Exploration Of The Role And The Therapeutic Potential Of Androgen In Th17 Severe Asthma

Introduction:Bronchial asthma(asthma)is a complex,chronic,and heterogenous airway inflammatory disease affecting about 21%of the world population with the estimation of about 300 million adults are being affected by it.It shows a heterogenous character because it presents with different phenotypes(clinical presentations)and endotypes(pathobiological mechanisms).Asthma can also be characterized as an allergic and nonallergic asthma.In general,we see two asthmatic peaks in a person’s life.Childhood asthma peaks first,followed by adult-onset/old age asthma.CD4+Th2 cells are primarily activated during the first peak of the disease and also known as T2 asthma(T2 high asthma).IL-4,IL-5,and IL-13 are among a set of Th2 cytokines that are essential for the start of an allergic reaction and are primarily involved in the pathophysiology of asthma.Their ongoing production may have an impact on the physiology of allergic reactions in the lungs,and research has indicated that blocking or silencing the genes for IL-4 and/or IL-13 will only prevent the onset of allergic asthma.According to a study,expression of IL-4Ra is necessary to start the onset of allergic asthma in an animal model in response to the Th2 antigen,while IL-5 is crucial for the differentiation,maturation,and adherence of eosinophils.The second peak of asthma,non-T2,is still less well understood.Non-T2 asthma(T2 low),affects adults and older people more frequently,is a late-onset neutrophilic,pauci-granulocytic,or mixed heterogeneous type.IL-6,IL-17,and IL-23 are the main mediators of inflammation,and comorbid conditions like obesity and gastroesophageal reflux disease(GERD)are also linked to non-T2 asthma.Because non-T2 asthma is frequently insensitive or resistant to inhaled corticosteroids(ICS),hence it is also referred to as severe asthma.The cause is that a significant portion of this asthma is mostly neutrophilic,which can also lead to severe or refractory asthma because standard medication is less effective at controlling the symptoms.According to research,the Th17 cell is the primary influencing element in the etiology of severe asthma,earning it the nickname "the kernel of severe asthma." Retinoic acid receptor-related orphan nuclear receptor gamma t(RORγt),a master regulator for the production and differentiation of Th17 cells,is activated by T cell receptor stimulation and accessory factors,which in turn activate the expression of signal transducer and activator of transcription 3(STAT3).Transforming growth factor β(TGF-β),which is primarily in charge of bronchial hyperreactivity and structural alterations of the bronchial wall in asthma and other conditions,is produced by activated Treg cells.Treg cells also promote Th17 cell differentiation via TGF-β,and the transfer of Treg cells enhanced Th17 cell(IL-17)production,which is linked to systemic autoimmune disease in the animal model.Treg cells may be disrupted by the unintended increase of Th17 cells,and the disturbance of Th17/Treg cell balance is linked to asthma severity and exacerbation.Collectively,the interaction of Th17 cells(IL-17),STAT3,RORyt,TGF-β,and Treg cells are more complex that makes asthma puzzle mysterious.The protein Methyl-CpG-binding domain(MBD)regulates the transcriptional status of the epigenome and is thought to play a significant role in genetics and a variety of other medical problems.Methyl CpG binding protein 2(MeCp2)was the first MBD-containing protein to be identified in the past.Subsequently,MBD 1-6 proteins were linked to the MeCP2 domain via sequence homology.The MBD2 protein possesses the capacity to interpret DNA methylation and is recognized as a key player in the epigenetics process.MBD2 is considered to be a significant mediator in the asthma epigenetics pathway because it interprets DNA methylation,modifies chromatin structure,and produces posttranscriptional histone modification.It is well known that a loss of MBD2 results in Th2 cellular polarization and a lack of differentiation of the Th17 cell population.An unrepressed Tbet/Hlx axis prevents the formation of Th17 cells in MBD2-/-mice,protecting them from experimental autoimmune encephalomyelitis(EAE).In a preliminary investigation of patients,it is discovered that in peripheral blood mononuclear cells,Th17-mediated severe asthma had greater levels of MBD2 expression than the mild asthma and the control participants.Additionally,it is also found that after accepting stimulus differentiation,splenic CD4+T-cells expressed MBD2 more strongly in animal model study.These results imply that MBD2 participates in