【作者】 钱乾；

【导师】 徐薇； 熊思东；

【作者基本信息】 苏州大学 ， 免疫学， 2014， 博士

【摘要】 病毒性心肌炎（Viral myocarditis，VMC）是一类由病毒感染引起的以心肌局灶性或弥漫性炎症为主要特征的临床常见心血管系统疾病。VMC全球发病率估计在0.12-12%之间，近年来我国VMC发病率呈上升趋势，发病率约为5％～15％，已成为青少年猝死的主要原因之一。急性VMC患者起病隐秘性和缺乏治疗手段，因此其真实发病率往往被低估。急性VMC还可迁延进展为慢性心肌炎或致死性扩张性心肌病（Dilated cardiomyopathy，DCM）。VMC已成为影响人类健康的一类重要的心血管感染性疾病，然而迄今为止尚无有效的治疗方法，根本原因在于VMC的发病机制尚未明确。因此，阐明VMC的发病机制具有重要的理论和实践意义。病毒感染是VMC发病的主要病因。肠道病毒、腺病毒、流感病毒、巨细胞病毒等都能诱发VMC，其中肠道病毒尤其是B3型柯萨奇病毒（Coxsackievirus B type3，CVB3）感染引起的VMC占肠道病毒感染诱发的心肌炎病人数一半以上。CVB3为微小无衣壳正链RNA病毒，属于小核糖核酸病毒科。CVB3经粪口途径感染食道和胃肠道之后，经由柯萨奇-腺病毒受体（Coxsackie-adenovirus receptor，CAR）和辅助受体衰变加速因子（Decay accelerating factor，DAF）感染肠道，病毒入血后感染胰腺和心肌细胞，在感染的第7-10天诱导急性胰腺炎和急性心肌炎，并在感染一个月后可诱导慢性心肌炎。病毒性心肌炎的发病机制长期受到关注：已知CVB3的裂解性复制直接破坏心肌细胞；CVB3还通过编码Pro2A和衣壳蛋白VP2诱导心肌细胞凋亡。研究报道VMC易感性不同的雌雄小鼠在病毒感染后第7天，心肌中的病毒载量相当。此外，利用基因敲除小鼠（TLR3-/-, TRIF-/-, CD4-/-, MyD88-/-等）证明易感性不同的小鼠却有着相似的心肌病毒载量，提示病毒复制在CVB3诱导的心肌炎发病中并非至关重要的因素。越来越多的研究结果表明，虽然CVB3在感染早期可直接导致心肌细胞坏死，但CVB3感染诱导的心肌局部炎症细胞浸润和炎症损伤是VMC发病的主要原因。心肌局部浸润的免疫细胞包括Th1、Th17淋巴细胞、M1型巨噬细胞和嗜中性粒细胞，以巨噬细胞为主。上述免疫细胞分泌的炎症细胞因子如IFN、TNF、IL-6、IL-17是主要的心肌炎炎症相关细胞因子。我们实验室前期研究给VMC小鼠输注Th2型细胞因子如IL-4和IL-13和M2型巨噬细胞或体内诱导Th2型极化具有治疗VMC的作用。众所周知，固有免疫是机体抗感染过程中的第一道防线。近年来，固有免疫越来越受到关注，因其在启动和调控后续适应性免疫应答格局起重要作用，并且固有免疫也不再是人们之前认为的“非特异”免疫。固有免疫细胞如巨噬细胞和DC表面或胞内的多种模式识别受体（PRRs）通过识别病原体特定的病原相关分子模式（PAMPs），激活固有免疫信号通路，启动促炎因子和I型干扰素（）分泌和抗原提呈相关分子（MHC II和B7）高表达，因此固有免疫对特异性免疫应答T、B淋巴细胞的活化不可或缺。而固有免疫一旦失控往往导致各类炎性疾病，如炎症性肠病（IBD）和自身免疫病（SLE）。以往对VMC的研究重点大多放在适应性免疫应答，特别是Th1、Th17、Treg等在VMC发病中的作用及机制，而针对VMC固有免疫的研究较少。因此，本研究提出如下假设：在炎症性Th1/Th17应答诱导之前，心脏局部的炎症性固有免疫对于炎症的诱导和放大、对于Th1/Th17应答的激活发挥了关键的作用，经心脏局部PRR信号通路激活的炎症性固有免疫在VMC发病中扮演关键的初始作用。研究CVB3感染早期在肠道和心脏局部激活的关键的模式识别受体，及其启动的促炎信号和炎症效应及抗病毒效应，进而对炎性Th1/Th17细胞浸润至心肌组织造成免疫损伤的影响，对于阐明VMC的发病机制提出了创新性的思路。过去的研究提示TLR3/TLR4/TLR7/8/9和MDA5都参与了抗CVB3的固有免疫的激活，其中TLR3激活上调的表达发挥了显著的心肌炎保护作用而TLR4激活则加剧心肌炎症和小鼠死亡，MDA5在VMC中的作用仍存在争议。除了上述PRRs，我们认为应全局性地动态检测CVB3感染后肠道和心脏局部所有TLRs和病毒识别RLRs的表达谱系改变，从中筛选出与VMC发病最为相关的PRR，进行深入研究非常重要。由此，我们发现了过去未曾报道的新PRR---TLR13。TLR13是近年才发现的一类表达于细胞内体膜上的新型PRR，迄今为止其功能未完全阐明。2012年发现TLR13的天然配体是革兰氏阳性和阴性细菌23S rRNA上的一段保守序列；TLR13还被报道参与对水疱口炎病毒（VSV）的识别。为了深入研究固有免疫受体在CVB3感染早期在肠道和心脏局部的激活及其炎症效应对于病毒性心肌炎发病的关键作用，本研究中，我们通过CVB3感染雄性BALB/c小鼠建立的VMC小鼠模型，首先以实时定量PCR技术跟踪分析了15种PRRs在CVB3感染过程中的表达变化，由此发现TLR13的表达最为显著性地上调。在此基础上，通过体内外TLR13表达下调，研究了TLR13在VMC发病中的关键作用，并对其激活的抗病毒机制和炎症机制进行了深入研究。此外，考虑到CVB3隶属于肠道病毒属，而肠道细菌格局可影响机体免疫系统及感染性疾病的发病。我们提出初步假设：是否CVB3感染通过影响肠道正常菌群的格局发生改变，依次诱导胃肠道、肠系膜淋巴结和心肌局部的免疫反应，从而影响了病毒性心肌炎的发生。拟在VMC易感性不同的雌雄小鼠模型基础上，首先用高通量测序方法分析了雌雄小鼠肠道菌群的多样性及丰度差异，并通过灌服肠道液体的方法，初步探讨了肠道菌群改变对VMC致病的影响，该部分为次要研究和探索性研究，尚未形成明确的结论。第一部分CVB3诱导心肌炎急性期心肌组织PRRs动态表达谱一、CVB3致小鼠病毒性心肌炎模型的建立以CVB3感染小鼠建立的VMC动物模型与临床VMC病患特征相似，主要的判断指标有生存率、体重减轻率、血清学、病理学等。以103TCID50的CVB3经腹腔感染BALB/c雄小鼠，发现感染后第3天开始小鼠体重明显下降，感染第7天体重下降率达20%；且7天小鼠死亡率为50%。从血清学指标看，病毒感染后第7天小鼠血清中肌酸激酶（CK）和肌酸激酶同工酶（CK-MB）水平显著性升高。