Bee venom acupuncture alleviates trimellitic anhydride-induced atopic dermatitis-like skin lesions in mice

There have been few objective ways to determine the optimum dose of BV in clinics and animal studies. According to unpublished reports from Oriental clinics, the best outcome with BVA can be achieved with the maximum dose of BV, with substantial heat and pain around the injection site. Because the injection of excessive amounts of BV sometimes induces anaphylactic symptoms, physicians using BVA gradually increase its dose according to the patient’s verbal response to treatment. One possible way to objectively optimize the injection method or daily dose is to measure the skin temperature around the BV injection site. We measured the temperature at the injection site after intradermal, subcutaneous, and intramuscular injection of BV into the backs of mice, and eventually adopted subcutaneous injection for BVA in this study (Additional file 1: Figure S1).

In the present study, BV therapy was used to treat atopic dermatitis, a typical allergic disease in which the intrinsic immune system is over-activated in response to various environmental factors. To investigate anti-inflammatory activities of BVA, we produced chronic TMA-induced T cell-dependent skin inflammation in mouse ears using the protocol of Schneider et al. [19]. In this model, the repeated challenge of ear skin with 2 % TMA for 12 days after one-shot sensitization with 5 % TMA on day 0 caused atopic dermatitis-like skin symptoms, such as increased skin thickness, change in skin morphology, and infiltration of immune cells [28]. It is widely accepted that eosinophils, mast cells, and CD4⁺ T cells mainly infiltrated the dermis and CD8⁺ T cells mainly infiltrated the epidermis [29]. And our results were also coincident with the previous findings. From histological findings that the majority of skin-infiltrating T cells in active atopic dermatitis lesions in humans are CD4⁺ T cells, CD4⁺ T cells are considered pivotal to the development of eczema and skin eosinophilic inflammation, because they produce a mixed pattern of Th1 and Th2 cytokines in the pathological development of atopic dermatitis [26]. CD8⁺ T cells are also dominant effector cells. They are responsible for allergen-induced skin inflammation, and thus their infiltration is required for the development of atopic dermatitis-like lesions and the initiation of mixed Th1/Th2 skin inflammation in rodent models.

Local lymph node weight is also increased by topical application of allergens including TMA or haptens such as dinitrofluorobenzene, 2,4-dinitrochlorobenzene, and trinitrochlorobenzene [30]. Hence, local lymph node weight was recently validated as a main index of allergic and immune responses in murine models. In the present study, lymph node weight also significantly increased after repeated challenge of ear skin with TMA (Fig. 3b).

Repeated topical application of allergens or haptens causes significant increases in blood IgE and IL-4 levels in a rodent allergy model [23, 31]. Topical exposure of haptens including dinitrofluorobenzene, 2,4-dinitrochlorobenzene (DNCB), and trinitrochlorobenzene (TNCB) also developed severe contact hypersensitivity in the mice. However, unlike allergens including TMA, the significant increases in eosinophil and T-cell infiltration, Th2 cytokine production and IgE levels were not observed in the serum of these hapten-induced hypersensitivity animal models, although a minor increase was reported previously [32].

In the present study, TMA treatment directly triggered a T cell-mediated contact hypersensitivity reaction and also caused distinct changes in the innate and adaptive immune systems, primarily indicated by increases in the secretion of Th1 and inflammatory cytokines, and the production of Th2 cytokines and IgE, respectively.

Serological and tissue investigations also showed an increased serum IgE level and Th2 cytokine dominance in the serum, ear skin, and auricular lymph node, despite the simultaneous dominance of Th1 cytokines such as TNF-?, IL-1?, IL-2, IL-12, and IFN-? in ear or lymph node tissues. In ear and auricular lymph node tissues in our atopic animal model, Th1 cytokines were strongly secreted, although Th2 cytokines dominated. This might be due to a longer period of 2 % TMA challenge on both ears (3 days longer than in Schneider’s protocol) to elicit an intensified immune response.

As an alternative therapy, BV was injected to stimulate a specific skin point behind the knee. The stimulation point is located far away from the ears where allergic inflammation is induced by repeated exposure to TMA, and a specific component of BV can hardly be responsible for alleviation of skin symptoms. Thus, our results demonstrate that stimulation of the specific acupoint by BV significantly inhibited TMA-induced skin inflammation in the ears, probably due to modulation of the systemic immune system. Although the detailed mechanism has not yet been elucidated, the improvement in symptoms must be attributed to certain systemic changes being able to affect every part of the body as well as being triggered by local BV stimulation.

