TLR13和肠道菌群在柯萨奇病毒性心肌炎发病中的作用及其机制
|
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。
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 of
s 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 CVB3infection
1. Establishment of murine model of CVB3-induced myocarditis
Murine 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 mice
To 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 myocarditis
1. Validation of TLR13knockdown efficiency in vitro and in vivo
Down-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 TLR13
To 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 production
To 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 function
Murine 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 VMC
1. Regulation of NF-κB inflammatory signaling pathway by TLR13afterCVB3infection
To 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 pathway
2. Anti-viral effect of TLR13through IFN-β signaling pathway after CVB3infection
In 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 sex
1. Sex differences in susceptibility to CVB3-induced myocarditis
Male 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 mice
Unlike 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 mice
Microflora 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.
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。
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 of
s 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 CVB3infection
1. Establishment of murine model of CVB3-induced myocarditis
Murine 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 mice
To 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 myocarditis
1. Validation of TLR13knockdown efficiency in vitro and in vivo
Down-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 TLR13
To 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 production
To 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 function
Murine 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 VMC
1. Regulation of NF-κB inflammatory signaling pathway by TLR13afterCVB3infection
To 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 pathway
2. Anti-viral effect of TLR13through IFN-β signaling pathway after CVB3infection
In 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 sex
1. Sex differences in susceptibility to CVB3-induced myocarditis
Male 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 mice
Unlike 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 mice
Microflora 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.
引文
1. Kawai T, Akira S: Innate immune recognition of viral infection. Nat Immunol7,131-137(2006).
2. Bowles NE, Ni J, Kearney DL, et al.: Detection of viruses in myocardialtissues by polymerase chain reaction. evidence of adenovirus as a common cause ofmyocarditis in children and adults. J Am Coll Cardiol42,466-472(2003).
3. Gupta S, Markham DW, Drazner MH, Mammen PP: Fulminant myocarditis.Nat Clin Pract Cardiovasc Med5,693-706(2008).
4. Kyto V, Saraste A, Voipio-Pulkki LM, Saukko P: Incidence of fatalmyocarditis: a population-based study in Finland. Am J Epidemiol165,570-574(2007).
5. Dennert R, Crijns HJ, Heymans S: Acute viral myocarditis. Eur Heart J29,2073-2082(2008).
6. Remes J, Helin M, Vaino P, Rautio P: Clinical outcome and left ventricularfunction23years after acute coxsackie virus myopericarditis. Eur Heart J11,182-188(1990).
7. Feldman AM, McNamara D: Myocarditis. N Engl J Med343,1388-1398(2000).
8. Curnen EC: Human disease associated with the Coxsackie viruses. Bull N YAcad Med26,335-342(1950).
9. Easton AJ, Eglin RP: The detection of coxsackievirus RNA in cardiac tissue byin situ hybridization. J Gen Virol69(Pt2),285-291(1988).
10. Blumohr T, Klocking R, Sprossig M, Wockel W:[The coxsackievirus-infectedmouse: an animal model for the study of virus myocarditis]. Acta Biol Med Ger35,103-109(1976).
11. Badorff C, Fichtlscherer B, Rhoads RE, et al.: Nitric oxide inhibits dystrophinproteolysis by coxsackieviral protease2A through S-nitrosylation: A protectivemechanism against enteroviral cardiomyopathy. Circulation102,2276-2281(2000).
12. Henke A, Nestler M, Strunze S, et al.: The apoptotic capability ofcoxsackievirus B3is influenced by the efficient interaction between the capsid proteinVP2and the proapoptotic host protein Siva. Virology289,15-22(2001).
13. Woodruff JF, Woodruff JJ: Involvement of T lymphocytes in the pathogenesisof coxsackie virus B3heart disease. J Immunol113,1726-1734(1974).
14. Hirasawa K, Tsutsui S, Takeda M, et al.: Depletion of Mac1-positivemacrophages protects DBA/2mice from encephalomyocarditis virus-inducedmyocarditis and diabetes. J Gen Virol77(Pt4),737-741(1996).
15. Leipner C, Grun K, Borchers M, Stelzner A: The outcome of coxsackievirusB3-(CVB3-) induced myocarditis is influenced by the cellular immune status. Herz25,245-248(2000).
16. Sagar S, Liu PP, Cooper LT, Jr.: Myocarditis. Lancet379,738-747(2012).
17. Fairweather D, Stafford KA, Sung YK: Update on coxsackievirus B3myocarditis. Curr Opin Rheumatol24,401-407(2012).
18. Huber SA: Increased susceptibility of male BALB/c mice to coxsackievirusB3-induced myocarditis: role for CD1d. Med Microbiol Immun194,121-127(2005).
19. Jiang Z, Xu W, Li K, et al.: Remission of CVB3-induced viral myocarditis byin vivo Th2polarization via hydrodynamics-based interleukin-4gene transfer. J GeneMed10,918-929(2008).
20. Xie Y, Chen R, Zhang X, et al.: The role of Th17cells and regulatory T cells inCoxsackievirus B3-induced myocarditis. Virology421,78-84(2011).
21. Yuan J, Cao AL, Yu M, et al.: Th17cells facilitate the humoral immuneresponse in patients with acute viral myocarditis. J Clin Immunol30,226-234(2010).
22. Yuan J, Yu M, Lin QW, et al.: Neutralization of IL-17inhibits the productionof anti-ANT autoantibodies in CVB3-induced acute viral myocarditis. IntImmunopharmacol10,272-276(2010).
23. Opavsky MA, Penninger J, Aitken K, et al.: Susceptibility to myocarditis isdependent on the response of alphabeta T lymphocytes to coxsackieviral infection. CircRes85,551-558(1999).
24. Li K, Xu W, Guo Q, et al.: Differential macrophage polarization in male andfemale BALB/c mice infected with coxsackievirus B3defines susceptibility to viralmyocarditis. Circ Res105,353-364(2009).
25. Gorbea C, Makar KA, Pauschinger M, et al.: A role for Toll-like receptor3variants in host susceptibility to enteroviral myocarditis and dilated cardiomyopathy. JBiol Chem285,23208-23223(2010).
26. Frisancho-Kiss S, Davis SE, Nyland JF, et al.: Cutting edge: cross-regulationby TLR4and T cell Ig mucin-3determines sex differences in inflammatory heartdisease. J Immunol178,6710-6714(2007).
27. Triantafilou K, Orthopoulos G, Vakakis E, et al.: Human cardiac inflammatoryresponses triggered by Coxsackie B viruses are mainly Toll-like receptor (TLR)8-dependent. Cell Microbiol7,1117-1126(2005).
28. Wang JP, Cerny A, Asher DR, et al.: MDA5and MAVS mediate type Iinterferon responses to coxsackie B virus. J Virol84,254-260(2010).
29. Huhn MH, McCartney SA, Lind K, et al.: Melanoma differentiation-associatedprotein-5(MDA-5) limits early viral replication but is not essential for the induction oftype1interferons after Coxsackievirus infection. Virology401,42-48(2010).
30. Fairweather D, Yusung S, Frisancho S, et al.: IL-12receptor beta1andToll-like receptor4increase IL-1beta-and IL-18-associated myocarditis andcoxsackievirus replication. J Immunol170,4731-4737(2003).
31. Triantafilou K, Orthopoulos G, Vakakis E, Ahmed M: Human cardiacinflammatory responses triggered by Coxsackie B viruses are mainly Toll-like receptor(TLR)8-dependent. Cell Microbiol7,1117-1126(2005).
32. Weinzierl AO, Szalay G, Wolburg H, et al.: Effective chemokine secretion bydendritic cells and expansion of cross-presenting CD4-/CD8+dendritic cells define aprotective phenotype in the mouse model of coxsackievirus myocarditis. J Virol82,8149-8160(2008).
33. Abston ED, Coronado MJ, Bucek A, et al.: Th2regulation of viral myocarditisin mice: different roles for TLR3versus TRIF in progression to chronic disease. ClinDev Immunol2012,129486(2012).
