The Regulatory Mechanism Of Circadian Gene Nr1d1 In Cigarette Smoke-induced Macrophages Polarization In The Airway Inflammation Of COPD

Objective:Chronic Obstructive Pulmonary Disease（COPD）is one of Chronic respiratory Diseases characterized by persistent and incompletely reversible airway limitation.By 2030,COPD is expected to be the third leading cause of death worldwide, severely affecting the quality of life and lobar ability of patients.Tobacco smoking is currently known to be an independent risk factor of COPD.Cigarette smoking activates oxidative stress,promotes the release of inflammatory mediators,and recruits inflammatory cells into airways,thereby amplifying the inflammatory response.Persistent and abnormal airway inflammation causes repeated injury and repair,resulting in structural abnormalities in the airway and alveoli destruction,ultimately leading to persistent airflow restriction and progressive deterioration of lung function.Therefore,smoking-induced chronic airway inflammation is an important pathological feature and the main pathological basis of COPD.The immune system plays a key role in cigarette smoke-induced airway inflammation involving in COPD.Mononuclear phagocytes（MNP）are an important part of the lung immune system.MNPs are mainly composed of lung macrophages（LMs）,that can be activated and polarized into different phenotypes when they are stimulated by the irritants so as to play different roles and functions.M1-like macrophages mainly produce inflammatory cytokines,chemokines and high levels of oxidative stress products to kill bacteria and form a Th1-type inflammatory response,which is called classical activated（pro-inflammatory）macrophages.M2-like macrophages secrete anti-inflammatory mediators participating in immune regulation,and are mainly involved in tissue remodeling to promote the proliferation of macrophages,which is called selectively activated macrophages.In recent years,studies have shown that lung macrophages in smoking or COPD patients displayed different phenotypes and functions.Recent study showed that alveolar macrophages in smokers or patients with COPD were mainly M1 and M2 dual-positive,which were correlated with the severity of COPD.Some studies also suggested that the alveolar macrophages were mainly in the non-polarized state or in the M2 polarized state.Thus,the effect of cigarette smoking on the polarization of macrophages to“M1 or M2”type is still controversial.Recently,more attention has been paid to the role of circadian pathways in the regulation of cigarette smoke-induced airway inflammation.Current studies have shown that the circadian regulation of genes in the circadian system is crucial to maintain the normal function of various organ systems.Studies have shown that circadian genes may be involved in the development of smoking-related airway inflammation or COPD and researches have shown that the Circadian gene Nr1d1regulates the polarization of macrophages.However,the effect of the circadian gene Nr1d1 on cigarette smoking-induced airway inflammation or the polarization of macrophages in COPD and its regulatory mechanism remain unclear.Therefore,this study intends to explore the role of circadian gene Nr1d1 in the regulation of cigarette smoke-induced macrophage polarization and its possible mechanism in the airway inflammation involving in COPD.Materials and Methods:1.COPD mouse model was constructed using the snuff-smoked device that designed by our group.Transcriptome sequencing was used to detect the expression of Nr1d1 of lung tissue in mice with COPD and control group at ZT0 and ZT12.To identify the purity of macrophages that was induced by M-CSF from mononuclear cells in murine bone marrow,flow cytometry was used.CCK-8 approach was used to measure cell viability of BMDMs that were stimulated with different concentration of cigarettes smoking-extracts（CSE）at different time.Meanwhile, reverse transcription-polymerase chain reaction（RT-PCR）was used to detect the m RNA level of Nr1d1 after the stimulation of CSE.2.Wild type and Nr1d1^-/-mice were obtained from Jackson laboratory.BMDM of wild type and Nr1d1^-/-mice were cultured in vitro and stimulated with CSE.RT-PCR,Western blot and Luminex assay was used to measure the markers of“M1-like”or“M2-like”macrophages after treatment with CSE.3.After stimulation BMDMs of wild-type and Nr1d1^-/-mice with CSE, transcriptome sequencing was conducted to explore the possible pathways of Nr1d1 in the regulation of macrophages polarization toward“M1-like”or“M2-like”subtypes,and