【作者】 刘超；

【导师】 徐峰；

【作者基本信息】 浙江大学 ， 内科学（呼吸系病）， 2019， 博士

【摘要】 金黄色葡萄球菌(金葡菌,Staphlococcus aureus)是引起人类化脓性感染最常见的病原菌,既可以导致皮肤软组织感染、肺炎、胸膜炎、心包炎等局部感染,也可以导致败血症等全身性感染。金葡菌具有极强的致病力,能产生大量侵袭性物质,具有很高致死率,是引起重症肺炎最常见的致病菌之一。近年来的一份国内细菌耐药的检测报告显示:甲氧西林耐药金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus,MRSA)的检出率已经上升到35.8%,而且大多数MRSA菌株呈多重耐药。近年来还出现对糖肽类中介敏感的金黄色葡萄球菌(Glycopeptides-intermediate resistant Syaphylococcus aureus,GISA)。因此,除加快新型抗生素的研发以外,进一步明确机体天然免疫系统抵御金葡菌感染的调控机制,是感染免疫学领域的重要科学问题。肥大细胞(Mast cells,MC)大多分布于皮肤和内脏粘膜,作为一种免疫细胞不仅可以监视病原体的入侵,而且其来源的细胞介质也能快速作用于其他炎性细胞和血管内皮细胞,并经血液循环分布到全身各处。在急性炎症中,一系列细胞因子通过级联反应促使肥大细胞数量增加并且活化。肥大细胞活化后会产生脱颗粒现象,可以释放蛋白酶和诸如白介素(interleukin,IL)、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)等细胞因子。另一方面,也有研究发现肥大细胞具有吞噬和清除细菌的能力,并且随着细菌刺激量增加,肥大细胞分泌TNF-α水平也相应增多。TNF-α能够将中性粒细胞等炎性细胞招募到炎症部位,以加强机体对病原体的清除能力。当前,多项研究发现肥大细胞在不同病原体感染中发挥着重要的免疫功能,然而关于肥大细胞在金葡菌感染中的作用机制却未见深入阐明。因此探索肥大细胞在金葡菌感染免疫中的新作用,有助于寻找金葡菌感染新的干预靶点,为疾病诊疗提供新的手段。第一部分肥大细胞参与金黄色葡萄球菌肺炎肺肠轴的调控目的:从肺部肥大细胞介导肺肠轴的角度,探讨肥大细胞在金黄色葡萄球菌肺炎发病中的作用和机制。方法:用野生型C57BL/6小鼠(WT)和肥大细胞缺陷小鼠(KitW-sh/W-sh)构建金葡菌肺炎模型。取肺组织病理切片进行中性粒细胞计数和炎症评分;ELISA检测各组肺组织炎症因子和抗菌肽表达;免疫荧光明确肺部肥大细胞来源的CRAMP的表达量;CFU平板计数法测定肺组织细菌载量;16S rRNA测序对肠道粪便进行检测,分析肠道菌群的构成。此外,分别给予WT小鼠和KitW-sh/W-sh小鼠进行骨髓来源的肥大细胞(bone marrow mast cells,BMMC)和 Cathelicidin 相关抗菌肽(Cathelicidin-related antimicrobial peptide,CRAMP)的回输:ELISA 法检测各组小鼠肺部炎症因子和抗菌肽的表达;CFU测定肺组织细菌载量;16S rRNA测序对肠道粪便进行检测,分析肠道菌群的组成。结果:与WT小鼠相比,肥大细胞缺陷小鼠(KitW-sh/W-sh)感染金葡菌后肺组织中性粒细胞浸润降低,炎症评分下降,炎症因子IL-6,KC,MIP-2,TNF-α表达下调,抗菌肽CRAMP的表达降低,肺组织细菌载量则明显增高。同时免疫荧光显示感染组WT小鼠肺组织肥大细胞表达CRAMP明显增高。16S rRNA测序结果表明与WT小鼠相比,KitW-sh/W-sh小鼠肠道有害菌变形菌门的丰度增高,益生菌包括拟杆菌门和厚壁菌门的丰度下降。回输BMMC后,KitW-sh/W-sh小鼠肺组织IL-6,KC,MIP-2,TNF-α,CRAMP表达较未回输组上调,细菌负荷显著下降,肠道菌群失调得到纠正。气道给予CRAMP后,尽管KitW-sh/W-sh小鼠炎症因子IL-6,KC,MIP-2,TNF-α的表达水平没有明显变化,但肺组织细菌负荷显著降低。结论:肥大细胞参与了金葡菌肺炎肺肠轴的调控;肥大细胞激活可以改善金葡菌肺炎伴发的肠道菌群紊乱;金葡菌肺炎中CRAMP参与肺部肥大细胞介导的抗菌免疫。第二部分TNF-α参与肥大细胞介导的抵御皮肤金葡菌感染的免疫反应目的:探讨肥大细胞参与金葡菌皮肤感染的抗菌免疫机制,为金葡菌皮肤感染的诊疗提供新的思路。方法:用WT小鼠和KitW-sh/W-sh小鼠构建金葡菌皮肤感染模型。观察小鼠背部疤痕的大小;CFU平板计数法测定皮肤组织细菌载量;取皮肤组织病理切片进行中性粒细胞计数和炎症评估;qRT-PCR和ELISA检测皮肤组织炎症因子和抗菌肽表达;免疫荧光检测皮肤肥大细胞来源的TNF-α的表达量。此外,分别对WT小鼠和KitW-sh/W-sh小鼠进行BMMC或TNF-α回输:观察小鼠背部疤痕大小的变化;CFU平板计数法测定皮肤组织细菌载量;取皮肤组织病理切片进行中性粒细胞计数和炎症评估;ELISA检测皮肤组织炎症因子和抗菌肽表达。结果:与WT小鼠相比,KitW-sh/W-sh小鼠在感染金葡菌后皮肤疤痕面积增大,细菌载量增高,皮肤组织中性粒细胞聚集减少,炎症因子(IL-6,IL-17A,TNF-α)和抗菌肽(CRAMP)水平降低,其中TNF-α下降最为明显。免疫荧光显示WT小鼠感染后皮肤组织肥大细胞表达TNF-α明显增高。回输BMMC或TNF-α后,KitW-sh/W-sh小鼠皮肤损伤减轻,组织细菌负荷降低,皮肤组织中性粒细胞聚集增多,部分炎症因子和抗菌肽表达上调。结论:肥大细胞参与了抗皮肤金葡菌感染的宿主免疫;肥大细胞激活可以减轻金葡菌造成的皮肤损伤和细菌负荷;TNF-α参与皮肤肥大细胞介导的抗菌免疫。第三部分C-kit受体信号通路介导肥大细胞抗菌免疫目的:通过体外实验观察c-kit受体信号通路对肥大细胞抗金葡菌感染的影响,揭示肥大细胞抗菌免疫分子机制。方法:选择骨髓来源的肥大细胞BMMC以及肥大细胞株P815建立金葡菌体外感染模型,干预组给予c-kit受体特异性抑制剂Masitinib。qRT-PCR检测各组炎症因子(IL-1β,IL-6,IL-17A,KC,MIP-2,TNF-α)和抗菌肽(CRAMP,RegⅢγ)的表达;观察肥大细胞的杀菌作用;Western blot检测ERK,JNK,P38,PI3K,AKT,P65-NF-κB的磷酸化情况。分别应用Wortmannin和PDTC干预,qRT-PCR和ELISA法观察炎症因子及抗菌肽的变化。结果:与未感染组相比,感染组肥大细胞BMMC和P815释放炎症因子(IL-1β,IL-6,KC,MIP-2,TNF-α)和抗菌肽(CRAMP)增多,应用Masitinib后炎症因子(IL-6,TNF-α)和抗菌肽(CRAMP)表达下降。细胞杀菌实验发现肥大细胞P815具有杀菌功能。Western blot结果表明金黄色葡萄球菌感染后肥大细胞c-kit受体下游PI3K,AKT,P65-NF-κB发生了磷酸化,应用Masitinib后,磷酸化受抑制。应用Wortmannin或PDTC干预后,肥大细胞的TNF-α和CRAMP表达下降。结论:C-kit依赖的PI3K/AKT/P65-NF-κB的信号通路参与肥大细胞对金葡菌的抗菌免疫反应。更多还原