34. Roberts B, Dragon J, Moussawi M, Huber S: Sex-specific signaling throughToll-Like Receptors2and4contributes to survival outcome of Coxsackievirus B3infection in C57Bl/6mice. Biol Sex Differ3,25. doi:10.1186/2042-6410-1183-1125.(2012).
35. Riad A, Westermann D, Escher F, et al.: Myeloid differentiation factor-88contributes to TLR9-mediated modulation of acute coxsackievirus B3-inducedmyocarditis in vivo. Am J Physiol Heart Circ Physiol298, H2024-2031(2010).
36. Fields B: Virology. Third Edition. Lippincott-Raven Publisher.(1996).
37. Guarner F, Malagelada JR: Gut flora in health and disease. Lancet361,512-519(2003).
38. Wostmann BS, Larkin C, Moriarty A, Bruckner-Kardoss E: Dietary intake,energy metabolism, and excretory losses of adult male germfree Wistar rats. Lab AnimSci33,46-50(1983).
39. Mazmanian SK, Round JL, Kasper DL: A microbial symbiosis factor preventsintestinal inflammatory disease. Nature453,620-625(2008).
40. Chen LW, Chen PH, Hsu CM: Commensal microflora contribute to hostdefense against Escherichia coli pneumonia through Toll-like receptors. Shock36,67-75(2011).
41. Medzhitov R, Janeway C, Jr.: Innate immunity. N Engl J Med343,338-344(2000).
42. Akira S, Uematsu S, Takeuchi O: Pathogen recognition and innate immunity.Cell124,783-801(2006).
43. Janeway CA, Jr.: Approaching the asymptote? Evolution and revolution inimmunology. Cold Spring Harb Symp Quant Biol54Pt1,1-13(1989).
44. Akira S, Takeda K: Toll-like receptor signalling. Nat Rev Immunol4,499-511(2004).
45. Mogensen TH: Pathogen recognition and inflammatory signaling in innateimmune defenses. Clin Microbiol Rev22,240-273, Table of Contents (2009).
46. Mogensen TH, Paludan SR: Reading the viral signature by Toll-like receptorsand other pattern recognition receptors. J Mol Med (Berl)83,180-192(2005).
47. Aretz HT, Billingham ME, Edwards WD, et al.: Myocarditis. Ahistopathologic definition and classification. Am J Cardiovasc Pathol1,3-14(1987).
48. Netea MG, Van der Meer JW, Sutmuller RP, Adema GJ, Kullberg BJ: Fromthe Th1/Th2paradigm towards a Toll-like receptor/T-helper bias. Antimicrob AgentsChemother49,3991-3996(2005).
49. Tohse N, Sperelakis N: cGMP inhibits the activity of single calcium channelsin embryonic chick heart cells. Circ Res69,325-331(1991).
50. Cohn RD, Durbeej M, Moore SA, et al.: Prevention of cardiomyopathy inmouse models lacking the smooth muscle sarcoglycan-sarcospan complex. J Clin Invest107, R1-7(2001).
51. Bobin AN, Parkhommenko Iu G:[Pathomorphology and morphogenesis ofmyocarditis in typhoid fever]. Arkh Patol67,38-40(2005).
52. Zhang SY, Jouanguy E, Ugolini S, et al.: TLR3deficiency in patients withherpes simplex encephalitis. Science317,1522-1527(2007).
53. Feldmann J, Prieur AM, Quartier P, et al.: Chronic infantile neurologicalcutaneous and articular syndrome is caused by mutations in CIAS1, a gene highlyexpressed in polymorphonuclear cells and chondrocytes. Am J Hum Genet71,198-203(2002).
54. Smirnova I, Mann N, Dols A, et al.: Assay of locus-specific genetic loadimplicates rare Toll-like receptor4mutations in meningococcal susceptibility. ProcNatl Acad Sci U S A100,6075-6080(2003).
55. Tao K, Fujii M, Tsukumo S, et al.: Genetic variations of Toll-like receptor9predispose to systemic lupus erythematosus in Japanese population. Ann Rheum Dis66,905-909(2007).
56. Tabeta K, Georgel P, Janssen E, et al.: Toll-like receptors9and3as essentialcomponents of innate immune defense against mouse cytomegalovirus infection. ProcNatl Acad Sci U S A101,3516-3521(2004).
57. Roach JC, Glusman G, Rowen L, et al.: The evolution of vertebrate Toll-likereceptors. Proc Natl Acad Sci U S A102,9577-9582(2005).
58. Shi Z, Cai Z, Sanchez A, et al.: A novel Toll-like receptor that recognizesvesicular stomatitis virus. J Biol Chem286,4517-4524(2011).
59. Takeuchi O, Akira S: Pattern recognition receptors and inflammation. Cell140,805-820(2010).
60. Li XD, Chen ZJ: Sequence specific detection of bacterial23S ribosomal RNAby TLR13. Elife1, e00102(2012).
61. Oldenburg M, Kruger A, Ferstl R, et al.: TLR13recognizes bacterial23SrRNA devoid of erythromycin resistance-forming modification. Science337,1111-1115(2012).
62. Nissen P, Hansen J, Ban N, Moore PB, Steitz TA: The structural basis ofribosome activity in peptide bond synthesis. Science289,920-930(2000).
63. McCusker KP, Fujimori DG: The chemistry of peptidyltransferasecenter-targeted antibiotics: enzymatic resistance and approaches to countering resistance.ACS Chem Biol7,64-72(2012).
64. Yajima T, Knowlton KU: Viral myocarditis: from the perspective of the virus.Circulation119,2615-2624(2009).
65.Kawai T, Akira S: The role of pattern-recognition receptors in innate immunity:update on Toll-like receptors. Nat Immunol11,373-384(2010).
66. O'Neill LA, Golenbock D, Bowie AG: The history of Toll-like receptors-redefining innate immunity. Nat Rev Immunol13,453-460(2013).
67. Sen R, Baltimore D: Multiple nuclear factors interact with the immunoglobulinenhancer sequences. Cell46,705-716(1986).
68. Li Q, Verma IM: NF-kappaB regulation in the immune system. Nat RevImmunol2,725-734(2002).
69. Goren N, Cuenca J, Martin-Sanz P, Bosca L: Attenuation of NF-kappaBsignalling in rat cardiomyocytes at birth restricts the induction of inflammatory genes.Cardiovasc Res64,289-297(2004).
70.刘慧强:核因子kB在病毒性心肌炎小鼠发病中的作用.吉林大学(2006).
71. Berer K, Krishnamoorthy G: Commensal gut flora and brain autoimmunity: alove or hate affair? Acta Neuropathol123,639-651(2012).
72. Nell S, Suerbaum S, Josenhans C: The impact of the microbiota on thepathogenesis of IBD: lessons from mouse infection models. Nat Rev Microbiol8,564-577(2010).
73. Round JL, Mazmanian SK: The gut microbiota shapes intestinal immuneresponses during health and disease. Nat Rev Immunol9,313-323(2009).
74. Cani PD, Delzenne NM, Amar J, Burcelin R: Role of gut microflora in thedevelopment of obesity and insulin resistance following high-fat diet feeding. PatholBiol (Paris)56,305-309(2008).
75. Brandt LJ: Fecal transplantation for the treatment of Clostridium difficileinfection. Gastroenterol Hepatol (N Y)8,191-194(2012).
76. Schwan A, Sjolin S, Trottestam U, Aronsson B: Relapsing clostridium difficileenterocolitis cured by rectal infusion of homologous faeces. Lancet2,845(1983).
77. Paterson DL, Iredell J, Whitby M: Putting back the bugs: bacterial treatmentrelieves chronic diarrhoea. Med J Aust160,232-233(1994).
78. Borody TJ:"Flora Power"--fecal bacteria cure chronic C. difficile diarrhea.The American journal of gastroenterology95,3028-3029(2000).
