| Background: Bronchiectasis is a chronic respiratory disease with typical clinical manifestations of cough,excessive sputum and radiologically by abnormal dilatation of the bronchi.Pseudomonas aeruginosa(P.aeruginosa)is a conditionally pathogenic gram-negative bacterium,which is one of the most important pathogens in bronchiectasis.Respiratory colonization of P.aeruginosa could lead to severe destruction of lung tissue and a decrease in lung function.At present,antibiotics are still the main clinical treatment for P.aeruginosa infection.However,the excessive application of broad-spectrum antibiotics has significantly increased the resistance rate of P.aeruginosa to antibiotics.Clinicians are still lack of effective intervention methods for P.aeruginosa colonization,which is the main clinical problem that needed to be solved urgently.As a common disease that seriously endangered the public health of patients,the pathogenesis of idiopathic bronchiectasis is still unclear.At present,the "vicious circle" hypothesis was widely accepted,in which chronic infection and airway inflammation played an important role in the development of the disease.Some markers in exhaled breath condensate(EBC)had been proven to be able to reflect airway inflammation.Currently,p H is considered to be one of the most promising variables of EBC,and airway acidification have been reported in many respiratory diseases such as chronic obstructive pulmonary disease,cystic fibrosis and asthma.However,when it comes to bronchiectasis,studies are relatively scarce.There is an urgent need for prospective and high-quality studies to clear the microenvironment p H in the airway of bronchiectasis and its relationship with disease severity and prognosis.Bacteria release membrane vesicles(membrane vesicles,MVs)with a diameter of 20 to 200 nm,which affect a variety of biological processes.MVs are produced by controlled blistering of the outer membrane of gram-negative bacteria,thus they are called outer membrane vesicles(OMVs).P.aeruginosa is a classic gram-negative bacterium that secretes OMVs,which contain lipopolysaccharides(LPS),deoxyribonucleic acid(DNA),ribonucleic acid(RNA),etc.It had been reported that OMVs activated a series of immune signals and amplified the inflammatory response in host cells,aggravating exacerbating injury.The release of bacterial OMVs could be regulated by some physical and chemical factors.However,whether environmental p H could also affect the release of PA＿OMVs or its pathogenicity has not been reported.Previous studies had shown that PA infection was often accompanied by high levels of type I interferonβ(IFN-β)production,which promoted the formation of biofilm and aggravated lung damage by increasing neutrophil extracellular traps and reactive oxygen species production.These were beneficial for the bacteria to escape the immune clearance.Besides,many studies had shown that purified OMVs activated caspase-11 dependent immune responses,and OMVs associated peptidoglycan could activate NOD-like receptor signaling and NFk B signaling.However,whether OMVs could induce host type I interferons response is unclear.Together,the study aimed to explore the influence of airway acidification on the disease progression of bronchiectasis and its underlying molecular mechanisms.Methods: The main research contents of this project included identifying the status of airway microenvironment in patients with stable idiopathic bronchiectasis and its relationship with disease severity and prognosis;elaborating the molecular mechanism of acidic microenvironment impaired host defense against P.aeruginosa infection;Exploring new intervention methods for the treatment of bronchiectasis complicated with refractory P.aeruginosa infection.Based on the above three main research contents,patients with stable idiopathic bronchiectasis and healthy controls were included in a prospective cohort study from the clinical point of view.The baseline characteristics of two groups were collected,including age,gender,height,weight and BMI index.At the same time,the important clinical indicators of the patient group were recorded in detail,including lung function,arterial blood gas parameters,peripheral blood inflammation indicators,sputum bacterial culture results,BSI score and SGRQ score.The exhaled breath condensate was collected and standardized p H test was performed in both groups.All patients were followed up for one year,including the time to first acute exacerbation and the number of acute exacerbations.In terms of mechanism research,effects of normal and acidic cell culture environments on the adhesion and invasiveness ability of P.aeruginosa were determined by in vitro experiments,and the effects of acidic microenvironment on the release of P.aeruginosa outer membrane vesicles and its induced type 1 interferonβproduction were further explored.Interferon alpha receptor 1(IFNAR1)knockout mice and interferon regulatory factor 3(IRF3)knockout mice were used in this study.Effects of intratracheally acid pretreatment on mouse P.aeruginosa lung infection and downstream important target genes regulated by acidic microenvironment were lung tissue,bacterial load of lung tissue,lung inflammation and the survival of mice were observed in this study.Results: A total of 82 patients with stable idiopathic bronchiectasis and 40 healthy controls were included in the study.One patient was lost to follow-up during the oneyear period,so 81 patients with stable idiopathic bronchiectasis and 40 healthy controls were included in the final data analysis.Results showed that the standardized EBC p H of bronchiectasis patients was significantly lower than that of healthy controls(7.67(0.68)vs 8.03(0.33),p<0.001),and there was a significant negative correlation between standardized EBC p H and the severity score of bronchiectasis disease(r =-0.4822,p<0.001).The standardized EBC p H of patients with severe bronchiectasis was significantly lower than that of patients with mild and moderate bronchiectasis(7.14(1.67)vs 7.72(0.39),p=0.003;7.14(1.67)vs 7.79(0.35),p<0.001).Patients with EBC p H<7.755 had significantly shorter time to first acute exacerbation than those with EBC p H≥7.755(217.000(95%CI 185.270-248.730)vs257.379(95%CI 211.897-302.862),p=0.039),indicating that airway acidification was closely related to the severity and prognosis of bronchiectasis.Mechanistically,it was found that acidic airway microenvironment could impair host defense against P.aeruginosa pulmonary infection through promoting the release of P.aeruginosa outer membrane vesicles in vitro and enhancing its induced the activation of IRF3 protein,leading to a significant increase in IFN-β production.Excessive IFN-β production promoted the adhesion and intracellular survival ability of P.aeruginosa.Results of animal experiments showed that the lung tissue damage,bacterial load and IFN-βexpression level in lung tissue of mice were significantly increased following by P.aeruginosa pulmonary infection with intratracheally acid pretreatment compared with those without.Flow cytometric staining analysis of inflammatory cells in alveolar lavage fluid of mice showed that intratracheally acid pretreatment promoted the late apoptosis and necrosis of neutrophils after P.aeruginosa infection in mice,and accelerate the death of mice after infection.Targeted knockout of IRF3 or IFNAR1 alleviated lung injury and mortality of mice after P.aeruginosa infection that exacerbated by acidic airway microenvironment.Conclusion: Standardized EBC p H was closely related to disease severity and prognosis,which was a good candidate for biomarker of disease.The study revealed a novel mechanism of the effect of airway acidic microenvironment on the IFN-βresponse in the host after P.aeruginosa pulmonary infection,suggesting that,in addition to airway alkalizing therapy,inhibiting IFN-β production may be another novel intervention for the treatment of chronic airway inflammatory disease.The study provided new ideas for explaining the potential mechanism of acidic airway microenvironment in aggravating certain chronic airway inflammatory diseases and provided one more reference parameter for drug selection and new drug discovery for bronchiectasis. |
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