【作者】 李媛媛；

【导师】 罗征秀；

【作者基本信息】 重庆医科大学 ， 临床医学（专业学位）， 2024， 博士

【摘要】 第一部分新生期肺炎链球菌感染对肠道菌群及肺部维生素A水平的影响目的:生命早期是肠道菌群、肺功能发育的关键时间窗,研究发现在此期间抗菌药物暴露可影响肠道微生态发育,参与哮喘等过敏性疾病发生。众所周知,临床实践中细菌感染是抗菌药物应用指征,早期细菌感染是否对发育不成熟的肠道微生物菌群产生影响尚不清楚。研究发现,肠道菌群可通过影响维生素A转化、肠道上皮吸收、转运蛋白表达等方式影响其在体内的代谢。肺炎链球菌(Streptococcus pneumoniae,Sp)是儿童下呼吸道感染第一位细菌病原体,课题组既往研究发现新生期肺炎链球菌感染(Streptococcus Pneumoniae infection,S.p)导致小鼠肺部维生素A(Vitamin A,VA)持续降低,但机制尚不清楚,本部分研究拟分析新生期肺炎链球菌感染对小鼠肠道菌群的影响,及其与肺部维生素A水平的关系。方法:7日龄Balb/c小鼠随机分成对照组、S.p组,S.p组小鼠给与鼻腔滴注5*10^6 CFU肺炎链球菌D39菌株建立S.p感染模型,对照组给与等量PBS滴鼻。在哺乳期(感染后1周,1wpi)、婴儿期(感染后2周,2wpi)和成年期(感染后7周,7wpi)时收集小鼠盲肠粪便标本进行16S rRNA测序。同时,收集婴儿期小鼠肺组织、盲肠粪便标本进行非靶向代谢组学检测。结果:S.p组较对照组小鼠在哺乳期、婴儿期、成年期肠道菌群α、β多样性、群落结构、肠道微生物组成均发生显著改变。其中,在哺乳期、婴儿期时,S.p组小鼠肠道中乳酸菌属(Lactobacillus)丰度显著降低,而成年期时异杆菌属(Allobaculum)、变形菌门(Proteobacteria)丰度显著增加。对肠道菌群改变进行PICRUSt2功能预测发现,菌群改变对代谢、降解相关通路有显著影响,其中包括维生素合成通路。非靶向代谢组学发现,S.p组小鼠粪便、肺组织中代谢产物种类较对照组均有显著差异。尽管S.p组小鼠粪便中VA活性产物(全反式视黄酸、9-顺式-视黄酸)水平无明显差异,但粪便差异代谢物KEGG通路分析发现,维生素消化吸收通路显著富集;S.p组小鼠肺组织中9-顺式-视黄酸水平较对照组显著降低,肺组织差异代谢物KEGG通路分析发现PPAR通路显著富集。结论:新生期肺炎链球菌感染诱导小鼠肠道菌群持续性紊乱,包括肠道菌群群落结构及组成改变;推测可能通过影响肠道中VA的吸收,致肺部VA降低。第二部分新生期肺炎链球菌感染降低肺部维生素A,激活Notch1信号,抑制BC向Club细胞分化目的:前一部分研究结果发现,新生期S.p感染导致肠道菌群持久性改变,通过PICRUSt2进行功能预测,发现菌群改变对维生素合成通路有明显影响,S.p组小鼠肺组织中9-顺式-视黄酸水平较对照组显著降低。VA常转化为视黄酸(Retinoid acid,RA)发挥生物活性,9-顺式-视黄酸是RA的一种异构体形式。VA具有维持上皮细胞完整性和细胞损伤后修复功能。课题组前期研究发现S.p感染诱导小鼠肺部VA持续性降低及气道上皮损伤,促进哮喘发生;感染后补充VA抑制哮喘形成,VA如何参与气道上皮损伤修复尚不清楚。Club细胞是主要由基底细胞(Basal cell,BC)分化而来的气道多能干细胞,具有修复受损细胞维持气道上皮完整性功能。CC16是Club细胞特异性分泌的肺部保护性蛋白,临床、动物研究均证实,CC16水平降低可增加哮喘发生风险。Notch是高度保守的信号通路,可参与细胞增殖分化调节,Notch1已被证实可调控BC分化进程。研究报道,RA可与其受体结合形成复合物,进而结合于Notch1启动子区域,调控Notch1表达。S.p感染后肺部VA降低是否通过Notch1信号通路影响Club细胞水平,参与气道上皮损伤修复尚不清楚。故此部分主要研究新生期S.p感染后肺部VA水平与Notch1、Club细胞水平的关系。方法:7日龄Balb/c小鼠随机分成对照组、S.p组、S.p+VA组、S.p+DAPT组。S.p组小鼠给与鼻腔滴注5*10^6 CFU肺炎链球菌D39菌株建立S.p感染模型,对照组给与等量PBS滴鼻。S.p感染第0天,S.p+VA组小鼠给与20 IU/g全反式视黄酸灌胃(每天一次,共4天);S.p感染第7天,S.p+DAPT组小鼠给与鼻腔滴注0.3 mg/kg的Notch1抑制剂DAPT。分别于小鼠发育至幼年期、成年期时,RT-PCR 检测 Notch1-mRNA 表达水平,Western Blot 检测 Notch1 蛋白水平;ELISA检测BALF、血清CC16水平;收集肺组织,免疫荧光染色、流式细胞学检测Club细胞数量;流式细胞学检测Club细胞增殖水平;免疫荧光共聚焦检测BC向Club细胞分化水平;成年期监测肺功能、肺组织病理染色。结果:1.S.p组小鼠发育至幼年期、成年期时肺部Notch1表达水平显著升高、Club细胞数量明显降低,血清及BALF中CC16水平均显著降低,但Club细胞增殖水平无显著改变;2.S.p+VA组小鼠发育至成年期时肺部Notch1表达水平较S.p组明显降低,Club细胞数量较S.p组显著增多;3.S.p+DAPT组小鼠发育至成年期时Club细胞数量较S.p组增多,血清及BALF中CC16水平增加,气道周围炎症水平、气道高反应性较S.p组明显降低;4.S.p组小鼠肺部检测到一种BC向Club细胞分化的中间状态细胞(KRT5+CC16+细胞),KRT5+CC16+细胞在S.p组小鼠发育至幼年期、成年期时均较对照组显著增加,感染后阻断Notch1可致KRT5+CC16+细胞数量显著降低。结论:新生期肺炎链球菌感染降低肺部VA,激活Notch 1信号,阻碍BC向Club细胞分化进程,导致Club细胞数量及分泌CC16减少,促进气道上皮损伤、气道高反应性发生。第三部分Club细胞源性HMGB1通过TLR4/SMMHC诱导新生期肺炎链球菌感染后气道高反应性形成目的:第二部分研究结果发现,新生期S.p感染导致Club细胞数量减少,BALF、血清中CC16表达水平显著降低。研究发现,气道收缩性是决定气道高反应性的关键因素。高迁移率族蛋白 B1(High Mobility Group Box-1 Protein,HMGB1)是一种重要的损伤相关分子模式(Damage associated molecular patterns,DAMPs),分泌至细胞外不仅可发挥类炎症因子活性,还可与下游受体结合,调控气道平滑肌肌球蛋白重链(Smooth Muscle Myosin Heavy Chain,SMMHC)表达。Club 细胞是感染后表达病原相关分子模式的重要气道上皮细胞,在RSV感染小鼠模型中,Club细胞是气道HMGB1的主要细胞来源;同时可分泌炎症因子调控气道平滑肌蛋白表达水平,影响气道收缩力。课题组既往研究发现,S.p感染小鼠发育至成年期时SMMHC表达水平显著升高,但SMMHC高表达是否与Club细胞分泌HMGB1、促进SMMHC高表达相关尚不清楚。故本部分研究拟进一步分析S.p感染后气道HMGB1的表达水平及来源;并探讨HMGB1调节对SMMHC表达的机制。