79. Brandt LJ, Aroniadis OC, Mellow M, et al.: Long-term follow-up ofcolonoscopic fecal microbiota transplant for recurrent Clostridium difficile infection.Am J Gastroenterol107,1079-1087(2012).
80. Galama J: Enteroviral infections in the immunocompromised host. RevMedical Microbiol8,33-40(1997).
81. van der Waaij D: The ecology of the human intestine and its consequences forovergrowth by pathogens such as Clostridium difficile. Annu Rev Microbiol43,69-87(1989).
82. Markle JG, Frank DN, Mortin-Toth S, et al.: Sex differences in the gutmicrobiome drive hormone-dependent regulation of autoimmunity. Science339,1084-1088(2013).
1. Kawai T, Akira S. Innate immune recognition of viral infection. Nat Immunol.7,131-137(2006)
2. Bowles NE, Ni J, Kearney DL et al. Detection of viruses in myocardial tissuesby polymerase chain reaction. Evidence of adenovirus as a common cause ofmyocarditis in children and adults. J Am Coll Cardiol.42,466-472(2003)
3. Gupta S, Markham DW, Drazner MH, Mammen PP. Fulminant myocarditis.Nat Clin Pract Cardiovasc Med.5,693-706(2008)
4. Kyto V, Saraste A, Voipio-Pulkki LM, Saukko P. Incidence of fatalmyocarditis: a population-based study in Finland. Am J Epidemiol.165,570-574(2007)
5. Dennert R, Crijns HJ, Heymans S. Acute viral myocarditis. Eur Heart J.29,2073-2082(2008)
6. Blauwet LA, Cooper LT. Myocarditis. Prog Cardiovasc Dis.52,274-288(2010)
7. Chen X, Li B, Liao Y et al. Analysis on Coxsackievirus infection and onset ofviral myocarditis of doctors and nurses in department of cardiology. Chin J CardiovascRehab Med.13,328-330(2004)
8. Cooper LT Jr. Myocarditis. N Eng J Med.360,1526-1538(2009)
9. Tam PE. Coxsackievirus myocarditis: interplay between virus and host in thepathogenesis of heart disease. Viral Immunol.19,133-146(2006)
10. Bowles NE, Vallejo J. Viral causes of cardiac inflammation. Curr OpinCardiol.18,182-188(2003)
11. Yajima T, Knowlton KU. Viral myocarditis: from the perspective of the virus.Circulation.119,2615-2624(2009)
12. Feuer R, Mena I, Pagarigan R, Slifka MK, Whitton JL. Cell cycle status affectsCoxsackievirus replication, persistence, and reactivation in vitro. J Virol.76,4430-4440(2002)
13. Feuer R, Mena I, Pagarigan RR, Hassett DE, Whitton JL. Coxsackievirusreplication and the cell cycle: a potential regulatory mechanism for viralpersistence/latency. Med Microbiol Immunol.193,83-90(2004)
14. Huber S, Sartini D. T cells expressing the Vgamma1T-cell receptor enhancevirus-neutralizing antibody response during Coxsackievirus B3infection of BALB/cmice: differences in male and female mice. Viral Immunol.18,730-739(2005)
15. Abston ED, Coronado MJ, Bucek A et al. Th2regulation of viral myocarditisin mice: different roles for TLR3versus TRIF in progression to chronic disease. ClinDev Immunol.2012,129486(2012)
16. Opavsky MA, Penninger J, Aitken K et al. Susceptibility to myocarditis isdependent on the response of alphabeta T lymphocytes to Coxsackieviral infection. CircRes.85,551-558(1999)
17. Li K, Xu W, Guo Q et al. Differential macrophage polarization in male andfemale BALB/c mice infected with Coxsackievirus B3defines susceptibility to viralmyocarditis. Circ Res.105,353-364(2009)
18. Woodruff JF, Woodruff JJ. Involvement of T lymphocytes in the pathogenesisof Coxsackievirus B3heart disease. J Immunol.113,1726-1734(1974)
19. Haga T, Suzuki J, Kosuge H et al. Attenuation of experimental autoimmunemyocarditis by blocking T cell activation through4-1BB pathway. J Mol Cell Cardiol.46,719-727(2009)
20. Sagar S, Liu PP, Cooper LT Jr. Myocarditis. Lancet.379,738-747(2012)
21. Fairweather D, Stafford KA, Sung YK. Update on Coxsackievirus B3myocarditis. Curr Opin Rheumatol.24,401-407(2012)
22. Pauschinger M, Phan MD, Doerner A et al. Enteroviral RNA replication in themyocardium of patients with left ventricular dysfunction and clinically suspectedmyocarditis. Circulation.99,889-895(1999)
23. Reetoo KN, Osman SA, Illavia SJ et al. Quantitative analysis of viral RNAkinetics in Coxsackievirus B3-induced murine myocarditis, biphasic pattern ofclearance following acute infection, with persistence of residual viral RNA throughoutand beyond the inflammatory phase of disease. J Gen Virol.81,2755-2762(2000)
24. Kishimoto C, Kuribayashi K, Masuda T, Tomika N, Kawai C. Immunologicbehavior of lymphocytes in experimental viral myocarditis: significance of Tlymphocytes in the severity of myocarditis and silent myocarditis in BALB/c-nu/numice. Circulation.71,1247-1254(1985)
25. Huber SA. Increased susceptibility of male BALB/c mice to CoxsackievirusB3-induced myocarditis: role for CD1d. Med Microbiol Immunol.194,121-127(2005)
26. Jiang Z, Xu W, Li K et al. Remission of CVB3-induced viral myocarditis by invivo Th2polarization via hydrodynamics-based interleukin-4gene transfer. J Gene Med.10,918-929(2008)
27. Fairweather D, Frisancho-Kiss S, Yusung SA et al. Interferon-gamma protectsagainst chronic viral myocarditis by reducing mast cell degranulation, fibrosis, and theprofibrotic cytokines transforming growth factor-beta1, interleukin-1beta, andinterleukin-4in the heart. Am J Pathol.165,1883-1894(2004)
28. Konkel JE, Chen W. Balancing acts: the role of TGF-beta in the mucosalimmune system. Trends Mol Med.17,668-676(2011)
29. Xie Y, Chen R, Zhang X et al. The role of Th17cells and regulatory T cells inCoxsackievirus B3-induced myocarditis. Virology.421,78-84(2011)
30. Yuan J, Cao AL, Yu M et al. Th17cells facilitate the humoral immuneresponse in patients with acute viral myocarditis. J Clin Immunol.30,226-234(2010)
31. Yuan J, Yu M, Lin QW et al. Neutralization of IL-17inhibits the production ofanti-ANT autoantibodies in CVB3-induced acute viral myocarditis. IntImmunopharmacol.10,272-276(2010)
32. Frisancho-Kiss S, Nyland JF, Davis SE et al. Cutting edge: T cell Ig mucin-3reduces inflammatory heart disease by increasing CTLA-4during innate immunity. JImmunol.176,6411-6415(2006)
33. Lv K, Xu W, Wang C et al. Galectin-9administration ameliorates CVB3induced myocarditis by promoting the proliferation of regulatory T cells andalternatively activated Th2cells. Clin Immunol.140,92-101(2011)
34. Hwang JY, Jun EJ, Seo I et al. Characterization of infections of humanleukocytes by non-polio enteroviruses. Intervirology.55,333-341(2012)
35. Kling K, Rieger P, Mall G et al. Visualization of enteroviral replication inmyocardial tissue by ultrastructural in situ hybridization: identification of target cellsand cytopathic effects. Lab Invest.78,1227-1237(1998)
36. Kandolf R, Sauter M. Aepinus C et al. Mechanisms and consequences ofenterovirus persistence in cardiac myocytes and cells of the immune system. Virus Res.62,149-158(1999)
37. Opavsky MA, Martino T, Rabinovitch M et al. Enhanced ERK-1/2activationin mice susceptible to Coxsackievirus-induced myocarditis. J Clin Invest.109,1561-1569(2002)
38. Kim DS, Park JH, Kim JY, Kim D, Nam JH. A mechanism of immunoreceptortyrosine-based activation motif (ITAM)-like sequences in the capsid protein VP2inviral growth and pathogenesis of Coxsackievirus B3. Virus Genes.44,176-182(2012)
39. Barin JG, Rose NR, Cihakova D. Macrophage diversity in cardiacinflammation: a review. Immunobiology.217,468-475(2012)
40. Mantovani A, Sica A, Locati M. Macrophage polarization comes of age.Immunity.23,344-346(2005)
