Supplementary Material for: 1,2,4,5-Tetramethoxybenzene Suppresses House Dust Mite-Induced Allergic Inflammation in BALB/c Mice

<b><i>Background:</i></b> Atopic dermatitis (AD) is the most common allergic inflammatory skin disease. The activation of innate immunity by house dust mite (<i>Dermatophagoides farinae </i>extract, DFE) allergen plays an important role in the pathogenesis of AD. We previously showed the inhibitory effect of an extract of <i>Amomum xanthioides</i> on allergic diseases, and isolated 1,2,4,5-tetramethoxybenzene (TMB) as a major active component. In this study, we investigated whether TMB relieves DFE-induced allergic inflammation symptoms. <b><i>Methods:</i></b> We established a DFE-induced allergic inflammation model in BALB/c mice by repeated skin exposure to DFE. To define the underlying mechanisms of action, we used a tumor necrosis factor-α and interferon-γ-activated human keratinocytes (HaCaT cell line) and mouse keratinocytes (3PC cell line) cell line model. <b><i>Results:</i></b> Oral administration of TMB suppressed allergic inflammation symptoms, such as histopathological analysis and ear thickness, in addition to serum IgE, DFE-specific IgE and IgG2a levels. TMB decreased the serum histamine levels and tissue infiltration of inflammatory cells, including mast cells and eosinophils. TMB also inhibited CD4<sup>+</sup>IFN-γ<sup>+</sup>, CD4<sup>+</sup>IL-4<sup>+</sup>, and CD4<sup>+</sup>IL-17A<sup>+</sup> lymphocyte expansion in the draining lymph nodes and expression of the Th2 cytokines in the ear tissue. TMB significantly inhibited the expression of cytokines and chemokines by the downregulation of the mitogen-activated protein kinases and nuclear factor of activated cytoplasmic T cells in HaCaT cells. <b><i>Conclusions:</i></b> TMB improved DFE-induced allergic inflammation by suppressing the production of proinflammatory cytokines and chemokines. Our results suggest that TMB might be a potential therapeutic agent for AD.