【作者】 李俊；

【导师】 郑芳；

【作者基本信息】 华中科技大学 ， 免疫学， 2020， 博士

【摘要】 背景:心脏移植已成为治疗终末期心脏病的主要选择。尽管免疫抑制剂的发现确实很大程度上提高了受者的生存率,但其副作用,如感染仍然是移植后导致死亡的主要原因之一。肺部是心脏移植患者感染的主要部位。呼吸道病毒,如流感病毒,被认为是移植后肺部感染的潜在病因。高迁移率族蛋白1(High-mobility group box 1,HMGB1)是一种DNA结合蛋白,参与并维持核小体结构和调节基因的转录。HMGB1可以由免疫细胞和非免疫细胞释放到细胞外基质中,以应对各种刺激。释放的HMGB1参与到许多疾病的发生发展,包括心脏移植排斥反应中的移植物损伤和炎症反应。本实验室前期研究表明,中和HMGB1可延长移植物存活时间,减轻同种异体的移植排斥反应。为了研究中和HMGB1改善移植物存活同时,对受者应对病原体相关分子模式(Pathogen-associated molecular patterns,PAMPs)的影响,移植后受者鼠经气道给予poly(I:C)以模拟病毒感染。我们发现,受者鼠经poly(I:C)刺激后,支气管肺泡灌洗液内HMGB1水平升高,肺炎加重。而在中和HMGB1后,减轻其肺组织病理学损伤、中性粒细胞浸润和炎性细胞因子释放,并对IFN-β水平无影响。因此中和HMGB1或能为移植受者带来双重利好,即延长移植物存活同时,维持防御相关反应,而这一特点区别于传统的免疫抑制剂。基于以上前期研究基础,为进一步研究移植受者在中和HMGB1后应对呼吸道病毒挑战的应答,我们选用H1N1病毒进行后续研究。在过去的甲型H1N1流感大流行中,接受实体器官移植的患者与普通患者相比,在H1N1感染后出现并发症的风险更高,且在移植早期感染时更容易出现并发症。H1N1感染在移植受者中引起的死亡率明显高于普通人感染H1N1后的死亡率。目前,疫苗接种是预防流感病毒最有效的方式。然而,流感疫苗的效力在使用了免疫抑制剂的实体器官移植病人中明显降低。因此,寻找合适的药物在延长移植物存活的同时降低感染后发病率和死亡率对移植受者来说至关重要,而现有的免疫抑制剂无法达到这一效果。基于以上原因,我们进一步研究了HMGB1单克隆抗体在抑制排斥反应的同时,是否影响受者对病毒感染的反应,并设计了本课题。为模拟受者在使用免疫抑制剂时感染病毒的实际临床情境,我们建立了心脏移植+HMGB1单抗+病毒感染的小鼠模型,研究HMGB1单抗对受者鼠病毒感染整体表现(严重程度、生存、恢复等)的影响、对肺部炎症和抗病毒的固有免疫细胞的影响,以及HMGB1单克隆抗体造成上述影响的机制,以期为心脏移植并发感染的治疗提供新思路。目的:本研究拟探讨中和HMGB1对心脏移植小鼠流感肺炎的影响。方法:首先建立小鼠心脏移植急性排斥反应模型,BALB/c小鼠作为供者提供供心,C57BL/6小鼠作为受者进行腹部异位心脏移植。受者小鼠从移植当天开始接受抗HMGB1单克隆抗体处理,并在移植后第3天接受H1N1病毒感染,以模拟临床移植用药情况下的移植后早期感染。实验中,我们首先进行了全病程观察,后续利用苏木素-伊红染色法、实时荧光定量聚合酶链反应、酶联免疫吸附测定、蛋白质印迹法、免疫荧光、免疫组化、流式细胞术等技术对病程中免疫指标和HMGB1相关的发病机制进行了研究。结果:1.抗HMGB1单抗对H1N1病毒感染的心脏移植受者鼠起保护作用:中和HMGB1可延长移植物的存活时间,提高感染后生存率,并且加速受者鼠感染后恢复。2.HMGB1单抗的应用对肺部病理及炎症反应的影响:减轻流感肺炎受者鼠肺部病理损伤程度、肺内炎症细胞因子水平,降低中性粒细胞和间质巨噬细胞这类促炎细胞的比例,不影响保护性细胞因子和保护性免疫细胞(CD103~+DC)水平,提高保护性免疫细胞AMs(肺泡巨噬细胞)的比例。3.HMGB1水平在受者鼠H1N1病毒感染后升高。4.HMGB1在H1N1病毒感染的肺泡Ⅱ型上皮细胞中易位和释放。5.HMGB1受体TLR4和RAGE在受者鼠肺泡Ⅱ型上皮细胞中高表达。6.HMGB1可通过TLR4受体而非RAGE受体激活肺泡Ⅱ型上皮细胞中的MAPK通路和下游趋化因子的产生。7.心脏移植会升高受者鼠肺内HMGB1基础水平。体外实验中,微环境HMGB1升高导致肺泡Ⅱ型上皮细胞H1N1感染后释放趋化因子升高。这可能使得移植病人在应对H1N1感染时存在更高的肺损伤风险。结论:综上所述,中和HMGB1在心脏移植受者中有潜在的应用价值。它在延长移植物存活同时,减轻感染后肺部炎症反应,并提高生存率,加速感染后恢复,即在受者H1N1感染中起保护作用。并且我们发现,移植小鼠感染后体重下降的程度缓于感染后的普通小鼠,提示正常小鼠的感染情况不能完全反映移植后受者感染的情况。心脏移植受者的肺部HMGB1水平升高,同时微环境中HMGB1水平升高会加重肺泡Ⅱ型上皮细胞感染H1N1后释放的趋化因子KC和CCL2的水平,这可能是移植病人在面对感染时存在更高的肺损伤风险的原因之一。因此中和HMGB1为器官移植后用药提供了新思路,和以往的免疫抑制剂不同的是,它或能在抑制移植排斥反应的同时,在受者的病毒感染中起保护作用。更多还原