41. Mosser DM, Edwards JP. Exploring the full spectrum of macrophageactivation. Nat Rev Immunol.8,958-969(2008)
42. Holtmeier W, Kabelitz D. Gammadelta T cells link innate and adaptiveimmune responses. Chem Immunol Allergy.86,151-183(2005)
43. Huber SA. Depletion of gammadelta+T cells increases CD4+FoxP3(Tregulatory) cell response in Coxsackievirus B3-induced myocarditis. Immunology.127,567-576(2009)
44. Huber SA. Born W, O’Brien R. Dual functions of murine gammadelta cells ininflammation and autoimmunity in Coxsackievirus B3-induced myocarditis: role ofVgamma1+and Vgamma4+cells. Microbes Infect.7,537-543(2005)
45. Huber S, Sartini D. T cells expressing the Vgamma1T-cell receptor enhancevirus-neutralizing antibody response during Coxsackievirus B3infection of BALB/cmice: differences in male and female mice. Viral Immunol.18,730-739(2005)
46. Huber S, Sartini D, Exley M. Role of CD1d in Coxsackievirus B3-inducedmyocarditis. J Immunol.170,3147-3153(2003)
47. Huber SA, Graveline D, Born WK, O’Brien RL. Cytokine production byVgamma+-T-cell subsets is an important factor determining CD4+-Th-cell phenotypeand susceptibility of BALB/c mice to Coxsackievirus B3-induced myocarditis. J Virol.75,5860-5869(2001)
48. Kemball CC, Alirezaei M, Whitton JL. Type B Coxsackieviruses and theirinteractions with the innate and adaptive immune systems. Future Microbiol.5,1329-1347(2010)
49. Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linkinginnate and acquired immunity. Nat Immunol.2,675-680(2001)
50. Huang CH, Vallejo JG, Kollias G, Mann DL. Role of the innate immunesystem in acute viral myocarditis. Basic Res Cardiol.104,228-237(2009)
51. Sen GC. Viruses and interferons. Annu Rev Microbiol.55,255-281(2001)
52. Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD. How cellsrespond to interferons. Annu Rev Biochem.67,227-264(1998)
53. Frisancho-Kiss S, Davis SE, Nyland JF et al. Cutting edge: corss-regulation byTLR4and T cell Ig mucin-3determines sex differences in inflammatory heart disease. JImmunol.178,6710-6714(2007)
54. Gorbea C, Makar KA, Pauschinger M et al. A role for Toll-like receptor3variants in host susceptibility to enteroviral myocarditis and dilated cardiomyopathy. JBiol Chem.285,23208-23223(2010)
55. Wang JP, Asher DR, Chan M, Kurt-Jones EA, Finberg RW. Cutting edge:antibody-mediated TLR7-dependent recognition of viral RNA. J Immunol.178,3363-3367(2007)
56. Triantafilou K, Orthopoulos G, Vakakis E et al. Human cardiac inflammatoryresponses triggered by Coxsackie B viruses are mainly Toll-like receptor (TLR)8-dependent. Cell Microbiol.7,1117-1126(2005)
57. Riad A, Westermann D, Escher F et al. Myeloid differentiation factor-88contributes to TLR9-mediated modulation of acute Coxsackievirus B3-inducedmyocarditis in vivo. Am J Physiol Heart Circ Physiol.298, H2024-H2031(2010)
58. Hardarson HS, Baker JS, Yang Z et al. Toll-like receptors3is an essentialcomponent of the innate stress response in virus-induced cardiac injury. Am J PhysiolHeart Circ Physiol.292, H251-H258(2007)
59. Netea MG, Van der Meet JW, Sutmuller RP, Adema GJ, Kullberg BJ. Fromthe Th1/Th2paradigm towards a Toll-like receptor/T-helper bias. Antimicrob AgentsChemother.49,3991-3996(2005)
60. Roberts BJ, Moussawi M, Huber SA. Sex differences in TLR2and TLR4expression and their effect on Coxsackievirus-induced autoimmune myocarditis. ExpMol Pathol.94,58-64(2013)
61. Kato H, Sato S, Yoneyama M et al. Cell type-specific involvement of RIG-I inantiviral responses. Immunity.23,19-28(2005)
62. Yoneyama M, Kikuchi M, Natsukawa T et al. The RNA helicase RIG-I has anessential function in double-stranded RNA-induced innate antiviral responses. NatImmunol.5,730-737(2004)
63. Yoneyama M, Kikuchi M, Matsumono K et al. Shared and unique functions ofthe DExD/H-box helicases RIG-I, MDA5, and LGP2in antiviral innate immunity. JImmunol.175,2851-2858(2005)
64. Kato H, Takeuchi O, Sato S et al. Differential roles of MDA5and RIG-Ihelicases in the recognition of RNA viruses. Nature.441,101-105(2006)
65. Wang JP, Cerny A, Asher DR et al. MDA5and MAVS mediate type Iinterferon responses to Coxsackie B virus. J Virol.84,254-260(2010)
66. Huhn MH, McCartney SA, Lind K et al. Melanoma differentiation-associatedprotein-5(MDA-5) limits early viral replication but is not essential for the induction oftype I interferons after Coxsackievirus infection. Virology.401,42-48(2010)
67. Bopegamage S, Borsanyiova M, Vargova A et al. Coxsackievirus infection ofmice. I. Viral kinetics and histopathological changes in mice experimentally infectedwith Coxsackievirus B3and B4by oral route. Acta Virol.47,245-251(2003)
68. Vargova A, Bopegamage S, Borsanyiova M et al. Coxsackievirus infection ofmice. II. Viral kinetics and histopathological changes in mice experimentally infectedwith Coxsackievirus B3by intraperitoneal route. Acta Virol.47,253-257(2003)
69. Molin Y, Frisk P, Ilback NG. Viral RNA kinetics is associated with changes intrace elements in target organs of Coxsackievirus B3infection. Microbes Infect.11,493-499(2009)
70. Mazmanian SK, Round JL, Kasper DL. A microbial symbiosis factor preventsintestinal inflammatory disease. Nature.453,620-625(2008)
71. Chen LW, Chen PH, Hsu CM. Commensal microflora contribute to hostdefense against Escherichia coli pneumonia through Toll-like receptors. Shock.36,67-75(2011)
72. Gagliani N, Huber S, Flavell RA. The intestine: where amazing things happen.Cell Res.22,277-279(2012)
73. Lathrop SK, Bloom SM, Rao SM et al. Peripheral education of the immunesystem by colonic commensal microbiota. Nature.478,250-254(2011)
74. Ley RE, Peterson DA, Gordon JI. Ecological and evolutionary forces shapingmicrobial diversity in the human intestine. Cell.124,837-848(2006)
75. Stecher B, Chaffron S, Kappeli R et al. Like will to like: abundances of closelyrelated species can predict susceptibility to intestinal colonization by pathogenic andcommensal bacteria. PLoS Pathog.6, e1000711(2010)
76. Round JL, Mazmanian SK. The gut microbiota shapes intestinal immuneresponses during health and disease. Nat Rev Immunol.9,313-323(2009)
77. Decker E, Hornef M, Stockinger S. Cesarean delivery is associated with celiacdisease but not inflammatory bowel disease in children. Gut Microbes.2,91-98(2011)
78. Ivanov II, Atarashi K, Manel N et al. Induction of intestinal Th17cells bysegmented filamentous bacteria. Cell.139,485-498(2009)
79. Lavasani S, Dzhambazov B, Nouri M et al. A novel probiotic mixture exerts atherapeutic effect on experimental autoimmune encephalomyelitis mediated by IL-10producing regulatory T cells. PLoS One.5, e9009(2010)
80. Mueller S, Saunier K, Hanisch C et al. Differences in fecal microbiota indifferent European study populations in relation to age, gender, and country: across-sectional study. Appl Environ Microbiol.72,1027-1033(2006)
81. Gomez A, Luckey D, Yeoman CJ et al. Loss of sex and age driven differencesin the gut microbiome characterize arthritis-susceptible0401mice but notarthritis-resistant0402mice. PLoS One.7, e36095(2012)
2. Bowles NE, Ni J, Kearney DL, et al.: Detection of viruses in myocardialtissues by polymerase chain reaction. evidence of adenovirus as a common cause ofmyocarditis in children and adults. J Am Coll Cardiol42,466-472(2003).
