| Objective:Bronchial asthma is a chronic airway inflammation disease participated by airway inflammatory cells?such as eosinophils,neutrophils,macrophages,lymphocytes,etc?,structural cells?such as airway epithelial cells,dendritic cells,etc?and cells components.when allergen enters the airways,it was captured by antigen-presenting cells and presented to CD4+T cells.The most important condition for antigen presentation is the involvement of IL-4 at the site of presentation and certain co-receptors that determine the differentiation of T cells into Th2 cells.Th2 cells produce cytokines such as IL-4,IL-3,and IL-5.IL-4 and IL-3 induce plasma cells to produce IgE,which binds to mast cells and basophils through the high-affinity receptor Fc?RI.When the corresponding antigen binds to IgE,it generates an activation signal that activates cells?mainly mast cells?,thereby degranulating cells,releasing inflammatory mediators and leading to the release of various chemical media.Biochemical mediators,such as histamine and LTs,cause local tissue effects,including airway smooth muscle contraction,tissue edema,increased mucus production,neurostimulation symptoms,and itching and sneezing.IL-18 a pleiotropic proinflammatory cytokine in the IL-1 family plays a major role in innate as well as acquired immunity.The facts that IL-18 variants are significantly associated with asthma severity,and that IL-18 protein and IL-18R were strongly expressed in the lungs of fatal asthmatics implicate that IL-18 plays a role in asthma.On the other hand,a significant low IL-18 level was observed in sputum of asthmatics and in sputum supernatant of patients with severe refractory asthma,which seems to correlate with number of macrophages,inversely correlates with the percentage of neutrophils,but not correlate with eosinophil cationic protein?ECP?and eosinophils.In order to further understand potential role of IL-18 in asthma,we examined expression of IL-18,IL-18BP and IL-18R in blood basophils and sputum mast cells in the present study.It has been reported that Alternaria extract induced rapid release of IL-18 from cultured normal human bronchial epithelial cells and directly initiated Th2 differentiation of na?ve CD4+T cells via a unique NF-?B dependent pathway,and that IL-18 has been linked to induction of IL-13 production by mast cells and basophils,suggesting that allergens may cause allergic airway reactions through IL-18,and IL-18 can affect functions of mast cells and basophils.Since mast cells and basophils are key cell types in asthma,which can produce an array of different cytokines,they could be a major source of IL-18.We therefore examined potential contribution of mast cells and basophils to asthma via IL-18,IL-18BP and IL-18R associated mechanisms.The aim of the current study is to further investigate the role of IL-18 related mechanisms in the pathogenesis of asthma.Because the number of basophils is very small in human,we further study the mechanism of IL-18 and other cytokines on dendritic cells?DCs?.DCs are the initiators of asthma.They can drive the activation of CD4+naive T cells into Th2 cells and play a key role in maintaining immune response and immune tolerance.DCs monitor the environment in a variety of receptors,including Toll-like receptors?TLRs?.LPS is a TLR4 agonist and is also one of experimental and clinically effective adjuvants.TLR4 is distinct from other TLR receptors,and its downstream signal can be transmitted through two independent pathways.The first pathway depends on the MyD88 adaptor protein.The MyD88 signaling pathway plays a very important role in the release of pro-inflammatory factors,such as IL-6 and TNF-?,but studies have found that the up-regulation of co-stimulatory molecules does not require the MyD88 signaling pathway,thus finding the second pathway.This is a MyD88-independent signaling pathway that is dependent on the TRIF adaptor protein.Previous studies have shown that TRIF is critical for upregulation of co-stimulatory molecules after LPS activation.In this article,upregulation of co-stimulatory molecules is thought to be the result of the IFN-I receptor pathway.However,this article mainly focuses on the observation of macrophages.We do not know whether the same mechanism can produce the same results on DCs.In this experiment,we will investigate whether the Th2 responses triggered by dust mite allergens are completed through the MyD88 signaling pathway or through the TRIF pathway.Reveals that dendritic cells are involved in HDM-induced immune responses and provide a new basis,providing a new theoretical basis for the pathogenesis of asthma and other related allergic diseases.Methods:Part?:A total of 31 patients with asthma and 14 HC subjects were