The Role And Molecular Mechanism Of MiR-146a Regulating Type 2 Innate Lymphocytes (ILC2s) In Asthma

Part Ⅰ MiR-146a mimics attenuate allergic airway inflammation by impacted group 2 innate lymphoid cells(ILC2s)in ovalbumin(OVA)-induced mouse asthmaBackgroundAsthma is a complex and common chronic inflammatory disease,caused by a combination of geneticand environmental factors.The prevalence of allergic asthma has increased dramatically.The main changes in the lungs include intense leukocyte including eosinophilic infiltration,airway hyperresponsiveness(AHR),mucus hypersecretion and overexpression of T helper 2(Th2)cell-type cytokines including interleukin(IL)-4,IL-5 and IL-13.Innate lymphoid cells(ILCs),derived from common lymphoid progenitor,are a group of innate immune cells in the lymphoid lineage.Group 2 ILCs(ILC2,also termed natural helper cells)is valued for its important role in the pathogenesis of asthma.Upon stimulation with Th2 polarizing cytokines such as IL-25,IL-33 and thymic stromal lymphopoietin(TSLP),mature ILC2s start to produce Th2 cell-type cytokines that participate in the progression of allergic asthma.MicroRNA(miRNA or miR)is a subtype of 18–22 nt non-encoding RNA,which functions in the regulation of cell proliferation,differentiation and apoptosis.It has been found that multiple miRNAs are associated with the occurrence and development of allergic asthma.Previous studies have found that the MicroRNA-146a(miR-146a)expression in asthma inhibits cell proliferation and promotes apoptosis of bronchial smooth muscle cells(BSMCs).In addition,recent studies suggested that the expression level of miR-146a was significantly increased in the plasma of children with asthma.However,current research on the pathogenic mechanisms of miR-146a still remains at the cellular level in vitro,and needs to be further elucidated.Our present study aimed to investigate the effect of miR-146a on ovalbumin(OVA)-induced asthma mouse model.MethodsSpecific pathogen-free female BALB/c mice(6–8 weeks old;18–20 g)were used.The experimental groups were described as follows:normal control group(PBS),OVA group,miR-146a group,DEX group,anti-IL-17RB-ctrl group,anti-IL-17RB-OVA group,anti-IL-17RB-146a group,anti-ST2-ctrl group,anti-ST2-OVA group and anti-ST2-146a group.To determine the effect of miR-146a on airway inflammation,inflammatory cell infiltration(eosinophils,neutrophils,macrophages and lymphocytes)in bronchoalveolar lavage fluid(BALF)was measured by flow cytometry.Levels of OVA-specific immunoglobulin E(IgE)in serum and Th2 cell-type cytokines IL-4,IL-5 and IL-13 in BALF were examined by enzyme-linked immunosorbent assay.For monitoring the airway,Penh value(%baseline)was measured using a whole‐body plethysmograph.We examined the ILC2 compartment,defined by expression of ICOS in the absence of classical lineage markers(ICOS~+Lineage~–).As ILC2s are known to be activated by IL-25 and IL-33,the experiments of allergic inflammation were followed in mice treated with the antibody against IL-17RB(the IL-25-specific subunit of the receptor)or IL17RBxST2(the IL-25 and IL-33 receptors)to block the respective signaling pathways.ResultsIn OVA-induced asthmatic mice,miR-146a significantly suppressed the infiltration of eosinophils,neutrophils,macrophages and lymphocytes in BALF and decreased the levels of OVA-specific IgE and T helper 2(Th2)cell-type cytokines IL-4,IL-5 and IL-13.In addition,miR-146a remarkably alleviated the increased Penh value to an extent close to the positive DEX group.miR-146a inhibited the OVA-induced airway hyperresponsiveness(AHR)and the group 2 innate lymphoid cells(ILC2s)responses.a marked increase in the productions of IL-5 and IL-13 were found within this subset of allergic mice challenged by OVA.However,we found significant decreases in lung ILC2 IL-5 and IL-13 productions when miR-146a was co-administered,showing that miR-146a might inhibit the activation of ILC2.Moreover,the effects of miR-146a mimics were probably dependent on interleukin(IL)-33 stimulation.ConclusionsIn conclusion,we hereby describe suppression of allergic airway responses in an OVA-induced mouse asthma model by co-administration of miR-146a.Our data demonstrate that miR-146a significantly attenuates AHR and allergic airway inflammation through reducing the infiltration of inflammatory cells into lung tissues and suppressing the expressions of OVA-specific IgE and Th2 cytokines,which might correlate with its capacity to block the IL-33 signaling pathway.Thus,the ability of miR-146a to downregulate the allergic reactivity in OVA-induced mouse asthma observed in this paper might stimulate research for more specific molecular mechanisms,which may provide new routes for treating allergic diseases