3. Gupta S, Markham DW, Drazner MH, Mammen PP: Fulminant myocarditis.Nat Clin Pract Cardiovasc Med5,693-706(2008).
4. Kyto V, Saraste A, Voipio-Pulkki LM, Saukko P: Incidence of fatalmyocarditis: a population-based study in Finland. Am J Epidemiol165,570-574(2007).
5. Dennert R, Crijns HJ, Heymans S: Acute viral myocarditis. Eur Heart J29,2073-2082(2008).
6. Remes J, Helin M, Vaino P, Rautio P: Clinical outcome and left ventricularfunction23years after acute coxsackie virus myopericarditis. Eur Heart J11,182-188(1990).
7. Feldman AM, McNamara D: Myocarditis. N Engl J Med343,1388-1398(2000).
8. Curnen EC: Human disease associated with the Coxsackie viruses. Bull N YAcad Med26,335-342(1950).
9. Easton AJ, Eglin RP: The detection of coxsackievirus RNA in cardiac tissue byin situ hybridization. J Gen Virol69(Pt2),285-291(1988).
10. Blumohr T, Klocking R, Sprossig M, Wockel W:[The coxsackievirus-infectedmouse: an animal model for the study of virus myocarditis]. Acta Biol Med Ger35,103-109(1976).
11. Badorff C, Fichtlscherer B, Rhoads RE, et al.: Nitric oxide inhibits dystrophinproteolysis by coxsackieviral protease2A through S-nitrosylation: A protectivemechanism against enteroviral cardiomyopathy. Circulation102,2276-2281(2000).
12. Henke A, Nestler M, Strunze S, et al.: The apoptotic capability ofcoxsackievirus B3is influenced by the efficient interaction between the capsid proteinVP2and the proapoptotic host protein Siva. Virology289,15-22(2001).
13. Woodruff JF, Woodruff JJ: Involvement of T lymphocytes in the pathogenesisof coxsackie virus B3heart disease. J Immunol113,1726-1734(1974).
14. Hirasawa K, Tsutsui S, Takeda M, et al.: Depletion of Mac1-positivemacrophages protects DBA/2mice from encephalomyocarditis virus-inducedmyocarditis and diabetes. J Gen Virol77(Pt4),737-741(1996).
15. Leipner C, Grun K, Borchers M, Stelzner A: The outcome of coxsackievirusB3-(CVB3-) induced myocarditis is influenced by the cellular immune status. Herz25,245-248(2000).
16. Sagar S, Liu PP, Cooper LT, Jr.: Myocarditis. Lancet379,738-747(2012).
17. Fairweather D, Stafford KA, Sung YK: Update on coxsackievirus B3myocarditis. Curr Opin Rheumatol24,401-407(2012).
18. Huber SA: Increased susceptibility of male BALB/c mice to coxsackievirusB3-induced myocarditis: role for CD1d. Med Microbiol Immun194,121-127(2005).
19. Jiang Z, Xu W, Li K, et al.: Remission of CVB3-induced viral myocarditis byin vivo Th2polarization via hydrodynamics-based interleukin-4gene transfer. J GeneMed10,918-929(2008).
20. Xie Y, Chen R, Zhang X, et al.: The role of Th17cells and regulatory T cells inCoxsackievirus B3-induced myocarditis. Virology421,78-84(2011).
21. Yuan J, Cao AL, Yu M, et al.: Th17cells facilitate the humoral immuneresponse in patients with acute viral myocarditis. J Clin Immunol30,226-234(2010).
22. Yuan J, Yu M, Lin QW, et al.: Neutralization of IL-17inhibits the productionof anti-ANT autoantibodies in CVB3-induced acute viral myocarditis. IntImmunopharmacol10,272-276(2010).
23. Opavsky MA, Penninger J, Aitken K, et al.: Susceptibility to myocarditis isdependent on the response of alphabeta T lymphocytes to coxsackieviral infection. CircRes85,551-558(1999).
24. Li K, Xu W, Guo Q, et al.: Differential macrophage polarization in male andfemale BALB/c mice infected with coxsackievirus B3defines susceptibility to viralmyocarditis. Circ Res105,353-364(2009).
25. Gorbea C, Makar KA, Pauschinger M, et al.: A role for Toll-like receptor3variants in host susceptibility to enteroviral myocarditis and dilated cardiomyopathy. JBiol Chem285,23208-23223(2010).
26. Frisancho-Kiss S, Davis SE, Nyland JF, et al.: Cutting edge: cross-regulationby TLR4and T cell Ig mucin-3determines sex differences in inflammatory heartdisease. J Immunol178,6710-6714(2007).
27. Triantafilou K, Orthopoulos G, Vakakis E, et al.: Human cardiac inflammatoryresponses triggered by Coxsackie B viruses are mainly Toll-like receptor (TLR)8-dependent. Cell Microbiol7,1117-1126(2005).
28. Wang JP, Cerny A, Asher DR, et al.: MDA5and MAVS mediate type Iinterferon responses to coxsackie B virus. J Virol84,254-260(2010).
29. Huhn MH, McCartney SA, Lind K, et al.: Melanoma differentiation-associatedprotein-5(MDA-5) limits early viral replication but is not essential for the induction oftype1interferons after Coxsackievirus infection. Virology401,42-48(2010).
30. Fairweather D, Yusung S, Frisancho S, et al.: IL-12receptor beta1andToll-like receptor4increase IL-1beta-and IL-18-associated myocarditis andcoxsackievirus replication. J Immunol170,4731-4737(2003).
31. Triantafilou K, Orthopoulos G, Vakakis E, Ahmed M: Human cardiacinflammatory responses triggered by Coxsackie B viruses are mainly Toll-like receptor(TLR)8-dependent. Cell Microbiol7,1117-1126(2005).
32. Weinzierl AO, Szalay G, Wolburg H, et al.: Effective chemokine secretion bydendritic cells and expansion of cross-presenting CD4-/CD8+dendritic cells define aprotective phenotype in the mouse model of coxsackievirus myocarditis. J Virol82,8149-8160(2008).
33. Abston ED, Coronado MJ, Bucek A, et al.: Th2regulation of viral myocarditisin mice: different roles for TLR3versus TRIF in progression to chronic disease. ClinDev Immunol2012,129486(2012).
34. Roberts B, Dragon J, Moussawi M, Huber S: Sex-specific signaling throughToll-Like Receptors2and4contributes to survival outcome of Coxsackievirus B3infection in C57Bl/6mice. Biol Sex Differ3,25. doi:10.1186/2042-6410-1183-1125.(2012).
35. Riad A, Westermann D, Escher F, et al.: Myeloid differentiation factor-88contributes to TLR9-mediated modulation of acute coxsackievirus B3-inducedmyocarditis in vivo. Am J Physiol Heart Circ Physiol298, H2024-2031(2010).
