| 【Background】Allergic asthma is a common chronic respiratory disease characterized byreversible airflow limitation and bronchial inflammation. The development of thisdisease is composed by three phases, the phase of sensitization, resolution andchallenge, with the first symptoms occurring during childhood in most cases. Asthmahas a strong genetic component. Identification of the elements impacting the phases inasthma development will be helpful to understanding the underlying mechanisms ofallergic asthma and seeking treatments for this disease. Actually, more and morereports indicated the changes of some genes would increase the risk of asthma. Thesegenes are generally involved in modulating the sensitivity of airway epithelium toenvironmental stimulation or the sensitivity of immune system to allergen.Bone Morphogenetic Proteins (BMPs) are pleiotropic secreted cytokines,structurally related to transforming growth factor and activins. Except for BMP1, othermembers of BMPs are structurally similar to TGF-β. The BMPs are synthesize asproprotein and cleaved as activated mature BMPs. Thereafter mature BMPs aresecreted and regulate gene transcription by binding with the two kinds ofserine-threonine kinase receptor (BMPR-I and BMPR-II) in the form of homo-orhetero-dimer. BMPs play an important role in tissue regeneration and repair byaffecting cell proliferation, differentiation, migration and apoptosis. BMP4is one ofthe BMPs isoform expressed in endothelium, smooth muscle cells and epithelium andplays pivotal roles in embryo development. In recent years, more and more reportssupposed BMP4participated in modulating inflammatory responses. In asthma patients, the number of airway epithelial cells expressing BMP7was significantly increasedafter allergen challenge. That means BMPs mediated signaling is involved inregulating asthma associated lung inflammation. However, another group found that inan allergic asthma mouse model induced by ovalbumin as allergen, although the BMPmediated signaling in airway epithelium was activated with expression increase ofBMPR-I, BMP2and BMP6, the BMP4was reduced. But in a clinical investigation onasthma patients, the BMP4level in airway epithelium was similar between asthmapatients and normal population. Thus, the changes and functions of BMP4in allergicasthma are remained to be elucidated.Except for the secreted cytokines, the protein tethered on cell surface also has thepotential in regulating asthma. Mucin1（Muc1）is a transmembrane protein mainlyexpressed on apical surfaces of glandular epithelial cells and on the sueface of someimmune cells including T cells. Muc1consists of a large extracellular (EC) region, asingle-pass transmembrane region and an intracellular cytoplasmic tail (CT). The CTdomain contains evolutionally conserved tyrosine, the binding sites of GSK3β orβ-catenin. The phosphorylation of tyrosine or the binding between Muc1and theligands will lead to the downstream signaling change. In the past few years, more andmore evidence showed that Muc1had functions in regulating lung inflammation. In anacute lung inflammation mouse model induced by LPS inhalation, Muc1preventedextensive inflammatory responses, because the mice with Muc1elimination developedmore severe inflammation. However, it is still not known whether Muc1affect asthmadevelopment.Animal model is a useful tool in asthma research. The most widely used asthmaanimal model is established in mice. Although all characteristics of human asthmacould not be mimicked in the asthma animal model, some basic asthma-like symptoms,including bronchial inflammation, bronchial hyperresponsiveness, mucushypersecretion and airway remodeling, could be found in mouse asthma model. Thesuccess asthma model should process two principal symptoms, bronchial inflammation and bronchial hyperresponsiveness.【Objectives】This project focused on investigating the changes of BMP4and Muc1in asthmadevelopment and tried to illuminate the effects and underlying mechanism of BMP4and Muc1in asthma by establishing asthma model in BMP4heterozygous mutant miceand Muc1knockout mice. This research will contribute to developing noveltherapeutic targets of this disease.【Methods】1. Genotype determination:①. cut mouse tail tip tissue andput in lysis bufferovernight at55℃.②. Purify thegenomic DNA from the mouse tail tissue lysate.③. Run PCR with the genomic DNA as templates.④. The PCR products wereseparated by agarose gel electrophoresis and visualized under UV after PI staining.2. Establish asthma mouse model with various ovalbumin dosage:①, Theestablishment of asthma mouse model includes three phases, sensitization byintraperitoneally injection of mixture of ovalbumin and alum as adjuvant, the time forresolution (1to2weeks) and finally4-day constitutively intranasal allergen inhalationas challenge. The time for resolution is alternative depending on the geneticbackground of model mice.