![]() ![]() 5, CD4 + naïve T cells were profoundly proliferated and differentiated under each skewing condition, along with anti-CD3 stimulation. Then, the flow cytometry was employed to evaluate the precursor frequency in terms of the T cell differentiation. After 3 days, the cells were harvested and stained with CD4 and interferon (IFN)-γ for Th1, IL-4 for Th2, IL-17 for Th17 and Foxp3 for T reg cells, respectively. CFSE-labeled CD4 + T cells were cultured under various conditions, which polarized Th1, Th2, Th17, and T reg with or without spa spring water. To evaluate the in vitro suppressive capacity of spa spring water toward the helper T cell differentiation, magnetic sorted CD4 + T cells, from the spleen, were prepared from Balb/c mice. Because the effector T cells, such as Th1, Th2, and Th17 cells act on various skin resident cells and potentiate the immune reaction, regulating T cell differentiation may modulate pro-inflammatory responses. We subsequently attempted to ascertain the potential involvement of spa spring water in the differentiation of the helper T cells. ![]() Proliferation characteristics of T cells under spa spring water ![]() Stop solutions (2 N H 2SO 4) were added, and the absorbance was read at 450 nm within 30 minutes. After extensive washing, 100 µl of substrate solution (tetramethylbenzidine and hydrogen peroxide, BD Biosciences Pharmingen) was added to each well and the plates were incubated for 30 minutes at RT, in darkness. After washing, 100 µl of detection antibody and streptavidin-conjugated horseradish peroxidase reagent were added to the wells, which was then incubated for 1 hour at RT. After washing the wells with washing buffer (phosphate buffered saline with 0.05% Tween-20), the wells were blocked with assay diluents (10% FBS in PBS) for 1 hour at room temperature (RT), followed by the addition of 100 µl/well of cell supernatant and cytokine standard solutions for 2 hours at RT. In brief, the wells were coated with 100 µl of capture antibody in coating buffer (0.1 M sodium carbonate, pH 9.5) and the plates were incubated overnight at 4℃. IL-6 and TNF-α in mouse antigen presenting cells (APC) supernatant were also measured. Levels of IL-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), and TNF-α (BD OptEIA™, BD Biosciences Pharmingen, San Diego, CA, USA), IL-1α (Biolegend, San Diego, CA, USA) in HaCaT supernatant treated with TLR agonist in the presence or absence of hot spring water were quantified according to the manufacturer's protocol. ![]() Among them, we investigated the immunomodulatory or anti-inflammatory effect of thermal spring water on the expression of pro-inflammatory cytokines in the HaCaT cells under TLR stimulation, as well as the effect on differentiation of CD4 + T cells under spring water. The efficacy of spa therapy for inflammatory skin diseases and the mechanisms are only partly understood, and presumably incorporate chemical, thermal, mechanical and immunomodulatory effects 7, 8. Spa therapy is widely used for the treatment of inflammatory skin diseases, such as atopic dermatitis, psoriasis, pruritus, rosacea, seborrheic dermatitis and others 6. In addition to the antimicrobial peptides, keratinocytes secrete cytokines, including IL-1, IL-6, IL-10, IL-18 and tumor necrosis factor (TNF) 5. IL-17 and IL-22 increased antimicrobial peptides, β-defensins and cathelicidins, from keratinocytes 4. The Th17 cells were reported to contribute to defend against various fungal or bacterial infections, and possibly induced atopic dermatitis and epidermal changes in psoriasis 2, 3 via secretion of interleukin (IL)-17 and IL-22. For example, Th1-dominant immune reactions were reported to be associated with the autoimmunity or psoriasis, and Th2-dominant responses were related to that of asthma or atopic dermatitis. The three main types of CD4 + T cells can be found in the skin during inflammatory skin disease, i.e., Th1, Th2 and Th17. Epidermal keratinocytes express several TLRs, including TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7 and TLR9 1, and through the TLRs, keratinocytes initiate and promote immune responses in the skin.įor inflammatory skin disease, pathogenesis is due to the sum of the immune reactions of the keratinocytes, immune cells and soluble mediators. As immune sentinels, keratinocytes can recognize foreign or danger stimuli from the outside via pattern-recognition receptors, such as Toll-like receptors (TLRs), and release innate immune mediators, including cytokines and chemokines, under the stimulation of the keratinocytes. Skin contributes to the first line of defense against any foreign materials outside of our body, as a physical barrier. ![]()
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