病理学指标是VMC最重要的判断标准，感染第7天的小鼠心脏石蜡切片H.E染色显示：心脏组织中有大量炎性细胞浸润以及大片心肌细胞坏死，而正常小鼠心肌细胞完整，无炎症细胞浸润。综合上述指标，已成功建立了CVB3感染诱导的VMC小鼠模型。二、VMC小鼠心脏组织PRRs表达谱的动态改变为了寻找在VMC发病中可能起重要作用的PRRs，选择了15种PRRs（TLR1，TLR2，TLR3，TLR4，TLR5，TLR6，TLR7，TLR8，TLR9，TLR11，TLR12，TLR13，RIG-I，MDA5，LGP2），在CVB3感染诱导的VMC小鼠模型中，以实时定量PCR分析这些PRRs的动态表达变化，结果显示：病毒感染后第4天，心脏组织中大部分PRRs表达显著升高，至第7天，PRRs的表达均有所下调。其中以TLR13在感染第4天的上调表达最为显著（p<0.05），增幅近13倍。推测TLR13很可能被CVB3所激活，其介导的固有免疫炎症参与了VMC的致病过程。第二部分体内下调TLR13表达对于CVB3诱导的VMC的作用一、TLR13敲弱质粒效率的体外、体内验证由于CVB3感染极显著地上调TLR13的表达，因此下调TLR13水平对研究其在VMC发病中的作用至关重要。为有效下调TLR13的表达，设计了针对TLR13的shRNA序列，将其连接入pLL3.7构建了TLR13-ShRNA下调质粒。用Lipofectamine2000将质粒转染入NIH-3T3细胞中，以实时定量PCR和Western Blot分析了TLR13的mRNA和蛋白水平表达。结果显示该下调质粒能显著下调细胞中TLR13的表达，体外下调效率达50%以上。为研究TLR13在VMC小鼠中的真实作用，需在体内将小鼠心脏局部的TLR13水平下调。采用In vivo JET-PEI技术将下调质粒通过PEI以眼眶静脉注射途径注射入动物体内，检测基因表达干扰效果，发现，给予下调质粒第5天，心脏组织TLR13表达量已显著降低90%以上，且下调效率能持续至第7天。二、心脏局部下调TLR13水平对VMC炎症的影响为了研究TLR13在VMC发病中的作用，小鼠设立正常组（A组）、单独感染CVB3组（B组）、空质粒合并CVB3感染组（C组）以及TLR13下调合并CVB3感染组（D组），于-4天、0天给小鼠注射TLR13下调质粒二次，于0天感染CVB3病毒。结果显示感染后7天，对照组小鼠心肌组织炎性细胞浸润严重，而TLR13下调组生存率显著提高、体重保持不变、血清CK和CK-MB活性显著下降，最重要的，心肌组织炎性细胞浸润明显改善，表明在心脏局部下调TLR13表达后可显著缓解CVB3诱导的VMC小鼠心肌炎炎症程度。三、心脏局部TLR13下调对心脏病毒载量及心脏炎症细胞因子的影响为了研究TLR13下调对心肌组织病毒RNA载量及心肌细胞因子的影响，病毒感染第7天将小鼠处死。结果显示与空质粒组和对照组小鼠相比，TLR13下调组小鼠心脏病毒RNA载量显著降低；心脏炎症细胞因子水平较感染了CVB3的两组对照小鼠显著下降，其中Th1型细胞因子（IFN-γ、TNF-）降幅达50%以上，表明体内下调TLR13水平能显著降低CVB3诱导的炎症性Th1型应答，并降低病毒复制水平，对机体起保护作用。四、心脏局部TLR13下调对心脏功能的影响病毒感染第7天，采用超声诊断仪对小鼠心脏行超声检测。结果显示，与正常小鼠相比，TLR13下调组心脏左心室收缩末期内径显著增大，左室射血分数及左室短轴缩短率数值均显著降低，说明病毒感染后VMC小鼠心脏收缩功能减退，而对照小鼠则出现一定的心脏扩张显现。提示体内TLR13水平下调可降低CVB3诱导的病毒性心肌炎的后遗症-扩张性心脏病的可能。第三部分TLR13介导的炎症固有免疫和抗病毒反应在VMC发病中的分子机制探讨一、CVB3刺激TLR13介导的NF-κB信号通路激活的炎症效应为了研究CVB3感染后TLR13在炎性反应和抗病毒作用中的分子机制，我们选择了支持CVB3复制的Hela细胞（天然缺陷TLR13），利用真核表达质粒将TLR13过表达于人源Hela细胞株中。结果显示，我们成功构建了pTLR13真核表达质粒，并在细胞中显著表达。在转染并高表达TLR13的Hela细胞中，转染含有NF-κB启动子的荧光素酶报告质粒，以CVB3病毒和TLR激动剂ORN Sa19刺激后，检测荧光素酶活性。结果显示，以CVB3病毒感染过表达TLR13的Hela细胞后，NF-kB启动子活性比空质粒转染组活性显著提高近50%，提示TLR13识别CVB3后可激活下游NF-κB信号通路。二、CVB3刺激TLR13介导的IFN-β信号通路激活的抗病毒效应在转染并高表达TLR13的Hela细胞中，转染含有IFN-β启动子的荧光素酶报告质粒，以CVB3病毒和TLR激动剂ORN Sa19刺激后，检测荧光素酶活性。结果显示，以CVB3病毒感染过表达TLR13的Hela细胞后，IFN-β启动子活性比空质粒转染组活性显著提高。同时我们在心肌细胞株HL-1中过表达TLR13，并感染标记绿色荧光的CVB3-eGFP，感染24小时后，流式检测病毒载量。与空质粒组相比，TLR13过表达组中携带绿色荧光的HL-1细胞显著减少，提示TLR13识别CVB3后可激活下游IFN-β信号通路，发挥抗病毒效应。第四部分肠道菌群与雌雄小鼠VMC易感性差异的关系的初步探究一、CVB3感染后雌雄BALB/c小鼠心肌炎的差异以CVB3感染BALB/c雌雄小鼠，比较VMC发病率。发现感染后第7天雄小鼠死亡率为50%，而雌小鼠死亡率仅为10%。从心脏病理看，雄小鼠心脏组织中有大量炎性细胞浸润和心肌细胞坏死，而雌小鼠仅有少量炎性细胞浸润。从炎症细胞因子水平看，病毒感染后第7天雄小鼠心肌Th1型促炎细胞因子（IFN-γ、TNF-）显著高于雌小鼠（p<0.05），提示诱导Th1型免疫；而雌小鼠Th2型细胞因子（IL-10、IL-13）水平显著高于雄小鼠（p<0.05），提示诱导Th2型免疫。综上所述，与雌小鼠相比，雄小鼠对CVB3易感，可诱导显著的急性病毒性心肌炎，与其体内诱导的Th1型免疫相关。二、正常雌雄小鼠小肠组织中细胞因子本底表达的差异雌雄小鼠在感染CVB3后心脏炎症细胞因子水平差异显著，而肠道细胞因子表达无性别差异。为排除不同性别小鼠肠道本底细胞因子水平的影响，检测正常雌雄小鼠肠道细胞因子的本底水平，发现雌小鼠肠道细胞因子（IFN-γ、TNF-、IL-10、IL-13）的本底表达量均高于雄小鼠，特别是Th2型细胞因子水平显著高于雄鼠（IL-10、IL-13），因肠道本底细胞因子水平与感染无关，而与肠道菌群和性别相关，推测雌雄小鼠对VMC易感性可能与肠道菌群调节的肠道细胞因子水平有一定关系。三、正常雌雄小鼠肠道菌群谱系的差异肠道菌群不仅可阻止病原菌入侵肠道，还能与肠道免疫细胞相互作用影响机体免疫功能。推测正常雌雄小鼠的肠道细胞因子水平差异很可能与肠道菌群差异相关。利用高通量测序技术，我们对雌雄小鼠肠道菌群进行多样性分析，结果显示无论是从科水平还是属水平，未发现在某一性别小鼠中特异性表达的肠道细菌，但某些细菌在雌雄小鼠肠道菌群中存在丰度差异，说明并非单一的肠道细菌而有一群肠道菌群在雌雄小鼠肠道存在差异，可能影响小鼠肠道免疫功能。更多还原

