Lung MIWI2 and the host defense against Influenza A virus

摘要 流感是全球最常见的呼吸道感染之一，已导致惊人的死亡人数 每年。季节性疫苗和疗法在高风险患者中的疗效各不相同， 作为唯一的对策。这就迫切需要进一步了解宿主对 流感，以揭示可以使用新疗法靶向的途径。其中一种靶细胞 甲型流感病毒（IAV）是肺气道的多纤毛细胞。我们的实验室之前发现了一个 由MIWI 2表达定义的多纤毛细胞，MIWI 2是Argonaute家族蛋白质， 哺乳动物的睾丸MIWI 2与PIWI相互作用RNA（皮尔纳）结合并抑制逆转录转座子活性， 生殖细胞以维持基因组完整性。然而，MIWI 2在体细胞中的表达并不清楚。 为了了解肺MIWI 2，我们使用了MIWI 2-TdTomato杂合子（单倍充足）和 纯合子（缺陷型）敲入报告基因小鼠模型。MIWI 2缺陷的小鼠表现出显著降低 提示MIWI 2在调节病毒发病机制中起关键作用， 免疫反应通过对HA-mNeon记者IAV的采访，我们排除了MIWI 2多分支的可能性。 细胞被优先感染，因为我们观察到MIWI 2单倍足够的细胞之间的病毒嗜性没有差异。 和缺陷小鼠。最近，我们发现了一种免疫细胞群的存在， MIWI 2，这提高了前景，即这种迄今未被认识的造血细胞可能是MIWI 2的关键 依赖免疫功能。综上所述，我们假设肺中表达MIWI 2的细胞严重地 调节宿主对IAV感染的反应。我们将通过追求两个目标来检验我们的中心假设。在 第一个目的，我们将使用骨髓移植和细胞特异性缺失的MIWI 2小鼠模型， 确定在免疫应答期间调节免疫应答的表达MIWI 2的细胞群体的身份， IAV感染。在第二个目标中，我们将采用单细胞测序来阐明MIWI 2依赖的途径 关键性地调节肺部的抗病毒免疫反应。总的来说，这里提出的工作将扩大我们的 了解IAV的病毒发病机制，并发现参与肺部疾病的新途径， 宿主免疫反应这些研究还将提供关于Argonaute作用的功能信息 体细胞中的蛋白质。最重要的是，这是一个平台，一个综合和令人信服的培训计划 这将支持我作为一个独立研究员的发展。

Abstract Influenza is one of the most common respiratory infections globally and has led to an alarming number of deaths annually. Seasonal vaccines and therapies vary in efficacy among higher risk patients although they currently serve as the only countermeasures. This presents a critical need to further understand the host response to influenza in order to uncover pathways that can be targeted using novel therapies. One of the target cells of Influenza A virus (IAV) are the multiciliated cells of the lung airway. Our lab had previously discovered a subset of multiciliated cells defined by the expression of MIWI2, an Argonaute family protein most commonly studied in mammalian testes. MIWI2 binds to PIWI-interacting RNA (piRNA) and suppresses retrotransposon activity in germline cells to maintain genome integrity. However, MIWI2 expression in somatic cells is not well understood. In order to understand lung MIWI2, we used a MIWI2-TdTomato heterozygote (haplo-sufficient) and a homozygote (deficient) knock-in reporter mouse model. Mice deficient in MIWI2 exhibited a markedly decrease burden of Influenza A virus suggesting that MIWI2 plays a critical role in regulating viral pathogenesis and the immune response. Incorporating an HA-mNeon reporter IAV, we ruled out the possibility that MIWI2 multiciliated cells were preferentially infected as we observed no differences in viral tropism between MIWI2 haplo-sufficient and deficient mice. Recently, we identified the presence of an immune cell population that also expresses MIWI2, raising the prospect that this heretofore unrecognized hematopoietic cell could be key to MIWI2 dependent immune function. Taken together, we hypothesize that a MIWI2 expressing cell in the lung critically modulates the host response to IAV infection. We will examine our central hypothesis by pursuing two aims. In the first Aim, we will use bone marrow transplantations and cell-specific deletion of MIWI2 mouse models to determine the identity of the MIWI2 expressing cell population that modulates the immune response during an IAV infection. In the second Aim, we will employ single cell sequencing to elucidate MIWI2-dependent pathways that critically regulate the lung’s antiviral immune response. Overall, the work proposed here will expand our understanding of the viral pathogenesis of IAV as well as uncover novel pathways that are involved in the lung’s host immune response. These studies will also provide functional information regarding the action of Argonaute proteins in somatic cells. Most importantly, this serves as a platform for an integrated and cogent training plan that will support my development as an independent researcher.