Dynamic Changes Of Type 3 Innate Lymphoid Cells In Lung Tissues Of Mice With Bronchopulmonary Dysplasia

ObjectiveWe established the lipopolysaccharide(LPS)-induced bronchopulmonary dysplasia(BPD)model to investigate dynamic changes of type 3 innate lymphoid cells(ILC3)and their role in lungs of mice with bronchopulmonary dysplasia(BPD).MethodsThe BPD model was replicated by intra-amniotic of LPS(200μg/kg).Twenty male and twenty female mice were selected at a ratio of 1:1 into twenty cages.The next day,the vaginal secretions of female mice were smeared,and the mice with sperm-bearing vaginal secretions detected by smear microscopy were recognized as pregnant.Intra-amniotic injection of lipopolysaccharide was given to some of the pregnant mice,and normal saline was given to the other pregnant mice.Neonatal mice born to mothers injected with LPS were recognized as the LPS group,while mice born to mothers injected with normal Saline were recognized as the control group.Five mice in each group were sacrificed 1,3,7,14 d after they were born for procurement of fresh lung tissues.HE staining was used to observe the pathological changes of lung tissues.Flow cytometry was used for measuring the proportion of ILC3 and NKP46-ILC3 in lymphocytes,and the proportion of IL-17~+ILC3 in the lung.ELISA was used to detect the protein content of downstream cytokines interleukin-17(IL-17)and interleukin-22(IL-22)as well as granulocyte-macrophage colony stimulating factor(GM-CSF)in lung homogenate.In the intervention research,recombinant IL-23 was injected into the intraperitoneal cavity of neonatal mice to stimulate ILC3 amplification,and then lung tissues were collected for HE staining to observe morphological changes.In order to further study the role of ILC3 in BPD,mice were intraperitoneally injected with anti-CD90 antibody to knock down ILC3.The proportion of ILC3 was detected to evaluate whether ILC3 was knocked down.In order to evaluate the pathological changes of lung tissues after ILC3 knockdown,lung tissues of mice in the anti-CD90 antibody treatment group were selected for HE staining on the 14th day.The ratio of IL-17+ILC3 was detected by flow cytometry.The neonatal mice were intraperitoneally injected with anti-IL-17 neutralizing antibody to block the effect of IL-17.Lung tissues were taken for HE staining on the14th day after birth to observe the pathological changes of lung tissues in the treated BPD group.Results(1)In the control group,the alveolar wall of the lung tissue became thinner and the alveolar structure became complete gradually.Simultaneously,the LPS group showed significantly increased alveolar volume,decreased alveolar number,simplified alveolar structure,increased inflammatory cells,and increased average alveolar count.The number and proportion of ILC3 in lymphocytes of lung tissues from mice in control group increased gradually from day 1 to 7 after birth,reached the peak on day 7,and then decreased gradually.Similarly,the number and proportion of ILC3 in lung tissues of mice in the LPS group increased gradually from day 1 to 7after birth,reached the peak on 7 days,and decreased significantly on 14 days.Compared with the control group,the number and the ratio of ILC3 in lymphocytes in lung tissues of mice in LPS group were significantly increased(P<0.05).(2)After birth,expression level of IL-17 in lung tissues of mice in control group and LPS group first rose and then fell.The expression of IL-17 showed an increasing trend from day 1 to day 7,reached the highest level on day 7,and showed apparent declination on day 14.Compared with the control group,the expression of IL-17 in LPS group was increased significantly at the same time point,and the difference was statistically significant(P<0.05).The expression of IL-22 in control group and LPS group was increased gradually from postnatal day 1 to day 14.Compared with the control group,expression of IL-22 in LPS group was increased at the same time point,with statistically significant difference(P<0.05).Compared with the control group,the trend of GM-CSF expression in LPS group was not obvious from postnatal day 1to the day 14.(3)Compared with the control group,lung tissues of mice treated with recombinant IL-23 showed more severe alveolar damage and inflammation with the increase of ILC3 ratio.The proportion of ILC3 in lung tissues of BPD mice treated with anti-CD90 antibody was significantly lower than that in lung tissues of BPD mice treated with normal saline.Compared with the BPD group,the anti-CD90antibody group showed improved lung pathology,significantly reduced inflammation and improved alveolar destruction.We used flow cytometry to detect IL-17+ILC3,and results showed that the proportion of IL-17+ILC3 in BPD group was significantly increased compared with the control group at the same time point(P<0.05).Microobservation showed that lung inflammation and alveolar destruction were significantly reduced in the anti-IL-17A neutralizing antibody treatment group compared with the BPD group,suggesting that lung pathology was improved in the mice whose effect of IL-17 was blocked.Conclusion(1)In the LPS-induced BPD model,the increasing number of ILC3 in lung tissues of mice in the LPS group may be closely correlated with the development of BPD.(2)In the early stage of BPD,ILC3 may recruit neutrophils to accumulate bysecreting IL-17,thus promoting the secretion of inflammatory mediators,and aggravating the inflammatory response.The dynamic changes of ILC3 in BPD suggest that ILC3 may be a potential target for new therapy of bronchopulmonary dysplasia.