The Investigation Of Interleukin-27 In Regulating Airway Inflammation And Airway Hyper-responsiveness By Acting On Dendritic Cells CD39/ATP Axis In A Mouse Model Of Asthma

Background and Objectives Interleukin-27(IL-27),a member of IL-6/IL-12 family,is a heterodimeric cytokine composed of EBV-induced gene 3(EBI3)and IL-27p28 subunits.The major producers of IL-27 are monocytes,dendritic cells(DCs),and macrophages.IL-27 receptor(IL-27R)consists of two chains,WSX-1 and gp130,expressed on several types of immune cells and structural cells,such as DCs.IL-27Ra,IL-27R specific subunit,can recognize the signal of IL-27.Previous studies have demonstrated that IL-27 is involved in the pathogenesis of asthma.Hence,we took advantage of IL-27Ra-deficient(IL-27Rα-/-)mice and induced experimental asthma to explore the role of IL-27 in regulating airway inflammation and airway hyper-responsiveness in a mouse model of asthma.DCs are the key initiators to induce allergen-specific immune responses in asthma.CD39 is a cell surface-located prototypic member of the ecto-nucleoside triphosphate diphosphohydrolase(E-NTPDase)family.CD39,expressed on DCs,monocytes,natural killer cells and subsets of activated T cells,can hydrolyze ATP to ADP and AMP,thus suppressing the pro-inflammatory effects of ATP which acting as an endogenous danger signal.NLRP3 inflammasome,an important family member of pattern recognition receptors(PRR)in innate immunity,is composed of NLRP3,the adaptor ASC and effector protein Caspase-1.Endogenous danger signals,such as ATP,can trigger the activation of NLRP3 inflammasome and induce the release of large amount of interleukin(IL)-1β and IL-18,augmenting Th2 and Th17 immune responses.A recent study showed that IL-27 and CD39/ATP axis in DCs are closely related in a mouse model of experimental autoimmune encephalomyelitis(EAE).It was demonstrated that the effects of IL-27 suppressing the established relapsing-remitting EAE were mediated at least in part through induction of the immunoregulatory molecule CD39 in DCs.We speculate that it is possible that IL-27 and CD39 may be in a similar association in a mouse model of asthma.Therefore,in this study,we investigate the effects of IL-27 on CD39/ATP axis and the expression and function of NLRP3 inflammasome in DCs to clarify the potential mechanism of IL-27 in regulating airway inflammation and airway hyper-responsiveness in a mouse model of asthma.Methods(1)Part I:A mouse model of allergic asthma was developed with ovalbumin(OVA)-Imject Alum sensitization and OVA challenge in female wild type(WT)C57BL/6 mice(6-8 weeks old)and IL-27Ra knock-out(IL-27Rα-/-)(C57BL/6 background).The control mice,WT C57BL/6 mice and IL-27Rα-/-mice,were only challenged with OVA without OVA-Imject Alum sensitization.Airway hyper-responsiveness and airway inflammation were evaluated at 48 h after the final challenge.The airway resistance and dynamic lung compliance were recorded by FinePointe RC system.Lung tissue inflammation and goblet cell hyperplasia were assessed by hematoxyin and eosin(HE)and perioic-acid Schiff(PAS)staining,respectively.The expression of Mucin 5AC(Muc5AC)in lung tissue was detected by immunohistochemical analysis.Cellular profiles in the BALF were counted by cytospin with Wright-Giemesa staining.The levels of total IgE and OVA-specific IgE(OVA-IgE)in serum and cytokine(IFN-y,IL-4,IL-5,IL-13,IL-17A and IL-10)in the BALF were measured by enzyme-linked immunosorbent assay(ELISA).The mRNA levels of transcript factors(T-bet,GATA3,RORyt and Foxp3)were determined by quantitative polymerase chain reaction(qPCR).