【Abstract】 Viral myocarditis (VMC) is a common infectious disease of cardiovascular systemresulting from virus infection, featured by focal or diffuse cardiac inflammatoryinfiltration. The incidence of VMC is estimated as0.12-12%worldwide, and is about5%-15%in China, which is continues increasing accounting for one of the main causesof sudden death among adolescents. As acute VMC is asymptomatic and lack of clinicaltreatment, the incidence of VMC is largely low-estimated. Acute VMC may developeinto chronic myocarditis and then lethal dilated cardiomyopathy (DCM) thus becomesan important cardiovascular infectious disease injurious to human health. Less is knownabout the clinical treatment of VMC since pathogenesis of VMC is still unclarified.Hence, it is of great significance to elucidate the mechanism of pathogenesis of VMC.Viral infection is the prototype pathogenetic factor of VMC. Various virusesincluding nteroviruses, adenoviruses, influenza viruses and cytomegaloviruses havebeen reported to cause VMC. Among these viruses, enteroviruses especiallyCoxsackievirus group B type3(CVB3) is known to be one of the dominant cause factorfor VMC which accounts for more than50%of enterovirus-caused VMC cases. CVB3belongs to family of Picornaviridae with nonenveloped, positive-sense ssRNA. CVB3usually is taken into esophageal and gastrointestinal tracts via fecal-oral route, it theninfects and propagates in intestinal cells through interacting with coxsackie-adenovirusreceptor (CAR) and Decay accelerating factor (DAF), and then spread through bloodinto pancreas and heart tissues. On7-10days post-infection, acute pancreatitis and acutemyocarditis are observed, and chronic myocarditis could be developed at1monthpost-infection.The pathogenesis of VMC has been studied for long time. CVB3, a strictintracellular virus, could directly damages myocytes when propagates, and it producePro2A and VP2protein which can induce apoptosis of myocytes. However, it has beenreported that different mice sexes, which have different VMC susceptibility at day7 post-infection, have similar viral loads in the heart. And various gene knock-out mice(TLR3-/-, TRIF-/-, CD4-/-,-/-, IL-4-/-et al) which show quite differential VMCsusceptibility, also have comparable cardiac viral loads at day7, indicating CVB3replication is not a pivotal fator for the pathogenesis of CVB3-myocarditis.Accumulating evidences indicate that direct damage of myocytes by CVB3may occurat very early phase of infection (day1-3), however CVB3-induced cardiac