Coxsackieviral pancreatitis: autophagy, proteolysis, and inflammation

描述(申请人提供)：柯萨奇病毒B3(CVB3)是微小核糖核酸病毒家族中的一种肠道病毒，是胰腺炎的常见感染性原因，胰腺炎是一种常见、严重和昂贵的疾病。已知的一些形式的胰腺炎涉及自噬途径；例如，阻止途径后期步骤的毒素可能触发疾病，可能是因为阻断导致细胞内胰蛋白酶原的激活。矛盾的是，自噬的完全失活可以预防这些类型的胰腺炎。因此，至少这些形式的胰腺炎需要途径既是(I)活跃的，又(Ii)在晚期被阻断的。自噬在许多病毒和细菌感染期间被上调，并经常被抗击。可以预见的是，进化导致了几种病毒发展出逃避该途径抑制效应的机制，研究表明，一些病毒--包括CVB3--更进一步，积极利用自噬来增强自己的复制。我的实验室已经证明，在活体的腺泡细胞中，CVB3阻断了自噬途径的后期阶段。因此，我们假设在CVB3对自噬途径的这种影响与病毒引起胰腺炎的能力之间可能存在联系。我们希望在活体内(即在活体动物身上，而不仅仅是在组织培养细胞中)测试这一想法。为了实现这一目标，我们最近培育了条件性KO小鼠(Atg5f/f/Cre小鼠)，其中自噬途径的关键蛋白ATG5仅在胰腺腺泡细胞中缺失。使用这些小鼠，我们已经证明了CVB3受益于一个完整的自噬途径，没有这个途径，CVB3在胰腺中的复制被严重抑制。此外，在Atg5f/f/Cre小鼠中，病毒诱导的胰腺炎显著减少。因此，像一些其他类型的胰腺炎一样，CVB胰腺炎要求自噬是活跃的(就像在wt小鼠中一样)，但功能障碍(因为病毒阻断了途径)。此外，我们未发表的数据表明，CVB3感染诱导腺泡细胞内依赖于自噬的胰蛋白酶活性。这些和其他数据让我提出，所有的胰腺炎，无论是病毒的还是非病毒的，都有一个共同的原因；我认为它们是由于自噬途径的晚期阻断造成的，这导致细胞内胰蛋白酶原的裂解，释放细胞内的胰蛋白酶活性。这些问题将在AIMS 1和AIMS 2中进行研究。我进一步假设，T1IFN信号进入腺泡细胞会加剧胰酶启动的损伤；这将在AIMS 3中进行研究。如果这些概念得到验证，自噬和T1IFN将成为目前无法治疗的所有形式的疾病的关键治疗靶点。有三个具体目标。目的1.在体内更好地确定腺泡细胞中CVB3、自噬和胰酶原之间的相互作用。目的2.评价直接进入腺泡细胞的T1干扰素信号对病毒性胰腺炎的影响。目的3.探讨T1IFN在病毒感染的胰腺组织中募集免疫细胞的作用。

DESCRIPTION (provided by applicant): Coxsackievirus B3 (CVB3), an enterovirus in the picornavirus family, is a frequent infectious cause of pancreatitis, a common, serious, and costly disease. Some forms of pancreatitis are known to involve the autophagy pathway; e.g., toxins that block late steps in the pathway can trigger the disease, perhaps because the blockade causes activation of intracellular trypsinogen. Paradoxically, complete inactivation of autophagy protects against those types of pancreatitis. Therefore, at least those forms of pancreatitis require that the pathway be both (i) active and (ii) blocked at a late stage. Autophagy is upregulated during, and often combats, many viral and bacterial infections. Predictably, evolution has led to several viruses developing mechanisms by which to evade the inhibitory effects of the pathway, and studies have suggested that some viruses - including CVB3 - have gone one step further, actively exploiting autophagy to enhance their replication. My lab has shown that, in acinar cells in vivo, CVB3 interrupts a late stage of the autophagy pathway. We therefore hypothesized that there might be a link between this effect of CVB3 on the autophagy pathway, and the virus' ability to cause pancreatitis. We wished to test this idea in vivo, (i.e., n the living animal, not only in tissue culture cells). To achieve this goal, we have recently developed conditional KO mice (Atg5f/f/Cre+ mice) in which a key protein in the autophagy pathway, Atg5, has been deleted only in pancreatic acinar cells. Using these mice, we have shown that CVB3 benefits from an intact autophagy pathway without which CVB3 replication in the pancreas is severely curtailed. Furthermore, virus-induced pancreatitis is dramatically reduced in the Atg5f/f/Cre+ mice. Thus, like some other types of pancreatitis, CVB pancreatitis requires that autophagy be active (as it is in wt mice), but dysfunctional (because the virus blocks the pathway). Furthermore, our unpublished data show that CVB3 infection induces autophagy-dependent trypsin activity inside acinar cells. These and other data lead me to propose that there is a common cause for all pancreatitides, viral and non- viral; I propose that they result from a late blockade of the autophagy pathway, which leads to cleavage of intracellular trypsinogen, unleashing trypsin activity inside the cell. These issues will be investigated in Aims 1 & 2. I further hypothesize that the trypsin-initiated damage can be exacerbated by T1IFN signaling into acinar cells; this will be investigated in Aim 3. If these concepts are validated, autophagy and T1IFNs would become key therapeutic targets for all forms of this currently-untreatable disease. There are three Specific Aims. Aim 1. To better define interactions among CVB3, autophagy, and trypsinogen in acinar cells in vivo. Aim 2. To evaluate how T1IFN signaling directly into acinar cells affects viral pancreatitis. Aim 3. To evaluate the role of T1IFNs in recruiting immune cells to the virus-infected pancreas.