CD4+T-cell development and the immunological pathogenesis of neutrophilic severe asthma mediated by Th17 cells.Sex hormones,commonly referred to as sex steroids,are produced from cholesterol esters and are essential for sex differentiation and reproduction.Female sex hormones consist a group of hormones like 17 β-estradiol(E2),estriol,estrone,and so on,and androgen is a general term that refers to all male sex hormones like testosterone(TES),dihydrotestosterone(DHT),and dehydroepiandro sterone(DHEA).Through androgen receptors(ARs),androgen governs and regulates the emergence and maintenance of male features such as male secondary sexual character,and participates in reproductive system.Similarly,estrogen regulates secondary sex traits and the growth of the female reproductive system through estrogen receptors(ERs).When we look at the prevalence of sex hormones over the course of a human life,we can see how these hormones fluctuate at different periods of the pathophysiological conditions of the human body,including in some disease processes.During puberty and adolescence,the sex hormone concentration is higher;after that,it gradually declines reaching a much lower level in an old age.Menarche,the menstrual cycle,pregnancy,menopause,obesity,and the use of some hormone-containing medications,such as hormonal contraceptives,all can cause visible changes in the hormone prevalence graph.According to a study,female sex hormones like estrogen and progesterone increase Th17 cells development,IL-17A-mediated airway inflammation,and IL-23R production via suppressing let-7f microRNA expression.On the other hand,testosterone(TES)lessens the airway inflammation caused by neutrophils,which decreases the expression of the IL-17A protein.Male sex hormones even adversely regulate innate lymphoid cells 2(ILC2)and reduce allergic inflammation.It’s interesting to note that lower serum TES levels have been linked to premenstrual and menstrual asthma exacerbation,while lower androgen levels have been linked to older men’s higher rates of asthma.These findings led us to focus our preliminary work on how sex hormones interact in severe asthma and also what will be the hormonal prevalence?At present,we do not know the role of sex hormones especially in MBD2-mediated Th17 cells predominant neutrophilic severe asthma.Does sex hormone have the ability to treat severe asthma by downregulating MBD2 expression or by directly influencing the differentiation and expression of Th17 cells?In a recent review,the correlation of sex hormone fluctuation status,MBD2 expression,and Th17 cells was assessed and the androgenic therapeutic potential in Th17 cells predominant neutrophilic severe asthma via regulating MBD2 was hypothesized,and this study was carried out to test this hypothesis.First,levels of androgen,estrogen,and the ratio of androgen to estrogen in participants(normal control,asthma,and severe asthma groups)were assessed and linked with asthma status.In order to compare the therapeutic efficacy of male and female sex hormones in severe asthma,as well as how sex hormones affect the expression of Th17 cells via regulating MBD2 in BECs as a cellular level working mechanism of sex hormone in severe asthma,the severe asthma animal model and the bronchial epithelial cells(BECs)cellular model were established.Here,this study for the first time is intended to evaluate whether sex hormones(androgen)can offer a novel potential therapeutic role in the treatment of BECs regulated Th17 cells predominant neutrophilic severe asthma through regulating MBD2 expression.Part 1 Measurement of the concentration and the ratio of sex hormones in severe asthmaObjectives:The main aim of this part of the study was to measure the concentration and the ratio of androgen and estrogen in normal control,asthma,and severe asthma subjects and correlate it with severe asthma status in order to further evaluate the hormonal involvement and therapeutic potential in severe asthma in the subsequent parts of the study.Methods:After the completion of ethical approval from Xiangya Second Hospital of Central South University,subjects were enrolled in normal control,asthma,and severe asthma groups from the out-patient,emergency,and respiratory inpatient departments fulfilling the global initiatives of asthma(GINA)guidelines.A score from the asthma control test(ACT)was used to assess asthma control.Subjects were classified as having well-controlled asthma if their ACT score was between 20 and 25,while those with a score below 20 were classified as having poorly controlled asthma.Mild,moderate,and severe asthma were classified based on the combined analysis of the pulmonary