Analyzing the effects of type I interferons in the enterovirus-infected heart

 描述(申请人提供)：病毒性心肌炎是一种相对常见，有时严重，并具有潜在致命性的疾病，最常见的原因之一是肠道病毒柯萨奇病毒B3(CVB3)。心脏的CVB3感染是高度局灶性的，即使在缺乏完整的适应性免疫系统的小鼠中也是如此，这表明先天免疫可能会控制病毒。I型干扰素(T1IFN)是一个明显的候选药物，已知对肠道病毒性心肌炎的预后有积极影响。然而，关于T1IFN的作用是如何介导的，我们还有很多不知道的地方。例如，一个非常受人尊敬的研究小组认为，T1IFN甚至可能不在心脏内起作用，它们的心脏保护作用可能是间接的，这是由于抑制了其他器官的病毒复制。在我们培育的小鼠中，T1IFN受体(T1IFNR)可以在体内被特异性地从心肌细胞中去除，我们未发表的数据明确地表明，T1IFN确实在CVB3感染期间直接作用于心肌细胞，并在病毒滴度和CVB诱导的疾病中发挥重要作用。但仍有许多问题存在。它们是如何做到这一点的--在感染的(和未感染的)心肌细胞中，T1IFN激活了哪些基因？我们将在活体内确定对T1干扰素信号有直接反应的心肌细胞基因。CVB3还会导致慢性心肌炎和扩张性心肌病，这与RNA在心脏中的持续存在有关，我们的可诱导基因敲除模型在这方面提供了关键的实验优势；我们可以在没有发生疾病的遗传完整小鼠中建立持续的CVB3感染，然后阻断T1IFN信号进入心肌细胞，以问：这些小鼠现在是否发展成疾病，即对慢性心肌炎/DCM的抵抗/易感性是否与心肌细胞中的T1IFN信号有关？我们的初步数据表明，在CVB3感染心脏期间，T1IFN的作用可能是双相的，我们假设这两个时相是由T1IFN的不同细胞来源定义的。因此，目标2和目标3扩展了我们的重点，以解决T1IFN的生产问题。哪些细胞是T1IFN的来源？血浆细胞样树突状细胞(PDC)是明显的候选细胞，但最近的研究对其在病毒感染过程中的体内真实作用提出了质疑，并表明依赖于TLR3的T1IFN的产生在控制病毒感染方面可能更重要。该提案有三个具体目标：1.研究1型干扰素信号在急性和持续CVB3感染过程中对心肌细胞的体内影响。目的2.探讨pDC在CVB3感染中的作用。目的3.探讨TLR3在CVB3所致心肌炎中的关键作用。

 DESCRIPTION (provided by applicant): Viral myocarditis is a relatively common, sometimes severe, and potentially fatal disorder, and one of the most frequent causes is the enterovirus coxsackievirus B3 (CVB3). CVB3 infection of the heart is highly focal, even in mice lacking an intact adaptive immune system, suggesting that innate immunity may hold the virus in check. Type I interferons (T1IFNs) are an obvious candidate, and are known to positively affect the outcome of enteroviral myocarditis. However, there is much that we do not know about how the effects of T1IFNs are mediated. For example, one very well-respected group has argued that T1IFNs may not even act within the heart, and that their cardioprotective effects may be indirect, resulting from the suppression of viral replication in other organs. We have generated mice in which the T1IFN receptor (T1IFNR) can be ablated in vivo specifically from cardiomyocytes, and our unpublished data show unequivocally that T1IFNs do, in fact, act directly on cardiomyocytes during CVB3 infection, and play a substantial role in viral titers and CVB-induced disease. But many questions remain. How do they do this - what genes are activated by T1IFNs in infected (and in uninfected) cardiomyocytes? We shall identify the cardiomyocyte genes that are directly responsive to T1IFN signals in vivo. CVB3 also causes chronic myocarditis and dilated cardiomyopathy, which are related to RNA persistence in the heart, and our inducible gene knockout model confers a key experimental advantage in this regard; we can establish persistent CVB3 infection in genetically-intact mice that do not develop disease, and afterwards ablate T1IFN signaling into cardiomyocytes, to ask: do these mice now develop disease, i.e. might resistance/susceptibility to chronic myocarditis / DCM be related to T1IFN signaling in cardiomyocytes? Our preliminary data indicate that the effects of T1IFN during CVB3 infection of the heart may be biphasic, and we hypothesize that the two temporal phases are defined by differing cellular sources of the T1IFNs. So, Aims 2 and 3 extend our focus to address the production of T1IFNs. What cells serve as the source of the T1IFNs? Plasmacytoid DCs (pDCs) are obvious candidates, but recent work has raised questions about their true in vivo role during viral infection, and also has shown that TLR3-dependent production of T1IFNs may be more important in controlling virus infection. The proposal has three Specific Aims Aim 1. To investigate the in vivo consequences of type 1 IFN signaling in cardiomyocytes during acute and persistent CVB3 infection. Aim 2. To evaluate the role of pDCs during CVB3 infection Aim 3. To determine why TLR3 plays a key role in CVB3-induced myocarditis.