On the other side, as reported previously, BV contains several biologically active peptides, including melittin, apamin, and adolapin [33, 34]. Many studies indicate that BVA can modulate a systemic immune response by inhibiting inflammatory mediators, similar to non-steroidal anti-inflammatory drugs (NSAIDs) [35]. Other studies reported that BV has in vitro anti-inflammatory activity which is ascribed to the transcriptional downregulation of NF-?B and MAPKs in target tissues [36]. One can speculate that Th1 cytokine-based inflammatory symptoms in atopic dermatitis were alleviated by BV components and that this pharmacological anti-inflammatory effect was exerted via modulation of NF-?B and MAPK signal transduction pathways.

Since atopic dermatitis is primarily characterized by skin symptoms such as erythema, edema, excoriation, and scaling, we first investigated the therapeutic effects of BVA on ear edema and thickness in our animal model [37]. At the maximum severity of AD-like symptoms, we started BVA treatment at a dose of 0.3 mg/kg at the BL40 acupoints on both legs and found significant reductions in ear edema and thickness 2 days after starting BVA treatment. We thereafter confirmed that BVA treatment was very effective at inhibiting activation of resident mast cells and infiltration of immune cells by histological analysis. Because mast cells play a key role in mediating allergic inflammation in asthma, atopic dermatitis, and sinusitis, we also focused on changes in mast cells in the dermis of inflamed skin. BV treatment significantly decreased mast cell infiltration in ear tissues [38]. Thus, it is likely that BV regulates the infiltration of mast cells and inhibits immune cells, thereby normalizing the inflammatory response. Aggregation of Fc?RI triggers AD. After induction of AD, mast cells are activated by crosslinking of adjacent IgE molecules [39]. Notably, IgE is responsible for both acute and chronic symptoms in atopy-like skin diseases, and repeated injection of TMA increases serum IgE levels in the mice [40]. We found that total serum IgE levels in mice were significantly increased by repeated TMA treatment. BV treatment significantly reduced total serum IgE levels in TMA-treated mice.

Many studies suggest that an imbalance between Th1 and Th2 responses is closely associated with many immune disorders, including atopic dermatitis [41]. In typical allergic diseases, there are increases in the levels of Th2 cytokines and decreases in the levels of Th1 cytokines [42]. Many studies also support the assertion that restoration of the balance between Th1 and Th2 cell numbers, which can be achieved by either stimulation of Th1 cell development or inhibition of Th2 cell development, has remedial value in AD treatment [43]. However, interestingly, in our mouse model of atopy-like skin dermatitis, in which the expression levels of both Th1 and Th2 cytokines simultaneously increased, the alleviation of atopic skin symptoms by BV treatment was attributed to downregulation of both Th1 and Th2 cytokines. Th2-dominated cytokine profile plays a central role in the initiation of AD symptoms in the early stage, and in the chronic stage of human atopic dermatitis, cytokine profile shift from Th2 to Th1 is quite critical for aggravation of AD leading to severe dermal inflammation. Although our atopic model of TMA-induced hypersensitivity for 2 weeks seems to show such a mixed profile of both Th1 and Th2 dominance and thus be close to human chronic dermatitis of atopy, we do not have a confidence because we did not analyze time-course profiles of Th1- and Th-2 expression.

Local injection of BV exhibited simultaneous anti-inflammatory and anti-allergy effects for the treatment of atopic skin symptoms. This phenomenon might be caused by the double-sided character of BV, as mentioned earlier, although we did not clearly elucidate its mechanism in this study.

Unlike usual immunotherapies for immune diseases, venom therapy not only induces a rapid shift in cytokine expression from Th2 (IL-4) to Th1 (IFN-?) cytokines, but also leads to increased production of immunosuppressive cytokine IL-10 during the initial phase of the treatment [44]. Furthermore, it was reported that although low-dose BV treatment initially upregulated Th1 cytokine expression in experimental immunotherapy, aggravating inflammation, pain, and scratching behavior, it eventually relieved those symptoms [45].

In the present study, BV stimulation at a non-acupuncture point also exhibited a significant suppressive effect on TMA-induced edema formation, T-cell activation and the levels of inflammatory cytokine mRNAs and proteins. It means that BV itself showed certain levels of anti-inflammatory and anti-allergic activities, regardless of the use of specific acupuncture point.

Although we addressed that BVA significantly inhibited the TMA-induced expression of Th2- and Th1- cytokines in the present study, the detailed mechanisms of how BVA modulated the expression levels of Th1- and Th2-cytokines together should be elucidated in the future.