【Abstract】 Background:Heart transplantation has become the major therapy choice for advanced heart disease.Although the advances of immunosuppressant,such as tacrolimus and cyclosporine,do improved the survival of recipients to a large extent,infections remain a leading cause of post transplantation mortality,especially in the early stage after transplantation.Pneumonia is the major cause of infection in heart transplant patients.Respiratory viruses,such as influenza virus,have worked up as potential cause of pneumonia after transplantation.Recipients of solid-organ transplants with H1N1,the cause of most recent influenza pandemic,are at increased risk of complications compared to the general population,especially in the early post-transplant period.Mortality caused by H1N1 is higher in transplant recipients compared to healthy individuals.Thus,exploring appropriate drugs to reduce morbidity and mortality while prolonging the survival of graft is paramount in recipients.High-mobility group box 1(HMGB1),one of the DAMPs,initially identified as a DNA-binding protein involved in maintaining nucleosome structure and regulating gene transcription.It can be released into the extracellular matrix from immune and non-immune cells in response to varieties of stimuli.Released HMGB1 also contributes to the pathogenesis of numerous diseases,including graft injury and the inflammatory response in heart transplantation.HMGB1 blockaded significantly prolongs the survival of allograft and alleviates the allograft rejection.Preliminary studies in our laboratory showed that neutralizing HMGB1 could maintain recipient mice defenseassociated response against LPS and poly(I:C)challenges.Therefore,we propose the following scientific question: whether neutralizing HMGB1 can prolong the graft survival without affecting the recipient’s defense respond to influenza virus.Objective:To investigate the effect of anti-HMGB1 monoclonal antibody on influenza virus induced pneumonia in recipient mice.Methods:In order to simulate the clinical situation,the recipient mice were treated with HMGB1 monoclonal antibody from the day of transplantation,and then infected with H1N1 virus on the 3rd day after heart transplantation.In the experiment,we first observed the whole course of the disease,and then used H&E,Q-PCR,ELISA,Western blot,immunofluorescence,immunohistochemistry,flow cytometry etc technologies to study the immune system and the pathogenesis of HMGB1 during the course of the disease.Results:In the current study,we probed the protective effects of anti-HMGB1 m Ab in the cardiac transplant model challenged with H1N1 virus.1.Blockade of HMGB1 prolonged the survival time of graft and accelerated the recovery of infected recipient mice.2.The administration of HMGB1 m Ab display attenuated severity of inflammatory injury and accumulation of inflammatory cells.The neutralization of HMGB1 mitigated inflammatory injury and reduced lung leucocyte and interstitial macrophages in the lung of H1N1-infected HTx mice.3.Influenza virus infection in recipient mice resulted in the increase of HMGB1.4.H1N1 virus infection induces the translocation and release of HMGB1 in AEC II.5.TLR4 and RAGE,the receptors of HMGB1,were highly expressed in AEC II.6.In AEC II,HMGB1 could activate MAPK pathway through TLR4 signaling,but not RAGE.7.Heart transplantation changed the basic level of HMGB1 in lung of recipients.In vitro,the increase of HMGB1 in microenvironment led to the increase of chemokines released from type Ⅱ alveolar epithelial cells after H1N1 infection.This may lead to a higher risk of lung injury in transplant patients in the face of infection.Conclusion:Together,HMGB1 m Ab has potential application value in heart transplant recipients.It not only prolonged the graft survival,but also reduced the pulmonary inflammatory response after infection,protected the resistance of recipients to pathogenic microorganism infection,improved the survival rate and accelerated the recovery after infection.In addition,we found that the weight of the infected recipient mice was different from that of the infected normal mice,suggesting that the infection of the normal mice could not fully reflect the infection of the transplanted mice.We found that the increased level of HMGB1 in the microenvironment could aggravate the level of chemokine KC and CCL2 released by alveolar type II epithelial cells after H1N1 infection,which may lead to a higher risk of lung injury in the challenge of infection.Therefore,blocking HMGB1 provides a new idea for drug use after organ transplantation.Different from the previous immunosuppressants,HMGB1 may be able to inhibit the rejection of transplantation without affecting the recipient’s defense against virus infection.更多还原