方法:1.7日龄Balb/c小鼠随机分成对照组、S.p组,S.p小鼠给与鼻腔滴注5*10^6 CFU肺炎链球菌D39菌株建立S.p感染模型,对照组给与等量PBS滴鼻。于哺乳期、幼年期、成年期收集小鼠BALF检测HMGB1表达水平;成年期RT-PCR检测TLR4-mRNA表达水平,Western Blot检测TLR4、TLR2、RAGE蛋白表达水平;免疫组织化学、免疫荧光染色检测TLR4、HMGB1表达水平及组织定位。2.建立对照组、S.p组、S.p后TLR4阻断(S.p+TAK-242)组,S.p+TAK-242组小鼠在S.p感染后第14天给与腹腔注射3 mg/kg的TAK-242(两天一次,共三周),对照组、S.p组给与等量PBS+DMSO腹腔注射。于成年期检测小鼠肺功能,RT-PCR检测Myh11-mRNA表达水平,Western Blot检测TLR4、SMMHC蛋白表达水平;免疫组织化学检测气道上皮SMMHC表达水平;免疫荧光染色检测TLR4与SMMHC表达相关性。结果:1.S.p后小鼠BALF中HMGB1表达水平持续升高至成年期;免疫组化定位显示HMGB1主要表达于气道上皮;进一步共聚焦检查发现,气道上皮中高表达HMGB1的细胞主要为Club细胞;2.S.p组小鼠发育至成年期时,肺组织中TLR4-mRNA、蛋白表达均显著升高,且主要表达于气道上皮细胞;但TLR2、RAGE表达无显著差异;3.与S.p组相比,S.p感染TAK-242组小鼠肺组织中TLR4蛋白表达显著降低,肺组织中Myh11-mRNA、SMMHC蛋白表达显著降低;且TLR4与SMMHC表达水平负相关;4.S.p组小鼠阻断TLR4后AHR显著降低。结论:新生期肺炎链球菌感染后,Club细胞源性HMGB1通过TLR4诱导SMMHC高表达,促进气道高反应性发生。更多还原

【Abstract】 PART ONE THE EFFECT OF NEONATAL STREPTOCOCCUS PNEUMONIAE INFECTION ON GUT MICROBIOME AND LUNG VITAMIN AObjective:Early life represents a "critical window" for gut micriboita and lung function development.Antibiotic exposure during this period potentially leads to long-lasting effects on gut microbiota and asthma development.In clinical practice,bacterial infection is the indication of antibiotics,while the influence of early-life bacterial infection on gut microbiome still unclear.It has been observed that gut micriboita can influence the metabolism of vitamin A(VA)in vivo by affecting its transformation,intestinal epithelium absorption,and transporter protein expression.Streptococcus pneumoniae(S.p)is the prominent bacterial pathogen causing lower respiratory tract infections in children.Our previous study demonstrated that neonatal Streptococcus pneumoniae infection(S.p)could induce persistent lung VA decrease in adulthood,while the mechanisms remains unclear.Therefore,this part mainly studies the effects of S.p infection on mouse gut microbiome and its relationship with lung VA levels.Methods:Neonatal(7-day-old)Balb/c mice were randomly allocated into control and S.p groups.The S.p group mice were inoculated with 5*10^6 CFU D39 to establish non-lethal S.pneumoniae pneumonia model,while the Control group received an equal volume of PBS.At breastfeeding period(1 week post infection,lwpi),infancy(2 week post infection,2wpi),adulthood(7 week post infection,7wpi),feces in the cecum were prepared for 16S rRNA sequencing.Eventhough,cecum fecal specimens and lung tissues in infancy were collected for untargeted metabolomics.Results:The S.p group mice exhibited significant alterations in the alpha and beta diversities of the entire gut microbiota,as well as changes in community structure throughout the breastfeeding period,infancy,and adulthood.Furthermore,the gut microbial composition was modified after S.p infection,with a decreased relative abundance of Lactobacillus during breastfeeding period and infancy;in adulthood,the relative abundance of Allobaculum decreased while that of Proteobacteria increased.Further functional prediction using PICRUSt2 revealed that S.p infection-induced gut microbial