function test(PFT),ACT,and medication use.Negative methacholine(Mch)provocation test,normal spirometry results,no any obvious respiratory conditions were present in normal control subjects,and were without any history of use of oral or inhaled corticosteroids and leukotriene antagonists.Mild and moderate asthma was labelled as "asthma" in order to separate it from severe asthma.The demographic information of all subjects like age,sex,body mass index(BMI),smoking history,and etc.were recorded in all the three groups of study(normal control,asthma,and severe asthma).According to the established aseptic methodology,peripheral blood samples were taken,and eosinophils(EOS),neutrophils(NEU),and IgE concentrations were measured.Lung spirometry analysis was performed to evaluate the lung function like FEV1(L),FEV1/FVC(%),FEV1%(predicted%),and all parameters were evaluated and compared with each assigned group.Finally,androgen(dihydrotestosterone as DHT),estrogen(17β-estradiol as E2),and DHT:E2 ratio were measured from normal control,asthma,and severe asthma group of subjects by enzyme linked immunosorbent assay(ELISA)according to the instruction of the manufacturer and the results were correlated with severe asthma status.Results:(1)Subject demography:In terms of age,compared with the normal control group(51.73 ± 6.64),the age of asthma group(57.82 ± 10.51)and severe asthma group(62.31±10.14)were older,and the age of severe asthma group was greater than that of asthma group,with statistical difference(P=0.002).Similarly,a significant statistical difference was noted in subjects(male and female)ratio,number,and percentage in the groups(P<0.001).The Male and female numbers and the percentage was 9/10(47.4/52.6)in the normal control group,11/18(37.9/62.1)in the asthma group,and 26/3(89.7/10.3)in the severe asthma group.BMI in the asthma group(25.23 ± 3.95)and the severe asthma group(22.54 ± 3.28)increased compared with the normal control group(23.99 ± 2.68),with statistical difference(P=0.015).The smoking history as ex-smoker or never smoker was also accessed and calculated,and 68.4%(n=13)were never smoker and 31.6%(n=6)were ex-smokers in the normal control group,23(79.3%)were never smoker and 6(20.7%)were ex-smoker in asthma group,and severe asthma group had 44.8%(n=13)of never smoker and 55.2%(n=16)of ex-smoker.The asthma control test(ACT)was significantly lower in severe asthma group(12.93 ± 3.16)than that in asthma group(22.75±1.29),with statistical difference(P<0.001).(2)Lung function indexes:All the lung function indexes presented in the interquartile range(IQR)in this section showed significant statistical differences(p<0.001)when compared with other groups.For FEV1,compared with the normal control group[2.6(2.4-3.3)],the asthma group[1.8(1.6-2.0)]and the severe asthma group[1.0(0.8-1.2)]were all decreased,and the severe asthma group was significantly decreased(P<0.001).Similarly,FEV1/FVC(%)was lower in asthma group[69.3(57.8-74.2)]and severe asthma group[39.2(30.0-50.7)]than that in normal control group 81.2(77.6-82.4),and significantly lower in severe asthma group(P<0.001).The predicted value of FEV1%(%)was lower in the asthma group[74.8(65.7-87.5)]and the severe asthma group[32.8(26.2-42.0)]compared with the normal control group[101.8(98.1103.0)],and significantly lower in the severe asthma group(P<0.001).(3)Biochemical indexes:The biochemical indexes(interquartile range,IQR)like IgE,blood eosinophils(EOS),and neutrophils(NEU)were also measured and showed the distinctive pattern of presentation.IgE was increased in both asthma group[104.0(58.2-228.4)]and severe asthma group[205.8(161.5-1214.5)]compared with normal control group[58.6(27.0-442.0)],and significantly increased in severe asthma group(P=0.008).In EOS,there was an increase in both the asthma group(0.20 ± 0.19)and the severe asthma group(0.36 ± 0.19)compared with the normal control group(0.15 ± 0.12),and a significant increase in the severe asthma group(P<0.001).At the same time,similar expression levels of neutrophils were also observed.Compared with the normal control group(3.75 ± 0.86),both the asthma group(4.33 ± 1.32)and the severe asthma group(4.94 ± 1.75)were increased,and the severe asthma group was significantly increased(P=0.020).