The Role And Mechanism Of IL-33/ST2 In Skin Fibrosis Of Systemic Sclerosis

Background Systemic sclerosis(SSc)is a chronic immune-mediated autoimmune disease that affects the skin and various internal organs(including the lungs,gastrointestinal tract,heart,and kidneys).It has the highest mortality rate among the connective tissue diseases.SSc is characterized by fibrotic changes of the skin and internal organs,which in turn leads to distortion of tissue structure and gradual loss of organ function.So far,there is still no treatment allows full recovery from this severe disorder.Therefore,it is of great social significance to study the pathogenesis of this disease and find new targets for treatment.At present,there is a certain understanding of the pathogenesis of SSc,previous studies have shown that the clinical and pathological manifestations of the disease may be the result of the three different processes:(1)innate and adaptive immune system abnormalities leading to production of autoantibody and cell-mediated autoimmunity,(2)microvascular endothelial cells and small vessel fibroproliferative vasculopathy,and(3)fibroblast dysfunction leading to excessive accumulation of collagen and other matrix components in skin,blood vessels and internal organs.These three processes interact and affect each other.The Th2 pathway is thought to play a very important role in the process of fibrosis,and interleukin 33(IL-33),which is a potent inducer of type 2 immune response,has also been confirmed to be involved in the development and progression of multiple fibrotic diseases.However,the role and mechanism of IL-33 in SSc-related fibrosis remains unclear.Object To determine the levels of IL-33 and its receptor Suppression of tumorigenicity 2(ST2)in peripheral blood and skin tissues of SSc patients,and to identify its association with relevant clinical parameters,so as to explore its clinical significance;to investigate the effects of IL-33 on the proliferation,migration,inflammatory factor secretion and collagen synthesis of human healthy skin fibroblasts in vitro,and to explore its direct role in fibroblast activation;to detect the expression of IL-33/ST2 in skin tissue of bleomycin(BLM)induced mouse skin fibrosis model,and to elucidate the similarities and differences of IL-33/ST2 expression patterns between human and mouse;to observe the difference of skin fibrosis severity in SSc model induced in WT mice and IL33-/-mice,therefore clarify the role of IL-33 in the skin fibrosis of SSc;to provides a new target for the treatment of fibrosis in patients with SSc.Methods 1.Plasma and skin samples from patients with SSc and healthy controls were collected.The levels of IL33 and s ST2 in plasma were measured by ELISA.Spearman’s correlation was calculated between the above results and patients’ clinical characteristics(disease activity,age,duration,etc.).The IL-33 and ST2 levels were compared between different groups with positive or negative organ involvement and autoantibodies.The receivers’ operating curve was used to determine the optimal diagnostic threshold.Immunohistochemistry was used to detect the skin tissue expression and distribution of IL-33 and its receptor ST2,and the cellular localization of IL-33/ST2 was detected by immunofluorescence.2.Human healthy skin tissue was collected,primary fibroblasts were extracted and cultured in vitro,and were stimulated by IL-33 recombinant protein.The proliferation rate of fibroblasts was measured by MTT and Ed U method,respectively.The cell scratch test was used to observe the effect of IL-33 on fibroblasts migration.Real-time PCR and ELISA,CBA assay were used to measure the m RNA and protein levels of inflammation-related factors and collagen-related protein in fibroblasts.3.C57BL/6 mice were injected subcutaneously with BLM for 3w to induce a mouse SSc skin fibrosis model.The body weight changes were monitored in the process.HE staining and Masson staining were used to observe the pathological severity of the lesioned skin tissue.Hydroxyproline level was measured to reflect skin collagen content.The expression and distribution of IL-33/ST2 in local tissues of mouse lesions were detected by immunohistochemistry,and Pearson correlation index was calculated with the skin fibrosis markers.The BLM skin fibrosis model was also generated using IL33-/-mice,and the severity of skin fibrosis was evaluated to identify the effect of IL-33 deletion on mice.Flow cytometry was used to detect peripheral spleen and lymph node T/B cells,and Th/Treg cell ratio.Toluidine blue staining was used to detect changes in mast cell numbers.Results 1.The plasma s ST2 levels were significantly elevated in patients with SSc than normal controls,and were not significantly associated with disease activity(m RSS score),age,and duration of disease.There was no significant relationship between antibody positivity and organ involvement,but plasma s ST2 may be a good serological parameter for the diagnosis of SSc.At the threshold of 3.508 ng/ml,the sensitivity and specificity are 77.42% and 93.94%,respectively.Immunohistochemistry and immunofluorescence results showed that the expression of IL-33 and its receptor ST2 were up-regulated in skin lesions of SSc patients.IL-33 was mainly expressed in the nucleus of keratinocytes,endothelial cells and infiltrating inflammatory cells,and IL-33 located in the nucleus of keratinocytes is clearly translocated to the cytosol,while ST2 was mainly expressed on the membrane of keratinocytes,endothelial cells,inflammatory cells,and fibroblasts.2.Human skin fibroblasts express both IL-33 and its receptor ST2.Compared with PBS,IL-33 can not promote the expression and release of inflammatory factors(IL-6,MCP-1),but does significantly promote the proliferation and migration of fibroblasts,induce the production of collagen in fibroblast.3.BLM can significantly induce skin fibrosis in mice,resulting in increased skin thickness and collagen deposition,and significantly reduce the overall body weight of mice.The expression of IL-33 was increased in the fibrotic skin of mice,and the ST2+ cell number per high power field was increased,which were both positively correlated with the degree of skin fibrosis(skin thickness,hydroxyproline content).The level of IL-33 in the skin tissue homogenate(interstitial fluid)was increased.4.After IL33 deficiency,the severity of skin fibrosis induced by BLM was significantly reduced,and the number of ST2+ cells in the skin was significantly reduced,but the number of mast cells did not change significantly.The deletion of IL33 partially affected the differentiation and proliferation of Th2 cells in the spleen of IL33-/-mice,and had no significant effect on the differentiation and proliferation of other T/B population and Th/Treg cells.Conclusion Plasma s ST2 levels can be a very useful laboratory indicator to assist in the diagnosis of patients with SSc.IL33 promotes skin fibrosis by directly activating fibroblasts,and IL33/ST2 may be an important target for the treatment of fibrosis in patients with SSc.