36. Fields B: Virology. Third Edition. Lippincott-Raven Publisher.(1996).
37. Guarner F, Malagelada JR: Gut flora in health and disease. Lancet361,512-519(2003).
38. Wostmann BS, Larkin C, Moriarty A, Bruckner-Kardoss E: Dietary intake,energy metabolism, and excretory losses of adult male germfree Wistar rats. Lab AnimSci33,46-50(1983).
39. Mazmanian SK, Round JL, Kasper DL: A microbial symbiosis factor preventsintestinal inflammatory disease. Nature453,620-625(2008).
40. Chen LW, Chen PH, Hsu CM: Commensal microflora contribute to hostdefense against Escherichia coli pneumonia through Toll-like receptors. Shock36,67-75(2011).
41. Medzhitov R, Janeway C, Jr.: Innate immunity. N Engl J Med343,338-344(2000).
42. Akira S, Uematsu S, Takeuchi O: Pathogen recognition and innate immunity.Cell124,783-801(2006).
43. Janeway CA, Jr.: Approaching the asymptote? Evolution and revolution inimmunology. Cold Spring Harb Symp Quant Biol54Pt1,1-13(1989).
44. Akira S, Takeda K: Toll-like receptor signalling. Nat Rev Immunol4,499-511(2004).
45. Mogensen TH: Pathogen recognition and inflammatory signaling in innateimmune defenses. Clin Microbiol Rev22,240-273, Table of Contents (2009).
46. Mogensen TH, Paludan SR: Reading the viral signature by Toll-like receptorsand other pattern recognition receptors. J Mol Med (Berl)83,180-192(2005).
47. Aretz HT, Billingham ME, Edwards WD, et al.: Myocarditis. Ahistopathologic definition and classification. Am J Cardiovasc Pathol1,3-14(1987).
48. Netea MG, Van der Meer JW, Sutmuller RP, Adema GJ, Kullberg BJ: Fromthe Th1/Th2paradigm towards a Toll-like receptor/T-helper bias. Antimicrob AgentsChemother49,3991-3996(2005).
49. Tohse N, Sperelakis N: cGMP inhibits the activity of single calcium channelsin embryonic chick heart cells. Circ Res69,325-331(1991).
50. Cohn RD, Durbeej M, Moore SA, et al.: Prevention of cardiomyopathy inmouse models lacking the smooth muscle sarcoglycan-sarcospan complex. J Clin Invest107, R1-7(2001).
51. Bobin AN, Parkhommenko Iu G:[Pathomorphology and morphogenesis ofmyocarditis in typhoid fever]. Arkh Patol67,38-40(2005).
52. Zhang SY, Jouanguy E, Ugolini S, et al.: TLR3deficiency in patients withherpes simplex encephalitis. Science317,1522-1527(2007).
53. Feldmann J, Prieur AM, Quartier P, et al.: Chronic infantile neurologicalcutaneous and articular syndrome is caused by mutations in CIAS1, a gene highlyexpressed in polymorphonuclear cells and chondrocytes. Am J Hum Genet71,198-203(2002).
54. Smirnova I, Mann N, Dols A, et al.: Assay of locus-specific genetic loadimplicates rare Toll-like receptor4mutations in meningococcal susceptibility. ProcNatl Acad Sci U S A100,6075-6080(2003).
55. Tao K, Fujii M, Tsukumo S, et al.: Genetic variations of Toll-like receptor9predispose to systemic lupus erythematosus in Japanese population. Ann Rheum Dis66,905-909(2007).
56. Tabeta K, Georgel P, Janssen E, et al.: Toll-like receptors9and3as essentialcomponents of innate immune defense against mouse cytomegalovirus infection. ProcNatl Acad Sci U S A101,3516-3521(2004).
57. Roach JC, Glusman G, Rowen L, et al.: The evolution of vertebrate Toll-likereceptors. Proc Natl Acad Sci U S A102,9577-9582(2005).
58. Shi Z, Cai Z, Sanchez A, et al.: A novel Toll-like receptor that recognizesvesicular stomatitis virus. J Biol Chem286,4517-4524(2011).
59. Takeuchi O, Akira S: Pattern recognition receptors and inflammation. Cell140,805-820(2010).
60. Li XD, Chen ZJ: Sequence specific detection of bacterial23S ribosomal RNAby TLR13. Elife1, e00102(2012).
61. Oldenburg M, Kruger A, Ferstl R, et al.: TLR13recognizes bacterial23SrRNA devoid of erythromycin resistance-forming modification. Science337,1111-1115(2012).
62. Nissen P, Hansen J, Ban N, Moore PB, Steitz TA: The structural basis ofribosome activity in peptide bond synthesis. Science289,920-930(2000).
63. McCusker KP, Fujimori DG: The chemistry of peptidyltransferasecenter-targeted antibiotics: enzymatic resistance and approaches to countering resistance.ACS Chem Biol7,64-72(2012).
64. Yajima T, Knowlton KU: Viral myocarditis: from the perspective of the virus.Circulation119,2615-2624(2009).
65.Kawai T, Akira S: The role of pattern-recognition receptors in innate immunity:update on Toll-like receptors. Nat Immunol11,373-384(2010).
66. O'Neill LA, Golenbock D, Bowie AG: The history of Toll-like receptors-redefining innate immunity. Nat Rev Immunol13,453-460(2013).
67. Sen R, Baltimore D: Multiple nuclear factors interact with the immunoglobulinenhancer sequences. Cell46,705-716(1986).
68. Li Q, Verma IM: NF-kappaB regulation in the immune system. Nat RevImmunol2,725-734(2002).
69. Goren N, Cuenca J, Martin-Sanz P, Bosca L: Attenuation of NF-kappaBsignalling in rat cardiomyocytes at birth restricts the induction of inflammatory genes.Cardiovasc Res64,289-297(2004).
70.刘慧强:核因子kB在病毒性心肌炎小鼠发病中的作用.吉林大学(2006).
71. Berer K, Krishnamoorthy G: Commensal gut flora and brain autoimmunity: alove or hate affair? Acta Neuropathol123,639-651(2012).
72. Nell S, Suerbaum S, Josenhans C: The impact of the microbiota on thepathogenesis of IBD: lessons from mouse infection models. Nat Rev Microbiol8,564-577(2010).
73. Round JL, Mazmanian SK: The gut microbiota shapes intestinal immuneresponses during health and disease. Nat Rev Immunol9,313-323(2009).
74. Cani PD, Delzenne NM, Amar J, Burcelin R: Role of gut microflora in thedevelopment of obesity and insulin resistance following high-fat diet feeding. PatholBiol (Paris)56,305-309(2008).
75. Brandt LJ: Fecal transplantation for the treatment of Clostridium difficileinfection. Gastroenterol Hepatol (N Y)8,191-194(2012).
76. Schwan A, Sjolin S, Trottestam U, Aronsson B: Relapsing clostridium difficileenterocolitis cured by rectal infusion of homologous faeces. Lancet2,845(1983).
77. Paterson DL, Iredell J, Whitby M: Putting back the bugs: bacterial treatmentrelieves chronic diarrhoea. Med J Aust160,232-233(1994).
78. Borody TJ:"Flora Power"--fecal bacteria cure chronic C. difficile diarrhea.The American journal of gastroenterology95,3028-3029(2000).
79. Brandt LJ, Aroniadis OC, Mellow M, et al.: Long-term follow-up ofcolonoscopic fecal microbiota transplant for recurrent Clostridium difficile infection.Am J Gastroenterol107,1079-1087(2012).
80. Galama J: Enteroviral infections in the immunocompromised host. RevMedical Microbiol8,33-40(1997).
81. van der Waaij D: The ecology of the human intestine and its consequences forovergrowth by pathogens such as Clostridium difficile. Annu Rev Microbiol43,69-87(1989).
82. Markle JG, Frank DN, Mortin-Toth S, et al.: Sex differences in the gutmicrobiome drive hormone-dependent regulation of autoimmunity. Science339,1084-1088(2013).
