Changes And Role Of Lung Type 2 Innate Lymphoid Cells In Bronchopulmonary Dysplasia Mice

ObjectiveTo detect the number of lung type 2 Innate lymphoid cells(ILC2s),alveolar macrophages and the expression of related cytokines and dynamic changes of them in the mice models of bronchopulmonary dysplasia(BPD)induced by lipopolysaccharide,to explore the functional roles of ILC2 s and the relationship between ILC2 s and alveolarization in BPD,the effects of expansion or depletion of ILC2 s on alveolarization in BPD,and whether dexamethasone attenuates lung injury in BPD by affecting ILC2 s or partially by affecting ILC2 s.MethodsNewborn mice were randomly divided into saline and lipopolysaccharide groups.The model of BPD was established in mice induced by lipopolysaccharide and lung tissues were respectively harvested at the postnatal days 1,3,7,14 and 21(P 1,P 3,P7,P 14 and P 21),10 mice in each group.The lung histomorphological changes were observed by hematoxylin-eosin(HE)staining.The number of ILC2 s,M1 and M2 macrophages in lung tissues was measured by flow cytometry.The expression levels of IL-33,IL-13 and IL-4 in lung tissue were detected by enzyme-linked immunosorbent assay(ELISA).The r IL-33,anti-ST2 antibody,anti-CD90.2 antibody and dexamethasone(DEX)were injected intraperitoneally into mice respectively and randomly divided into saline control group(Saline group),lipopolysaccharide group(LPS group),lipopolysaccharide+IL-33 group(LPS+IL-33 group),lipopolysaccharide+anti-ST2 antibody(LPS+anti-ST2 group),lipopolysaccharide+anti-CD90.2 group(LPS+anti-CD90.2 group),and lipopolysaccharide+dexamethasone group(LPS+DEX group),10 mice in each group.Lung tissues were respectively harvested at the postnatal days 21.The pathological pattern of lung tissue was detected by HE staining.The number of ILC2 s,M1 and M2 macrophages in lung tissue was detected by Flow cytometry.IL-13 and IL-4 expression in lung tissues were detected by real-time fluorescence quantitative PCR(RT-q PCR).Results(1)In the saline group,with the increase in age,the lung tissue developed gradually,the alveolar structure was regular,and the alveolar wall became thinner,while in the LPS group,the alveolar structure was disordered,the alveolar fusion became larger,the number decreased,the structure simplified,and the alveolar wall gradually thickened,which was similar to the pathological changes in human BPD,indicating the successful establishment of the model.ILC2s increased in LPS group as compared to that in control group and were significantly improved in mice during 7 and 14 days after birth(P<0.05).The level of IL-33 was high after birth and gradually increased with the increase in age up to 14 days after birth,and higher than that in the saline group,while the opposite was true on day 21 after birth(P<0.05).The detection of cytokines in lung tissues of mice showed that the expression levels of IL-4 and IL-13 in the LPS group were significantly higher than those in the saline group(P<0.05).The levels of IL-4 and IL-13 increased first and then decreased,and the peak value was at the end of rapid alveolation(day 14 after birth).Also,the level of IL-13 was distinctly higher than that of IL-4(P<0.05).The expression levels of IL-4 and IL-13 were higher in LPS group than the saline group(P<0.05).The number of M2 macrophages,increased first and then decreased after birth and peaked on the day 14 after birth,and was significantly higher than those in the saline group before 14 days after birth(P<0.05).The number of M1 macrophages increased at 3 days after birth and then decreased,and was higher than that in the saline group before 14 days after birth(P<0.05),which was generally consistent with the altered degree of inflammation of BPD.(2)In BPD mice treated with IL-33,the structure of alveoli was disordered,the fusion of alveoli was larger,the number was reduced,the structure was simplified,the alveoli wall was thickened,and the inflammatory cells were increased,while the opposite was true in BPD mice treated with anti-ST2 antibody.ILC2 s and the expression levels of IL-4 and IL-13 increased notably in BPD mice that interfered with IL-33,while the effect of intervention with anti-ST2 antibody was reversed(P<0.05).Similarly,M2 macrophages in BPD mice with IL-33 were markedly higher,while M1 macrophages were markedly lower,and those in BPD mice with anti-ST2 antibody were opposite(P<0.05).Anti-CD90.2 antibody-treated BPD mice showed a regular alveolar structure with an increased number of alveoli and a thinner alveolar wall as compared to Ig G-treated BPD mice.Flow cytometric analysis of ILC2 s in the lung confirmed significant depletion of ILC2 s in mice with anti-CD90.2 antibody treatment as compared to Ig Gtreated control mice.This was accompanied by a significant decrease in the expression levels of IL-4 and IL-13(P< 0.05)and a significant decrease in M2 macrophages(P<0.05),while the opposite change was observed for M1 macrophages(P<0.05).After DEX treatment,BPD mice showed reduced lung injury and pneumonia,convergence to normal alveolar structure and thinner alveolar wall.The number of ILC2 s and the expression of IL-4 and IL-13 were reduced(P<0.05)and the number of M2 macrophages was significantly reduced(P<0.05),while the number of M1 macrophages had no significant change.Conclusion(1)ILC2s may promote the polarization of M2 macrophages by mediating the secretion of IL-4 and IL-13,thus promoting the occurrence and development of BPD.(2)The increase of IL-33 level promotes the proliferation and activation of ILC2 s,and the enrichment of both aggravates the severity of BPD.(3)DEX reduces the severity of BPD in part by inhibiting the proliferation of ILC2 s.