dysbiosis had a profound impact on metabolic and degradation related pathways,with a notable enrichment of vitamin metabolism producing pathways.Non-targeted metabolomics revealed significant differences in the types of metabolites in the faeces and lung tissue of mice in the S.p group compared to the control group.Eventhough,S.p infection had no significant effect on the levels of VA active metabolite(all-trans-retinoid acid and 9-cis-retinoid acid)in feces,fecal differential metabolite KEGG pathway analysis revealed a significant enrichment of the vitamin digestive and absorption pathway;9-cis-retinoic acid levels in lung tissues of mice in the S.p infection group were significantly reduced compared with those of the Control group,and lung tissue differential metabolite KEGG pathway analysis revealed a significant enrichment of the PPAR pathway.Conclusion:Neonatal S.pneumoniae infection induced a long-lasting dysbiosis of gut microbiome,including changes in the structure and composition of the microbial community,which may affect the digestion and absorption of VA in the intestinal tract,possibly resulting in a reduction of VA in the lungs.PART TWO NEONATAL STREPTOCOCCUS PNEUMONIAE INFECTION REDUCES LUNG VITAMIN A,ACTIVATES NOTCH1 SIGNALLING AND INHIBITES BC DIFFERENTIATION TO CLUB CELLSObjective:The first part found that neonatal S.p infection induced long-lasting gut microbial dysbiosis in mice,functional prediction by PICRUSt2 revealed that gut microbial dysbiosis had a profound impact on metabolic and degradation related pathways,resulting in decreased 9-cis-retinoid acid in lung tissue of the S.p group.VA is often converted to retinoid acid(RA)for biological activity,9-cis-retinoid is an isomer of RA.VA has the ability to maintain epithelial cell integrity and repair after injury.Our previous studies found that S.p infection induces lung VA decrease and airway epithelial injury,promoting asthma development;VA supplementation after S.p infection significantly alleviates the AHR induced by S.p infection,but whether VA is involved in and its mechanisms still unclear.Club cells are airway pluripotent stem cells that differentiate primarily from basal cells(BC),which have the function of repairing damaged cells to maintain the integrity of the airway epithelium.CC16 is a protective protein specifically secreted by Club cells,has been demonstrated associated with the risk of asthma.The Notch pathway is a highly conserved signaling pathway that can be involved in the regulation of the proliferation and differentiation,and Notchl has been shown to influence the process of BC differentiation.Studies have reported that VA can bind to its receptor to form a complex,which in turn binds to the Notch1 promoter region and participates in the regulation of Notch1 expression.It is not clear whether decreased VA in the lungs after S.p infection is involved in club cell repairment and airway epithelial injury via Notch1.Therefore,this part of the study will focus on the relationship among reduced lung