GLCCI1 Is Involved In Corticosteroid Insensitivity By Negatively Regulating M1 Macrophage Activation In Asthma

Objective:To investigate the role of glucocorticoid-induced transcription protein 1(GLCCI1)deficiency in glucocorticoids resistance in a mouse model of allergic airway inflammation induced by chicken ovalbumin(OVA)and its effect on M1 macrophages effects on cell activation.Methods:To clarify that GLCCI1 is involved in OVA-induced allergic airway inflammation in mice,we first detected the changes of GLCCI1 in mouse lung tissue after OVA induction by western blot(WB).Subsequently,by selecting wildtype mice(WT)and GLCCI1 knockout mice(KO),a mouse model of allergic airway inflammation induced by OVA was constructed and intervened with glucocorticoids.The airway response of mice in each group to different concentrations of acetylcholine was observed and evaluated by using a small animal pulmonary function instrument.Bronchoalveolar lavage fluid(BALF)was detected by flow cytometry through bronchoalveolar lavage of each group of mice.The number and classification of various inflammatory cells in the infiltration levels of inflammatory cells around the airways and blood vessels of the mice in each group were evaluated by pathological embedding and sectioning of mouse lung tissue;enzyme linked immunosorbent assay(ELISA)was applied to detect various inflammatory cytokines in lung tissue;flow cytometry(intracellular cytokine staining,ICS)to further determine the type 1 helper T cells(Th1),type 2 helper T cells(Th2)in lung tissue;proinflammatory macrophages(M1)and alternatively activated macrophages(M1)were analyzed by colocalization with tissue immunofluorescence double staining.WB,flow cytometry,and real-time quantitative PCR were used to detect the molecular markers of macrophage subtypes in lung tissue after OVA-induced stimulation and glucocorticoids treatment of mice in each group,such as:Arg1,CD206,iNOS,CXCL10.At the same time,in order to further verify the specific mechanism of the decrease of GLCCI1 expression leading to OVA-induced allergic airway inflammation in mice and the specific mechanism of glucocorticoids resistance,we confirmed that the target cells with high GLCCI1 expression in normal lung tissue were macrophages by immunohistochemistry and immunofluorescence co-localization.cell.The adoptive transfer of macrophages was used to analyze the effect of GLCCI1 knockout macrophages on promoting hormone resistance in asthmatic mice.Results:In the lung tissue of OVA-induced wild-type mouse asthma model,the mRNA and protein levels of GLCCI1 were significantly lower than those of control mice,and the expression of GLCCI was up-regμlated after glucocorticoids treatment.After phenotypic analysis of the OVA-induced asthma inflammation model established in WT mice and GLCCI1-knockout mice,the OVA-induced GLCCI1-knockout asthmatic mice had aggravated lung tissue inflammation compared with wild-type asthmatic mice.The bronchial hyperresponsiveness(BHR)was significantly increased,and the relieving effect of hormones was not obvious.We also found that GLCCI1 knockout promoted the proportion of pro-inflammatory macrophages in the lung tissue of the mouse OVA asthma model,but had little effect on the proportion of alternatively activated macrophages.Glucocorticoids could not effectively reduce the increased pro-inflammatory macrophages in GLCCI1-knockout asthma model mice.Adoptive transfer of macrophages further found that GLCCI1 knockout-induced glucocorticoids resistance in allergic asthma mice was directly related to the increase in pro-inflammatory macrophages.Conclusion:Lack of GLCCI1 expression aggravates airway inflammation and promotes glucocorticoids resistance in mice by inducing activation of pro-inflammatory macrophages.Objective:To explore the regulation of GLCCI1 on M1 macrophage-type activation and its effect on glucocorticoid sensitivity.Methods:(1)Extracted and culture wild-type mouse bone marrow-derived macrophages and then use OVA,LPS+IFNγ,IL4,IL4+IL13,IL10 and other substances to intervene.Real-time fluorescence quantitative PCR and western blot were applied to determine expression of GLCCI1 in each group cells.At the same time,mouse and human-derived lung epithelial cell lines were used to replace mouse bone marrow-derived macrophages,and the above intervention measures were repeated to determine whether the expression changes of GLCCI1 under the condition of pro-inflammatory macrophage polarization were cell-specific.(2)Bone marrow-derived macrophages were extracted from wild-type mice and GLCCI1 knockout mice,and were intervened with LPS+IFNγ,LPS+IFNγ+Dex,and then,WB,flow cytometry,ELISA,and CCK8 cell proliferation experiments were conducted to detect expression of M1 macrophage-related markers(CD86,CD80,MHCII,iNOS,CXCL10)and apoptosis and proliferation of M1 macrophages.(3)Bone marrow-derived macrophages from wild-type mice and GLCCI1 knockout mice were extracted and intervened with LPS+IFNγ,LPS+IFNγ+Dex and/or GR activation inhibitor mifepristone(RU486),and WB was applied to detect M1 markers iNOS;WB and cellular immunofluorescence were conducted to detect the total GR level,GR phosphorylation level,and GR intracellular localization in macrophages in each group to evaluate the effect of GLCCI1 on GR phosphorylation and glucocorticoids.