inflammatoryimmune cell infiltration and so-caused inflammatory injury is the major cause of VMC.Various immune cells have been observed in the cardiac tissue, including Type ICD4+Th (Th1), Th17cells, type I macrophages (M1) and neutrophils, withmacrophages being the most abundant one. Inflammatory cytokines secreted by theseimmune cells, such as IFNγ, TNF, IL-6, IL-17are all related with myocarditis.Therefore transfer of Th2cytokines (IL-4and IL-13), or M2macrophages, or in vivoinducing Th2polarization, have been reported by our group before to have the effect toalleviate myocarditis.Innate immunity is long recognized as the first line of host defense againstpathogens. However, recent years more and more attention has been focused on innateimmunity since it is so critical to prime and shape the type of subsequent adaptiveimmunity. People recognize that innate recognition is not previously-thoughtnon-specific, various pattern-recognizing receptors (PRRs) expressed on membrane orin cytosol of innate cells, such as macrophages and dendritic cells (DCs), recognizecertain groups of pathogen-assocoated molecular patterns (PAMPs) of pathogens thenactivate innate signaling pathway which triggers proinflammatory cytokines production,-related moleculeup-regulation (MHC II, B7). Therefore innate immunity is indispensable for theinduction of specific T and B cell immune response for combat of virus. Meanwhile,uncontrolled innate inflammation is usually correlated with immune-inflammatorydiseases such as Inflammatory Bowl Disease (IBD) and auto-immune diseases like SLE.Most of the previous study of VMC focus their attention into adaptive immune responseespecially Th1, Th17and Treg cells in the pathogenesis of VMC, and role of innateimmunity in VMC is low-speculated. We hypothesize that before activation of Th1andTh17responses, cardiac inflammatory innate immunity plays an important role in theinduction and amplification of inflammatory responses as well as activation and shaping pathogenesis of VMC. Thus, study the key PRRs involved in the intestinal and cardiacinnate immunity activation in the early phase of CVB3infection, and theproinflammatory and anti-viral effects they triggered, and influence on the subsequentlyinfiltrated Th1and Th17cells, if of great significant.Accumulating data indicated that TLR3/4/7/8/9and MDA5may be all involved inthe activation of innate immunity to CVB3. Activation of TLR3and production ofs myocarditisand mice mortality. Role of MDA5is still controversial. Besides these PRRs, it is worthto follow the expression profiles of PRRs (TLRs, RLRs and others) dynamically both inthe heart tissue and intestinal tissue. Screening a key PRR which is most closely relatedwith VMC and performing indepth study is of