Control of placental infection by decidual NK cell secreted granulysin

 描述(申请人提供)：在怀孕期间，母体免疫细胞需要建立对胎儿和胎盘组织的免疫耐受性，但也要保护免受感染。蜕膜NK细胞(DNK)是母胎界面上最大的母体免疫细胞群，是母胎组织相互作用的场所。这项提议将探索一种新颖而令人兴奋的机制，通过这种机制，DNK可以杀死感染胎儿滋养细胞的细菌，但不会感染胎儿细胞。人类dNK高度表达颗粒溶素，这是一种抗菌肽，优先破坏微生物的膜，但对哺乳动物膜的活性较低。颗粒溶素是一种无活性的15 kDa前体肽，在杀伤细胞细胞毒颗粒中加工成9 kDa的膜解肽。我们的初步数据表明，DNK细胞内有两个颗粒溶素池-一个池包含细胞毒性颗粒中活性和非活性形式的颗粒溶素以及穿孔素和颗粒酶，而另一个池仅包含非活性颗粒溶素前体。初步数据表明，DNK结构性地分泌高水平的颗粒溶素，而不分泌其他细胞溶解分子。文化 来自dNKs的培养上清，而不是外周血NK细胞(PNK)，在不杀死滋养层细胞的情况下，杀死细胞外单核细胞增生性李斯特氏菌和胎儿滋养层细胞系内的单核细胞增多性乳杆菌。基于这些初步数据，我们假设颗粒溶素的分泌在不损害胎儿细胞的情况下杀死了细胞内的病原体。为了研究这一假设， 我们建议证实我们的初步数据，即细胞内的微生物是由DNK分泌的颗粒溶素杀死的，而不是穿孔素和颗粒酶；确定细菌或细菌产物或炎性细胞因子是否上调颗粒溶素的表达和分泌；并了解滋养层细胞如何抵抗杀伤。我们还将确定颗粒溶素的分泌是结构性的还是受调控的，并研究dNKs对感染的胎儿绒毛外滋养层细胞(EVT，蜕膜中侵袭性最强的胎儿细胞)的反应是否与母体蜕膜基质细胞(DSC)不同。我们将使用成像来确定DNK细胞是否与这些感染细胞形成免疫突触，突触的类型是否不同，以及这种接触是否会触发细胞毒颗粒的释放。使用的模型将是3种在怀孕期间导致并发症的病原体-单核细胞增多性李斯特菌、B组链球菌(GBS)和弓形虫。

 DESCRIPTION (provided by applicant): During pregnancy maternal immune cells need to establish immune tolerance of fetal and placental tissues, but also protect against infection. Decidual NK cells (dNK) are the largest maternal immune cell population at the maternal-fetal interface, where maternal and fetal tissues interact. This proposal will explore a novel and exciting mechanism by which dNK kill bacteria that infect fetal trophoblasts, but spare the fetal cells. Human dNKs highly express granulysin, an antimicrobial peptide that preferentially disrupts the membranes of microbes, but is less active against mammalian membranes. Granulysin is produced as an inactive 15 kDa pro-peptide that is processed in killer cell cytotoxic granules to a 9 kDa membranolytic peptide. Our preliminary data suggest that dNK cells have two intracellular pools of granulysin - one pool contains both the active and inactive forms of granulysin in cytotoxic granules together with perforin and granzymes, while the other pool contains only the inactive granulysin precursor. Preliminary data demonstrate that dNK constitutively secrete high levels of granulysin without the other cytolytic molecules. The culture supernatants from dNKs, but not peripheral blood NK cells (pNK), kill extracellular L. monocytogenes as well as L. monocytogenes within fetal trophoblast cell lines without killing the trophoblast. Based on these preliminary data, we hypothesize that granulysin secretion kills intracellular pathogens in fetal cells without harming those cells. To investigate this hypothesis, we propose to confirm our preliminary data showing that intracellular microbes are killed by dNK secretion of granulysin, independently of perforin and granzymes; determine whether bacteria or bacterial products or inflammatory cytokines upregulate granulysin expression and secretion; and understand how the trophoblast resists killing. We will also define whether granulysin secretion is constitutive or regulated and investigate whether dNKs respond differently to infected fetal extravillous trophoblasts (EVT, the most invasive fetal cells in the decidua) than t maternal decidual stromal cells (DSC). We will use imaging to determine whether dNK cells form an immune synapse with these infected cells, whether the type of synapse differs and whether the encounter triggers cytotoxic granule release. The models used will be 3 pathogens that cause complications during pregnancy - L. monocytogenes, Group B Streptococci (GBS) and Toxoplasma gondii.