Anti-inflammatory and immunomodulatory effects of Aquaphilus dolomiae extract on in vitro models

Abstract

Background: Atopic dermatitis (AD) is a common skin disease characterized by recurrent pruritic inflammatory skin lesions resulting from structural and immune defects of the skin barrier. Previous studies have shown the clinical efficacy of Avène thermal spring water in AD, and a new microorganism, Aquaphilus dolomiae was suspected to contribute to these unique properties. The present study evaluated the anti-inflammatory, antipruritic, and immunomodulatory properties of ES0, an original biological extract of A. dolomiae, in immune and inflammatory cell models in order to assess its potential use in the treatment of AD.

Materials and methods: An ES0 extract containing periplasmic and membrane proteins, peptides, lipopolysaccharides, and exopolysaccharides was obtained from A. dolomiae. The effects of the extract on pruritus and inflammatory mediators and immune mechanisms were evaluated by using various AD cell models and assays.

Results: In a keratinocyte model, ES0 inhibited the expression of the inflammatory mediators, thymic stromal lymphopoietin, interleukin (IL)-18, IL-4R, IL-8, monocyte chemoattractant protein-3, macrophage inflammatory protein-3α, and macrophage-derived chemokine and induced the expression of involucrin, which is involved in skin barrier keratinocyte terminal differentiation. In addition, ES0 inhibited protease-activated receptor-2 activation in HaCaT human keratinocytes stimulated by stratum corneum tryptic enzyme and T helper type (Th) 1, Th2, and Th17 cytokine production in Staphylococcal enterotoxin B-stimulated CD4+ lymphocytes. Lastly, ES0 markedly activated innate immunity through toll-like receptor (TLR) 2, TLR4, and TLR5 activation (in recombinant human embryonic kidney 293 cells) and through antimicrobial peptide induction (psoriasin, human beta-defensin-2, and cathelicidin), mainly through TLR5 activation (in normal human keratinocytes).

Conclusion: Overall, these in vitro results confirm the marked regulatory activity of this A. dolomiae extract on inflammatory and immune responses, which may be of value by virtue of its potential as an adjunctive treatment of AD inflammatory and pruritic lesions.

Keywords: anti-inflammation; atopic dermatitis; barrier function; in vitro and in vivo activities; inflammation; microorganism.

Figure 1

Stimulation of PAR-2 receptors by SCTE in HaCaT keratinocyte cells. Notes: Dose-dependent inhibition by ES0 (30 and 100 µg/mL). ^*P<0.05; ^***P<0.001 versus SCTE; data are representative of three independent experiments. Abbreviations: ES0, extract from A. dolomiae; PAR-2, protease-activated receptor-2; SCTE, stratum corneum tryptic enzyme.

Figure 2

Spectrophotometric measurement of TLR2, TLR4, and TLR5 activation in HEK 293 cell lines. Notes: HEK were stimulated with ES0 (3–100 ng/mL) or positive controls: Pam2CSK4 = TLR2 ligand (A), LPS = TLR4 ligand (B), and flagellin = TLR5 ligand (C). ^***P<0.001 versus control; data are representative of three independent experiments. Abbreviations: ES0, extract from A. dolomiae; HEK, human embryonic kidney; LPS, lipopolysaccharide; OD, optical density; TLR, toll-like receptor.

Figure 3

Relative quantity of hBD-2, CAMP, and psoriasin mRNA in NHK cells. Notes: NHKs were stimulated by IL-1β (20 ng/mL, positive control), ES0 (6.5 µg/mL), flagellin (100 ng/mL), or alone (negative control). Pretreatment was performed for 1 h with anti-TLR2 ( ), anti-TLR4 ( ), anti-TLR5 (■) (1 µg/mL), or without antibody (□) to test whether stimulation was blocked in ES0- and flagellin-treated samples; data are the mean of three independent experiments. ^*P<0.05; ^**P<0.01; ^***P<0.001 versus control or comparator. Abbreviations: CAMP, cathelicidin antimicrobial peptide; ES0, extract from A. dolomiae; hBD2, beta-defensin 2; NHK, normal human keratinocyte; TLR, toll-like receptor.

Figure 4

Lymphocyte stimulation by Staphylococcal enterotoxin B (SEB; 300 ng/mL). Notes: Dose effect of ES0 (30, 60, and 100 µg/mL) on the production of Th1, Th2, and Th17 cytokines; data are representative of three independent experiments. ^***P<0.001 versus SEB-treated cells. Abbreviations: ES0, extract from A. dolomiae; IL, interleukin; SEB, Staphylococcal enterotoxin B; Th, T helper cell type; TNFα, tumor necrosis factor alpha.