Investigation Of The Roles And Targeted Intervention Of P38 MAPK In The Pathogenesis Of NTHi Induced Murine Neutrophilic Asthma Model

Bronchial asthma is a heterogeneous airways disease characterized by chronic airway inflammation.There are approximately 334 million people worldwide are suffering from asthma,including over 30 million Chinese patients,which makes China one of the countries with the highest asthma mortality rate.An excessive accumulation of eosinophils in the airways is a typical feature of asthma,and it is believed that Th1/Th2 immune imbalance is the main mechanism of asthma development.However,in recent years,studies have found large number of neutrophils accumulating in the airways of up to 50%of asthma patients.Therefore,in the previous study,we have proposed the concept of neutrophil asthma based on the technique of inducing sputum cell classification and counting,and confirmed that the acute attack of adult asthma is mainly neutrophil inflammatory subtype.Most of those patients’symptoms are not effectively controlled even when higher doses of steroids are given,clinically called glucocorticoid-resistant asthma.Althoμgh such patients account for only 5-10%of asthma,cost of treatment is approximately 50%of all asthma-related medical resources,and the pathogenesis of neutrophil asthma is still not yet clear.Numerous studies have shown that respiratory pathogens such as respiratory syncytial virus,rhinovirus,Chlamydia pneumoniae and Influenza virus are important causes of neutrophilic asthma.The infection leads to an abnormal accumulation of airway neutrophils,accompanied by an aggravated asthma symptoms and steroid resistance.Non-typeable Haemophilus influenzae(NTHi)as the most common opportunistic pathogen in the upper respiratory tract,has been proven to be one of the major pathogens of Chronic obstruction pulmonary disease(COPD)in adults.In our previous study,we have also confirmed that NTHi infection can lead to an imbalance in the Th17/Treg immune response in the lung of COPD mice,leading to an acute onset of COPD.NTHi infection has also been shown to be involved in the progression of asthma in clinical researches.NTHi has been detected in 60%of patients with neutrophilic asthma in induced sputum,and the detection rate in glucocorticoid-resistant asthma patients was as high as 78%of all asthmaics.However,the association between NTHi infection,neutrophil asthma and the mechanism of glucocorticoid resistance was also unclear.Although similar murine studies have focused on the association between NTHi infection and the pathogenesis of asthma,there were significant differences between them.NTHi infection in the early-life has been shown to induce AAD exacerbation,accompanied by an increase in Th2 immune response.In contrast,NTHi infection before OVA sensitization induced a Th17-mediated neutrophilic inflammation that was accompanied with steroid-resistance.This indicates that different timing of NTHi infection result in different immunological profiles in AAD.However,the pathogenesis of NTHi infection after asthma has not been reported.MAPK signaling pathway plays an important role in the pathogenesis of asthma and chronic obstructive pulmonary disease(COPD).It has been shown that over-activation of MAPK is closely related to the steroid resistance of COPD,but the roles of MAPK in the mechanism of neutrophilic asthma has not been clearly clarified.Therefore,this paper aims to establish a murine asthma model,then NTHi was inoculated intratracheally to simulate the pathogenesis of clinical neutrophil asthma,to elucidate the effect of NTHi infection on the progression of asthmatic disease.Furthermore,this paper also aims to investigate the roles of p38 MAPK signaling pathway in NTHi-induced neutrophilic asthma and to provide a new strategy for the clinical treatment of neutrophil asthma.OBJECTIVE:To elucidate the pathogenesis of neutrophilic asthma and provide a new strategy for the clinical treatment of glucocorticoid-resistant asthma.This paper aims to investigate the roles of P38 MAPK signaling pathway in NTHi-induced neutrophilic asthma and to determine if NTHi infection is the primary driver of neutrophilic asthma.1.Establishment of a murine model of NTHi infectionMETHODS:Different concentrations of NTHi were inoculated intranasally into the bronchi of mice to establish a model of respiratory infection.Bacterial plate culture method was used to detect the clearance of NTHi in the lung tissues of mice;pulmonary function was tested to detect the damage of lung function caused by NTHi infection;flow cytometry was utilized to determine the inflammatory cells in the alveolar lavage fluid(BALF)of mice and the number and proportion of Th17 cells in lung tissues;ELISA and HE staining were carried out to detect the contents of pro-inflammatory factors including IL-6,TNF-α,IL-1β,and the inflammation levels in lung