In this study, we identified a novel function of P. linteus as a potent therapeutic modulator for atopic dermatitis (AD) and elucidated the underlying action mechanisms of it in alleviation of AD symptoms. Treatment of water soluble extract of P. linteus (WA) significantly reduced serum IgE and the levels of AD related pathogenic cytokines and chemokines.
Atopic dermatitis is thought to be a typical Th2 type immune disorder which shows elevated serum IgE level and increment of Th2 type cytokines such as IL-4, IL-5 and IL-13
[23, 24]. Th1 type response also plays key role in pathogenesis and maintenance of AD
. Clinical trials have been performed to modulate Th2 and Th1 type responses as well as chemokines levels. General immune suppressants have been used to treat AD, which cause numerous side effects with short period of efficacy. Recently herbal medicines have become a major part of CAMs (Complementary and Alternative Medicines) for treatment various kinds of diseases including cancer, allergy and diabetes
. These plant derived products have been used as drugs and food additives for a long time. However, scientific evidence is required for the further development of herbal medicines and their clinical application to treat diverse diseases. P. linteus is a member of Hymenochaetaceae, which has been used as a traditional medicine in oriental countries for the treatment of various diseases such as gastroenteric disorder, inflammation, tumors and lymphopathic disease
[26, 27]. Its pharmacological activities, especially anti-tumor and anti-inflammatory activities, have been documented
[28–30]. However no information is available on the effect of mycelium of P. linteus in modulation of allergic skin disorders such as atopic dermatitis.
In this study we aimed to test and identify effective fraction of the mycelium of P. linteus in modulation of atopic dermatitis. Since P. linteus has limited production in nature, we used mycelium culture of P. linteus which can be easily available in large quantities. To elucidate whether P. linteus has therapeutic potential for atopic dermatitis, we first tested inhibitory effect on IgE production by B cells since serum IgE levels were considered as an authentic marker of AD. Indeed, 70 ~ 80% of AD patients showed significantly increased serum IgE level compared to non-AD patients. After determining the non-cytotoxic concentration we applied total extract of P. linteus into IgE producing U266B1 B cell lines and found that the extract significantly inhibited IgE production under the LPS and IL-4 stimulation which is the well known IgE class switching condition
[13, 31] as well as W/O stimulation (Figure
1B). To identify potent inhibitory fraction we further fractionated P. linteus by diverse solvents including chloroform, methanol, water and boiling water (Figure
2A). Among the extracts, water soluble extract of P. linteus (WA) showed the most potent inhibitory activity to block IgE production in both U266B1 cell line and primary B cells (Figure
2B). In accord with in vitro result, among the three fractions only water soluble extract of P. linteus (WA) significantly decreased total serum IgE levels from AD induced mice (data not shown). Based on these results, we investigated the therapeutic effect of soluble extract of P. linteus (WA) in ongoing AD model. We also used ceramide
 as a positive control to elucidate remedial value of soluble extract of P. linteus (WA). It has been reported that ceramide content is decreased in the skin of AD patients
 and ceramide content in the stratum corneum showed a correlation with skin barrier function in AD patients. Indeed, topical application of ceramide derivatives suppressed AD-like skin lesions in NC/Nga mice by inhibiting infiltration of leukocytes and mast cells and reduced IL-4, TNF-α expression from ear cells
. As expected, topical application of ceramide significantly reduced AD symptoms, ear thickness and increment of clinical scores compared to PBS treatment (Figure
3). Interestingly, treatment of water soluble extract of P. linteus (WA) showed comparable therapeutic effects with ceramide in reducing AD symptoms such as ear thickness and clinical score (Figure
3). Histological analysis of ear tissues further indicated that treatment water soluble extract of P. linteus (WA) significantly decreased tissue infiltration of lymphocytes and granulocytes compared with ear tissues from PBS treated control mice (Figure
3E). In addition, treatment of water soluble extract of P. linteus (WA) significantly reduced total IgE levels (Figure
4A) as well as in antigen (mite)-specific IgE levels (Figure
4B) without affecting total IgG levels (Figure
4C). Interestingly, water soluble extract of P. linteus (WA) was more potent than ceramide in reducing mite-specific IgE levels. These data indicated that P. linteus can suppress allergic responses in an allergen specific manner; consequently, this could be one of the merits of P. linteus for the treatment of atopic dermatitis.