in human.Our results suggest miR-146a might serve as an attractive candidate for further pre-clinical studies as an anti-inflammatory treatment of asthma.Part Ⅱ MicroRNA-146 a negatively regulates IL-33 in activated group 2 innate lymphoid cells by inhibiting IRAK1 and TRAF6Background Asthma is a chronic inflammation that is driven by T helper 2(Th2),an immune response characterized by airway stenosis or overreaction,obstruction,inflammation and mucus secretion,which can be life threatening.Type II innate lymphoid cells(ILC2)play a very important role in the pathogenesis of allergic asthma.However,some studies reports that there are much more neutrophils in the airway and BAL of severe asthma patients and that IFN-γ or IL-17 A signal pathways are involved in the phenomenon.Micro RNA(mi RNA)are a class of 18～22nt small noncoding RNAs,playing important roles in gene expression regulation.They usually degrade their target m RNAs and/or inhibit their translation at the posttranscriptional level.mi RNAs have important functions in regulating cell proliferation,differentiation and apoptosis.Our previous study found that mi R-146 a administration alleviated the symptoms of OVA-induced asthma in mice and found that mi R-146 a may act through the IL-33-ILC2 signaling pathway.However,it is unclear whether mi R-146 a inhibits the occurrence of asthma associated with IL-33,and the function of mi R-146 a in ILC2 is still unclear.This study aims to investigate whether mi R-146 a inhibition of asthma is related with IL-33 signaling pathway in ILC2 and the underlying mechanism.Methods Asthma mice model was induced by ovalbumin(OVA).To investigate mi R-146 role in ILC2,we built allergic asthma mice model by OVA to detect whether in vivo administration of mi R-146 a resulted in a decrease in IL-33 secretion by epithelial cells leading to a decrease in the ability of ILC2 s to secrete IL-5,13.mi RNA146 a mimics was administrated to asthma mice or transfected to activated ILC2 purified from asthma mice lung.RT-PCR was used to detect mi RNA146 a level in lung tissue and ILC2.To further investigate mi R-146 a effect in ILC2,we detected IL-13 and IL-5 expression under the conditions of in vitro culture.IL-5 and IL-13 levels in culture supernatant were detected by flow cytometry.In order to demonstrate that mi R-146 a regulated the ST2 signaling pathway,we treated mi R-146 a in vitro.IRAK1,TRAF6,STAT1 protein expression levels were detected by western blot.Results No significant difference between the mi R-146 a group and the OVA group was found,and neither in IL-25 levels.IL-5 and IL-13 secretion of ILC2 was reduced remarkably,indicating that mi R-146 a can directly inhibit the function of ILC2.mi R-146 a directly inhibited ILC2 function and suppressed ILC2 proliferation both in vivo and in vitro.However,there was no obvious effect on apoptosis.During stimulation of ILC2,mi R-146 a expression gradually increased with a decrease of cell proliferation.Modulation of ILC2 function by mi R-146 a may depend on IL-33/ ST2 signaling through inhibiting IRAK1 and TRAF6.Conclusion In this study,OVA-induced allergic asthmamice model was used to determine whether mi R-146 a administration resulted in a decrease in the secretion of IL-33 and IL-25,affecting the ability of ILC2 s to secrete IL-5,13.The results showed that mi R-146 a had no significant effects on IL-33 and IL-25 secretion.Therefore,we suspected that mi R-146 a may act directly on ILC2 to inhibit its function.The results showed that IL-5 and IL-13 expression was significantly reduced in ILC2 sorted from OVA-induced asthma mice and culture with IL-7,TSLP,and IL-33,indicating that mi R-146 a can directly inhibit the function of ILC2.We also found that mi R-146 a expression increased with IL-33 stimulation while ILC2 proliferation gradually decreased in ILC2 in vitro culturewith the presence of IL-7,TSLP,and IL-33.Meanwhile,mi R-146 a inhibitor increased the ability of ILC2 to secrete IL-13 and IL-5.These results suggested that mi R-146 a may be an intrinsic regulatory mechanism for ILC2.We used mi R-146 a to co-culture with IL-33-stimulated activated ILC2 and found that its function was reduced,but we found that mi R-146 a had no significant effect on IL-25-activated ILC2 function as IL5,13 had no significant changes.It is suggested that mi R-146 a may affect ILC2 function by regulating IL-33-ST2 signaling.In our experiments,when treated ILC2 with mi R-146 a mimics,ILAK1 and TRAF6 expressions significantly decreased.mi R-146 a can inhibit IRAK1 and TRAF6,downstream molecules of ST2 signal pathway,thereby negatively regulate IL-33/ST2-activated ILC2 to inhibit asthma.Targeting mi R-146 maybe a novel strategy for the treatment of allergic asthma.