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.
Infections at the maternal-fetal interface during pregnancy (e.g. Listeria monocytogenes, Group B Streptococci, Toxoplasma gondii) can lead to fetal loss, premature labor, congenital anomalies, or fetal distress and raise the question of how the human immune system deals with the conflict between tolerance to semi-allogeneic fetal tissues and immunity to infections in this site. Preliminary observations indicate that granulysin secreted by decidual NK cells is able to kill intracellular bacteria within trophoblasts (that form the feta side of the maternal- fetal interface) without harming the host cells. Understanding the mechanism involved may lead to insights that can be applied to the development of therapies to reduce the frequency of severe maternal health problems, for example, therapies that enhance secretion of granulysin by decidual natural killer cells.