great value. We thereafter discovered anundocumented PRR---TLR13. TLR13is a novel and uncharacterized endosomal PRR.Recently, two research groups simultaneously reported that TLR13can detect a highlyconserved sequence at the catalytic center of the23S rRNA of both gram-positive andgram-negative bacteria. TLR13is also reported to be involved in the recognition ofVesicular Stomatitis Virus (VSV).To investigate the role of innate sensors (receptors) stimulation and so-causeinflammatory respose in intestinal and heart tissue in the pathogenesis of VMC. In ourstudy BALB/c mice model of VMC was first established. The dynamic expressionlevels of15characteristic PRRs were detected by real-time PCR following CVB3infection, and TLR13was identified to be most quantitatively up-regulated during thecourse of acute myocarditis. Then through down-regulation of TLR13level in vivo andin vitro, role of TLR13and mechanism of its effect on inflammation and viralreplication were carefully studied after CVB3infection.Meanwhile, considering CVB3as an enteroviruse, and the pattern of microfloramay modulate the status of host systemic immune system and influence the outcome ofinfectious diseases, we hypothesize that CVB3infection may cause viral myocarditisthrough changing pattern of gut microflora, inducing inflammatory response in thegut-intestinal tract first which migrate to MLNs and then blood thus influencing cardiacimmune response and the onset of VMC. We plan to analyze the diversity of intestinalmicroflora between male and female mice which are sex-biased in VMC susceptibilityusing high-throughput sequencing technology. Then through oral transfer of intestinal juice, role of microflora pattern in the pathogensis of VMC would be evaluated. Thispart is an immature and exploratory work, and no conclusion has been drawn.Part One: Dynamic expression profiles of PRRs in the acute phaseof VMC after CVB3infection1. Establishment of murine model of CVB3-induced myocarditisMurine model of CVB3-induced myocarditis is very similar in several levels toclinical VMC, and the indices of diagnosis include serology, pathology and body weightloss rate as well as mortality.103TCID50dose of CVB3was applied intraperitoneally tomale BALB/c mice. On day3-4post-infection, the weight loss is very obvious. On day7, the body weight decreased about to20%, and the mortality mounts to50%-80%.Concerning on serological indicates, the activity of serum CK and CK-MB were bothsignificantly increased in CVB3infected mice on day7. As the most importantdiagnosis criteria of VMC, H.E staining of paraffin section of heart tissue of miceshowed massive inflammatory infiltration and myocytes necrosis in CVB3-infectedmice on day7post-infection while no signs was observed in normal