【Abstract】 Staphylococcus aureus(S.aureus),which is the most common pathogen causing human pyogenic infection,could lead to skin and soft tissue infection,pneumonia,pleurisy,pericarditis,septicemia and other systemic infections.S.aureus with high pathogenicity and fatality rates,is one of the most common pathogen that cause severe pneumonia.Recently,one national bacterial resistance monitoring report showed that the detection rate of Methicillin-resistant S.aureus(MRSA)climbed to 35.8%,and most MRSA strains had multiple drug resistance.In recent years,Glycopeptides-intermediate resistant S.aureus(GISA),which is sensitive to glycopeptide intermediates,has also appeared.Therefore,in addition to development of new antibiotics,how to resist S.aureus infection by regulating the innate immune system,is also an important scientific issue in immunology of infectionMast cell(MC)is a kind of immune cells widely distributed in the skin tissue and internal organs.It can not only monitor the invasion of pathogens all the time,but also release medium and cytokines which rapidly affect the inflammatory cells in vascular endothelial cells and blood,and distribute to various tissues and organs through blood.In acute inflammation,a cascade of cytokines lead to the activation of MCs.Activated MCs could release a lot of inflammatory factors,such as interleukin and tumor necrosis factor-a.In addition,some studies have found that MCs play an important role in phagocytosis and sterilization.With the increase of bacteria,MCs-derived TNF-a increased accordingly.TNF-a could recruit inflammatory cells such as neutrophils to inflammatory sites,and enhance the ability to clear bacteria.Currently,several studies have found that MCs regulate series of immune responses in different pathogen infections.However,the mechanism of MCs in S.aureus infection has not been fully elucidated at present.Therefore,exploring the new role of MCs in the immune response to S.aureus infection will be helpful in finding new intervention target for S.aureus infection and providing new mean for disease treatment.Part I Mast Cells Participate in Regulation of Lung-gut Axis during Staphylococcus aureus PneumoniaObjective:We examined the role and mechanism of lung mast cells(MCs)in the regulation of the lung-gut axis during S.aureus pneumonia.Methods:We created a mouse model of S.aureus pneumonia using wild type mice(WT)and MC-deficient mice(KitW-sh/W-sh).H&E-stained lung