recruited in the study.Immediately after admission?acute exacerbation stage?,the blood from each patient with asthma was collected.Blood from HC subjects were collected in the outpatient clinic.From each individual,10 mL of peripheral blood was taken into an EDTA containing tube.The patients were then asked to make a deep cough,and induced sputum was discharged into a bacteria free cup.A minimum of 5 mL sputum was collected from each patient.Flow cytometry analysis of IL-18,IL-18BP and IL-18R in human basophils and mast cells.Flow cytometry analysis of expression of IL-18,IL-18BP and IL-18R in sputum mast cells from asthmatics.BALB/c mice?5-6 w,18-22 g?were sensitized on days 0,7,14 and 21 with an subcutaneous multi-point injection of 50?g OVA and 1.5 mg of Al?OH?3 suspended in normal saline to a total volume of 0.5 m L.On day 25,sensitized mice were challenged with intratracheal injection of 0.1 m L of 5%OVA solution with or without 10 ng/mL of IL-18 for 3 h,and collecting bronchoalveolar lavage fluid?BALF?.Blood from mice were collected by enucleation of eyeball.Kill mice by Cervical dislocation and digest lung tissue.Flow cytometry analysis of IL-18,IL-18BP and IL-18R in mouse basophils and mast cells.Flow cytometry analysis of expressions of IL-18BP and IL-18R in mast cells of mouse BALF and lung tissues Determination of levels of IL-18BP in mouse plasma.Level of IL-18BP in mouse plasma was detected by a sandwich ELISA.Part?:Serum from healthy controls and HDM+asthma patients were collected in this study,and CD14+monocytes from human peripheral blood were sortted by CD14+immunomagnetic beads to induce differentiation into DCs.DCs were incubated with serum from healthy controls and allergic patients respectively.LPS was used as a positive control.The cells were incubated for 24 hours.MDDC cells were collected and the secretion of IL-6 in the supernatant of MDDC cells was detected by ELISA.The expression of TLR4,TLR2,MyD88,MAPKs and NF-?B on MDDC cells were detected by Western blot.Results:Part?:1.It was observed that approximately 53 and 51%of basophils in HC and asthmatic blood expressed IL-18,and more than 85 and 81%basophils in HC and asthmatic blood expressed IL-18BP.However,only 19.8 and 8.6%basophils in HC and asthmatic blood expressed IL-18R,which counts for a 56.4%reduction of IL-18R+basophils in asthmatic blood compared with HC blood.2.CD34-Fc?RI?+CD117+mast cells consisted of only approximately 0.39%dispersed nucleated cells,and only 3.5,14.3and 2.4%of them expressed IL-18,IL-18BP and IL-18R,respectively in asthmatic sputum.3.Compared with non-sensitized mice,sensitized mice had markedly decreased proportion of IL-18BP+basophils?Fig.3a,3c?,but dramatically increased IL-18R+basophils in their blood.4.sensitization and IL-18 elicited mast cell numbers in BALF,and IL-18 enhanced IL-18BP+mast cells.5.IL-18 enhanced number of mast cells and percentage of IL-18R+mast cells in lung of OVA-sensitized mice.6.IL-18 injection increased IL-18BP levels in plasma of non-sensitized and OVA-sensitized mice by 48.0%and 16.1%.Part?:1.In the microenvironment of HDM+asthma patients serum,MDDC cells recognize HDME through TLR4 receptor.2.MyD88 in MDDC cells could be upregulated by the serum of HDM+asthma alone.Both high dose of HDME and asthma serum increased the expression of MyD88 in MDDC cells.3.Both healthy serum and asthmatic serum increased the phosphorylation of ERK and p38 in MDDC cells.U0126and SB203580 can inhibit this reaction.4.The serum of HDM+asthma patients has an effect on MDDC and promotes the translocation of NF-?B.Conclusions:Part?:1.Greater than 50%and 80%of human peripheral blood basophils express IL-18 and IL-18BP,suggesting that the contribution of basophils to human asthma pathogenesis of asthma might be IL-18-independent.2.Only 0.39%of mast cells in sputum were nucleated suggests that mast cells are unlikely to be the primary source of IL-18 in asthmatic sputum,which may help to explain the low level of IL-18 present in the sputum of asthmatics.3.Sensitization did not alter the number of mast cells,the proportion of IL-18BP+mast cells or the percentage of IL-18R+mast cells in the lung tissue of mice.4.Extrinsic IL-18 enhances mast cell as well as IL-18R+mast cell numbers in the BALF and lung tissue of OVA-sensitized mice,these findings implicate mast cells participate in the pathogenesis of asthma via an IL-18 related mechanism.Part?:1.Co-culture of Allergic asthma serum and MDDC cells can simulate the changes of DC cells in the asthma microenvironment as a whole.2.HDME activates TLR4 signaling pathway in MDDC cells and then induce inflammatory response through MyD88/ERK/p38/NF-?B signaling pathway. |
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