mice. Takentogether, CVB3-induced myocarditis animal model was successfully established.2. Dynamic expression profiles of PRRs in myocardial tissue of VMC miceTo figure out the vital PRRs in the pathogenesis of VMC, the expression of15PRRs (TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR11, TLR12,TLR13, RIG-I, MDA5, LGP2) were detected by Real-time PCR in myocardial tissue ofmice during day0to day7following CVB3infection. It was found that most PRRslevels were elevated significantly in myocardial tissue on day4post-infection, thendown-regulated till day7post-infection. Among those PRRs, expression of TLR13wasup-regulated up to13folds on day4post-infection, being the most significantlychanged one. We speculate that TLR13innate pathway may be activated by CVB3andTLR13-mediated innate inflammatory response may be closely involved in thepathogenesis of VMC. Part Two; Role of TLR13in CVB3-induced myocarditis1. Validation of TLR13knockdown efficiency in vitro and in vivoDown-regulation TLR level is meaningful to evaluate it role in pathogenesis ofVMC since its level is significantly enhanced during VMC. In order to decrease theexpression of TLR13, shRNA sequences of TLR13were designed and cloned intopLL3.7vector. pLL3.7-shRNA-TLR13plasmids were transfected into NIH-3T3cellsby aiding of Lipofectamine2000. Real-time PCR and Western Blot assay showed thatthe knockdown plasmid can significantly reduce the expression of TLR13up to50%invitro. To investigate the role of TLR13in mice during VMC, we need to knockdown theexpression of TLR13in the myocardial tissue in vivo. By using In vivo JET-PEItransfection reagent, pLL3.7-shRNA-TLR13plasmids were retroorbitally injected. Itwas found that the expression of myocardial TLR13were obviously diminished up to90%on day5post-injection, and the knockdown effect can lasts7days.2. Development of VMC after cardiac knockdown of TLR13To investigate the role of TLR13in the pathogenesis of VMC, mice wererandomly divided into4groups, normal micegroup (A), no treatment group (B),pLL3.7vector control group (C) and TLR13knockdown group (D). Mice were injectedwith pLL3.7or pLL3.7-shRNA-TLR13knockdown plasmids at day-4and day0, andchallenged with CVB3at day0. It was found that mice with no treatment develop severemyocarditis on day7post-infection, while mice injected with TLR13-shRNA plasmidexhibited significant increased survival rate, less body weight loss, significant decreasedserum CK and CK-MB activity, and most importantly, less inflammatory infiltrationcompared with control mice. These data suggest that knockdown of expression ofcardiac TLR13significantly protect VMC development.3. Effect of cardiac TLR13knockdown on cardial viral loads andinflammatory cytokines productionTo see the change of viral