Establishment Of An Atopic Dermatitis-like Mouse Model With House Dust Mite Sensitization And Observation Of The Curative Effect Of Allergen-specific Immunotherapy

Research background and purposeAtopic dermatitis（AD）is a chronic,relapsing,inflammatory skin disease in which patients often have a personal and family history of other allergic diseases,including allergic rhinitis,conjunctivitis,and asthma.The main clinical manifestations of AD patients are dry skin,severe itching and chronic eczema-like skin lesions.The pathogenesis of AD is complex,including genetic environmental factors,skin barrier dysfunction,microbial flora imbalance,and abnormal immune responses.Allergens derived from house dust mite（HDM）are one of the important causes of AD pathogenesis.Clinically,most AD patients can detect high titers of HDM-specific IgE,and HDM can promote skin Th2-type immune response.Due to the widespread presence of HDM in the environment,it is difficult for patients to completely avoid exposure to HDM.Therefore,the establishment of an atopic dermatitis-like mouse model with house dust mite sensitization can largely simulate the real situation of clinical patients,which is of practical significance.Some researchers used extracts of Dermatophagoides farinae（DfE）to repeatedly stimulate NC/Nga mice to establish an AD mouse model,but the skin lesions of the AD model were limited to the head and neck,and they applied DfE to stimulate BALB/c mice without obvious skin lesions.Dinitrofluorobenzene（DNFB）,a common hapten,induces contact dermatitis in mice and disrupts the function of the skin barrier.Based on the skin barrier dysfunction in AD patients,which makes it easy for external protein antigens（like HDM）to get through the skin and participate in AD pathogenesis,we speculate that the skin dysfunction caused by DNFB can enhance the effect of DfE on BALB/c mice.Thus,the combination of DNFB and DfE can better establish an AD mouse model with house dust mite sensitization,which can not only induce Th2-type inflammatory response,but also produce typical chronic eczematous lesions.Allergen-specific immunotherapy（ASIT）,also known as desensitization therapy,is the only method that is thought to alter the natural history of allergic diseases.Its administration routes include subcutaneous,sublingual,and lymph node injections.Subcutaneous immunotherapy（SCIT）is a traditional and widely used specific immunotherapy administration method.Some studies have confirmed the safety and efficacy of ASIT in the treatment of AD.In recent years,it has been increasingly used in AD patients sensitized to dust mites,but the related mechanisms are still less studied.Antigen-presenting cells（APC）are an important link in the control of peripheral immunity and tolerance.Although some studies have analyzed some changes in mucosa-associated APC after sublingual immunotherapy for AD,there are few studies focusing on cutaneous APC after SCIT.In clinical practice,human specimens are not easy to obtain,and the course of specific immunotherapy for patients is rather long.These factors make it difficult to study the related mechanism of specific immunotherapy in AD patients,so people pay more attention to AD mice model of specific immunotherapy.In mouse skin,APC has a complex composition,including monocyte-derived dendritic cell,Macrophages,Langerhans cells,conventional dendritic cells and so on.In conclusion,in order to better observe the efficacy of AD-specific immunotherapy and explore the relevant mechanisms,we first compared the similarities and differences of the models established by the repeated stimulation of BALB/c mice with DNFB alone,DfE alone,and alternating use of both.Furthermore,we performed SCIT on AD mice,evaluated the efficacy of SCIT through clinical manifestations and related immune indicators,and preliminarily analyzed the changes in the number of skin APCs and immune phenotypes during this process.Research methods1.BALB/c mice were stimulated with DfE or DNFB,and the combination of the two for8 consecutive weeks.AD-related indicators were observed,including the frequency of scratching;the severity of skin lesions;the number of inflammatory cells（including eosinophils and basophils,etc.）in the skin lesions;the expression levels of serum specific IgE,total IgE,IL-4,IL-13,IL-2,and IFN-γ;the expression levels of IL-4,IL-13,IL-2 and IFN-γin skin lesions.The above indicators are used to evaluate whether the model is established successfully and confirm the rationality of the model.2.SCIT was performed on AD mice induced by DfE combined with DNFB.The treatment started after 3 weeks of DfE