VA after Sp infection,Notch1 and club cells.Methods:Neonatal(7-day-old)Balb/c mice were randomly allocated into control,S.p infection,S.p+VA,S.p+DAPT groups.The S.p group of mice were inoculated with 5*10^6 CFU D39 to establish non-lethal S.pneumoniae pneumonia model,while the Control group received an equal volume of PBS.On the day 0th S.p infection,S.p+VA group were administrated orally with 20 IU/g all-trans retinoid acid every day for 4 days;on the day 7th of S.p infection,S.p+DAPT group were inoculated with 0.3 mg/kg DAPT,while the Control,S.p and S.p+VA group received an equal volume of PBS.At infancy and adulthood,RT-PCR was used to detect Notchl-mRNA expression level and Western Blot to detect Notchl protein level;ELISA was used to detect CC16 concentrations in BALF and serum;immunofluorescence staining and flow cytometry were used to detect the number of Club cells;flow cytometry and confocal assay were used to detect the proliferation of Club cells and the differentiation of Basal cells to Club cells.In adulthood,lung pathological changes and airway hyperresponsiveness were assessed.Results:1.The S.p group presented significantly higher Notchl expression,lower Club cell quantification and lower CC16 concentration in infancy and adulthood;while showed no significant changes in the Club cells proliferation;2.The S.p+VA group of mice has reduced Notchl expression and increased club cell quantification in adulthood compared to S.p group;3.Compared to the S.p group,S.p+DAPT mice had higher numbers of club cells and increased levels of CC16,while had alleviated peribronchiolar inflammation and airway hyperresponsiveness;4.An immature Club cell(KRT5+CC16+cell),was detected in the lungs of mice in S.p group,which was an intermediate differentiation state from BC to Club cells.The level of KRT5+CC16+cells in S.p group was significantly increased in both infancy and adulthood compared to the Control group,but then significantly decreased after Notchl inhibition.Conclusion:Neonatal S.pneumoniae infection reduces lung VA,which activates Notchl signalling,and hinders the process of BC differentiation to Club cells,leading to a reduction in the number of Club cells and the concentration of CC16,which induces airway epithelial damage and airway hyperresponsiveness.PART THREE CLUB CELL DERIVED HMGB1 INDUCES AIRWAY HYPERRESPONSIVENESS FOLLOWING NEONATAL STREPTOCOCCUS PNEUMONIAE INFECTION VIA TLR4/SMMHCObjective:The second part of this study found that the number and function of Club cells were impaired in mice after S.p infection.Airway contractility was found to be a key determinant of airway hyperresponsiveness.High Mobility Group Box-1 Protein(HMGB1)is a crucial damage associated molecular patterns(DAMP),not only exhibiting inflammatory factor like activity,but also binds to downstream receptors to regulate the expression of airway smooth muscle protein SMMHC.Club cells are important airway epithelial cells that express pathogen associated molecular patterns(PAMPs)after infection.In RSV-infected