Toxoplasma gondii is a major source of congenital disease worldwide, but the cellular and molecular factors associated with its vertical transmission are largely unknown. In humans, the placenta forms the key interface between the maternal and fetal compartments and forms the primary barrier that restricts the hematogenous spread of microorganisms. Here, we utilized primary human trophoblast (PHT) cells isolated from full-term placentas and human midgestation chorionic villous explants to determine the mechanisms by which human trophoblasts restrict and respond to T. gondii infection. We show that placental syncytiotrophoblasts, multinucleated cells that are in direct contact with maternal blood, restrict T. gondii infection at two distinct stages of the parasite lytic cycle—at the time of attachment and also during intracellular replication. Utilizing comparative transcriptome sequencing (RNA-seq) transcriptional profiling, we also show that human placental trophoblasts from both the second and third trimesters respond uniquely to T. gondii infection compared to trophoblast cell lines, typified by the upregulation of several immunity-related genes. One of the most differentially induced genes was the chemokine CCL22, which relies on the secretion of a parasite effector(s) either during or after invasion for its induction. Collectively, our findings provide new insights into the mechanisms by which the human placenta restricts the vertical transmission of T. gondii at early and late stages of human pregnancy and demonstrate the existence of at least two interferon-independent pathways that restrict T. gondii access to the fetal compartment.

Toxoplasma gondii is a globally ubiquitous pathogen that can cause severe disease in HIV/AIDS patients and can also cross the placenta and infect the developing fetus. We have found that placental and immune cells infected with T. gondii secrete significant amounts of a chemokine (called CCL22) that is critical for immune tolerance during pregnancy.

Toxoplasma gondii is an intracellular protozoan pathogen of humans that causes severe disease in immunocompromised patients and in the developing fetus. T. gondii specifically alters production of the immunomodulatory chemokine CCL22 in human placental cells during infection. Using a combination of bioinformatics and molecular genetics, we have now identified T. gondii GRA28 as the gene product required for CCL22 induction. GRA28 is strongly co-regulated at the transcriptional level along with other known secreted effectors and their chaperones. GRA28 is secreted into the host cell where it localizes to the nucleus, and deletion of this gene results in reduced CCL22 secretion from human monocytes and second trimester placental explants. The impact of GRA28 on CCL22 is also conserved in mouse immune and placental cells and the deletion of GRA28 results in increased inflammatory responses and reduced CNS burden during mouse infections

Field research can be an important component of the career trajectories for researchers in numerous academic fields; however, conducting research in field settings poses risks to health and safety, and researchers from marginalized groups often face greater risks than those experienced by other researchers in their fields; If these additional risks are not actively and thoughtfully mitigated, they are likely to hinder the participation of qualified investigators in field research and counteract efforts to improve and promote diversity, equity and inclusion in the field sciences. Here we provide, from our perspectives as co‐authors of a field safety manual for the Department of Biological Sciences at the University of Pittsburgh in Pennsylvania, United States, (A) background on risks and barriers that should be considered when planning and conducting field research and (B) suggestions on how to work as a collaborative team for developing an inclusive field safety manual. As an example of a manual this proposed process has yielded, we have included our own field safety manual written with diversity, equity and inclusion as a central focus. We hope this publication serves as a starting point for those interested in developing a similar document for use in their laboratory group, department or institution.

52Toxoplasma gondii is a major source of congenital disease worldwide, but the cellular and 53 molecular factors associated with its vertical transmission are largely unknown. In humans, the 54 placenta forms the key interface between the maternal and fetal compartments and forms the 55 primary barrier that restricts the hematogenous spread of microorganisms. Here, we utilized 56 primary human trophoblast (PHT) cells isolated from full-term placentas and human mid-gestation 57 chorionic villous explants to determine the mechanisms by which human trophoblasts restrict and 58 respond to T. gondii infection. We show that placental syncytiotrophoblasts, multinucleated cells 59 that are in direct contact with maternal blood, restrict T. gondii infection at distinct stages of the 60 parasite lytic cycle-at the time of attachment and also during intracellular replication. Utilizing 61 comparative RNAseq transcriptional profiling, we also show that human placental trophoblasts at 62 both mid-and late-stages of gestation induce the chemokine CCL22 in response to T. gondii 63 infection, which relies on the secretion of parasite effector(s). Collectively, our findings provide 64 new insights into the mechanisms by which the human placenta restricts the vertical transmission 65 of T. gondii at early and late stages of human pregnancy, and demonstrate the existence of at 66 least two interferon-independent pathways that restrict T. gondii access to the fetal compartment. 67 68 Significance statement 69 Toxoplasma gondii is a major source of congenital disease worldwide and must breach the 70 placental barrier to be transmitted from maternal blood to the developing fetus. The events 71 associated with the vertical transmission of T. gondii are largely unknown. Here, we show that 72 primary human syncytiotrophoblasts, the fetal-derived cells that comprise the primary placental 73 barrier, restrict T. gondii infection at two distinct stages of the parasite life cycle and respond to 74 infection through the induction of the chemokine CCL22. Collectively, our findings provide 75 important insights into the mechanisms by which human syncytiotrophoblasts restrict T. 76 3 gondii infection at early and late stages of human pregnancy and identify the placental-77 enriched signaling pathways induced in response to infection. Toxoplasma gondii is a major source of congenital disease, with ~200,000 global cases 104 of congenital toxoplasmosis reported each year (1). In the majority of instances (~80%), in utero 105 infections by T. gondii result in a range of severe birth defects, including ocular disease and 106 developmental delays, and can also result in fetal death (2). However, despite the clear impact of 107 T. gondii infections on fetal health, the mechanisms by which the parasite is transmitted from the 108 maternal bloodstream into the fetal compartment are largely unknown. 109In eutherian organisms, the placenta serves as the sole source of gas, nutrient, and waste 110 exchange from the fetal compartment and acts as a key barrie...