②, Lung function measurement and specimen collection.Twenty-four hours after the last challenge, the mice were subjected to lung functionmeasurement by useing a whole body plethysmography. Then the mice were dissecredto collect the bronchoalveolar lavage fluid, sera and lung tissues.3. Evaluate the lung inflammation:①, Counts the total and differential cellnumber in bronchoalveolar lavage fluid.②,Measure the mRNA level of the followinginflammatory elements in the whole lung: IL4, IL5, IL17A, IL1β, MCPT-8andCCL-11.③. The lung inflammatory symptom was observed and evaluated in lungparaffin sections received H&E staining. The goblet cell hyperplasia was observed in lung sections by using PAS staining.4. Monitor the changes of BMP4and Muc1:①, The protein level of secretedBMP4in bronchoalveolar lavage fluid and the total level of BMP4in whole lung weremeasured by Elisa and western blotting, respectively.②. The mRNA level of BMP4and Muc1in whole lung was measured by real-time RT-PCR.【Results】1. The change and function of BMP4during asthma development.1.1The BMP4expression level in lung tissue remained untouched. But the levelof secreted BMP4in bronchoalveolar lavage fluid was increased and coincident to thedose of allergen used in challenge.1.2The inflammatory phenotypes between BMP4+/+and BMP4+/-mice weresimilar when the asthmatic reactions were provoked by low dose allergen in thechallenge phase. In both BMP4+/+and BMP4+/-mice, the total cells in BAL fluidwere increased in which macrophages and eosinophils were predominant with minoramounts of neutrophils. The pulmonary inflammatory cell infiltration was observed.The levels of Th2and Th17cytokines in lung were upregulated. But the level of Th1cytokine was almost unchanged. All of the parameters mentioned above showed noobvious difference between BMP4+/+and BMP4+/-mice.1.3High dose allergen alone induced neutrophils increase in BAL fluid. In theasthma model caused by high dose allergen challenge, more severe increases on totalcells, macrophages, eosinophils and neutrophils in BAL fluid were found in BMP4+/+but not BMP4+/-mice. Higher level of Th1cytokine IL1β and less Th2cytokine IL4in lung was found in BMP4+/+mice than in BMP4+/-mice. The Th17cytokine wasupregulated in both kind of mice but no difference found between. Compared withasthma provoked with low dose allergen, high dose allergen caused more obviousneutrophils increase in BAL fluid. The difference on neutrophil number in BAL fluidwas more obvious between the two kinds of mice than under low dose allergen challenge. Concerning the cytokines in BAL fluid cells, cells from BMP4+/+miceexpressed more IL1β and less IL4, IL17A and CCL11than BMP4+/-mice.1.4The level of lung MCPT-8, the marker of basophils, was not changed inasthma model mice.2. The change and function of BMP4during asthma development.2.1The Muc1level in lung was downregulated in asthma.2.2The level of Muc5ac was increased during asthma, which was not impacted byMuc1elimination.2.3The level of Th1cytokine IL1β was reduced in mice received intraperitonealsensitization. But intraperitoneal sensitization alone did not induce lung inflammation.2.4Intranasal challenge alone did not cause lung inflammation.2.5In asthma model mice, Muc1+/+mice displayed more severe symptoms thanMuc1+/-mice manifested by more cells increase in BAL fluid, most of which waseosinophils, more serious lung inflammation, goblet cell hyperplasia and mucushypersecretion, and higher levels of cytokines IL5and IL17, chemokine CCL11. Butthe level of IL1β was lower in Muc1+/+mice than in Muc1+/-mice.【Conclusion】1, The secretion of BMP4in airway was increased in asthma development. Thelevel of airway BMP4was associated with the dose of allergen used in challenge.2. In asthma provoked by low dose allergen, no obvious difference on the severityof inflammation was observed between BMP4+/+and BMP4+/-mice. Unlike the lowdose allergen, high dose allergen challenge induced more cell increase, especiallyobvious neutrophils increase, in BAL fluid from BMP4+/+mice. These results hintedthat BMP4impacts the allergic lung inflammation related to asthma and probablyincline to control the neutrophils increase which served as a hallmark of severeasthma. 1.3Lung Muc1level was decreased in asthma. And mice without Muc1expression developed less severe symptoms. Thus Muc1has the functions triggeringasthma occurrence. The lung Muc1decrease might due to the loss of epithelial cellsin lung inflammation.1.4Since Muc1is also expressed on the T cells surface and participates inmodulating the sensitivity of activate T cells to allergen, and challenge alone did notcause any visible inflammatory symptom, then Muc1might impact the lunginflammation by regulating the intraperitoneal sensitization. |
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