(4)GR,GLCCI1,GR-dependent luciferase reporter gene,renilla luciferase reporter gene were transiently transfected into HEK-293 cells(without endogenous GR functional activity),and intervented with different concentration of glucocorticoids and RU486 to evaluate luciferin signal valuesin different groups of cells.(5)In order to explore the specific mechanism of GLCCI1 deficiency in reducing the effect of glucocorticoids in proinflammatory macrophages,the protein synthesis inhibitor cycloheximide(CHX)and the proteasome inhibitor MG132 were pretreated in LPS and IFNγpolarized macrophages,and then,WB was applied to detect the level of GR activation.Co-immunoprecipitation and cellular immunofluorescence to detect the binding of GR and GLCCI1 in macrophages in cytoplasm and nucleus of macrophages under M1 polarization stimulation;By using various kinase inhibitors(JNK inhibitor(SP600125)),p38 MAPK inhibitor(SB203580),MEK1/2 inhibitor(PD98059),GSK inhibitor(CHIR99021),AKT1/2 inhibitor(AKti-1/2))pretreat M1 polarization macrophages along with glucocorticoids,we detect transcription levels of glucocorticoids-regulated genes GILZ and MKP1 with RT-PCR.Results:(1)After OVA intervention and the activation of pro-inflammatory macrophages induced by the M1 classical polarization pathway(IFNγ+LPS),the expression of GLCCI1 was down-regulated.(2)There was no difference in the expression of GLCCI1 under inflammatory stimulation in mouse and human lung epithelial cell lines.Compared with wild-type mouse-derived macrophages,the GLCCI1-knockout macrophages had higher expression of the classic M1 markers iNOS,CD80,and MHCⅡ;stronger secretion of CXCL10 after stimμlation with OVA or IFNγ and LPS;higher proliferative capacity and resistance to glucocorticoids pro-apoptosis trends.The GR phosphate level of M1 macrophages after RU486 intervention was significantly down-regulated with higher expression of iNOS.When GLCCI1 was knocked out,the phosphorylation level of GR Ser211 in M1 macrophages was down-regulated,and the nuclear import of GR was reduced.Compared with HEK 293 cells transfected with GR overexpression plasmid alone,GR activity was increased in macrophages co-transfected with GLCCI1 overexpression plasmid in a glucocorticoids concentration-dependent manner,and RU486 could inhibit this promoting effect of GLCCI1.The protein levels of GR Ser211 and GLCCI1 were significantly down-regμlated in macrophages stimμlated with IFNγ and LPS by pretreatment with CHX and MG132,and the same trend was also observed under glucocorticoids intervention.Likewise,the addition of MG132 coμld not reverse the downregulation of GR Ser211 phosphorylation site by GLCCI1 knockdown in M1 macrophages.In resting macrophages,GLCCI1 and GR were weakly bound,and in M1 macrophages after glucocorticoids intervention,GR and GLCCI1 signal values overlapped at the nucleus.By isolating the nuclear and cytoplasmic proteins of M1 macrophages,the expression of GLCC1 in the nucleus was higher than that in the cytoplasm after M1 polarization and hormone stimulation,and the binding to GR was also increased in the nucleus.In the state of M1 polarization and glucocorticoids intervention,the expression of GILZ and MKP1 mRNA in GLCCI1 knockout macrophages was significantly decreased compared with that in wild-type mouse-derived macrophages;JNK inhibitor(SP600125),p38 inhibitor(SB203580),MEK1/2 inhibitor(PD98059),and GSK inhibitor(CHIR99021)all can reverse the transcriptional repression of hormone-responsive genes by GLCCI1 knockdown.Conclusion:GLCCI1 knockout promoted IFNγ and LPS induced activation of proinflammatory macrophages and exhibited glucocorticoids resistance.The mechanism is that the deficiency of GLCCI1 expression reduces the inhibitory effect of non-ligand and ligand-activated GR on pro-inflammatory macrophages by reducing GR Ser211 phosphorylation.Objective:The relationship between the expression of GLCCI1 in induced sputum of asthma patients and the activation of M1 macrophages was analyzed.Methods:The induced sputum of asthma patients were collected,and the expression of GLCCI1 in induced sputum cells was detected by real-time fluorescence quantitative PCR,and its correlation with asthma severity was analyzed.Flow cytometry was applied to detect the proportions of M1 macrophages(CD68+CD86+)and M2 macrophages(CD68+CD206+)in induced sputum,and analyze the correlation between them with GLCCI1 expression.Results:The level of GLCCI1 mRNA in induced sputum in patients with severe asthma was lower than that in patients with mild and moderate asthma.The improvement in FEV1%of asthma patients in the low GLCCI1 expression group was lower than that in the asthma patients in the high GLCCI1 expression group after application of airway dilators.The number of emergency department visits due to exacerbations of asthma in the former was significantly increased in the past 1 year.The level of GLCCI1 mRNA in induced sputum cells of asthma patients was negatively correlated with the proportion of M1-type macrophages,but no significant correlation was found between the levels of GLCCI1 mRNA in induced sputum cells of asthma patients and the proportion of M2-type macrophages.Conclusion:In asthmatic patients with reduced glucocorticoids sensitivity,the level of GLCCI1 mRNA in induced sputum was negatively correlated with the proportion of M1 macrophages.