(4)Concentration and ratio of DHT and E2 by ELISA:DHT concentrations in the normal control group were higher than those in the asthma and severe asthma groups.Compared with asthma group,DHT in severe asthma group was significantly decreased,and the difference was statistically significant(P<0.05).As opposed to DHT concentration,E2 was associated in all three groups in the opposite way.Compared with normal control group and asthma group,E2 concentration in severe asthma group was significantly increased.Compared with the normal control group,E2 in asthma group was increased,and the difference was statistically significant(P<0.05).The ratio of DHT/E2 was also evaluated,and compared with the normal control group,the ratio was lower in both the severe asthma group and the asthma group,and the severe asthma was significantly lower,the difference was statistically significant(P<0.05).Conclusion:Severe asthma is associated with comparatively higher estrogen and reduced androgen levels and this association also indicates that sex hormone fluctuation status might affect asthma prevalence,remission,and might have therapeutic potential.Part 2:Establishment of severe asthma model and preliminary evaluation of DHT and E2 effects on the modelObjectives:The main aim of this part of this study was to establish and confirm the animal and bronchial epithelial cells(BECs)severe asthma model,and evaluate the effects of DHT and E2 on the model.Methods:(1)C57BL/6J female mice,each 10 in numbers were allocated randomly in normal control group,asthma(conventional asthma)group,severe asthma group,severe asthma+dihydrotestosterone(severe asthma+DHT)group,severe asthma+17β-estradiol(severe asthma+E2)group,and severe asthma+DHT/E2 group.DHT(the most bioactive product of androgen),E2(17β-estradiol),and DHT:E2 was injected in the severe asthma+DHT group,severe asthma+E2 group,and severe asthma+DHT/E2 group of mice respectively as per doses according to the study published.The severe asthma model was also followed and established according to the published report.On days 0,1,and 2,the severe asthma,severe asthma+DHT,severe asthma+E2,and severe asthma+DHT/E2 group of mice were sensitized intraperitoneally by 100μg HDM(house dust mites)+100μg OVA(ovalbumin)+15μg LPS(lipopolysaccharides)+2 mg aluminum hydroxide(Al(OH)3).The same groups were also OVA atomized 30 minutes before intranasal HDM excitation on days 14,15,18,19,and 20.On days 0 and 7,the asthma group of each mouse were given an intraperitoneal injection of 25 μg OVA+1 mg Al(OH)3 and OVA atomized on days 14 to 20 for 30 minutes.Normal saline was only injected in the normal control group for sensitization and atomization on the same days,site,and dose as the severe asthma group.On day 21st,mice were sacrificed and the study was further proceeded.After 24 hours of the final challenge,asthmatic symptoms were observed and the variations in airway responses were measured by lung resistance(RL)through methacholine(Mch)inhalation test;broncho-alveolar lavage fluids(BALF)collected and BALF cell counts measured for total cells,neutrophils,and eosinophils;lung tissue slicing for H&E staining to observe the histology changes and immunohistochemistry also performed for eosinophilic cationic protein(ECP)and anti-granulocyte receptor 1(Gr-1).(2)After the isolation of splenic single nucleated cells,the CD4+T cells were isolated with immune-magnetic beads methods only after separation of red blood cells from splenic single cell suspension.Flow cytometric analysis for the Th2 and Th17 cells were performed from the isolated CD4+T cells and the concentration of IL-4 and IL-17 measured by ELISA from the serum collected from all the groups.At the same time,the effect of DHT,E2,and DHT/E2 on Th2/Th17 cells and IL-4/IL-17 was also evaluated.(3)After the isolation of bronchial epithelial cells(BECs)followed by its culture and cytokeratin and DAPI(4’,6-diamidino-2-phenylindole)staining confirmed the BECs,the BECs asthma and severe asthma model was established according to the published protocol.The isolated BECs were treated with 100μg/mL HDM for the induction of asthma(asthma group).The severe asthma,severe asthma+DHT,severe asthma+E2,and severe asthma+DHT/E2 groups were treated with 100μg/ml HDM and 100 ng/ml LPS.PBS was only used for normal control group.(4)BECs were pretreated with androgen(DHT),estrogen(E2),and androgen estrogen(DHT:E2)in the designated groups to evaluate the effect of sex hormones on the model.The BECs were pretreated with 10nM of DHT,1nM of E2,and 10:1nM of DHT:E2 in severe asthma+DHT,severe asthma+E2,and severe asthma+DHT/E2 group respectively for 24 hours and co-cultured with CD4+T cells in the ratio of 1:10 for 24 hours.