FIGUREure 8. Yinxieling(YXL) inhibits the Langerhans cell(LC) differentiation in vitro . A: Gating strategy, flow cytometry scatter plot and frequency of cytokine induced LC; B: YXL inhibits the Langerhans cell(LC) differentiation in vitro. n = 6, repreated at least twice with similar results. Comparing to induced LC group, * p <0.05.
DISCUSSION
Psoriasis is not just a disFIGUREuring and refractory skin disease, but also a systemic disease associated with metabolic syndrome and cardiovascular diseases [21]. In recent years, the incidence of psoriasis has gradually increased, which has brought great pressure to patients’ physical and mental health and economy[22]. In 2013, the annual meeting of the World Psoriasis Federation reached agreement on the new treatment strategy of psoriasis [23]. The main treatment strategies are to control the development, reduce recurrence, prevent complications and improve the quality of life, which is in line with the characteristics of traditional Chinese medicine in the treatment of diseases.
Prescription Yinxieling has been used in clinic for more than 20 years with good curative effect and no obvious adverse reactions. Based on this, this study used IMQ-induced psoriasis-like mouse model to investigate the pharmacological effects of YXL[24]. Light red spots appeared on the second day of modeling. With the continuous effect of modeling drugs, the red patches became darker, the scales increased and the skin thickened, and reached the peak on the fifth day, covering almost all the bare skin. It can be seen from the PASI score curve that both CsA and YXL can effectively improve the clinical symptoms of psoriasis in mice, and the PSAI score is almost lower than that of IMQ group, especially YXL-M. The experiment confirmed that although the peak time of psoriasis-like symptoms in YXL group was basically the same as that in IMQ group, both erythema and scales were relieved in the later stage, indicating that YXL did not delay the inflammatory eruption time of psoriasis-like skin lesions induced by IMQ, but alleviated psoriasis by inhibiting the intensity of inflammatory eruption in this model skin lesions.
DCs stimulate naive T cells through their antigen presentation function to initiate acquired immunity and trigger inflammation. The plasmacytoid dendritic cell B220+DCs, when stimulated by bacterial CpG or viral infection, is a potent producer of interferon-α (IFN-α ) and interferon-β (IFN-β ) [25]. Classical dendritic cells(cDC), otherwise known as myeloid dendritic cells, are the most abundant dendritic cell type in the dermis of healthy mice and are classified by expression of CD11b. Part of the dermal CD11b+cDCs in mice express Aldh1a2, the sole rate-limiting factor for retinoic acid production. Retinoic acid is a vitamin A metabolite that promotes the production of Treg cells from primitive CD4+T cells. The other part expressed the C-type lectin Langerin (also known as CD207 and CLEC4K), including CD103+ and CD103- cells. They developmentally related to CD8α +cDCs in secondary lymphoid tissues. When cDCs are activated by proinflammatory cytokines or pathogens, CD103 cDCs mainly secretes IL-12 and CD11b cDCs mainly secretes IL-23. These two cytokines share IL23p40 subunit and are involved in the development of psoriasis [26].
The experimental results showed that CsA had no effect on the total DCs ratio, but reduced the proportion of B220+ cells and CD207-CD11B+ cells, and promoted the proliferation of CD207-CD11B- cells. However, CsA had no obvious effect on CD103+CD207+ and CD103-CD207+ subsets. These results indicate that CsA may inhibit the secretion of IL-23 by reducing CD11b+ cells, while YXL has no effect on cDCs in lymph nodes. LC are stellate cells that protrude their dendrites through tight junctions toward the cuticle and thus can detect antigens of the epidermis without disrupting the permeability barrier. After birth, they acquire a typical DC phenotype of histocompatibility complex II (MHC-II) molecule and CD207.