AD pathogenesis is associated with recruitment of lymphocyte, granulocyte to the inflammatory skin region. During this multiple step process of leukocyte trafficking and migration, chemokine ligand-receptor interactions are considered as crucial factors. Several chemokines have association with AD phenotype and among them, CCL17, CCL22, CCR4 have pivotal roles in migration of pathogenic immune cells (mainly CD4+ T cells) to the site of inflammation
. Interestingly, treatment of water soluble extract of P. linteus (WA) significantly decreased the expression of CCL22 and CCR4 (Figure
5A). Since keratinocyte, epithelial cells and dendritic cells are major source of these chemokines, suppression of chemokine expression by the treatment of water soluble extract of P. linteus (WA) could lead to lowering of infiltration of pathogenic T cells at the site of inflammation (Figure
[35–38]. Decreased expression of CCR4 also indicates that water soluble extract of P. linteus (WA) could inhibit T cell differentiation into Th2 cells since CCR4 is mainly expressed on the surface of Th2 cells in AD patients. Inhibitory effect of water soluble extract of P. linteus (WA) on the chemokine expression was comparable with that of ceramide (Figure
5A). Like chemokines, cytokines are also crucial pathogenic factors in AD pathogenesis. Both Th1 and Th2 type cytokines contribute to the pathogenesis of AD and their expression pattern is not mutually exclusive
. IL-4, IL-5 and IL-13 are typical Th2 type cytokines which stimulates Th2 differentiation and IgE production by B cells. IL-12 and IFN-γ are typical Th1 type cytokines that induce differentiation and maturation of T cells into Th1 type cells. Development of AD is induced by Th2 type response, while the chronic inflammatory responses is dominantly mediated by Th1 type reactions
. In addition, house dust mite reactive T cells produce both Th1 and Th2 type cytokines which strongly supports this concept
. Therefore, for the treatment of AD, both Th1 and Th2 types of immune responses should be considered as therapeutic targets. To elucidate the underlying mechanism of water soluble extract of P. linteus (WA), we measured mRNA expression levels of AD-related pathogenic cytokines from ear tissues and ear residual CD4+ T cells (Figure
5C). In ear tissues, topical application of water soluble extract of P. linteus (WA) significantly reduced not only Th2 cytokines (IL-10 and IL-13) but also Th1 cytokines (IL-12 and IFN-γ) (Figure
5B). IL-13 is known as the major inducer of Th2 generation in the cutaneous microenvironment, independent of IL-4
. IL-12 may have critical roles to terminate Th2 cytokine expression but it also initiates expression of Th1 cytokine IFN- γ which induces CCL17 (TARC) and CCL22 (MDC) from keratinocyte and epithelial cells
[43–45]. Interestingly, treatment of water soluble extract of P. linteus (WA) significantly decreased expression of MHCII, B7.1 and B7.2 in ear tissues of AD mice compared to PBS treated mice (data not shown). Hence, treatment with water soluble extract of P. linteus (WA) may suppress activation of antigen presenting cells including dendritic cells, macrophage, keratinocyte and epithelial cells at the site of inflammation, which modulates activation and differentiation of CD4+ T cells into Th1 or Th2 type. Interestingly, treatment of water soluble extract of P. linteus (WA) more significantly down-regulated the expression levels of IL-2, IL-10, IL-12, and IFN-γ from ear cells compared to ceramide treatment (Figure
5B). Indeed, treatment with water soluble extract of P. linteus (WA) significantly decreased expression level of pathogenic cytokines including IL-12, IL-13 and IFN- γ in CD4+ T cells of atopic regions and the inhibitory effect was more efficient than ceramide treatment (Figure
5C). In addition, consistent with PMA/ionomycin stimulation, under the antigen specific stimulation by mite extract, WA also significantly decreased the expression levels of pathogenic cytokines (IL-4, IL-13 and IFN-γ) and chemokines (CCL22) and this effect was more effective than ceramide treatment (Additional file 1: Figure S
1). Among the subfractions, water soluble extract of P. linteus (WA) was the most effective in modulating AD-associated inflammatory responses, implying that P. linteus may contain active anti-inflammatory component(s) with relatively hydrophilic characters. Further characterization of active compound will lead to develop a potent AD modulating agent. Topical treatment of ointment containing extract of P. linteus significantly decreased total serum IgE levels. Furthermore, not only topical treatment of WA, oral administration of WA also improved AD symptoms including reduction of IgE levels, pathogenic cytokine expression and immune cell infiltrations (data not shown). P. linteus is well known to exhibit anti-cancer effects through immuno-potentiating effects and also anti-inflammatory effects. Exact action mechanisms of anti-tumor and anti-inflammatory effect might be mediated by different active component. In addition, routes of treatments such as oral administration or topical application and target cells of P. linteus under the certain disease environment may mediate diverse effect of P. linteus on different immune disorders.
Collectively, topical application of water soluble extract of P. linteus (WA) may inhibit hyper-activation of tissue residual antigen presenting cells, which subsequently block the initiation of immune cascade from innate to adaptive immunity.