tissue sections were used for neutrophil count and inflammation score.The protein levels of inflammatory factors and antimicrobial peptide in lung tissues were detected by ELISA.Immunofluorescence was used to examine the expression of CRAMP in lung MCs.The bacterial load of lung tissues was measured by CFU plate counting.16S rRNA sequencing was used to detect intestinal feces and analyze the composition of intestinal flora.In addition,WT mice and mice were given the bone marrow mast cell(BMMC)and cathelicidin-related antimicrobial peptide(CRAMP),respectively.The expressions of inflammatory factors and antimicrobial peptides were detected by ELISA.The bacterial load of lung tissue was measured by CFU plate counting.16S rRNA sequencing was used to detect intestinal feces and analyze the composition of intestinal flora.Results:After S.aureus challenge,KitW-sh/W-sh,compared with WT mice,displayed decreased the number of neutrophils,inflammatory score,the expression of inflammatory factors and CRAMP.KitW-sh/W-sh mice also experienced higher bacterial load in lung tissues.The expression of CRAMP in lung MCs of WT mice also increased.16S rRNA sequencing showed that compared with WT mice,KitW-sh/W-sh mice have increased abundance of Proteobacteria in the intestinal tract,while decreased abundance of the Bacteroidetes and Firmicutes.An adoptive MC transfer experiment was performed and constituted KitW-sh/W-sh mice experienced increased expression of inflammatory factors and CRAMP,and improved intestinal flora imbalance.Although there was no statistically significant difference in the expression of IL-6,KC,MIP-2,TNF-a in KitW-sh/W-sh mice after CRAMP administration,the bacterial load of the lung tissues decreased.Conclusion:MCs are involved in the regulation of lung-gut axis in S.aureus pneumonia;MCs activation could improve intestinal flora disorder associated with S.aureus pneumonia;CRAMP participates in the antibacterial immunity mediated by lung MCs in S.aureus pneumonia.Part Ⅱ TNF-α is Required for Mast Cell-mediated Immune Response against Cutaneous Staphylococcus aureus InfectionObjective:We explored the antibacterial immune mechanism of MCs participating in skin S.aureus infection and provided a new theoretical basis for the diagnosis and treatment of skin S.aureus infection.Methods:WT mice an KitW-sh/W-sh mice were selected to build skin infection model.The lesion area on the back of mice were observed.The bacterial load of skin tissue was measured by CFU plate counting.The skin histopathological sections were obtained for neutrophil counting and inflammation assessment.The expressions of inflammatory cytokines and antimicrobial peptides were detected by qRT-PCR and ELISA.Immunofluorescence was used for detecting the expression of MC-derived