combined with DNFB stimulation,and for 5consecutive weeks.100μL of dust mite solution（containing 100μg of DfE）per animal was subcutaneously injected on the back,2 times per week,and the curative effect of the treatment was observed.In addition to the mentioned indicators above,we also used flow cytometry to sort CD11b⁺CD24^-CCR2⁺CD64⁺moDC,CD11b⁺CD24^-CCR2^-CD64^highMacro,CD11c⁺CD207⁺LC and CD11c⁺CD207^-cDC in the skin,and their maturity was evaluated by the expression levels of CD83 and MHC-Ⅱ.Activation was assessed by expression of CD86or pro-inflammatory cytokines IL-1 and IL-12.Research results1.After repeatedly stimulating BALB/c mice with DfE alone,no obvious eczema-like skin lesions appeared in the mice,but the DfE-sIgE and IL-13 in the serum were higher than those in the control group;after repeated stimulation with DNFB alone,mild eczema-like skin lesions appeared on the back of mice,and serum DNFB-sIgE,total IgE,IL-4,IL-13,IL-2 and IFN-γwere higher than those in the control group,but no increase in the level of inflammatory factors was detected in the skin lesions;after stimulation of BALB/c mice with DfE and DNFB,typical and stable eczema-like skin lesions appeared on the entire back of the mice,accompanied by high-frequency scratching behavior.In DfE and DNFB treated mice,the infiltration of inflammatory cells in the skin lesions was significantly increased,and the DfE-sIgE,DNFB-sIgE,total IgE,IL-4 and IL-13 in serum were higher than those in the control group,and IL-13 in the skin lesions was also higher than that in the control group,P<0.05.2.After 5 weeks of SCIT in AD mice with HDM sensitization,compared with untreated AD mice,the severity of skin lesions,the frequency of scratching,and the infiltrating inflammation of the skin lesions was reduced.What`s more,the DfE-specific IgE,total IgE,IL-4 and IL-13 in serum and IL-13 in skin lesions also decreased significantly,P<0.05.3.Mo DC,Macro,LC and cDC were successfully sorted by flow cytometry,and their number and expression levels of CD83,MHC-Ⅱ,CD86,IL-1 and IL-12 were further compared.Compared with the control group,the number of moDC in AD mice was not increased,but the expressions of CD83,MHC-Ⅱ,IL-1 and IL-12 were increased.The number of cDC and the expression of CD83 and CD86 were not changed,but the expression of MHC-Ⅱwas lower than that of the control group.The number of Macro and LC increased,but the expression of CD83 and MHC-Ⅱdid not change.After SCIT in AD mice,the number of moDC and LC was increased,the expression levels of CD83,MHC-Ⅱ,IL-1 and IL-12 in moDC were significantly decreased,and the expression levels of CD86 and MHC-Ⅱin LC were also decreased compared with untreated AD mice.The number of cDC also increased,while the expression levels of CD83,CD86 and MHC-Ⅱdid not change.The number of Macro and the expression of MHC-Ⅱwere decreased,while the expression of CD83 was increased,P<0.05.Conclusions1.Repeated stimulation of BALB/c mice with DfE alone can induce a Th2-type immune response,but it is not enough to induce obvious eczema-like skin lesions and the eczema-like skin lesions induced by repeated stimulation of DNFB tend to be more Th1-mediated.The combination of DfE and DNFB can not only induce stable eczema-like skin lesions,but also show the characteristics of the Th2-type immune response induced by house dust mites,which is closer to the clinical characteristics and inflammatory response pattern of human AD.This model can be applied to the study of AD pathogenesis and therapeutic drugs.2.SCIT performed on AD mice sensitized to dust mites achieved good curative effect.Compared with untreated AD mice,the severity of skin lesions,the frequency of scratching,and the infiltrating inflammation of the skin lesions was reduced.What`s more,the DfE-sIgE,total IgE,IL-4 and IL-13 in serum and IL-13 in skin lesions also decreased.3.In the skin lesions of AD mice,the function of moDC was significantly enhanced（the expression levels of CD83,MHC-Ⅱ,IL-1 and IL-12 were increased）,suggesting that moDC plays an important role in the pathogenesis of AD.After SCIT treatment,the number of moDC in skin lesions increased significantly,and their maturation and activation were inhibited.Such partially mature moDC were more inclined to induce immune tolerance,suggesting that they were deeply involved ASIT,which may become a new target for AD treatment.In addition,the number of LC and cDC in skin lesions of AD mice increased significantly after SCIT,but their maturity did not change significantly,suggesting that LC and cDC may be transferred to lymph nodes rather than skin to induce immune tolerance.