shown that zinc salts decrease TLR2 surface expression by keratinocytes induced by FM extracts of P. acnes. Interestingly, recently, Kitamura et al., [21] showed a link between zinc and TLR on dendritic cells (DC), which are also present in the epidermis. Indeed, they have demonstrated that some TLR stimuli decrease intracellular free zinc in DCs, which is critical for DC maturation and, moreover, that activation of Toll/Il-1R gene homology (TIR)-containing adaptor-inducing interferon (IFN) (TRIF)-dependent signalling, alters the expression of zinc transporters and results in decreased intracellular free zinc. These results, added to ours concerning keratinocytes, demonstrate that innate immunity with TLR is an important target of zinc salts both on keratinocytes and DC. Concerning the activation of TLR2, the intracellular domain of TLR2 may activate NF-κB which then modulates the expression of many immune response genes [22]. By western-blot, we observed that NF-κB expression on explants was not modulated by P. acnes extracts or by zinc salts (1 μg/mL) (data not shown).By ELISA we firstly confirmed that FM extracts of P. acnes induced IL-8 secretion by NHEK. Previously, Nagy et al. [16] demonstrated that IL-8 secretion induced by P. acnes on keratinocytes are both TLR2 and TLR4 dependent. Our results shown that zinc salts (1 μg/mL) have no modulatory effect on IL-8 secretion (by explants or by NHEK) despite their inhibitory effect on TLR2 surface expression. Several studies have shown that Il-8 is not only produced by keratinocytes via TLRs stimulation but in response of pro-inflammatory cytokines stimulation. In particular, NHEK have been shown to produce IL-8 in response to IL-1 or TNF-α [23] and it has been shown that P. acnes produce IL-1α like and TNF-α like [3]. In healthy tissues, IL-8 is barely detectable, but is rapidly induced by ten- to 100-fold in response to pro-inflammatory cytokines such as TNF, IL-1, bacterial or viral products, and cellular stress [24]. Concerning the skin explants, we observed a high basal level of IL-8 secretion in normal skin. One hypothesis could be that in culture, cells present in explants as fibroblasts secrete cytokines such as IL-1 and TNF-α, inducing the production of IL-8 by keratinocytes. Thus it could explain the lack of modulation of IL- 8 by zinc salts, via inhibition of TLR. In conclusion, our results demonstrate for the first time, that zinc salts inhibit TLR2 expression by keratinocytes induced both by P. acnes FM extracts and LPS. Interestingly, this inhibition was not associated with an inhibition of interleukin-8 secretion. AcknowledgementsFinancial support and P. acnes extracts were provided by Pierre Fabre Laboratories (Toulouse, France) and Labcatal Laboratories (Montrouge, France). No conflicts of interest have been declared. We thank surgeons from the plastic surgery Department of Nantes Hospital for abdominal skin samples and the pediatric surgery Department of Nantes Hospital for foreskin samples.References1 Dreno B, Amblard P, Agache P, Sirot S, Litoux P. Low doses of zinc gluconate for inflammatory acne. Acta Derm Venereol 1989; 69: 541-3.2 Dreno B, Moyse D, Alirezai M, Amblard P, Auffret N, Beylot C, Bodokh I, Chivot M, Daniel F, Humbert P, Meynadier J, Poli F. Multicenter randomized comparative double-blind controlled clinical trial of the safety and efficacy of zinc gluconate versus minocycline hydrochloride in the treatment of inflammatory acne vulgaris. Dermatology 2001; 2003: 135-40. 3 Pawin H, Beylot C, Chivot M, Faure M, Poli F, Revuz J, Dreno B. Physiopathology of acne vulgaris: recent data, new understanding of the treatments. Eur J Dermatol 2004; 14: 4-12. 