【作者】 杨玲；

【导师】 徐金富；

【作者基本信息】 同济大学 ， 内科学， 2022， 博士

【摘要】 背景:铜绿假单胞菌(Pseudomonas aeruginosa)是一种经典的革兰氏阴性杆菌,肺部铜绿假单胞菌的感染会引起严重的肺组织破坏和肺功能下降。重要的是,在中国的成年支气管扩张症患者中,从肺中分离出的铜绿假单胞菌对支气管扩张症患者的预后有重要的指示作用。在以往的研究中,铜绿假单胞菌被定义为细胞外病原体。然而,近期的一些研究强调有不少的细胞外病原体可以进入宿主细胞的内部,让该菌在细胞内部拥有生存能力。另外,还有研究人员提出在铜绿假单胞菌急性感染期间,气道上皮细胞可以在细菌的生物膜形成之前将细菌吞入细胞中。此外,还有研究人员将铜绿假单胞菌和上皮细胞共培养后,展示了该细菌进入细胞的特定过程。另一项研究更加深入地揭示了铜绿假单胞菌在共培养的巨噬细胞中的命运,揭示了由细胞内部的铜绿假单胞菌驱动的巨噬细胞裂解过程。重要的是,与细胞外浮游条件下的铜绿假单胞菌相比,细胞内的铜绿假单胞菌对生长抑制剂或抗生素的作用不敏感,这些细胞内部的细菌能够穿透上皮内细胞,从而导致慢性感染的发生。众多的报道让学者们明白铜绿假单胞菌不仅仅是一种细胞外病原体。因此,揭示细胞内铜绿假单胞菌的免疫逃逸机制可能为清除肺部铜绿假单胞菌感染提供新思路。铜绿假单胞菌是一种机会性致病菌,在入侵宿主后,能够以多种方式激活固有免疫。I型干扰素也是固有免疫的重要组成部分。体内的多种细胞都可以在跨膜或者胞质受体的刺激下产生I型干扰素。在大多数细胞中,诱导I型干扰素产生的主要途径是通过激活细胞质中的相关受体,而这些受体可以识别病毒或自身的核酸。I型干扰素在抵御病毒感染中有重要的作用,然而关于I型干扰素是否能清除细菌感染,众多的研究说法不一。目前,较为公认的说法是I型干扰素通过诱导CCL2趋化因子,与免疫细胞上的CCR2受体结合,促进免疫细胞的招募,从而对李斯特菌和结核杆菌进行清除。但相反地,I型干扰素也可以抑制CXCR2的配体CXCL1和CXCL2的产生,从而减少中性粒细胞的招募,抑制宿主对继发性细菌感染的防御。迄今为止,关于I型干扰素和细胞内部铜绿假单胞菌的生存是否存在联系尚无相关报道。且调控这一现象的信号通路仍未知。在病原体入侵宿主免疫细胞时,细胞内部的炎性体会被激活,炎性体是一个蛋白复合体,其主要作用是激活Caspase-1的功能。一旦Caspase-1被激活,会切割IL-18前体(Pro-IL-18)和IL-1β的前体(Pro-IL-1β),Pro-IL-18和Pro-IL-1β被切割后,产生成熟体IL-18和IL-1β释放到细胞外,从而放大前炎症反应。而铜绿假单胞菌与炎性体之间的联系十分密切。铜绿假单胞菌的III型分泌系统将该细菌的鞭毛蛋白运输到细胞浆中,进入细胞内的鞭毛蛋白激活细胞中的NLRC4炎性体,促进IL-1β的释放。另外,铜绿假单胞菌对于NLRP3炎性体的激活也有所报道。近几年来有不少人在研究炎性体和IFN-β两者之间的关系。有报道指出在病毒感染的模型中,Caspase-1可以切割cGAS,使cGAS酶活性下降,达到降低IFN-β产生的目的。此外,NLRC4能促进TBK1的泛素化降解,进一步减少IFN-β产生。但是在铜绿假单胞菌感染的过程中,我们还不能确定是否也存在同样的调控机制,这一点值得深入的探索。因此在本课题中,我们以细胞内部铜绿假单胞菌的生存,I型干扰素和炎性体为研究背景,探索了这三者的相关性以及互相调控的信号通路。方法:细胞实验:用铜绿假单胞菌感染细胞不同的时间,用Western Blot方法测定细胞中TBK1,P-TBK1,IRF3,P-IRF3,P-AKT的表达量。q RT-PCR测定细胞中IFN-β的m RNA表达水平。ELISA试剂盒测定细胞上清中IFN-β蛋白表达水平以及细胞内部cGAMP的含量。共聚焦扫描显微镜观察细胞内部中铜绿假单胞菌的生存以及数量。利用庆大霉素处理细胞外的细菌,再加入细胞裂解液,将细胞内部的细菌进行稀释涂板,计算细胞内的菌荷量以及细菌生存的百分比。用RNASequence技术分析细胞中受影响的信号通路。动物实验:通过舌下呛咳的方式,将铜绿假单胞菌打进小鼠的肺中,构建铜绿假单胞菌急性肺部感染模型。感染24小时后取小鼠肺组织,提取小鼠肺组织RNA,通过反转录,q RT-PCR测定肺组织中IFN-β的m RNA表达水平。取肺组织进行H&E染色,对其病理损伤进行评分。研磨肺组织,对肺组织匀浆液稀释涂板,计算肺部菌荷量。离心肺组织匀浆液,留取上清,用ELISA试剂盒测定上清中IFN-β蛋白表达水平。结果:在本研究中通过共聚焦扫描显微镜的观察,我们看到铜绿假单胞菌可在细胞内部生存。IFN-β蛋白与没有细胞存在的铜绿假单胞菌共培养时,铜绿假单胞菌的生长无明显变化。但是IFN-β蛋白促进了细胞内部铜绿假单胞菌的生存。急性铜绿假单胞菌感染的小鼠肺组织中的IFN-β的m RNA表达水平显著上升。我们用铜绿假单胞菌感染cGAS基因敲除小鼠的腹腔巨噬细胞,STING基因敲除小鼠腹腔巨噬细胞和TBK1抑制剂的处理过的巨噬细胞,我们发现与对照组相比,这三个实验组中IFN-β的表达量呈断崖式下降。其次,为了验证铜绿假单胞菌可以通过NLRP3炎性体和NLRC4炎性体诱导IL-1β的产生,我们又引入NLRP3基因敲除小鼠和NLRC4基因敲除小鼠。在NLRP3基因敲除小鼠的腹腔巨噬细胞和NLRC4基因敲除小鼠腹腔巨噬细胞中,加入铜绿假单胞菌感染,IL-1β的产生是显著下调的,但是IFN-β的表达明显上升。与这一结果一致的是,在加入NLRP3激动剂后,感染过铜绿假单胞菌的细胞中IFN-β的表达量有所下降。此外,加入Caspase-1抑制剂后,感染过铜绿假单胞菌的细胞中IFN-β的表达量有所上升。紧接着我们再引入IL-1受体抑制剂和IL-1β蛋白,发现IL-1β减少了IFN-β的表达。而这其中的机制是IL-1β促进了AKT的磷酸化,减少细胞内部cGAMP和IFN-β的产生。将AKT抑制剂预处理细胞后,再加入铜绿假单胞菌感染,细胞内cGAMP和IFN-β的产生明显增加。在动物实验中,与WT小鼠对比,IFN-β敲除小鼠在感染铜绿假单胞菌后肺部的损伤明显减轻,肺部的病理评分下降,肺部的菌荷量也显著下调。结论:综上所述,我们证明了在铜绿假单胞菌感染巨噬细胞的过程中,该菌能够诱导IL-1β和IFN-β的产生。但是铜绿假单胞菌通过NLRP3、NLRC4炎症小体复合物诱导的IL-1β可以有效抑制IFN-β的产生。NLRP3基因敲除和NLRC4基因敲除的小鼠增强了铜绿假单胞菌诱导的IFN-β反应。而IFN-β有利于细胞内铜绿假单胞菌的生存。在机制上,IL-1β通过激活AKT激酶,进一步降低了cGAMP的生成,减少了IFN-β的产量。而下降的IFN-β又能够降低细胞内铜绿假单胞菌的生存。因此,我们发现了铜绿假单胞菌可以通过IL-1β-AKT-cGAMP-IFN-β轴调节了其在细胞内生存这一新途径。这一发现为开发针对肺部铜绿假单胞菌感染患者的临床新方法提供了理论基础。更多还原