(5)The flowcytometry analysis of BECs plus T cells co-culture was performed to observe the differentiation of Th2 and Th17 cells in various groups of BECs asthma and severe asthma model and ELISA was performed from BECs culture supernatant for the measurement of IL-17 and IL-4.At the same setting,the effect of DHT,E2,and DHT/E2 was also evaluated.Results:(1)Extensive changes in the asthmatic behavior of the mice especially in the severe asthma group was observed than the normal control and the asthma group.There was an exaggerated symptoms and behavioral changes in the severe asthma+E2 group and less in the severe asthma+DHT group as compared with the severe asthma group.The intermediate level of these symptoms was noted in the severe asthma+DHT/E2 group and the normal group mice were from these symptomatic changes.(2)After the MCh provocation test,dose-response curves for RL shifted upward in severe asthma group than the asthma and normal control group(p<0.05).Severe asthma+E2 group showed the most exaggerated upward shift of RL than the severe asthma+DHT/E2 and severe asthma+DHT group(p<0.05).(3)There was an obvious increment of BALF total cells and NEU in severe asthma than the normal control(p<0.01 for comparison with BALF total cells)and asthma group(p<0.05),and at the same time EOS was significantly higher in asthma group than the severe asthma group(p<0.05).Severe asthma+ E2 group showed much exaggerated increase in BALF total cell,NEU,and EOS while severe asthma+DHT group of mice showed the opposite status(p<0.05).Severe asthma+DHT/E2 group of mice showed an intermediatory response in BALF cells expression as compared with severe asthma+DHT and severe asthma+E2 groups.(4)Under a light microscope,normal bronchial and alveolar architecture of the lung tissues and intact mucosal epithelium of the bronchi without evidence of inflammatory cell infiltration were observed in the normal control group.Inflammatory cells such as NEU,EOS,and lymphocytes were present infiltrating the airway wall,mucosa,submucosal layer,and interstitial lung tissue of all other groups of animals.Compared to the severe asthma+DHT group,the severe asthma+E2 group was linked to greater inflammatory infiltration.(5)Lung immunohistochemistry for ECP and Gr-1 showed only few infiltrating EOS and NEU in the normal control group.When compared to the normal control group,the asthma group’s EOS and NEU were noticeably higher(p<0.05).In comparison to the asthma group,the severe asthma group had significantly higher EOS and NEU infiltration(p<0.05).In comparison to the severe asthma group,the addition of E2 as in severe asthma+E2 was linked to significantly greater EOS and NEU detection and expression(p<0.05).The levels of EOS and Gr-1 were observed decreased in the severe asthma+DHT group.Inflammatory cells were decreased in the severe asthma+DHT/E2 group but not to the same extent as in the severe asthma+DHT group(p<0.05).(6)According to a flow cytometric analysis(animal model),the differentiation of Th2 cells in the asthma model was found to be higher than that of Th17 cells.Greater differentiation of Th17 cells than Th2 cells were found in the severe asthma group.Compared to the severe asthma group,severe asthma+E2 was linked with greater Th17 cell differentiation.Comparatively less differentiation of Th17 cells were seen in the severe asthma+DHT group compared to the severe asthma+E2 group.In addition,the severe asthma+DHT/E2 group showed less differentiation of Th17 cells than the severe asthma+E2 group,but not as much as the severe asthma+DHT group.The ELISA measurement of IL-4 and IL-17 also coincided with the flow cytometric results.(7)Cytokeratin is expressed in epithelial cells and about 95%of epithelial cells are positive to it,and DAPI staining showed the membrane viability and fixation of cells.BECs treatment with HDM and LPS induced the asthma and severe asthma model and hormones(DHT,E2,and DHT/E2)pretreatment implicated for the observation of hormonal effect in asthma.The co-culture of BECs with CD4+T cells were implicated as the BECs can directly regulate the Th2 and Th17 cells differentiation.(8)Comparatively more Th17 cells differentiation were seen in severe asthma compared to asthma group in BECs model.Th2 cells were primarily elevated in the asthma group.In the culture supernatant of BECs,IL-4 and IL-17 concentrations coincided with Th2 and Th17 cells results.DHT,E2,and DHT/E2 pretreated groups showed the variations in the differentiation of Th2/Th17 cells and the expression of IL-4/IL-17.(Refer to this part result section for details).