4 Dreno B, Trossaert M, Boiteau HL, Litoux P. Zinc salts effects on granulocyte zinc concentration and chemotaxis in acne patients. Acta Derm Venereol 1992; 72: 250-2. 5 Strauss JS, Stranieri AM. Acne treatment with topical erythromycin and zinc: effect of Propionibacterium acnes and free fatty acid composition. J Am Acad Dermatol 1984; 11: 86-9. 6 Dreno B, Foulc P, Reynaud A, Moyse D, Habert H, Richet H. Effect of zinc gluconate on propionibacterium acnes resistance to erythromycin in patients with inflammatory acne: in vitro and in vivo study. Eur J Dermatol 2005; 15(3): 152-5. 7 Chvapil M, Stankova L, Zukoski CT, Zukoski 3rd C. Inhibition of some functions of polymorphonuclear leukocytes by in vitro zinc. J Lab Clin Med 1977; 89: 135-46. 8 Sainte-Marie I, Jumbou O, Tenaud I, Dreno B. Comparative study of the in vitro inflammatory activity of three nickel salts on keratinocytes. Acta Derm Venereol 1998; 78: 169-72. 9 Tenaud I, Sainte-Marie I, Jumbou O, Litoux P, Dreno B. In vitro modulation of keratinocyte wound healing integrins by zinc, copper and manganese. Br J Dermatol 1999; 140: 26-34. 10 Tenaud I, Leroy S, Chebassier N, Dreno B. Zinc, copper and manganese enhanced keratinocyte migration through a functional modulation of keratinocyte integrins. Exp Dermatol 2000; 9: 407-16. 11 Gueniche A, Viac J, Lizard G, Charveron M, Schmitt D. Protective effect of zinc on keratinocyte activation markers induced by interferon or nickel. Acta Derm Venereol 1995; 75: 19-23. 12 Sugimoto Y, Lopez-Solache I, Labrie F, Luu-The V. Cations inhibit specifically type I 5 alpha-reductase found in human skin. J Invest Dermatol 1995; 104: 775-8. 13 Aderem A, Ulevitch RJ. Toll-like receptors in the induction of the innate immune response. Nature 2000; 406: 782-7. 14 Jarrousse V, Quéreux G, Marques-Briand S, Knol AC, Khammari A, Dreno B. Toll-like receptors 2, 4 and 9 expression in cutaneous T-cell lymphoma (mycosis fungoides and Sezary syndrome). Eur J Dermatol 2006; 16(6): 636-41. 15 Kim J. Review of the innate immune response in acne vulgaris: activation of Toll-like receptor 2 in acne triggers inflammatory cytokine responses. Dermatology 2005; 211: 193-8. 16 Nagy I, Pivarcsi A, Koreck A, Szell M, Urban E, Kemeny L. Distinct strains of Propionibacterium acnes induce selective human beta-defensin-2 and interleukin-8 expression in human keratinocytes through toll-like receptors. J Invest Dermatol 2005; 124: 931-8. 17 Jugeau S, Tenaud I, Knol AC, Jarrousse V, Quereux G, Khammari A, Dreno B. Induction of toll-like receptors by Propionibacterium acnes. Br J Dermatol 2005; 153: 1105-13. 18 Kawai K, Shimura H, Minagawa M, Ito A, Tomiyama K, Ito M. Expression of functional Toll-like receptor 2 on human epidermal keratinocytes. J Dermatol Sci 2002; 30: 185-94. 19 Fraker PJ, Gershwin ME, Good RA, Prasad A. Interrelationships between zinc and immune function. Fed Proc 1986; 45: 1474-9. 20 Kirchner H, Ruhl H. Stimulation of human peripheral lymphocytes by Zn2+ in vitro. Exp Cell Res 1970; 61: 229-30. 21 Kitamura H, Morikawa H, Kamon H, Iguchi M, Hojyo S, Fukada T, Yamashita S, Kaisho T, Akira S, Murakami M, Hirano T. Toll-like receptor-mediated regulation of zinc homeostasis influences dendritic cell function. Nat Immunol 2006; 7: 971-7. 22 Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004; 4: 499-511. 23 Larsen CG, Anderson AO, Oppenheim JJ, Matsushima K. Production of interleukin-8 by human dermal fibroblasts and keratinocytes in response to interleukin-1 or tumour necrosis factor. Immunology 1989; 68: 31-6. 24 Hoffmann E, Dittrich-Breiholz O, Holtmann H, Kracht M. Multiple control of interleukin-8 gene expression. J Leukoc Biol 2002; 72: 847-55. |
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