【Abstract】 BackgroundPseudomonas aeruginosa(P.aeruginosa,PA)is a conditionally pathogenicGramnegative bacillus and connected with severe lung destruction and reduced lung function.And the isolation of P.aeruginosa in Chinese adult patients with bronchiectasis is a significant prognostic indicator.P.aeruginosa is defined as extracellular pathogen classically.But recent studies have highlighted the presence of an extracellular pathogen that can enter the interior of the host cell,where the bacterium has viability.Some researchers have proposed that during the acute infection period of P.aeruginosa,the airway epithelial cells could engulf the bacteria into the cells before the biofilm is formed.In addition,others have co-cultured P.aeruginosa and epithelial cells to show the specific process of bacteria entering the cell.Another study shed more light on the fate of P.aeruginosa in co-cultured macrophages,shedding light on the macrophage lysis process driven by P.aeruginosa inside cells.A study which traced the P.aeruginosa inside epithelial cells now makes it clear to researchers that P.aeruginosa is not simply an extracellular pathogen.To this end,revealing immune escape mechanism of intracellular P.aeruginosa may provide new ideas for combating pulmonary P.aeruginosa infection.As a kind of opportunistic pathogen,P.aeruginosa activates innate immunity in several ways.Interferon is an important part of innate immunity.A variety of cells in the host can produce type I interferons under the stimulation of transmembrane or cytoplasmic receptors.In most cell types,the primary pathway for inducing type I interferon production is the activation of associated receptors in the cytoplasm that recognize viral or other xenogeneic or own nucleic acids.Type I interferon plays an important role in defending against viral infections.However,there are different opinions about whether type I interferon could clear bacterial infection.At present,it is generally accepted that type I interferon induces CCL2 chemokine,which binds the chemokine to the CCR2 receptor on immune cells and promotes the recruitment of immune cells,thereby eliminating Listeria and Mycobacterium tuberculosis.But type I interferons also inhibit the production of CXCR2 ligands,CXCL1 and CXCL2,thereby reducing neutrophil recruitment and suppressing host defenses against secondary bacterial infection.Up to now,there has been no report on whether type I interferons are related to the survival of P.aeruginosa inside the macrophages.And the signaling pathway that regulates this phenomenon is still unknown.Therefore,in this topic,we took the intracellular survival of P.aeruginosa type I interferon and inflammasome as the background to explore the correlation of these three and the signaling pathways that regulate each other.MethodsIn vitro: The cells were infected with P.aeruginosa for different times,and the expression levels of TBK1,P-TBK1,IRF3,P-IRF3,and P-AKT in the cells were determined by Western Blot.The m RNA expression level of IFN-β in cells was determined by q RT-PCR.ELISA kits were used to measure the expression level of IFN-β protein in the cell supernatant and the content of cGAMP inside the cell.The survival and number of P.aeruginosa in the cell were observed by confocal scanning microscope.The bacteria outside the cells were treated with gentamicin,the cell lysate was added after the gentamicin,and the bacteria inside the cells were diluted and plated,and the bacterial load in the cells and the percentage of bacterial survival were calculated.Analysis of affected signaling pathways in cells using RNA-Sequence technology.In vivo: P.aeruginosa was injected into the lungs of mice by coughing under the tongue to construct an acute lung infection model of P.aeruginosa.Twenty-four hours after infection,the