The Role Of PNU-282987 (α7nAChR Agonist) In The Allergic Pulmonary Inflammation Mediated By ILC2

BackgroundAllergic asthma is a complex and chronic inflammatory disorder that is driven by Th2 cytokines.The traditional view is that the allergic inflammation is an eosinophilic inflammation caused by the activation of Th2 cells which is very important for the development of allergic asthma,starting with the antigen presenting cell(APCs)presenting allergens.Recent studies have shown that Group 2 innate lymphoid cells(ILC2s)are the main source of Th2 cytokines in the early allergic airway inflammation.ILC2s are directly activated by some innate signals from myeloid cells and cytokines and alarmin secreted by epithelial cells,such as IL-25,IL-33 and proteases,without requiring further differentiation.Following activation,ILC2s produce robust amounts of Th2 cytokines IL-5 and IL-13 to promote eosinophilic inflammation and airway hyperreactivity(AHR).A study showed that mice lacking T-cells and B-cells could be also induced acute allergic lung inflammation after they were sensitized by papain,which suggesting that other immune cells should play a key role in the early stages of allergic inflammation.And the further studies showed that the mice deficient in ILC2s could not be induced acute type 2 lung inflammation even after the stimulation of papain.These evidence indicate that ILC2s is involved in the pathogenesis of asthma and plays a key role.Therefore,regulating the function of ILC2s could be an ideal strategy for treating asthma.Theα7 nicotinic acetylcholine receptor(nAChR),which mediates rapid excitatory synaptic transmission,is shown to be a potential therapeutic target in neuropsychiatric,neurodegenerative and inflammatory diseases.In pulmonary allergic inflammation,an agonist for nAChR,nicotine decreases the levels of Th2cytokines,IgE and cysteinyl leukotrienes,thus reducing the incidence of allergic inflammation.However,the specific cellular mechanisms of nAChR regulating allergic inflammation have not yet been clearly defined.In view of its therapeutic potential in nervous system and inflammatory diseases,the receptor agonist developed byα7nAChR as a target has been used in the treatment of many diseases,show promising efficacy and have advanced to clinical trials.Therefore,we propose to use PNU-282987,a selective agonist forα7nAChR,to explore its role in ILC2s-dependent allergic pulmonary inflammation.ObjectiveIn this study,IL-33 was used as a stimulating agent for nasal instillation to establish a model of acute allergic airway inflammation in mice,and intraperitoneal injection of PNU-282987 was used to intervene.The changes in the number of ILC2and the levels of Th2 cytokines IL-5 and IL-13 in the bronchoalveolar lavage fluid and lung tissues were measured,and the expression levels of IL-5,IL-13,GATA3,and ST2 mRNA in lung tissues were detected by Quantitative Real-time PCR to investigate whether PNU-282987 inhibited the ILC2-mediated acute allergic pulmonary inflammation in mice,so as to provide a new basis for the prevention and treatment of allergic pulmonary inflammation.Materials and methodsTwenty-four 6-8 weeks old C57BL/6 female mice were randomly divided into PBS control group,IL-33 model group,IL-33 plus PNU-282987 intervention group,and PNU-282987 control group;6 mice in each group.Experiments were carried out on the days of 1st,2d,and 3d respectively,and mice were executed on day 5.The content of ILC2s and eosinophils(EOS)in lung tissue and bronchoalveolar lavage fluid(BALF)of mice were detected by flow cytometry.Hematoxylin and eosin(HE)stained lung tissue sections were used to observe the inflammatory response of each group,and the periodontal mucous secretion was observed by periodic acid-Schiff regent(PAS)staining.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of IL-5 and IL-13 in the supernatants of BALF and lung homogenate.The levels of IL-5~+ILC2s and IL-13~+ILC2s in lung tissues were determined by flow cytometry through Intracellular staining.And the expression of IL-5,IL-13,GATA3and ST2 mRNA were detected by Quantitative Real-time PCR..Results1.HE and PAS staining resultsThe staining results in PBS control group showed that there was no infiltration of inflammatory cells around the blood vessels and bronchioles of the mice,and there were no a large number of goblet cell proliferation and mucus secretion in the bronchial mucosa.The staining of PNU-282987 control group was similar to that of the PBS control group,without significant difference.Compared with the PBS control group,the IL-33 model group had a large number of inflammatory cells infiltrating around the blood vessels and bronchioles,and there was a large number of goblet cell proliferation and mucus secretion in the bronchial mucosa.Compared with IL-33 model group,the IL-33+PNU-282987 intervention group still had inflammatory cell infiltration in the lung tissues,but the degree of infiltration was significantly reduced.Although there was still goblet cell proliferation and mucus secretion in the bronchial mucosa,the mucus secretion was significantly reduced.2.ELISA resultsPNU-282987 control group had no significant difference in the concentrations of IL-5 and IL-13 in the supernatants of lung homogenate and BALF in the mice compared with the PBS control group(P>0.05),and those of the IL-33 model group were both increased significantly compared with the PBS control group(P<0.05),while those of the IL-33+PNU-282987 group were all decreased significantly compared with the IL-33 model group(P<0.05).3.Flow cytometry resultsCompared with the PBS control group,PNU-282987 control group had no significant difference in the percentages and total number of ILC2s and EOS in the lung tissues and BALF(P>0.05),while those of the IL-33 model group were increased(P<0.05).Compared with the IL-33 model group,the percentages and total number of ILC2s and EOS in the lung tissues and BALF of IL-33+PNU-282987 group were decreased(P<0.05).4.Intracellular staining resultsCompared with the IL-33 model group,the percentages of IL-5~+ILC2 and IL-13~+ILC2 in the lung tissues of IL-33+PNU-282987were significantly reduced(P<0.05).5.Real-time PCR resultsThe relative expression of IL-5,IL-13,GATA3 and ST2 mRNA in the lung tissues of mice treated with PNU-282987 alone had no significant difference compared with the PBS control group(P>0.05),while those of IL-33 model group were increased significantly compared with the PBS control group(P<0.05).And the relative expression of IL-5,IL-13,GATA3 and ST2 mRNA in the lung tissues of IL-33+PNU-282987 model group were decreased compared with the IL-33 model group(P<0.05).ConclusionPNU-282987 has a significant inhibitory effect on ILC2 mediated allergic pulmonary inflammation,which can reduce pulmonary inflammatory reaction and mucus secretion,and reduce the production of Th2 cytokines such as IL-5 and IL-13.