LC is the main source of IL-23 in psoriasis, and the reduction or absence of LC may lead to the attenuation of psoriasis-like lesions[27]. IL-23 /Th17 axis is the main known pathogenesis of psoriasis. IL-23 is a cytokine essential for the expansion and survival of pathogenic Th17 cells. IL-23 is highly expressed in psoriatic skin and mouse models, including models that rely on the local application of TLR7 agonist IMQ [28]. It was also found that the expression of LC in IMQ group was much higher than that in normal group.
LC differentiation is closely related to the expression of the transcription factor PU.1, an ETS family member transcription factor encoded by the Sfpi1 gene, which is an important regulator of many aspects of early hematopoietic and bone marrow cell differentiation [29]. Recently, PU.1 has been shown to control the expression of Flt3 in a dose-dependent manner, promoting the differentiation of cDC and pDC, and synergistically promoting the expression of RUNX3 with TGF-β , which further promotes the differentiation of LC[30]. Therefore, MHC-II and CD207 were used to label LC. The results showed that the ratio and number of LC and PU.1 were highly expressed in psoriatic lesions, while YXL and CsA significantly down-regulated the number and ratio of LC, but only decreased the number of PU.1, indicating that PU.1 was directly proportional to LC. Therefore, YXL may inhibit the expression of LC by reducing PU.1.
LC, as a unique antigen presenting cell in the epidermis, captures exogenous antigens and then passes them to T cells in the skin draining lymph nodes, activating CD8+T cells and CD4+T cells, further inducing the production of proinflammatory factors and leading to inflammation. The antigen presentation function of LC is powerful, and its ability to transfer from epidermis to local lymph nodes is important for inducing skin immune response [31].
Psoriasis is just an immune-mediated skin disease, one of its pathological features is keratinocyte proliferation[28]. It has been observed that keratinocytes under stress rapidly up-regulate ligands of lymphocyte activating receptor natural killer group 2D (NKG2D), leading to migration of LC populations out of the epidermis and subsequent emergence of αβ +T cells in the epidermis. Enhanced expression of the murine NKG2D ligand Rae-1εwas observed in injured skin, while mice lacking NKG2D showed delayed skin wound healing, confirming the importance of keratinocyte and LCs interactions during skin regeneration [32]. The activated LC expresses CCR7 and then travels through dermal lymphatics along the CCL19 and CCL21 chemokine gradients into parcutaneous cortical draining lymph nodes[33]. LC migration is associated with the synergistic effect of inflammatory cytokines IL-1β , IL-18, and TNF, so mice treated with these cytokines or with blocking antibodies against Casp1-/-, IL-1B -/-, and Tnfr2-/- reduced LC migration. Therefore, abnormal proliferation of epidermal keratinocytes in psoriasis may promote LC migration[34].
The LC carrying the neoantigen ”descends” into the dermis and, together with the draining lymph, drains into the local lymph node, forming a nest that attracts antigen-specific lymphocytes. These lymphocytes are activated in the lymph nodes, proliferate, and then propagate systemically back primarily to the skin to destroy keratinocytes[35]. The experimental data showed that in IMQ group, the proportion of epidermal LC migrating to lymph nodes increased, and the epidermal LC with antigen-presenting function reduced. LC carrying antigen induced T cell differentiation and produced proinflammatory factors to aggravate psoriasis. After YXL administration, the LC migrating to lymph nodes decreased significantly, while the LC maintaining antigen presentation function in epidermis increased dramatically.
Therefore, YXL may inhibit the differentiation of LC by inhibiting the expression of TGFβ, blocking the migration and maturation of LC and its antigen presentation, thereby inhibiting the release of inflammatory factors IL-23 and alleviating the development of psoriasis.
CONCLUSIONS
Yinxieling significantly alleviates psoriasis-like skin inflammation induced by IMQ in mice. Although it has no obvious effect on DC in skin draining lymph nodes, it can significantly reduce the expression of LC in skin lesions, inhibit the migration and maturation of LC, and prevent its antigen presentation by TGFβ1/PU.1 signaling axis, so as to effectively alleviates the psoriasis-like skin inflammation on mice.