(9)The measurement of the concentration of DHT and E2 in normal control,asthma,and severe asthma group of mice was also performed.As subjects with severe asthma have comparatively higher estrogen and lower androgen concentration as shown in part 1 of the study and the same pattern of result was also observed in this section.The normal control group’s DHT concentration was found to be significantly greater than that of the asthmatic and severely asthmatic groups(p<0.05)and(p<0.01)respectively.DHT levels decreased in the asthma group,whereas the severe asthma group experienced a substantially greater decline(p<0.05).Intriguingly,all experimental groups observed exactly the opposite correlation of E2 compared to DHT.Asthma and severe asthma groups had higher E2 concentrations than the normal control group(p<0.05 and p<0.01).,respectively).In comparison to the asthma group,severe asthma was related with a higher E2 concentration(p<0.05).Conclusion:(1)Animal and bronchial epithelial cells(BECs)severe asthma model was successfully established.(2))Severe asthma was associated with comparatively increased differentiation and expression of Th17 cells and IL-17,and Th2 cells differentiation and IL-4 expression were predominantly correlated with asthma.(3)The preliminary effect of DHT,E2,and DHT/E2 on the model was also explored and evaluated.Part 3:Role of DHT and E2 in severe asthmaObjectives:The main aim of this part of study was to measure the expression and detection of Th17 cells(IL-17),RORyt,and MBD2 in different groups of animals and BECs severe asthma model and also observe the effect of DHT,E2,and DHT/E2 on these parameters.Methods:As the methods of previous part of the study(part 2)is inter-linked with this part,the following methodological processes were undertaken in this part of the study only after the completion of methods of previous part of the study:(1)The lung tissues derived CD4+T cells were obtained and western blot analysis for the detection of GATA3 and RORyt from all the groups of animal model was performed.Similarly,the effect of DHT,E2,and DHT/E2 on GATA3 and RORyt detection was also measured.(2)After the flowcytometric analysis of Th2 and Th17 cells,and ELISA measurement of IL-4 and IL-17,western blot analysis was performed for the detection of GATA3 and RORyt in different groups of BECs model including the role of DHT,E2,and DHT/E2 on GATA3 and RORγt detection.(3)Lung immunohistochemistry(IHC)staining was used to investigate the cells of MBD2 positive stained infiltration in all the groups of animal model.Western blot technique was used to identify the detection and expression of MBD2 from fresh lung tissues grinded from all the groups and the effect of DHT,E2,and DHT/E2 on MBD2 detection and expression was also evaluated.(4)BECs were identified using cytokeratin and DAPI labeling,and then different dosages of HDM,LPS,and PBS were given to the cells to establish an asthma model.Four groups were created from the severe asthma group:severe asthma,severe asthma+DHT,severe asthma+E2,and severe asthma+DHT/E2.The severe asthma+DHT group received 10nM DHT for 24 hours,the severe asthma+E2 group received 1nM E2,and the severe asthma+DHT/E2 group received 10:1nM DHT:E2 during the same period of time.After 24 hours,BECs and CD4+T cells were cocultured in culture media at a 1:10(BECs:CD4+T cells)ratio.Finally,western blotting was used to identify the expression of the MBD2 protein across all BEC groups.Results:(1)Comparatively higher RORγt was detected in the lung tissuederived western blot(WB)analysis than in the GATA3 sample.At the same time,the asthma group had higher GATA3 detection than the other group.In comparison to the severe asthma group,the severe asthma+E2 group had higher RORγt detection.However,compared to severe asthma and severe asthma+E2 group,the addition of DHT to severe asthma+DHT group was linked with reduced detection of RORγt detection by WB.Additionally,compared to the severe asthma group,the severe asthma+DHT/E2 group displayed a relatively lower degree of RORγt detection.The detection,however,did not reach the same degree as that seen in the severe asthma+DHT group.DHT,E2,and DHT/E2 supplemented groups also showed some variations in the detection of GAT A3.Along with the western blot data,the relative levels of GATA3 and RORγt protein expression coincided with the WB results.(2)In BECs model,in comparison to the GAT A3,severe asthma was linked to greater RORγt detection and the asthma group showed more GATA3 detection than the other group.In comparison to the severe asthma group,the severe asthma+E2 group had higher RORγt detection.However,compared to severe asthma and severe asthma+E2 group,the addition of DHT(severe asthma+DHT)was linked to a lower detection of RORγt.Additionally,compared to the severe asthma group,the severe asthma+DHT/E2 group displayed a relatively lower degree of RORγt detection.The relative expression of the GATA3 and RORγt proteins matched the WB findings.