The Role And Mechanism Of MiR-223 Targeting NLRP3 Inflammasome To Regulate The Airway Inflammation Of Neutrophilic Asthma

Objectives Studies have found that the NLRP3/IL-1βaxis signaling pathway is involved in the pathophysiology of neutrophilic asthma.Mi R-223 is highly expressed in hematopioietic cells and is strictly regulated.It acts as a key regulator during the differentiation and activation of myeloid cells.The core role of mi R-223 in the differentiation and activation of neutrophils and macrophages has been extensively studied.Previous studies have shown that mi R-223 is involved in the pathophysiological process of asthma,but the specific pathophysiological mechanism is still unclear.This study explored whether mi R-223 is involved in the pathophysiology of neutrophilic asthma,and whether it is involved in regulation of airway inflammation in neutrophilic asthma through targeted regulation of NLRP3inflammasome.Methods（1）Part 1:C57BL/6 wild-type（WT）female mice were selected and randomly divided into eosinophilic asthma group（EA group）,neutrophil asthma group（NA group）and control group（NC group）.Mice in EA group were sensitized with chicken ovalbumin（OVA）combined with aluminum adjuvant and nebulized with 1%OVA to establish eosinophilic asthma model;mice in group NA group were sensitized with OVA combined with CFA and nebulized with 1%OVA established neutrophilic asthma;NC group mice were sensitized and challenged with PBS at the same dose as a control.Observe the symptoms of asthma in mice in each group.Twenty-four hours after the last challenge,the total number and classification of cells in the bronchoalveolar lavage fluid（BALF）of each group were detected.Lung tissue inflammation was assessed by HE staining and goblet cell hyperplasia was evaluated by PAS staining;Airway hyper-responsiveness（AHR）was assessed by detection the airway resistance（2）Part 2:A mouse model of neutrophil asthma was estabilished with OVA/CFA sensitization and challenge in mi R-223 knock-out(mi R-223^-/-)mice and WT mice with the same background.Real-time quantitative polymerase chain reaction（q PCR）was used to detect the expression of mi R-223 in lung tissue.HE staining and PAS staining were used to observe airway inflammation,bronchial epithelial goblet cell hyperplasia and mucus secretion;the total number of cells in BALF was counted,and the cellular profiles were counted after Wright-Giemesa staining;the AHR of mice were detected.The levels of Th1（IFN-γ）,Th2（IL-4,IL-5,IL-13）and Th17（IL-17A,IL-22,IL-23）and IL-1βand IL-18 in BALF were detected by ELISA.（3）Part 3:A mouse model of neutrophil asthma was estabilished in mi R-223^-/-mice and WT mice as described.The highly selective NLRP3 blocker（MCC950）or IL-1βreceptor antagonist（anakinra）were given to the OVA/CFA-sensitized mi R-223^-/-mice immediately after each challenge,respectively.Control mice were treated with the same volume of PBS for comparison.The level of NLRP3 inflammasome（NLRP3,ASC,Caspase-1）,IL-1βand IL-18 were detected by q PCR and Western blot at 24 hours after the last challenge.HE staining and PAS staining were used to observe airway inflammation,bronchial epithelial goblet cell proliferation and mucus secretion.The total number of cells in BALF was counted,and cellular profiles were counted after Wright-Giemesa staining;The AHR was assessed by detection the airway resistance.The levels of Th1（IFN-γ）,Th2（IL-4,IL-5,IL-13）and Th17（IL-17A,IL-22,IL-23）and IL-1βand IL-18 in BALF were determined by ELISA.（4）Part 4:WT mice were subsequently treated with mi R-223agomirs or negative control agomirs before each challenge.The level of mi R-223 in the lung was detected by q PCR.q PCR and Western blot were used to detect the levels of NLRP3 inflammatory bodies（NLRP3,ASC,Caspase-1）,IL-1βand IL-18 m RNA and proteins in lung tissue,respectively.HE staining and PAS staining were used to observe airway inflammation,bronchial epithelial goblet cell proliferation and mucus secretion.The total number of cells in BALF was counted,and cellular profiles were counted after Wright-Giemesa staining.The AHR was assessed by detection the airway resistance.The levels of Th1（IFN-γ）,Th2（IL-4,IL-5,IL-13）and Th17（IL-17A,IL-22,IL-23）and IL-1βand IL-18 BALF were determined by ELISA.Results（1）The EA group and the NA group mice showed varying degrees of irritability,arched back,shortness of breath,nod breathing,dull hair and other symptoms and signs.The total number of cells in BALF of EA and NA group were significantly increased.The proportion of eosinophil was mainly increased in EA group,and the proportion of neutrophil was mainly increased in NA group.The EA group was dominated by eosinophil infiltration,and the NA group was dominated by neutrophil infiltration.Both eosinophilic asthma and neutrophilic asthma show goblet cell hyperplasia and mucus over-production,but the changes in neutrophilic asthma are more serious.Airway hyper-responsiveness of both groups increased,but the increase in neutrophilic asthma was more significant.（2）The expression of mi R-223was upregulated in lung tissues of experimental mice model.Furthermore,mi R-223^-/-mice led to aggravated neutrophilic airway inflammation with heightened histopathological,inflammatory cells and cytokines readouts.（3）Mi R-223^-/-mice presented with enhanced NLRP3 inflammasome level with elevated IL-1β.Blocking NLRP3 or IL-1βdiminished this phenotype.（4）Overexpression of mi R-223 via treatment with mi R-223 agomirs attenuated airway inflammation,NLRP3 levels and IL-1βrelease.Conclusions The expression of mi R-223 was increased in the lungs of neutrophilic asthma mice,and mi R-223 konck-out can aggravate airway inflammation and AHR in neutrophilic asthma.The potential mechanism was that the deletion of mi R-223 leads to activate NLRP3 inflammasome with elevated IL-1βrelease,which promotes the polarization of Th17 cells and Th2 cells,and exacerbates airway inflammation and airway hyperresponsiveness in neutrophilic asthma.