1. Kawai T, Akira S. Innate immune recognition of viral infection. Nat Immunol.7,131-137(2006)
2. Bowles NE, Ni J, Kearney DL et al. Detection of viruses in myocardial tissuesby polymerase chain reaction. Evidence of adenovirus as a common cause ofmyocarditis in children and adults. J Am Coll Cardiol.42,466-472(2003)
3. Gupta S, Markham DW, Drazner MH, Mammen PP. Fulminant myocarditis.Nat Clin Pract Cardiovasc Med.5,693-706(2008)
4. Kyto V, Saraste A, Voipio-Pulkki LM, Saukko P. Incidence of fatalmyocarditis: a population-based study in Finland. Am J Epidemiol.165,570-574(2007)
5. Dennert R, Crijns HJ, Heymans S. Acute viral myocarditis. Eur Heart J.29,2073-2082(2008)
6. Blauwet LA, Cooper LT. Myocarditis. Prog Cardiovasc Dis.52,274-288(2010)
7. Chen X, Li B, Liao Y et al. Analysis on Coxsackievirus infection and onset ofviral myocarditis of doctors and nurses in department of cardiology. Chin J CardiovascRehab Med.13,328-330(2004)
8. Cooper LT Jr. Myocarditis. N Eng J Med.360,1526-1538(2009)
9. Tam PE. Coxsackievirus myocarditis: interplay between virus and host in thepathogenesis of heart disease. Viral Immunol.19,133-146(2006)
10. Bowles NE, Vallejo J. Viral causes of cardiac inflammation. Curr OpinCardiol.18,182-188(2003)
11. Yajima T, Knowlton KU. Viral myocarditis: from the perspective of the virus.Circulation.119,2615-2624(2009)
12. Feuer R, Mena I, Pagarigan R, Slifka MK, Whitton JL. Cell cycle status affectsCoxsackievirus replication, persistence, and reactivation in vitro. J Virol.76,4430-4440(2002)
13. Feuer R, Mena I, Pagarigan RR, Hassett DE, Whitton JL. Coxsackievirusreplication and the cell cycle: a potential regulatory mechanism for viralpersistence/latency. Med Microbiol Immunol.193,83-90(2004)
14. Huber S, Sartini D. T cells expressing the Vgamma1T-cell receptor enhancevirus-neutralizing antibody response during Coxsackievirus B3infection of BALB/cmice: differences in male and female mice. Viral Immunol.18,730-739(2005)
15. Abston ED, Coronado MJ, Bucek A et al. Th2regulation of viral myocarditisin mice: different roles for TLR3versus TRIF in progression to chronic disease. ClinDev Immunol.2012,129486(2012)
16. Opavsky MA, Penninger J, Aitken K et al. Susceptibility to myocarditis isdependent on the response of alphabeta T lymphocytes to Coxsackieviral infection. CircRes.85,551-558(1999)
17. Li K, Xu W, Guo Q et al. Differential macrophage polarization in male andfemale BALB/c mice infected with Coxsackievirus B3defines susceptibility to viralmyocarditis. Circ Res.105,353-364(2009)
18. Woodruff JF, Woodruff JJ. Involvement of T lymphocytes in the pathogenesisof Coxsackievirus B3heart disease. J Immunol.113,1726-1734(1974)
19. Haga T, Suzuki J, Kosuge H et al. Attenuation of experimental autoimmunemyocarditis by blocking T cell activation through4-1BB pathway. J Mol Cell Cardiol.46,719-727(2009)
20. Sagar S, Liu PP, Cooper LT Jr. Myocarditis. Lancet.379,738-747(2012)
21. Fairweather D, Stafford KA, Sung YK. Update on Coxsackievirus B3myocarditis. Curr Opin Rheumatol.24,401-407(2012)
22. Pauschinger M, Phan MD, Doerner A et al. Enteroviral RNA replication in themyocardium of patients with left ventricular dysfunction and clinically suspectedmyocarditis. Circulation.99,889-895(1999)
23. Reetoo KN, Osman SA, Illavia SJ et al. Quantitative analysis of viral RNAkinetics in Coxsackievirus B3-induced murine myocarditis, biphasic pattern ofclearance following acute infection, with persistence of residual viral RNA throughoutand beyond the inflammatory phase of disease. J Gen Virol.81,2755-2762(2000)
24. Kishimoto C, Kuribayashi K, Masuda T, Tomika N, Kawai C. Immunologicbehavior of lymphocytes in experimental viral myocarditis: significance of Tlymphocytes in the severity of myocarditis and silent myocarditis in BALB/c-nu/numice. Circulation.71,1247-1254(1985)
25. Huber SA. Increased susceptibility of male BALB/c mice to CoxsackievirusB3-induced myocarditis: role for CD1d. Med Microbiol Immunol.194,121-127(2005)
26. Jiang Z, Xu W, Li K et al. Remission of CVB3-induced viral myocarditis by invivo Th2polarization via hydrodynamics-based interleukin-4gene transfer. J Gene Med.10,918-929(2008)
27. Fairweather D, Frisancho-Kiss S, Yusung SA et al. Interferon-gamma protectsagainst chronic viral myocarditis by reducing mast cell degranulation, fibrosis, and theprofibrotic cytokines transforming growth factor-beta1, interleukin-1beta, andinterleukin-4in the heart. Am J Pathol.165,1883-1894(2004)
28. Konkel JE, Chen W. Balancing acts: the role of TGF-beta in the mucosalimmune system. Trends Mol Med.17,668-676(2011)
29. Xie Y, Chen R, Zhang X et al. The role of Th17cells and regulatory T cells inCoxsackievirus B3-induced myocarditis. Virology.421,78-84(2011)
30. Yuan J, Cao AL, Yu M et al. Th17cells facilitate the humoral immuneresponse in patients with acute viral myocarditis. J Clin Immunol.30,226-234(2010)
31. Yuan J, Yu M, Lin QW et al. Neutralization of IL-17inhibits the production ofanti-ANT autoantibodies in CVB3-induced acute viral myocarditis. IntImmunopharmacol.10,272-276(2010)
32. Frisancho-Kiss S, Nyland JF, Davis SE et al. Cutting edge: T cell Ig mucin-3reduces inflammatory heart disease by increasing CTLA-4during innate immunity. JImmunol.176,6411-6415(2006)
33. Lv K, Xu W, Wang C et al. Galectin-9administration ameliorates CVB3induced myocarditis by promoting the proliferation of regulatory T cells andalternatively activated Th2cells. Clin Immunol.140,92-101(2011)
34. Hwang JY, Jun EJ, Seo I et al. Characterization of infections of humanleukocytes by non-polio enteroviruses. Intervirology.55,333-341(2012)
35. Kling K, Rieger P, Mall G et al. Visualization of enteroviral replication inmyocardial tissue by ultrastructural in situ hybridization: identification of target cellsand cytopathic effects. Lab Invest.78,1227-1237(1998)
36. Kandolf R, Sauter M. Aepinus C et al. Mechanisms and consequences ofenterovirus persistence in cardiac myocytes and cells of the immune system. Virus Res.62,149-158(1999)
37. Opavsky MA, Martino T, Rabinovitch M et al. Enhanced ERK-1/2activationin mice susceptible to Coxsackievirus-induced myocarditis. J Clin Invest.109,1561-1569(2002)
38. Kim DS, Park JH, Kim JY, Kim D, Nam JH. A mechanism of immunoreceptortyrosine-based activation motif (ITAM)-like sequences in the capsid protein VP2inviral growth and pathogenesis of Coxsackievirus B3. Virus Genes.44,176-182(2012)
39. Barin JG, Rose NR, Cihakova D. Macrophage diversity in cardiacinflammation: a review. Immunobiology.217,468-475(2012)
40. Mantovani A, Sica A, Locati M. Macrophage polarization comes of age.Immunity.23,344-346(2005)
41. Mosser DM, Edwards JP. Exploring the full spectrum of macrophageactivation. Nat Rev Immunol.8,958-969(2008)
42. Holtmeier W, Kabelitz D. Gammadelta T cells link innate and adaptiveimmune responses. Chem Immunol Allergy.86,151-183(2005)