(3)In an animal model of severe asthma,lung immunohistochemistry staining revealed that the severe asthma group had higher levels of MBD2 expression in the lung tissue compared to the asthma and normal control groups.A greater percentage of MBD2 staining positive cells were seen in the severe asthma+E2 group,but the severe asthma+DHT group had a relatively lower degree of peribronchial inflammation and positive MBD2 cells.The severe asthma+DHT/E2 showed a moderate level of peri-bronchial inflammation with MBD2 stained positive cells and the effect was in between the independent effect of severe asthma+DHT and severe asthma+E2.(4)In both animal and BECs asthma model,while compared to the asthma group,severe asthma was linked to a higher detection of MBD2.Severe asthma was associated with comparatively higher detection of MBD2 than the asthma group.When compared to the normal control group,a comparatively higher percentage of MBD2 was also found in the asthma group.While compared the severe asthma group,severe asthma+DHT group,and severe asthma+DHT/E2 group with the E2 addition group(severe asthma+E2),a relatively higher level MBD2 was detected in severe asthma+E2 group.The severe asthma+DHT group showed a lower MBD2 detection than the severe asthma,severe asthma+E2,and severe asthma+DHT/E2 groups.In contrast to the severe asthma+DHT and severe asthma+E2 groups,the severe asthma+DHT/E2 group displayed an intermediate degree of MBD2 detection.The relative MBD2 protein expression in all the groups were also similar to WB results.Conclusion:(1)Increased detection and expression of RORγt and MBD2 were associated with severe asthma.(2)DHT decreased the differentiation and expression of RORγt and MBD2 in severe asthma while E2 increased the detection and expression of all of them.DHT/E2 effect was in between the independent effect of DHT and E2.Part 4:Androgens attenuates the BECs regulated Th17 cells differentiation and function via MBD2 in Th17 cells predominant severe asthmaObjectives:The main aim of this part of the study was to evaluate and observe the association of MBD2 with Th17 cells of BECs model of asthma and observe the effect of DHT,E2,and DHT/E2 on MBD2 Th17 cells correlation.Methods:(Reminder:The methods of part 2 and 3 are inter-related with methods of this part of the study)(1)Bronchial epithelial cells(BECs)isolation and cytokeratin and DAPI(4’,6-diamidino-2-phenylindole)staining was performed as the process explained in part 3 of the study.(2)From RiboBo(Guangzhou,China),siRNAs targeting MBD2(siR-MBD2)and a control siRNA(siR-NC)were ordered.The sequence that interfered with MBD2 was 5’-GCAAGATGATGCCTAGTAA3’.From Honor Gene(Hunan,China),plasmids for mouse MBD2,OEMBD2,and negative control(OE-NC)were bought.Using Lipofectamine 3000,small interfering MBD2 RNA and plasmid were transfected in BECs for 48 hours.(3)The isolated and transfected BECs were treated with100μg/ml HDM and 100 ng/ml of LPS for induction of severe asthma.Only the normal control group was needed to use PBS.(For reminder,the following groups were made:normal control,severe asthma,severe asthma+DHT,severe asthma+E2,and severe asthma+DHT/E2).(4)In order to evaluate the effects of sex hormones on the model,the isolated cells were pretreated with androgen and estrogen in addition to the formation of the BECs severe asthma model.The severe asthma model transfected BECs were pretreated with 10nM of DHT,1nM of E2,and 10:1nM of DHT:E2 for 24 hours in the severe asthma+DHT,severe asthma+E2,and severe asthma+DHT/E2 groups respectively.(5)The method of isolation of splenic single nucleated cells and splenic CD4+T cells isolation had already been explained in part 3 of the study and also in the methods of this part.(6)After the mice’s splenic CD4+T lymphocytes were extracted by magnetic bead separation(Miltenyi Biotec)and DHT,E2,DHT/E2 pretreatment was conducted in severe asthma+DHT,severe asthma+E2,and severe asthma+DHT/E2 groups with 10nM of DHT,1nM of E2,and 10:1nM of DHT:E2 respectively in the asthma model transfected BECs for 24 hours.Then the BECs were then cocultured with CD4+T cells at a ratio of 10:1(TCs:BECs)for 24 hours in complete RPMI 1640 culture medium(Gibco,Australia)supplemented with 10%FBS,1%penicillin and streptomycin,soluble anti-CD28(1.0μg/ml),soluble anti-cd3e(0.5μg/ml),and IL-2(20 ng/ml).(7)Th2 and Th17 cells flow cytometry analysis from transfected BECs plus CD4+T cell mixture and under the DHT,E2,and DHT/E2 effect in BECs severe asthma model was also conducted.The methods for flowcytometry have been described in pervious parts of the study.Similarly,ELISA for measurement of IL-17 and IL-4 from transfected BECs plus CD4+T cells mixture supernatant and under the effect of DHT,E2,and DHT/E2 in BECs severe asthma model was performe...