loads and cardiac cytokines after knock-down of TLR13,mice were sacrificed on day7post-infection. Compared to normal or non-treated mice,the viral loads of TLR13-knock-down mice was significantly reduced. In addition,cardiac inflammatory cytokines of this group of mice were also significantly decreased.Among them, Th1cytokine (IFN-γ, TNF-) levels were above50%reduced. These data indicated that TLR13knockdown in vivo protect host from VMC by significantlyreducing the production of CVB3-induced pathogenic Th1inflammatory cytokines andCVB3replication.4. Role of cardiac TLR13knockdown on heart tissue functionMurine transthoracic echocardiography was performed on day7post-infection andfound that TLR13knockdown mice had significantly elevated left ventricular internaldimension systole (LVID-s), while significantly reduced left ventricular ejectionfraction (LVEF%) and Left Ventricular Fraction Shortening (LVFS%) compared withnormal and control mice, demonstrating that while CVB3infection result in the failureof systole, knockdown of cardiac TLR13level in vivo could reduce the susceptibility todilated cardiomayopathy of VMC mice.Part Three; The molecular mechanism of TLR13in regulatinguring VMC1. Regulation of NF-κB inflammatory signaling pathway by TLR13afterCVB3infectionTo study the molecular mechanism of TLR13in reducing inflammation and viralduring CVB3infection, CVB3replication supportive cell line, Hela (which has noTLR13expression), was selected to overexpress TLR13using eukaryotic expressionvector pTLR13. The results indicated that the TLR13expressing plasmid wasconstructed, and TLR13was significantly highly expressed in Hela cells.In Hela cells with high expression of TLR13, fluorescent reporter plasmidharnessing NF-κB promoter was co-transfected into the cell. Upon CVB3infection orTLR13agonist, ORN Sa19, stimulation, luciferase activity was analyzed. Dataindicated that compared with vector-transfected cells, the activity of NF-κB promoterwas improved50%in TLR13-forced-expressed cells upon CVB3infection, indicatingTLR13recognition of CVB3could trigger activation of NF-κB signaling pathway2. Anti-viral effect of TLR13through IFN-β signaling pathway after CVB3infectionIn Hela cells with high expression of TLR13, Fluorescent reporter plasmidharnessing IFN-β promoter was co-transfected into the cell. Upon CVB3infection and TLR13agonist, ORN Sa19, stimulation, luciferase activity was analyzed. Comparedwith vector-transfected cells, the activity of IFN-β promoter was significantly elevatedin TLR13forced-expressed cells upon CVB3infection. Meanwhile, HL-1cardiac cellswere transfected with pTLR13plasmid to over-express TLR13then infected withCVB3-eGFP. After24hour, CVB3loads were detected by Flow Cytometry.Significantly reduced green