tissues;immunohistochemical staining was utilized to determine the level of airway mucin.The expression levels of TLR2/4,MyD88,p38 MAPK,ERK1/2 and JNK1 and their activation levels(by phosphorylation)were detected by Western blot,and the role of MAPK signaling pathway in NTHi respiratory infection was explored.RESULTS:High dose(1×10~8 CFU/mL)of NTHi infection resulted in a significant increase in mortality rate in mice(p<0.01),which may be related to the decreased clearance of NTHi in lung tissues;NTHi could cause lung function decrease,high airway mucin secretion,and lower airway neutrophil inflammation in a dose-dependent manner,which might be possibly related to the excessive activation of Th17 cells;NTHi infection could lead to a higher expression of TLR4,MyD88 and promote the phosphorylation of p38 MAPK.2.Investigation of the effect of NTHi on OVA-sensitized asthmatic miceMETHODS:A murine model of asthma induced by OVA was well established.NTHi(1×108 CFU/mL,30 uL)was inoculated intratracheally 24 h after the last OVA challenge.After 1 h of NTHi infection,dexamethasone(DEX,2 mg/kg)was intraperitoneally injected.The effect of NTHi infection on airway hyperresponsiveness(AHR)in OVA mice was detected by small animal pulmonary function test;the expression levels of MUC5AC and MUC5B in the airway of OVA mice were detected via ELISA and immunohistochemistry;cell counting and flow cytometry analysis was utilized to detect the number and proportion of inflammatory cells in mouse BALF;HE staining was performed in mice lung tissues;Western blot was used to analyse the MAPK-related protein expression and phosphorylation level.RESULTS:OVA-induced asthmatic mice showed increased AHR,imbalanced mucus secretion,and Th2-mediated eosinophilic inflammation.However,the symptoms were significantly improved after treatment with DEX;the infection of NTHi 24 hours after OVA challenge further led to the deterioration of airway hyperresponsiveness and an excessive secretion of mucus;NTHi infection inhibited Th2 inflammatory response but promoted Th17 inflammatory response in airway;DEX treatment did not improve the symptoms of OVA/NTHi mice.The phosphorylation level of p38 MAPK was significantly increased in the lung tissues of OVA/NTHi mice.3.Targeted inhibition of p38 MAPK activity in the treatment of NTHi-induced neutrophil asthmaMETHODS:A murine model of OVA asthma was established.NTHi was inoculated intratracheally,24 hours after OVA challenge.Mice were then treated with p38 MAPK inhibitor SB203580 alone or with DEX(SB203580+DEX),and the effect of neutrophilic asthma was observed.Small animal lung function was tested to observe airway hyperresponsiveness in mice;flow cytometry and cell counting methods were used to detect the number and proportion of inflammatory cells in BALF;HE staining was applied to observe the level of lung inflammation in mice;immunohistochemistry was performed to see the expression of MUC5AC and MUC5B;Western blot was used to analyse the protein expression level of NF-κB.RESULTS:SB203580 alone treatment could significantly inhibit airway hyperresponsiveness and the secretion of MUC5AC/MUC5B in asthmatic mice,but failed to improve the Th17 neutrophilic response induced by NTHi infection effectively;the combination use of SB203580+DEX treatment significantly inhibited airway hyperresponsiveness,high mucus secretion and Th17-mediated neutrophil inflammatory response in OVA/NTHi mice;in addition,western blot results showed that treatment with SB203580+DEX could significantly inhibit p65 NF-κB protein expression and its phosphorylation level.Conclusion:1.NTHi infection induced neutrophil inflammatory response is associated with the activation of TLR4-MyD88-p38 MAPK signaling pathway.2.After NTHi infection,the Th2 inflammatory response in allergic asthma could be converted into Th17 inflammatory response,which in turn might induce the occurrence of neutrophilic asthma.3.Combined treatment of p38 MAPK inhibitor and glucocorticoid could effectively improve airway hyperresponsiveness,mucus hypersecretion and inflammatory response in neutrophilic asthma.Targeted inhibition of p38 MAPK could significantly improve glucocorticoid-resistant neutrophil asthma.Innovation:1.Our study established the NTHi infection-induced neutrophilic asthma model,simulating the pathogenesis of clinical neutrophil inflammatory subtype asthma.Our study showed that the Th2 inflammatory response of allergic asthma could be converted into Th17 inflammatory response after NTHi infection,which in turn might induce the occurrence of neutrophilic asthma.2.Our study discovered that combined treatment of p38 MAPK inhibitors and glucocorticoids could effectively improve airway hyperresponsiveness,mucus hypersecretion and inflammatory response in neutrophilic asthma,proposing that p38MAPK might be a potential treatment target for the glucocorticoid-resistant neutrophil asthma.