43. Huber SA. Depletion of gammadelta+T cells increases CD4+FoxP3(Tregulatory) cell response in Coxsackievirus B3-induced myocarditis. Immunology.127,567-576(2009)
44. Huber SA. Born W, O’Brien R. Dual functions of murine gammadelta cells ininflammation and autoimmunity in Coxsackievirus B3-induced myocarditis: role ofVgamma1+and Vgamma4+cells. Microbes Infect.7,537-543(2005)
45. Huber S, Sartini D. T cells expressing the Vgamma1T-cell receptor enhancevirus-neutralizing antibody response during Coxsackievirus B3infection of BALB/cmice: differences in male and female mice. Viral Immunol.18,730-739(2005)
46. Huber S, Sartini D, Exley M. Role of CD1d in Coxsackievirus B3-inducedmyocarditis. J Immunol.170,3147-3153(2003)
47. Huber SA, Graveline D, Born WK, O’Brien RL. Cytokine production byVgamma+-T-cell subsets is an important factor determining CD4+-Th-cell phenotypeand susceptibility of BALB/c mice to Coxsackievirus B3-induced myocarditis. J Virol.75,5860-5869(2001)
48. Kemball CC, Alirezaei M, Whitton JL. Type B Coxsackieviruses and theirinteractions with the innate and adaptive immune systems. Future Microbiol.5,1329-1347(2010)
49. Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linkinginnate and acquired immunity. Nat Immunol.2,675-680(2001)
50. Huang CH, Vallejo JG, Kollias G, Mann DL. Role of the innate immunesystem in acute viral myocarditis. Basic Res Cardiol.104,228-237(2009)
51. Sen GC. Viruses and interferons. Annu Rev Microbiol.55,255-281(2001)
52. Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD. How cellsrespond to interferons. Annu Rev Biochem.67,227-264(1998)
53. Frisancho-Kiss S, Davis SE, Nyland JF et al. Cutting edge: corss-regulation byTLR4and T cell Ig mucin-3determines sex differences in inflammatory heart disease. JImmunol.178,6710-6714(2007)
54. Gorbea C, Makar KA, Pauschinger M et al. A role for Toll-like receptor3variants in host susceptibility to enteroviral myocarditis and dilated cardiomyopathy. JBiol Chem.285,23208-23223(2010)
55. Wang JP, Asher DR, Chan M, Kurt-Jones EA, Finberg RW. Cutting edge:antibody-mediated TLR7-dependent recognition of viral RNA. J Immunol.178,3363-3367(2007)
56. Triantafilou K, Orthopoulos G, Vakakis E et al. Human cardiac inflammatoryresponses triggered by Coxsackie B viruses are mainly Toll-like receptor (TLR)8-dependent. Cell Microbiol.7,1117-1126(2005)
57. Riad A, Westermann D, Escher F et al. Myeloid differentiation factor-88contributes to TLR9-mediated modulation of acute Coxsackievirus B3-inducedmyocarditis in vivo. Am J Physiol Heart Circ Physiol.298, H2024-H2031(2010)
58. Hardarson HS, Baker JS, Yang Z et al. Toll-like receptors3is an essentialcomponent of the innate stress response in virus-induced cardiac injury. Am J PhysiolHeart Circ Physiol.292, H251-H258(2007)
59. Netea MG, Van der Meet JW, Sutmuller RP, Adema GJ, Kullberg BJ. Fromthe Th1/Th2paradigm towards a Toll-like receptor/T-helper bias. Antimicrob AgentsChemother.49,3991-3996(2005)
60. Roberts BJ, Moussawi M, Huber SA. Sex differences in TLR2and TLR4expression and their effect on Coxsackievirus-induced autoimmune myocarditis. ExpMol Pathol.94,58-64(2013)
61. Kato H, Sato S, Yoneyama M et al. Cell type-specific involvement of RIG-I inantiviral responses. Immunity.23,19-28(2005)
62. Yoneyama M, Kikuchi M, Natsukawa T et al. The RNA helicase RIG-I has anessential function in double-stranded RNA-induced innate antiviral responses. NatImmunol.5,730-737(2004)
63. Yoneyama M, Kikuchi M, Matsumono K et al. Shared and unique functions ofthe DExD/H-box helicases RIG-I, MDA5, and LGP2in antiviral innate immunity. JImmunol.175,2851-2858(2005)
64. Kato H, Takeuchi O, Sato S et al. Differential roles of MDA5and RIG-Ihelicases in the recognition of RNA viruses. Nature.441,101-105(2006)
65. Wang JP, Cerny A, Asher DR et al. MDA5and MAVS mediate type Iinterferon responses to Coxsackie B virus. J Virol.84,254-260(2010)
66. Huhn MH, McCartney SA, Lind K et al. Melanoma differentiation-associatedprotein-5(MDA-5) limits early viral replication but is not essential for the induction oftype I interferons after Coxsackievirus infection. Virology.401,42-48(2010)
67. Bopegamage S, Borsanyiova M, Vargova A et al. Coxsackievirus infection ofmice. I. Viral kinetics and histopathological changes in mice experimentally infectedwith Coxsackievirus B3and B4by oral route. Acta Virol.47,245-251(2003)
68. Vargova A, Bopegamage S, Borsanyiova M et al. Coxsackievirus infection ofmice. II. Viral kinetics and histopathological changes in mice experimentally infectedwith Coxsackievirus B3by intraperitoneal route. Acta Virol.47,253-257(2003)
69. Molin Y, Frisk P, Ilback NG. Viral RNA kinetics is associated with changes intrace elements in target organs of Coxsackievirus B3infection. Microbes Infect.11,493-499(2009)
70. Mazmanian SK, Round JL, Kasper DL. A microbial symbiosis factor preventsintestinal inflammatory disease. Nature.453,620-625(2008)
71. Chen LW, Chen PH, Hsu CM. Commensal microflora contribute to hostdefense against Escherichia coli pneumonia through Toll-like receptors. Shock.36,67-75(2011)
72. Gagliani N, Huber S, Flavell RA. The intestine: where amazing things happen.Cell Res.22,277-279(2012)
73. Lathrop SK, Bloom SM, Rao SM et al. Peripheral education of the immunesystem by colonic commensal microbiota. Nature.478,250-254(2011)
74. Ley RE, Peterson DA, Gordon JI. Ecological and evolutionary forces shapingmicrobial diversity in the human intestine. Cell.124,837-848(2006)
75. Stecher B, Chaffron S, Kappeli R et al. Like will to like: abundances of closelyrelated species can predict susceptibility to intestinal colonization by pathogenic andcommensal bacteria. PLoS Pathog.6, e1000711(2010)
76. Round JL, Mazmanian SK. The gut microbiota shapes intestinal immuneresponses during health and disease. Nat Rev Immunol.9,313-323(2009)
77. Decker E, Hornef M, Stockinger S. Cesarean delivery is associated with celiacdisease but not inflammatory bowel disease in children. Gut Microbes.2,91-98(2011)
78. Ivanov II, Atarashi K, Manel N et al. Induction of intestinal Th17cells bysegmented filamentous bacteria. Cell.139,485-498(2009)
79. Lavasani S, Dzhambazov B, Nouri M et al. A novel probiotic mixture exerts atherapeutic effect on experimental autoimmune encephalomyelitis mediated by IL-10producing regulatory T cells. PLoS One.5, e9009(2010)
80. Mueller S, Saunier K, Hanisch C et al. Differences in fecal microbiota indifferent European study populations in relation to age, gender, and country: across-sectional study. Appl Environ Microbiol.72,1027-1033(2006)
81. Gomez A, Luckey D, Yeoman CJ et al. Loss of sex and age driven differencesin the gut microbiome characterize arthritis-susceptible0401mice but notarthritis-resistant0402mice. PLoS One.7, e36095(2012)