Changes And Effects Of Helios~+ Regulatory T Cells In Emphysema

PartⅠChanges and effects of Helios~+regulatory T cells in mice with emphysemaObjective To observe the changes of Helios~+regulatory T cells in mice with emphysema exposed to cigarette smoke,and to explore the role of Helios~+regulatory T cells in the development of emphysema in mice exposed to cigarette smoke.Methods Healthy male C57BL/6 mice aged 8 weeks were randomly divided into air exposure group(control group)and tobacco smoke exposure group(smoke group).Tobacco smoke exposure group was exposed to tobacco smoke for 24 weeks.Air exposure group was exposed to air for 24 weeks.Hematoxylin-eosin(HE)staining was used to observe the pathological changes of lung tissue.The average alveolar lining interval(MLI)was calculated to evaluate the degree of emphysema in mice.The peripheral blood,spleen and lung of mice were isolated.Mononuclear cells(PBMCs)in tissues were used to detect the expression of Helios~+regulatory T cells in blood,spleen and lung tissues of the two groups by flow cytometry.The expression levels of IL-10,TGF-βand TNF-αin lung tissue of mice were detected by RT-PCR.Western blotting was used to detect neutrophil elastase protein expression in mouse lung tissue.Treg cells in the spleen were sorted by immunomagnetic beads,and Real-time polymerase chain reaction was performed.The expression levels of Foxp3 and GITR mRNA in Treg cells were detected.And the expression of Foxp3 protein was also detected by Western blot.The correlation analysis method was used to analyze the results of the detection,and the correlation between the expression level of Helios~+regulatory T cells and the concentration of corresponding inflammation-related factors and Treg cell function indexes in mice with emphysema was analyzed.Results(1)The average internal village interval(MLI)of the smoked group was(27.76±8.18)μm,which was significantly higher than that of the control group(16.57±3.71)μm.The difference was statistically significant(P<0.01).The emphysema model was successfully replicated.(2)Helios~+regulatory T cells:compared with the control group,the Helios~+Treg cells in the peripheral blood,spleen and lung tissues of the smoked group were significantly increased(P<0.05).(3)Compared with the control group,the expression of neutrophil elastase in the lung tissue of the smoked group increased,and the expression of IL-10 mRNA in the lung tissue increased,TNF-αand TGF-βexpression decreased,the difference was statistically significant(P<0.05).(4)The expression of Foxp3 and GITR in the spleen Treg cells of the smoked group was significantly higher than that of the control group(P<0.05).(5)Correlation analysis showed that Helios~+Treg cells in the smoked group were positive correlated with IL-10 in lung tissue(r=0.63,P<0.05),and there was no significant correlation with TGF-βand TNF-αmRNA in lung tissue(r=0.21,0.58,P>0.05),and expression of neutrophil elastase(NE)in lung tissue,there was no significant correlation between the levels(r=-0.48,P>0.05);The proportion of Helios~+Treg cells in the spleen of the smoked group was positively correlated with Foxp3 and GITR in spleen Treg cells(r=0.67,0.78,P<0.05).Conclusion The proportion of Helios~+regulatory T cells in the exposed mice with tobacco smoke is increased,and positively correlated with the inflammatory cytokine IL-10,and also positively correlated with Foxp3 and GITR in Treg cells.Helios regulatory T cells may be involved in the development of emphysema caused by tobacco smoke exposure.Part Ⅱ Changes of Helios~+ Regulatory T Cells in Peripheral Blood of Patients with Chronic Obstructive Pulmonary DiseaseObjective To study the changes and significance of Helios~+ regulatory T cells in peripheral blood of patients with chronic obstructive pulmonary disease in emphysema.Methods Seventeen patients with chronic obstructive pulmonary disease(COPD)who were admitted to the First Affiliated Hospital of Guangxi Medical University from June 2018 to December 2018 were enrolled.At the same time,14 healthy people who did not smoke(nonsmokers)and 15 patients with normal smoking lung function(healthy smokers)were collected as control group.The above three groups were included as subjects,and the general clinical data of each group of subjects were compared and analyzed.The peripheral blood of each group was extracted,the peripheral blood mononuclear cells(PBMCs)were separated by density gradient centrifugation,and the proportion of Helios~+regulatory T cells(Helios~+ Treg cells)in each group was detected by flow cytometry.At the same time,the concentrations of IL-10,TNF-α,TGF-β and IFN-γ in the serum of each group were also detected by enzyme-linked immunosorbent assay(ELISA).Pulmonary function tests were performed on each group of subjects.Correlation analysis was used to analyze the correlation between the expression of Helios~+ regulatory T cells in peripheral blood and the levels of IL-10,TNF-α,TGF-β,IFN-γ and lung function test in patients with chronic obstructive pulmonary disease.Results(1)Compared with non-smokers,the proportion of Helios~+ regulatory T cells in peripheral blood of COPD patients was significantly higher,the difference was statistically significant(P<0.05).The proportion of Helios~+ regulatory T cells in peripheral blood of patients with COPD The difference was statistically significant(P<0.01).Compared with those with normal smoking lung function,there was no significant difference in the expression of Helios~+ regulatory T cells in peripheral blood of patients with COPD.(2)Compared with non-smokers,the expression of TNF-α in serum of patients with normal lung function was increased(P<0.05),the expression of TGF-β was decreased(P<0.05),and the levels of IL-10 and IFN-γ were decreased.The difference was not statistically significant(P>0.05).The expression levels of IL-10,TNF-α and IFN-γ in serum of patients with COPD were increased(P<0.05),and the expression level of TGF-β was significantly decreased(P<0.05).Compared with those with normal lung function,the expression of IL-10 in serum of patients with COPD was increased(P<0.05),and the expression of TNF-α,IFN-γ and TGF-β was not significantly different(P>0.05).(3)Correlation analysis showed that Helios~+ regulatory T cells in peripheral blood of patients with COPD were positively correlated with the levels of IL-10,TNF-α,TGF-β and IFN-γ in serum(r=0.71,0.80,0.53,0.54,P<0.05),correlation analysis with lung function showed that the proportion of Helios~+ regulatory T cells in peripheral blood of patients with COPD was negatively correlated with FEV1% pred(r=-0.61,P<0.05),and with FEV1/FVC.There was no significant correlation between smoking index(r=-0.37,-0.15,P>0.05).Conclusion The proportion of Helios~+ regulatory T cells in peripheral blood of stable chronic obstructive pulmonary disease is increased,which is positively correlated with proinflammatory and anti-inflammatory factors,and negatively correlated with pulmonary function FEV1% pred,suggesting Helios~+ regulatory T Cells may be involved in the development of chronic obstructive pulmonary disease.