Ann Dermatol.  2018 Jun;30(3):322-330. 10.5021/ad.2018.30.3.322.

Investigation of Immune-Regulatory Effects of Mageumsan Hot Spring via Protein Microarray In Vitro

• Hahn HJ
• Kim JS
• Kim YH
• Lee YB
• Yu DS
• Kim JW

• KMID: 2419174
• DOI: http://doi.org/10.5021/ad.2018.30.3.322

Abstract

BACKGROUND
Empirical evidences for efficacy of hot spring (HS) water in inflammatory skin disorders have not been substantiated with sufficient, immunological "hard evidence". Mageumsan HS water, characterized by its weakly-alkaline properties and low total dissolved solids content, has been known to alleviate various immune-inflammatory skin diseases, including atopic dermatitis (AD).
OBJECTIVE
The trial attempted to quantitatively analyze in vitro expression levels of chemical mediators in cutaneous inflammation from HaCaT cell line treated with Mageumsan HS, and suggest the likely mode of action through which it exerts the apparent anti-inflammatory effects in AD.
METHODS
Using membrane-based human antibody array kit, customized to include 30 different, keratinocyte-derived mediator proteins, their expression levels (including interleukin [IL]-1, IL-6, IL-8, thymic stromal lymphopoietin, thymus and activation-regulated chemokine, and granulocyte macrophage colony-stimulating factor) were assessed in vitro. Selected key proteins were further quantified with enzyme-linked immunosorbent assay.
RESULTS
There was a clear pattern of overall suppression of the mediators, especially those noted for their pro-inflammatory role in AD (monocyte chemoattractant protein [MCP]-1, regulated on activation, normal T cell expressed and secreted, cutaneous T-cell-attracting chemokine, Eotaxin, and macrophage inflammatory protein-1α, etc.). Also, reduced expression of involucrin and cytokeratin 1 was also reduced in the HS-treated group.
CONCLUSION
The present study has shown that Mageumsan HS water may exert its effects on inflammatory skin disorders through regulation of proinflammatory cytokines. These evidences are to be supported with further future investigations to elucidate immunological mechanism behind these beneficial effects of HS water in the chronically inflamed skin of AD.

Keyword

Atopic dermatitis; Hot spring water; Immune-regulation; Keratinocyte-derived cytokines/chemokines; Protein array analysis

MeSH Terms

Cell Line
Chemokine CCL17
Chemokine CCL27
Cytokines
Dermatitis, Atopic
Enzyme-Linked Immunosorbent Assay
Granulocytes
Hot Springs*
Humans
In Vitro Techniques*
Inflammation
Interleukin-6
Interleukin-8
Interleukins
Keratins
Macrophages
Protein Array Analysis*
Skin
Skin Diseases
Water
Chemokine CCL17
Chemokine CCL27
Cytokines
Interleukin-6
Interleukin-8
Interleukins
Keratins
Water

Figure

• 

  Fig. 1 Arrangement of custom-made cytokine/chemokine-specific antibody microarray of 30 chemical mediators of keratinocyte origin. POS: positive control, NEG: negative control, IL: interleukin, TNF: tumor necrosis factor, IFN: interferon, TSLP: thymic stromal lymphopoietin, IP: interferon-γ induced protein, TARC: thymus and activation-regulated chemokine, MCP: monocyte chemoattractant protein, RANTES: regulated on activation, normal T cell expressed and secreted, CTACK: cutaneous T-cell-attracting chemokine, MIP: macrophage inflammatory protein, MIG: monokine induced by γ-interferon, I-TAC: interferon-inducible T-cell α chemoattractant, MCP: monocyte chemoattractant protein, GM-CSF: granulocyte macrophage colony-stimulating factor.

• 

  Fig. 2 Cytokine protein microarray in the human keratinocyte cell cultured after 24 hours. (A) Dulbeco's Modified Eagle Medium (DMEM; Gibco-BRL, USA) media (without fetal bovine serum, osmorality 336 mOSM/kg). (B) DMEM media with Magumsan hot spring (HS) water (osmorality 350 mOSM/kg). (C) DMEM media treated with toll-like receptor 3 agonist poly (I:C) (10 µg/ml). (D) DMEM media with Magumsan HS water treated with poly (I:C) (10 µg/ml).

• 

  Fig. 3 The ratios of the 30 mediator molecules expressed at different levels. (A) is the ratio of hot spring (HS)-treated group to the control. The ratio ranged from 0.58 (Fractalkine) to 1.70 (IL-8). (B) is the ratio of HS and poly (I:C) group to poly (I:C) group. The ratio ranged from 0.77 (CTACK) to 1.26 (IL-18). IL: interleukin, MIG: monokine induced by γ-interferon, MIP: macrophage inflammatory protein, RANTES: regulated on activation, normal T cell expressed and secreted, TARC: thymus and activation-regulated chemokine, TNF: tumor necrosis factor, IP: interferon-γ induced protein, MCP: monocyte chemoattractant protein, TSLP: thymic stromal lymphopoietin, CTACK: cutaneous T-cell-attracting chemokine, IFN: interferon, GM-CSF: granulocyte macrophage colony-stimulating factor.

• 

  Fig. 4 (A~D) The concentrations of four key molecules as measured by enzyme-linked immunosorbent assay in human keratinocyte cells treated with hot spring (HS), poly (I:C), HS with poly (I:C), and controls (n=3). The density of TARC expression was significantly decreased in HS with poly (I:C)-treated group versus poly (I:C) only group (p<0.001). The density of IP-10 was also measured to be lower in HS with poly (I:C)-treated group versus poly (I:C) only group (p<0.001), thus the ratio (0.84) was much smaller than that observed for TARC (0.18). RANTES followed the downward trend as well. The absolute concentration of Eotaxin was much lower than those of TARC, IP-10, or RANTES. TARC: thymus and activation-regulated chemokine, IP-10: interferon-γ induced protein-10, RANTES: regulated on activation, normal T cell expressed and secreted.

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