| Part Ⅰ The role of Chop in the pathogensis of OVA-induced allergic airway inflammation and M2 polarization.Objective:To investigate the effect of Chop in the airway inflammation,airway remodeling and airway hyper-responsiveness after OVA challenge and the role of Chop in the polarization of macrophage.Method:ER stress markers such as Chop,p-eIF2α,Atf4 and Bip were detected in the lung tissue after OVA-induction by western blot.Allergic airway inflammation mouse model was induced in WT and Chop-/-mice(8-10 weeks old)by sensitized and challenged with OVA.Airway responsiveness to acetylcholine(Ach)was assessed after the last OVA challenge using a computer-controlled small animal ventilator system.And then BALF was collected by cannulating the trachea and lavaging the lung with PBS.BALF was centrifuged at 300 g for 5 min,and the supernatants were stored at-80℃ prior to cytokine measurements by ELISA.And the Cells were counted using a haemocytometer.Differential cell counts were measured after Diff-Quick staining.Pathological staining was used to evaluate the airway inflammation and airway remodeling.The macrophages markers in the allergic airway inflammation model like CD206,CD11c,F4/80,Arginase 1,Yml and Fizzl were detected by flow cytometry,Western blot and RT-PCR.To confirm the protective role of Chop deficiency to allergic airway inflammation was depend on attenuating macrophage M2 programme,we adopt adoptive transfer studies,macrophages derived from WT or Chop deficiency mice were stimulated by IL-4 for 12 h and then transferred by intratracheal injection into the lungs of clodronate liposome-treated WT or Chop deficiency mice at a density of 1×106 cells/mouse.To demonstrate the therapeutic potential of Chop in OVA-induced mouse modle,we constructed lentiviruses expressing a Chop shRNA.And then airway inflammation,airway remodeling and airway hyper-responsiveness following OVA challenge were evaluated.Results:The expression of Chop,p-eIF2a,Atf4 and Bip were increased in allergic mice compared with control mice.Compared with WT mice loss of Chop provides protection for mice against OVA-induced allergic airway inflammation,airway remodeling and airway hyper-responsiveness.Chop was predominantly localized within the F4/80+ macrophages in OVA-challenged WT mice compared with saline-treated controls,and similar results were observed by analysis of lung sections.The M2 macrophages markers were attenuated in Chop deficiency mice following OVA challenge by flow cytometry,Western blot and RT-PCR.While,there was no different with M1 macrophage between WT and Chop deficiency mice.Adoptive transfer studies showed that Chop deficiency-mediated protection for mice against OVA-induced allergic airway inflammation is dependent on the attenuation of M2 programme in macrophages.Moreover,knockdown of Chop protects mice against OVA-induced allergic airway inflammation by suppressing the production of M2 macrophages.Conclusion:ER stress was existed in the pathogensis of allergic airway inflammation,and Chop deficiency or knockdown provides protection for mice against OVA-induced allergic airway inflammation,airway remodeling and airway hyper-responsiveness dependent on M2 programme.Part II The role of Chop in the polarization of M2 macrophage and the mechanism studyObjective:To investigate the role of Chop in the polarization of M2 macrophage in vitro and the study for the signal pathways through which Chop involved in the regulation of macrophage alternative activation.Method:Peritoneal macrophages were cultured from WT and Chop-/-mice.And then macrophages were induced by IL-4 for alternative activated macrophages.Western blot was used for detection the expression of Chop in the macrophages induced by IL-4 for different time points.The M2 macrophages marker Arginase 1 was detected by western blot.The related signaling pathways p-STAT6/STAT6 was next investigated to demonstrate the underlying mechanisms by western blot for different time points.Next,Tfec,a specific transcription factor for M2 macrophages,was tested by RT-PCR.To confirm that IL-4 recptor alpha(IL-4Ra)may be the target gene for Tfec,CHIP was adopted.And then,luciferase reporter was adopted to detect the transcription of IL-4Ra by Tfec.Results:Chop was almost undetectable in macrophages before stimulation,while a steady increase was noted upon IL-4 stimulation,and the highest expression levels were detected after 12h of IL-4 stimulation.The M2 macrophages markers were attenuated in Chop deficiency macrophages following IL-4 stimulated by western blot.Next the signal pathway for IL-4 was detected by western blot and the phosphorylated form of STAT6 was decreased in Chop deficiency macrophages.Tfec and IL-4Ra expression were increased in WT macrophages compared with Chop deficiency macrophages.CHIP result showed that Tfec manifested binding activity to the IL-4Rα promoter in a region of-571 bp upstream of the transcriptional start site and disruption of the Tfec binding site resulted in a 1.7-fold reduction in IL-4-induced luciferase reporter activities.Conclusion:Chop was high expression in the macrophages induced by IL-4.While,Chop promotes macrophage M2 programme by regulating an IL-4 positive feedback loop,in which IL-4 induces Chop expression,which then promotes STAT6 signalling to transcribe Tfec expression,and Tfec next transcribes IL-4Ra expression to promote macrophage M2 programme.Part III Sputum CHOP+CD206+ macrophages were positively correlated with asthma severityObjective:To observe the expression of CHOP protein in induced sputum macrophages in asthmatic patients and to explore the correlation between CHOP+CD206+ macrophages and lung function in asthmatic patients.Method:Induced sputum samples were obtained from healthy volunteers(n = 15)and asthmatic patients(n = 18)enrolled in Tongji Hospital.The expression of CHOP and BIP in induced sputum samples obtained from asthmatic patients were detected by western blot.immunofluorescent staining was conducted in induced sputum samples inflammation cells.To analyze the correlation between CHOP+CD206+ macrophages and lung function in asthmatic patients,we analyzed the relationship between the number of CHOP’CD206-macrophages and the FEV1 and FEV1/FVC%of asthmatic patients.Results:There were no significant differences in age,sex ratio,BMI,and FVC between the asthma and healthy controls.The FEV1,FEV1/FVC%,PEF of asthmatic patients were significantly lower than those of healthy controls.Immunofluorescence staining of CHOP and CD 14 showed that the expression of CHOP protein in asthmatic patients induced sputum macrophages was significantly increased compared with the healthy control.Immunofluorescence of CHOP and CD206 co-staining showed that the expression of CHOP protein in the alternative activated macrophages was significantly increased compared with the healthy control,the number of sputum CHOP+CD206+ macrophages was positively correlated with asthma severity,as manifested by the detection of negative correlation with FEV1/FVC ratios(R2 = 0.4061,p<0.01)and FEV1 values(R2 = 0.4382,p<0.01).Conclusion:The number of sputum CHOP+CD206+ macrophages is positively correlated with asthma severity. |
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