lung tissue of mice was collected,and RNA was extracted from the lung tissue of mice.The m RNA expression level of IFN-β in lung tissue was determined by reverse transcription and q RT-PCR.Lung tissue was taken for H&E staining and the pathological damage was scored.Lung tissue was ground,and the lung tissue homogenate was diluted and plated,and the bacterial load in the lung was calculated.The lung tissue homogenate was centrifuged,the supernatant was collected,and the expression level of IFN-β protein in the supernatant was measured by ELISA kit.ResultsThrough confocal scanning microscopy observations in this study,we saw that P.aeruginosa can survive inside cells.When IFN-β protein was co-cultured with P.aeruginosa without cells,the growth of P.aeruginosa did not change significantly.But the IFN-β protein promoted the survival of P.aeruginosa inside the cell.The m RNA expression level of IFN-β in the lung tissue of mice infected with acute P.aeruginosa was significantly increased.When we infected peritoneal macrophages of cGAS knockout mice,STING knockout mouse peritoneal macrophages and TBK1 inhibitortreated macrophages with P.aeruginosa,we found that,compared with controls,the expression of IFN-β in each experimental group decreased sharply in these three groups.Second,to verify that P.aeruginosa can induce IL-1β production through NLRP3 inflammasome and NLRC4 inflammasome,we introduced NLRP3 knockout mice and NLRC4 knockout mice.The production of IL-1β was significantly down-regulated,but the expression of IFN-β was significantly increased in the peritoneal macrophages of NLRP3 knockout mice and NLRC4 knockout mouse after infection with P.aeruginosa infection.Consistent with this result,IFN-β expression was decreased in P.aeruginosainfected cells after the addition of NLRP3 agonists.In addition,the expression of IFN-β in cells increased after the addition of caspase-1 inhibitor.Following the introduction of IL-1 receptor inhibitor and IL-1β protein,we found that IL-1β reduced the expression of IFN-β.Mechanistically,IL-1β promotes phosphorylation of AKT and reduces intracellular cGAMP and IFN-β production.After the cells were pretreated with AKT inhibitor and then infected with P.aeruginosa,the production of intracellular cGAMP and IFN-β was significantly increased.In animal experiments,compared with WT mice,IFN-β knockout mice significantly reduced lung damage after infection with P.aeruginosa,the pathological score of the lungs decreased,and the bacterial load in the lungs was also significantly down-regulated.ConclusionIn conclusion,we demonstrated that P.aeruginosa can induce the production of IL-1β and IFN-β.However,IL-1β induced by P.aeruginosa through the NLRP3 and NLRC4 inflammasome complexes can effectively inhibit the production of IFN-β.Similarly,NLRP3 knockout and NLRC4 knockout mice enhanced P.aeruginosainduced IFN-β responses.IFN-β was beneficial to the survival of intracellular P.aeruginosa.Mechanistically,IL-1β further reduces cGAMP production and IFN-βproduction by activating AKT kinase.The decreased IFN-β could reduce the intracellular survival of P.aeruginosa.Therefore,we discovered a novel pathway that P.aeruginosa can promote its intracellular survival through the IL-1β-AKT-cGAMPIFN-β axis.This finding provides a rationale for developing a new clinical approach to patients with P.aeruginosa infection in the lungs.更多还原