The Role And Mechanism Of C-type Lectin Receptor, DC-SIGN In The Pathogenesis Of Atopic Dermatitis

Atopic dermatitis (AD) is a chronic, relapsing, inflammatory disease, characterized by the presence of eczematous lesions and pruritus. AD affects nearly15-30%of children and2-10%of the adult population worldwide. Innate and adaptive immunity have definitive roles in the development, maintenance and flare-up of AD. Skin barrier dysfunctions of AD make the entry of antigens and pathogens into the skin possible. Once they come into skin, they would interact with DCs, and then create a milieu that shapes the immune response to allergy reaction Dendritic cells (DCs) which are the most powerful antigen presenting cells(APCs) mainly distributed in sub-epidermal and mucosal, where in those places, DCs are easy to attach allergens, then uptake, processing and presenting antigens in the draining lymph node, where they become able to stimulate naive T cells. Recently, the pattern-recognition receptor(PRRs) on APC surface were found one after another including C-type lectin receptors (CLRs), Toll-like receptors, or nucleotide-binding oligomerization domain proteins(NODs), and CLRs is one of an important PRRs among them that suggest to functionally interact with allergens and are critical in controlling immune response.DC-SIGN is a type Ⅱ membrane receptor that was expressed on immature DCs in peripheral tissue and mature DCs in lymphoid tissues, initially cloned and characterized from human placenta as a receptor for the HIV envelope glycoprotein gp120. However, it was not until much later that DC-SIGN was rediscovered as a cell-adhesion receptor as well as antigen uptake and signaling, that supports primary immune responses and enhanced HIV infection of CD4+T cell.The primary function of DC-SIGN is the recognition of glycans through its carbohydrate recognition domain (CRD). The CRD of DC-SIGN is a C-type lectin-like domain with an EPN motif that binds fucose and mannose and has two Ca2+coordination sites. Thus, DC-SIGN recognizes fucosylated glycans, such as the blood-type Lewis antigens and high-mannose structures. This relatively broad glycan specificity confers on the receptor the ability to recognize a wide variety of ligands, pathogens, self-glycoproteins, Fc portion of immunoglobulins and allergens. Internalization of DC-SIGN into the classical endolysosomal pathway is initially described to mediate efficient antigen processing and presentation into MHC class Ⅱ. DC-SIGN cross-presentation of allergens is enhanced by the simultaneous triggering of Toll-like receptors (TLRs). DC-SIGN is not only an antigen-uptake receptor, but also facilitates the intercellular adhesion of DCs with T cells. DC-SIGN also behaves as a signaling receptor.Recent studies displayed different pathogens interactions between DC-SIGN would be delivered to promote secretion of different cytokines. Such as Helicobacter pylori interaction with DC-SIGN promoted secretion of Th2-type pro-cytokines. While, Neisseria meningitidis variants delivered by DC-SIGN biased toward Thl-type immune response. Interestingly, Bacillus acidi lactic interacted with DC-SIGN promotes Treg polarization. The appearance of DC-SIGN+cells in the decidua was a pregnancy-associated event involved in maternal immune system accepted the allogeneic embryo.Huang reported oral delivery of Man51-BSA targed SIGNR-1reduced the BSA-induced anaphylactic response.Common allergens and pathogens that associated with the onset of AD were rich in polysaccharides, and the cellular basis of allergens uptake and processing by DCs through DC-SIGN and mechanisms of inducing naive T cells polarization were largely unknown. In this study, we had carried intensive investigations on the following aspects:①Clarified the expressed level of DC-SIGN within the lesions of AD patients;②ested the hypothesis that glycan-containing allergens and pathogens were natural ligands for DC-SIGN;③Determined the role of DC-SIGN to induce innate and adaptive immune responses.These studies promoted us to investigate the efficiency and specificity of the allergen-DC-SIGN interaction might be important in determining the immune outcomes of AD. Firstly, we analysed the DC-SIGN expression levels within lesions from AD patients and same samples from healthy individuals and psoriasis patients were used as controls at the same time. Secondly, because of the restricted expression of DC-SIGN on DCs, we speculated that DC-SIGN participate in antigens recognition, uptake, processing and presenting. We conducted a series of researches on the binding ability of DC-SIGN with allergens and pathogens. ELISA-based binding assay were performed to determine the binding activity of purified allergens and pathogens with soluble DC-SIGN. DC-SIGN transfected HEK-293T cells and human monocyte-derived DCs (MDDCs) were investigated as models for testing the cellular binding activity and functional effects of allergen-DC-SIGN interaction by using confocal microscopy and flow cytometry. Thirdly, we assessed the contribution of DC-SIGN and TLRs to DCs activation and maturation after Der p2stimulation, including the activation molecular markers (CD86/CD80and CD83) and proimflamation cytokines(TNF-α、IL-1β、IL-6and IL-12). Finally, in order to make clear the polarization of T cells, we co-cultured Der p2allergens stimulated DCs with naive T cells, and tested some cytokines (IL-4/IFN-y/IL-22/IL-17A) related with T cell types.Our studies found that①The expression of DC-SIGN within AD lesions was higher than that of healthy controls and psoriasis;②The best concentration for binding of allergens with Soluble DC-SIGN by Titration test was1ug/ml;③DC-SIGN mediated the internalization of Der p2and Gal d2from house dust mite and egg white through their carbohydrate moieties, DC-SIGN expressed on HEK293T cells and human MDDCs was the major receptor for Der p2and Gal d2recognition and endocytosis. These effects were dose-dependent and could be inhibited for the most part by DC-SIGN blocking antibody or mannan pretreatment;⑤CD86/CD80/CD83were predominantly expressed on MDDCs stimulated with crude extracts of house dust mite and egg white from AD patients for24h and the expression was partly inhibited by DC-SIGN blocking antibody and/or MyD88blocking antibody. The supernatant of proinflammatory cytokines in cultured DCs activated by house dust mite were assayed. Significantly increased TNF-a and IL-6productions in the supernatant were noted, and pretreated by DC-SIGN blocking antibody and/or MyD88blocking antibody could partly inhibit their productions.⑤When actived mature DCs were co-cultured with naive T cells, the IL-4and IL-22expression levels were increased significantly, and these effects could be inhibited by DC-SIGN blocking antibody also. Early events at the interface of DCs and allergens were clearly of great importance in determining T cell polarization. Our work demonstrated a major role for DC-SIGN in glycoallergen recognition and in the regulation of Th2/Th22responses.This study may provided evidences for the pathogenesis of atopic dermatitis and possible intervention for AD treatment.