Western blot was used to verify the results.4.Wild type and Nr1d1^-/-mice were exposed to cigarette smoke with whole body for consecutive 10 days or intermittent 4 weeks to build acute and subacute airway inflammation model.HE staining was used to evaluate the pathology change of lung tissue.Total cell counting and differential counting of neutrophils,macrophages of bronchoalveolar lavage fluid（BALF）was performed,and the concentration of mediators related to M1 and M2-phenotype was measured by Luminex assay.Flow cytometry and immunofluorescence test was used to detect protein levels related to lung macrophage activation and polarization.Results:1.There was circadian rhythmicity of Nr1d1 m RNA expression in the lung tissue of control group,and the expression at ZT12 was significantly higher than ZT0（p<0.001）,and the expression of ZT12 was significantly lower than ZT0 after cigarette smoke exposure（p<0.001）,and cigarette smoke can increase the expression of Nr1d1 at ZT0（p<0.05）and down-regulate the expression of Nr1d1at ZT12（p<0.0001）.In vitro experiments showed that CSE could down-regulate the expression of Nr1d1 m RNA in BMDM（p<0.01）.2.We found that M1 markers（i NOS,IL-1βand CXCL2）were up-regulated after treatment BMDM with CSE for 6h in vitro（all p<0.05）,and increase the m RNA and protein levels of the anti-inflammatory marker of M2 subtype（IL-10）（p<0.01）.Nr1d1 knockout could further increase the expression of M1-related markers induced by CSE（i NOS,IL-1βand CXCL2）（all p<0.01）,while the anti-inflammatory mediator of M2-related marker（IL-10）were down-regulated（p<0.05）.3.Transcriptome sequencing results showed that genes in BMDM of WT group that were up-regulated by CSE stimulation,and further up-regulated or down-regulated by CSE stimulation after Nr1d1 konckout,were mainly enriched in MAPK,MMPs,chemokines or interferon pathways.Cigarette smoke induced the up-regulation of p-p38 expression in BMDM（p<0.001）.Nr1d1 knockout further increased the expression of IRF5,a key protein in the Type I interferon pathway,and the expression of p-p38 was also up-regulated（all p<0.01）.4.Compared with wild type mice exposed to smoking,Nr1d1^-/-mice were intolerant to high concentration of nasal cigarette smoke exposure and showed higher mortality（p<0.05）.However,in the acute airway inflammation models induced by cigarette smoke exposure with whole body in 10 days,Nr1d1^-/-mice showed no significant changes in lung pathology,as well as total number of macrophages,and no significant differences in the M1-related cytokines IL-12,CXCL1 and CXCL2（all p>0.05）,except for increased proportion of macrophages（p<0.01） and decreased IL-10 in the BALF of Nr1d1^-/-mice（p<0.001）,in comparison with WT mice.In addition,the proportion of M1-like macrophages（CD86⁺IL-10^-）was not significantly different（all p>0.05）,but the proportion of M2-like macrophages（CD86^-IL-10⁺）was statistically down-regulated（p<0.05）.In the subacute airway inflammation models induced by cigarette smoke exposure with whole body in 4weeks,there was on statistical difference in the counts of macrophages,M1-related cytokines IL-12,CXCL1 and CXCL2,as well as M2-related cytokine IL-10（all p>0.05）,except for increased ratio of M1-like macrophages（CD68⁺i NOS⁺）（p<0.05）,between Nr1d1^-/-mice and WT mice.Conclusion:1.Cigarette smoke altered the circadian rhythm of Nr1d1 expression in lung tissues of healthy mice,suggesting that the clock gene Nr1d1 may play an important role in the cigarette smoke-induced COPD mouse model.2.In vivo cigarette smoke exposure or in vitro cigarette smoke-extract stimulation of macrophages can promote the polarization of macrophages to M1 phenotype and the anti-inflammatory M2 phenotype,suggesting that cigarette smoke plays an important role in macrophages polarization in the development of smoking-related COPD.3.To the best of our knowledge,this study is the first to show that Nr1d1 knockout primarily inhibiting cigarette smoke-induced polarization of mouse lung macrophages to anti-inflammatory M2 macrophages,suggesting that circadian gene Nr1d1 may be involved in the immune-inflammatory response of smoking-related COPD by regulating macrophage polarization.4.As far as we know,for the first time we found that Nr1d1 knockout mainly promote cigarette smoke-induced BMDM polarization to M1-like macrophages through activation of IRF5/P38 MAPK pathway,thus participating in the regulation of cigarette smoke-induced inflammation.Intervention with Nr1d1 may provide new insights for the treatment of smoking-related COPD.