(2)Part II:A mouse model of allergic asthma was established in WT mice and IL-27Rα-/-mice as described.CD39 and the components of NLRP3 inflammasome(NLRP3,ASC and Caspase-1)mRNA and protein expression in lung tissues were determined by qPCR and western blot,respectively.The levels of ATP and cytokines(IL-1β and IL-18)in BALF were measured by Luminescence ATP Detection Assay System and ELISA.(3)Part III:A mouse model of allergic asthma was established in WT mice and IL-27Rα-/-mice as described.The expression of CD39 on lung DCs was determined by flow cytometry in vivo.Bone marrow mononuclear cells were prepared from femur bone marrow suspensions of WT mice and IL-27Rα-/-mice,stimulated by recombinant murine granulocyte-macrophage colony-stimulating factor(GM-CSF)and recombinant murine IL-4 to induce bone marrow-derived DCs(BMDCs)in vitro.CD39 and the components of NLRP3 inflammasome(NLRP3,ASC and Caspase-1)mRNA and protein expression were determined by qPCR and Western blot in BMDCs in the presence of recombinant murine(rmIL-27)or/and lipopolysaccharide(LPS).The level of ATP in the supernatant of BMDCs was detected by Luminescence ATP Detection Assay System.In addition,the Janus kinase/signal transducer and activator of transcriptions(JAK/STAT)signaling pathway in WT-BMDC and IL-27Rα-/--BMDC in the presence of rmIL-27 was analyzed by Western blot.Results(1)In OVA-induced asthma models,IL-27Rα-/-mice showed marked enhancement of airway responsiveness with pulmonary eosinophil infiltration,goblet cell hyperplasia and overexpression of Muc5AC,increased levels of total IgE and OVA-IgE in serum,as well as the number of total cells and the proportion of eosinophils in BLAF.Furthermore,compared with WT asthmatic mice,the levels of IL-4,IL-5,IL-13 and IL-17A in BALF and the mRNA expression of transcription factor GATA-3 and RORyt in lung tissue were also increased in IL-27Rα-/-asthmatic mice.(2)Compared with WT asthmatic mice,the mRNA and protein expression of CD39 in lung tissue of IL-27Rα-/-asthmatic mice were reduced,while the components of NLRP3 inflammasome(NLRP3,ASC and Pro-caspase-1/Caspase-1)were augmented in IL-27Raα-/-asthmatic mice.The levels of ATP and cytokines(IL-1β and IL-18)in BALF were also increased in IL-27Rα-/-asthmatic mice than that of WT asthmatic mice.(3)The expression of CD39 on lung DCs in IL-27Rα-/-asthmatic mice was lower than that of WT asthmatic mice in vivo.Compared with WT-BMDC,the expression of CD39 was reduced in IL-27Raα-/--BMDC in vitro.And the level of ATP in the supernatant of IL-27Rα-/--BMDC was higher than that of WT-BMDC.The expressions of NLRP3,ASC and Pro-caspase-1/Caspase-1 were also increased in IL-27Rα-/--BMDC.The presence of rmIL-27 could induce the expression of CD39,but reduce the expressions of NLRP3,ASC and Caspase-1 in WT-BMDC.The levels of ATP,IL-1β and IL-18 in the supernatant of WT-BMDC were also reduced in the presence of rmIL-27.In addition,augmented expressions of phosphorylated(p)-JAK1,p-JAK2,p-STATl and p-STAT3 in WT-BMDC were triggered by rmIL-27.Conclusions Compared with WT asthmatic mice,augmented airway responsiveness and airway inflammation were exhibited in IL-27Rα-/-asthmatic mice.The potential mechanism was that the functional defect of IL-27 leads to lower expression of CD39 on lung DCs and higher level of extracellular ATP in IL-27Rα-/-asthmatic mice,inducing the activation of NLRP3 inflammasome in DCs,releasing of pro-inflammatory cytokines,IL-1β and IL-18,in large and ultimately aggravating the bias of Th2 and Th17 responses.