fluorescent cells were seen after pTLR13-transfectioncompared with empty vector. These data indicate that TLR13recognition of CVB3maytrigger activation of IFN-β signaling pathway thus exerting its anti-viral effect.Part Four; Preliminary data of the relationship betweenmicroflora and VMC susceptibility in different mice sex1. Sex differences in susceptibility to CVB3-induced myocarditisMale and female mice were infected with CVB3and the VMC incidence wascompared. On day7post-infection, the mortality was50%in male mice while10%infemale mice. In perspective of cardiac pathology, massive cardiac inflammatoryinfiltration and myocytes necrosis were observed in heart of male mice but only slightinflammatory infiltration seen in that of female mice. In terms of inflammatorycytokines production, cardiac pathogenic Th1cytokine (IFN-γ and TNF-) levels weresignificantly higher in male mice than in female mice, indicating Th1immune bias.While cardiac Th2cytokine (IL-10and IL-13) levels were significantly higher in femalemice than in male mice, indicating Th2immune bias. Taken together, male mice weremore susceptible to VMC than female mice after CVB3infection which was closelyrelated to in vivo Th1polarization.2. Basal intestinal cytokine level difference between male and female miceUnlike huge disparity of cardiac cytokines between male and female mice afterCVB3infection, the intestinal cytokines production is not sex-biased. To exclude theinterference of basal cytokine level, intestinal cytokines were analyzed in normal maleand female mice. It was found that basal intestinal cytokine (IFN-γ, TNF-, IL-10, andIL-13) levels in female mice were significantly higher than those in male mice,especially Th2cytokines---IL-10and IL-13. Considering basal intestinal cytokine levelis not related to pathogen infection, but more associated with microflora and sex, we hypothesize that the differential VMC susceptibility of male and female mice may berelated to the intestinal cytokines] levels which are closely modulated by microflora.3. The diversity of microflora in male and female miceMicroflora not only prevent intestinal pathogen invasion but also influencefunction of host immunity by interacting with host intestinal immune cells. Wehypothesize that the basal intestinal cytokine level difference between male and femalemice may be related to the different diversity of microflora. Using highthough-putsequencing method, the diversity of microflora was analyzed. No single sex-biasedbacterium in family level or genus level was found, but the abundance of some groupsof microflora was different between male and female mice. These data indicated thatintestinal immunity might be influenced by a cluster of sex-biased bacteria but not asingle bacterium.更多还原