Elevated Level Of IL-33 Enhanced Airway Remodeling Of Asthma By Up-regulating Profibrotic Function Of Fibrocytes

BackgroundThe main pathological characteristics of bronchial asthma are airway inflammation,dysfunction of airway smooth muscle and airway remodeling.The characteristics of airway remodeling is the pathological basis of airflow obstruction and lung function damage.The pathology of airway remodeling includes the thicking of reticular basement membrane,epithelial shedding,sub-epithelial frosis,goblet cell hyperplasia,the changes of epithelium function and structure,remodeling of micro-vessels and proliferation and hypertrophy of smooth muscle.It is reported that the airway epithelium is much more susceptible to different stress factors and repairs abnormally after damage in asthma.The epithelial cell is regarded as one of the most active secretory tissue cells when the tissue is injured.When the mature epithelial cells try to repair the tissue injury,they secret many kinds of proinflammatory factors such as interleukin 33(IL-33),interleukin 25(IL-25),thymic stromal lymphopoietin(TSLP),and so on to stimulate the remodeling process of matrix.And IL-3 3 has been recognized as a critical cytokine involed in asthma after the airway epithelium injury.IL-3 3 is widely expressed in many tissues,but its expression level is different in different kinds of cells.In contrast,the expression of IL-33 is higher in respiratory tract,digestive tract and other mucosal systems which are connected with the outside world.Some studies have found that IL-33 has dual functions:on the one hand,IL-33 is located in the nucleus and plays a role in regulating transcription;on the other hand,IL-33 as a cytokine can be secreted out of the cell and play a role in cytokine by binding and interacting with its specific receptor ST2.IL-33 can be produced by many structure cells and inflammatory cells after injury.IL-33 could induce the adoptive Th2 immunity and signaling pathway through a complex that includes ST2 protein and IL-IRs regulatory protein,and ST2 is a member of IL-1 receptor(IL-1R)family.It has been reported that IL-33/ST2 signaling pathway was very important in allergen-induced chronic inflammation of airway and hyperresponsiveness.Increasing evidence has demonstrated that IL-33 was responsible for the initiation of asthmatic airway remodeling and IL-33 was critical to enhance expression of IL-13 and IL-5 by fibrocytes in chronic inflammation.It has been reported that IL-33/ST2 signaling pathway was very important in allergen-induced chronic inflammation via the mast cells,airway epithelial cells,smooth muscle cells and Th2 cells,thus it plays an important role in the development of asthma.Previous studies demonstrated that IL-33 could enhance the synthesis of IL-5 and IL-13 from innate immune cells without antigen stimulation that might contribute to allergic pulmonary inflammation.Because IL-13 can stimulate the activation and proliferation of fibroblasts,increase the contractility of smooth muscle cells,and promote the deposition of extracellular matrix,thus IL-33 also plays an important role in airway remodeling.Our recent study showed that IL-33/ST2 signaling pathway was vital in promoting asthmatic airway remodeling,and it has specifically clinical therapeutic efficacy in asthmatic airway remodeling to inhibit IL-33/ST2 signaling pathway.Meanwhile studies have demonstrated that activeted IL-33/ST2 could promote the production of Th2 cytokines through NF-?B and MAPK signal pathway,thus plays an important role in the development of many diseases such as allergic shock,atopic dermatitis,rheumatoid arthritis,autoimmune disease and so on.Fibrocytes are unique bone marrow-derived mesenchymal progenitor cells that are characterized by expression of hematopoietic cell markers(CD34,CD45,and leukocyte specific protein 1)and stromal cell markers(collagen I and proly-4-hydroxylase).The fibrocytes were originally identfied in cultures of peripheral blood mononuclear cells(PBMCs)and in the wounds as CD45+CD45RO+CD34+CD11b+CD 13+HLA-DR+cells that constitutively produce fibronectin and collagens and express the myofibroblast marker a-smooth muscle actin upon stimulation with TGF-?1.There is increasing evidence that these cells are circulating mesenchymal progenitors,serving as a renewable source of fibroblasts and myfibroblasts in the repair of extensive tissue injuryand in fibrotic disorders associated with chronic inflammation.Fibrocytes had multiple functions and were involved in chronic inflammation and would healing.It has been postulated that fibrocytes could transit into fibroblasts and myofibroblasts after lung injury,thus this process contributed to allergen-induced asthmatic airway remodeling.Fibrocytes were originally distinguished by expressing both CD34 and collagen I or pro-collagen I in most settings.Lineage tracing studies show only minimal contribution of fibrocytes to a-SMA production in several models it is likely that fibrocytes possess other properties that promote tissue remodeling.Notably,it has been found that fibrocytes from asthmatic patients respond to IL17A by secreting proinflammatory mediators,and respond to TH2 cytokines by producing collagen.Thus fibrocytes play an important role in the development of asthma.Fibrocytes were generated in vitro from peripheral blood mononuclear cells by culturing in the presence of platelet derived growth factors and the cells were stimulated with IL-33.IL-33 enhanced cell proliferation,a-SMA expression,and pro-MMP-9 activity by the fibrocytes without increasing endogenous transforming growth factor-?1 production.Fibrocytes constitutively expressed IL-13 and IL-5,and their production was augmented