TNF-a.In addition,BMMC and TNF-a were injected into WT mice and KitW-sh/W-sh mice,respectively.The lesion area of mice were observed.The bacterial load of skin tissue were measured by CFU plate counting.The skin histopathological sections were obtained for neutrophil counting and inflammation assessment.Inflammatory cytokines and antimicrobial peptides were detected by ELISA.Results:Compared to WT mice,KitW-sh/W-sh mice developed significantly larger skin lesions after the cutaneous S.aureus challenge,while MC dysfunction reduced the inflammation response to S.aureus.The level of TNF-α in skin tissues were significantly decreased in KitW-sh/W-sh mice upon infection.Moreover,the exogenous administration of MCs or recombinant TNF-α effectively restored the immune response against S.aureus in KitW-sh/W-sh mice via the recruitment of neutrophils to the infected site.Conclusion:MCs are involved in host immunity against skin S.aureus infection;MCs activation could relieve skin damage and bacterial load caused by S.aureus;TNF-a is involved in anti-acterial immunity mediated by MCs in skin S.aureus infectionPart Ⅲ C-kit Receptor Signaling Pathway Mediates Mast Cell Antimicrobial ImmunityObjective:We explored the effect of c-kit receptor signaling pathway of mast cells in anti-S.aureus infection in vitro in order to determine the antibacterial immune mechanism of mast cellsMethods:The in vitro model of S.aureus infection were build by BMMC and P815 cells.C-kit receptor specific inhibitor Masitinib was used in intervention group.qRT-PCR was used to detect the expression of inflammatory factors(IL-1β,IL-6,IL-17A,KC,MIP-2,TNF-a)and antimicrobial peptides(CRAMP,RegⅢγ).The bactericidal effect of mast cells was also observed.The phosphorylation of ERK,JNK,P38,PI3K,AKT and P65-NF-κB were detected by Western blot.The expression of inflammatory factors and antimicrobial peptides were examined by qRT-PCR and ELISA after treatment with Wortmannin or PDTC.Results:Compared with the uninfected groups,infected BMMC and P815 showed increased release of inflammatory factors(IL-1β,IL-6,KC,MIP-2,TNF-α)and CRAMP,while decreased expression of inflammatory factors(IL-6,TNF-α)and CRAMP after the application of Masitinib.The result of bactericidal experiment indicated that P815 cells have direct bactericidal function.Result from Western blot showed that PI3K,AKT,and P65-NF-κB were phosphorylated after Staphylococcus aureus infection and the level of phosphorylation decreased after treatment with Masitinib.The expression of mast cell-derived TNF-a and CRAMP were decreased after treatment with Wortmannin and PDTC,respectively.Conclusion:C-kit-dependent PI3K/AKT/P65-NF-κB signaling pathway participates in mast cells-mediatedantimicrobial immune response to S.aureus.更多还原