The Effects Of Iloprost On Type 2 Innate Lymphoid Cells

Background and objectiveAsthma is a complex heterogeneous disease of the airways characterized by lung inflammation,airway hyperreactivity（AHR）,mucus over-production,and remodeling of the airways.Group 2 innate lymphoid cells（ILC2s）play a crucial role in the initiation and propagation of type 2 inflammatory programs in allergic asthma models,independent of adaptive immunity.In response to allergen,helminths or viral infection,damaged airway epithelial cells secrete IL-33,IL-25,and thymic stromal lymphopoietin（TSLP）,which activate ILC2s to produce type 2 cytokines such as IL-5,IL-13,and IL-9.Furthermore,ILC2s coordinate a network of cellular responses and interact with numerous cell types to propagate the inflammatory response and repair lung damage.PGs are generated from arachidonic acid via the cyclooxygenase（COX）-1/2pathways through two unstable intermediate forms:PGG2 and PGH2,and by subsequent specific PG synthase enzymes.PGI₂.Friedrich C.Luft et al found that COX production can improve the inflammation in lung tissue of asthma mouse model,and COX production can inhibit the allergic inflammation that induced by allergen.the ligation of the PGI2 receptor;IP being preferentially a Gs-coupled receptor,leads to an elevation of intracellular cAMP levels and subsequent activation of Epac protein,The PGI₂ analogue iloprost has been used in the treatment of pulmonary hypertension.Inhibition of COX in murine model can enhance the inflammatory situation in lung tissue and the expression of type-2 cytokines produced by CD4⁺Th2.It showed that COX production can inhibit the inflammatory response induced by allergen.As ILC2s has the familiar effects with Th2 and also act as key factor in pathological process in allergic asthma,it has not been reported that PGI₂ analog,iloprost,can inhibit the inflammatory response by restrain the function of ILC2through PGI2/IP signaling pathway.In our previous experiments in vivo,the inflammatory response in lung tissue on acute airway inflammatory murine model induced by IL-33 can relieve by iloprost,it also alleviated infiltration of inflammatory cells,hypersevere plasia of goblet cells and mucus secretion.In addition,iloprost could also reduce the number of ILC2s and eosinophils,the levels of IL-5 and IL-13in BALF and the mRNA expression of IL-5、IL-13、GATA3 and ST2 in lung tissues.In order to verify the idea from cellular level and molecular level,ILC2s are sorted from retired C57BL6/J mice,then stimulated in vitro in four groups,the proliferation of ILC2s and the ability to secret IL-5 and IL-13 were tested to detect the effect of iloprost on ILC2s,providing a new therapeutic basis for the treatment of asthma.Methods1.Methods in vivoWe choose C57BL/6J mice aged 6 to 8 weeks,randomly divided into four groups:vehicle group,IL-33 group,IL-33+iloprost group,single iloprost group.Administration IL-33 intranasal is adopted to establish the airway inflammatory model in mice.We assess the small airway and perivascular inflammatory infiltration by HE staining.PAS staining was used to detect the hyperplasia and mucus secretion of airway epithelium.2.Methods in vitroTo detect the effect of iloprost on ILC2s,ILC2s are sorted by magnetic beads selection combine with flow cytometry separation,then ILC2s are divided into 4groups:vehicle,IL-33,IL-33+iloprost,iloprost,ILC2s were cultured with different environment for 72h,pSTAT5 were tested at 24h;then measure the level of IL-5,IL-13 in supernatant by ELISA,and count the cell,detected the levels of IL-5,IL-13,GATA3,ST2,PPAR gamma mRNA by Real-time quantitative PCR.Result1.Results in vivoHE and PAS staining of lung sections revealed that the infiltration of inflammatory cells,hyperplasia of goblet cells and mucus secretion in IL-33 model group was more severe than vehicle group.The proliferation of goblet cells and the increase of mucus secretion in IL-33+iloprost group were significantly decreased.2.Results in vitro2.1 Cell count count the cell after culture for 72h,we found that the count of IL-33group（11170.41±2210.20 per well）compared to vehicle（8334.46±2020.11 per well）and iloprost control group（6972.57±850.13 per well）is increased significantly（P<0.05）,while combined with iloprost（6972.57±850.13 per well）,cell counts significantly decrease immediately（P<0.05）.2.2 Intracellular staining results The intracellular pSTAT5 were measured at 24h,compared to vehicle（MFI:2051.63±794.10）and iloprost control（MFI:2429.37±635.00）group,the MFI of pSTAT 5 on IL-33 group（MFI:4706.91±1343.11）increased significantly,while stimulate combine with iloprost,the MFI of pSTAT 5（MFI:2706.01±579.29）decreased immediately（P<0.05）,and we also examed the expression level of GATA3 at 72h by intracellular staining,the MFI of GATA3 in IL-33 group（MFI:22120.60±5740.00）were significantly higher（P<0.05）than vehicle（MFI:14680.34±5631.11）and iloprost group（MFI:11503±3251.41）,when compared to IL-33 plus iloprost group（MFI:10340.18±3293.21）,the MFI of GATA3decreased obviously（P<0.05）.the percentage of Ki-67+ILC2s were detected after culture for 72h,and the results shows that the percentage of Ki-67+ILC2s in IL-33group（68.28±9.45%）were significantly higher than vehicle group（54.48±7.43%）and iloprost control group（50.36±5.83%）,but when cultured combined with iloprost the percentage of Ki-67+ILC2s（46.22±15.81%）decreased distinctly（P<0.05）.3.Molecular experiment results3.1.ELISA results The level of IL-5 and IL-13 in culture supernatant of IL-33group（IL-5:397.00±53.57 pg/mL,IL-13:284.85±36.92 pg/mL）is significantly higher（P<0.05）than vehicle（IL-5:21.43±3.64 pg/mL,IL-13:20.25±2.19 pg/mL）and iloprost control group（IL-5:23.05±4.09 pg/mL,IL-13:20.70±4.46 pg/mL）,and when combined with iloprost,the levels of IL5,IL-13（IL-5:277.48±27.94,IL-13:19.90±3.44）decreased immediately（P<0.05）.3.2.Real-time quantitative PCR results The expression level of IL-5,IL-13,GATA3,PPAR gamma mRNA are increase significantly（P<0.05）in IL-33 group（IL-5:1.69±0.46,Il-13:1.90±0.56,GATA3:0.78±0.36）compared to vehicle group（IL-5:1,Il-13:1,GATA3:1）and iloprost control group（IL-5:0.39±0.20,IL-13:0.18±0.12,GATA3:0.24±0.09）,while the expression level of PPAR gamma is decrease in IL-33 group（0.42±0.26）.When combined with iloprost（4.20±0.4）,the expression of PPAR gamma obviously increased（P<0.05）.Conclusion1.The proliferation of ILC2s that induced by IL-33 can be inhibited by iloprost.2.The secretion of IL-5,IL-13 can be reduced by iloprost.Furthermore,iloprost may have the effect on inhibit the development of inflammation in airway inflammatory model in mice.