by stimulation with IL-33.Dexamethasone inhibited the functions of fibrocytes,but IL-33 made fibrocytes slightly refractory to the inhibitory effect of dexamethasone in terms of IL-13 production.Others found that IL-33 promotes the migration and proliferation of circulating fibrocytes from patients with allergen-exacerbated asthma.However,the interaction and involvement of IL-33-activated fibrocytes in asthmatic airway remodeling was still not clear.Substantial evidences have demonstrated that airway epithelial cells and fibrocytes both participate in airway remodeling in asthma.and IL-33 plays an important role in the inflammatory response of asthma.However,the crosstalk between the airway epithelial cells and fibrocytes is still not clear,and its mechanism in the process of IL-33 induced airway remodeling through fibrocytes are not clear yet and needed to be proved.Objective1.To investigate the role of IL-33 in airway remodeling in asthma and characerize the crosstalk between airway epithelial cells and fibrocytes regulated by IL-33 through the signaling pathway of IL-33/ST2-MAPK.2.To evaluate the effect of blocking the IL-33/ST2-MAPK signal pathway on airway remodling,and to determine whether targeted IL-33/ST2-MAPK signal pathway treatment can reduce the productions of airway remodeling through fibrocytes isolation an culture in vitro test.3.To evaluate the effect of blocking ST2 and p38 MAPK on airway remodling,and to determine whether targeted ST2 and p38 MAPK through fibrocytes treatment can relieve airway remodeling in a mouse asthma model.Methods1.We observed airway remodeling in asthma patients'airway epithelium stained by hematoxylin and eosin(H&E).Human biopsy specimens were stained with immunohistochemistry(IHC)to observe the expression of IL-33,Fibronectin-1(FN1),collagen I and alpha-smooth muscle actin(a-SMA).2.We detected the expression of IL-33 in the serum of patients with asthma by enzyme-linked immuno sorbent assay(ELISA).And Flow cytometry(FCM)was used to detect the expression of fibrocytes in peripheral blood mononuclear cell(PBMC).And the correlations were analyzed by SPSS.3.Fibrocytes generated in vitro from PBMCs were stimulated in presence or absence of IL-33.The protein expression of collagen I,FN1 and a-SMA were detected by Western blotting and immunofluorescenc.The expression of fibrocytes stimulated by IL-33 was detected by FCM.4.Fibrocytes generated in vitro from PBMCs were stimulated in presence or absence of anti-ST2 Ab and the specific p38 MAPK inhibitor(SB203580).The protein expression of collagen I,FN1 and a-SMA were detected by Western blotting and immunofluorescenc.Then,the effects of IL-33/ST2-p38MAPK axis on fibrocytes function that related with airway remodeling were analyzed by FCM.5.Airway epithelium were stained with immunohistochemistry(IHC)to observe the expression of collagen I,FN1 and a-SMA in asthma mice model.Anti-ST2 Ab and the specific p38 MAPK inhibitor(SB203580)was injected intraperitoneally to asthma mice model to investigated that whether blocking the IL-33/ST2-p38MAPK axis could alleviate asthma airway remodeling by IHC,Western blot and qPCR,and FCM to detect the expression of firbocytes by blocking the IL-33/ST2-p38MAPK axis.Results1.The pathology of airway remodeling such as basement membrane thickening,the epithelial cells infiltrate and the subepithelial deposition of collagen in the airway,etc was found in asthmatic patients through HE staining obviously.Compared with the control group,the expression of IL-33,collagen I,FN1 and a-SMA were obviously enhanced in epithelium of asthma patients.2.Compared with the control group,the expression of IL-33 and the proportion of fibrocytes in PBMC were obviously increased in asthma patients.And they are positively correlated.3.After the fibrocytes generated in vitro from PBMCs were stimulated by IL-3 3,the protein expression of collagen I,FN1 and a-SMA were obviously enhanced.The expression of fibrocytes stimulated by IL-33 detected by FCM was up-regulated.4.ST2 Ab and SB203580 could decrease the expression of collagen I,FN1 and a-SMA in fibrocytes in vitro.3.The expression of fibrocytes was also down-regulated.5.In vivo,HE showed airway structure change such as goblet cell hyperplasia,ASM cells hyperplasia,basement membrane thickening and so on in airway tissue of OVA-challenged mice.And airway structure change was attenuated by anti-ST2 Ab and SB203580 compared with OVA challenged mice.Compared with the control group,IHC showed that the expression of IL-33,collagen I,FN1 and a-SMA were obviously enhanced in epithelium of OVA challenged mice.The number of the fibrocytes in BALF of OVA challenged mice was upregulated.And the expression of IL-33,collagen I,FN1 and a-SMA were attenuated by anti-ST2 Ab and SB203580 compared with OVA challenged mice.And the number of the fibrocytes in BALF was also reduced.Conclusions1.The production of IL-33 was enhanced in airway epithelium of asthmatic patients and associated with fibrocytes%of PBMCs.It can binds to ST2 on the surface of fibrocytes,then activeted the p38MAPK axis.The expression of collagen I,FN 1 and?-SMA in fibrocytes were obviously enhanced,and the rusult was that the airway remodeling of asthma enhance.2.The expression of collagen I,FN 1 and a-SMA were repressed in fibrocytes pre-treated with Inhibition of IL-33/ST2-p38MAPK axis in vitro and vivo,and could improve the clinical manifestations of airway remodeling in animal model of asthma.3.IL-33 could enhance the airway remodeling of asthma by regulating profibrotic function of fibrocytes through ST2-p38MAPK axis.Blockade of IL-33/ST2-p38MAPK axis could alleviate airway remodeling which might provide a new therapeutic target for asthma.