mice model,Club cells have been found to be the main cell source of airway HMGB1;they also secrete inflammatory factors that regulate the level of airway smooth muscle protein expression and affect airway contractility.Our previous study observed a significant up-regulation of airway smooth muscle myosin heavy chain(SMMHC)expression in S.p mice in adulthood,but whether high SMMHC expression is associated with the secretion of HMGB1 from Club cells,which promotes high SMMHC expression,is not clear.Therefore,in this part,we will further investigate the expression level and source of airway HMGB1 after S.p infection;and to explore the mechanism of HMGB 1 regulation on SMMHC expression.Methods:1.Neonatal(7-day-old)Balb/c mice were randomly allocated into Control,S.p groups.The S.p group of mice were inoculated with 5*106 CFU D39 to establish non-lethal S.pneumoniae pneumonia model,while the Control group received an equal volume of PBS.At breastfeeding period,infancy and adulthood,HMGB1 in BALF were analyzed by ELISA.When reached adulthood,the mice were sacrificed for lung tissue collecting.The mRNA expression levels of TLR4 was assessed using RT-PCR,the protein expression levels of TLR4,TLR2 and RAGE were analyzed by Western Blot,the expression and localization of TLR4 and HMGB 1 were detected by immunohistochemistry and immunofluorescence staining.2.Mice were randomly allocated into Control,S.p,S.p+TAK-242 groups.On the day 14th of S.p infection,S.p+TAK-242 group were administrated intra-peritoneally with 3mg/kg TAK-242 every other day for 3 weeks;while the Control and S.p group received an equal volume of PBS+DMSO.In adulthood,the mRNA level of Myhll was assessed using RT-PCR,the protein expression levels of SMMHC was assesed by Western Blot and immunohistochemistry,AHR was evaluated by penh.In addition,the expression correlation between TLR4 and SMMHC expression was measured by immunofluorescence staining.Results:1.HMGB1 expression levels in BALF of mice after S.p infection continued to be elevated until adulthood;immunohistochemistry localized that HMGB1 was predominantly expressed in the airway epithelium;further confocal assay revealed that the overexpressed HMGB1 was mainly from the Club cells;2.In adulthood,the expression of TLR4-mRNA and protein was significantly elevated in lung tissues and mainly expressed in airway epithelial cells in S.p group;while we found no significant differences in TLR2 and RAGE expression between the two groups.3.Compared with the S.p group,TLR4 expression was significantly inhibited in the lung tissues of S.p+TAK-242 mice,with significantly lower Myhll-mRNA and SMMHC protein expression.The expression of TLR4 was negatively correlated with SMMHC,and both of them were significantly elevated in the airway epithelium;4.TLR4 inhibition significantly reduced AHR induced by S.p infection.Conclusion:After neonatal Streptococcus pneumoniae infection,Club cell-derived HMGB1 induces SMMHC overexpression through TLR4 pathway,promoting airway remodeling development.更多还原