The female reproductive tract must maintain normal reproductive functions while also being able to elicit immune responses to sexually transmitted microbes and viral pathogens. In response to virus exposure, the balance of these two functions can determine the outcome of infection and disease through alteration of vaginal mucosa integrity. Recent in vivo research in our laboratory revealed that vaginal infection by Zika virus (ZIKV), an emerging mosquito-transmitted flavivirus that is also sexually transmitted among humans, induces epithelial cell-specific innate immune response that accelerates a homeostatic form of cell death known as cornification. We hypothesize that specific processes in vaginal epithelial cells mediate cornification in response to ZIKV infection, and that this cornification may alter barrier properties, innate-adaptive immune crosstalk, and ultimately, the degree to which ZIKV disseminates from the female reproductive tract. We therefore analyzed and compared vaginal epithelial cell infection by both African and Asian lineage ZIKV strains using traditional 2-dimensional cell culture and organotypic 3-dimensional culture infection models. Differences in viral infection and replication kinetics, and innate immune response were characterized through RT-qPCR, immunoblot analysis, cell imaging, and viral plaque assay. Ongoing analyses are expected to reveal the application of organotypic 3-dimensional cultures in capturing in vivo qualities of vaginal epithelial cell/ZIKV infection and response compared to 2-dimension cultures. These studies will provide insights for application of vaginal epithelial cell culture models of ZIKV infection that encapsulates the complex functional and structural aspects present in vivo.

Tuft cells are a lineage of chemosensing epithelial cells that contribute to intestinal “type 2” immune responses during parasitic worm (helminth) infection. Tuft cells are thought to monitor the intestinal lumen, looking for type 2 agonists. While rare at steady state, during a helminth infection the number of tuft cells in the gut increases greatly (a phenomenon called hyperplasia), directly proportionate to the underlying immune response. Nippostrongylus brasiliensis is a common model of hookworm infection. This parasite is injected under the skin, migrates to the intestine, and can be cleared within 10 days, with a tuft cell response mediated by the cytokine interleukin (IL) 25 and largely independent of the IL33 receptor, ST2. In contrast, the roundworm Heligmosomoides polygyrus is ingested orally and has a lifecycle in which it matures inside the intestinal tissue rather than in the lumen, then re-enters the lumen and establishes chronic infection. Given their distinct lifecycles, we hypothesized that N. brasiliensis and H. polygyrus are sensed differently by tuft cells. Since the kinetics of tuft cell hyperplasia during N. brasiliensis are well defined, we created a comprehensive timeline of tuft cell hyperplasia in wild type mice infected with H. polygyrus for comparison. Intestinal tissue was collected from three mice on every other day of infection, until 20 days post infection. We used immunofluorescent staining to quantify the tuft cells on each harvest day and discovered distinct waves of tuft cell hyperplasia that align with the two separate times that H. polygyrus worms enter the lumen. This timecourse highlighted two peak timepoints of hyperplasia, which we studied further to test the hypothesis that IL33 is critical in the H. polygyrus immune response. Through this work, we strive to better understand how and why our bodies respond to the various type 2 agonists, and how this response can be most effective.

Breast milk is essential to the health and development of a child, containing antibodies that protect infants from common illnesses. However, exclusive breastfeeding is not always possible, and no infant formula substitutes for maternal antibodies. Previous studies on mice showed high germinal center (GC) B cell levels in response to the absence of maternal antibodies from breast milk early in life. Germinal centers B cells are involved in the adaptive immune system by secreting high affinity antibodies. However, little is known about the consequence of this, making characterizing the isotype, location, and duration of the antibody response in neonates (newborns) lacking maternal breast milk antibodies essential. For this project, I designed and optimized a tissue preparation and flow cytometry panel to assess memory B cells, GC B cells, and plasma cells (cells that secrete antibodies to fight infections and disease). The flow panel uses the cell markers CD138 and B220 to identify plasma cells by isolating the cells that are positive for CD138 and negative for B220. However, the marker CD138 can be sensitive to collagenase, resulting in the potential failure to identify plasma cells successfully. For this reason, I tested a range of collagenases, including Collagenase A, D, and IV. Concluding that CD138 was being cleaved off by all tested collagenases, I then used TACI as a new type of plasma cell differentiation marker. I evaluated these protocols by the viability of cells and the plasma cells' frequency. This protocol allows for the determination of localization, persistence, and isotype of early life B cells activated in the absence of breast milk.

We are currently investigating how neonates establish a symbiotic relationship between their developing immune system and the microbes colonizing their gut immediately post birth, and what role factors (e.g. antibodies) from the mother play in establishing this relationship. Lactobacillus is a genus of commensal bacteria that is commonly found in the neonatal intestine of both mice and humans. To track immune responses in the gut, I will create a fluorescent Lactobacillus species by transforming (genetically altering a cell through uptake and incorporation of outside DNA) a plasmid encoding a fluorescent protein into the bacterial cell. By engineering a fluorescent commensal species, we can track a normal, healthy immune-commensal interaction in vivo (in an animal model). Using this novel tool, we will study and compare how neonatal mice that do and do not receive antibodies from the mother post-birth differ in their immune response against these commensal species.

Gastrointestinal helminth infections are among the most common infections worldwide affecting about 1.5 billion people. Previous work shows that during infection with Trichuris muris, a mouse model of human whipworm in the large intestine, basophils, rare innate immune cells, accumulate at the site of infection and upregulate the expression of Notch receptors, part of a signaling pathway that regulates gene expression. Due to this discovery, we are investigating the importance of Notch receptor expression in basophils for the anti-helminth immune response and parasite clearance during infection. With single-cell RNA sequencing (scRNAseq) we are able to analyze the expression of individual cells to better understand immune cell response to infection. To investigate how Notch signaling in basophils impacts CD4 T cell function during infection, we performed scRNAseq on CD4 T cells from the intestine of naive and T. muris infected wild-type mice and mice without Notch signaling specifically in basophils. Using this approach, for the first time, we have characterized the gene expression profile of CD4 T cells at the site of infection, allowing us to identify different CD4 T cell subpopulations and their differential gene expression. This will provide insight into how T. muris infection impacts CD4 T cell gene expression and function, and the importance of Notch signaling in basophils for promoting T cell function during infection. Ongoing work is addressing how CD4 T cells and basophils interact in the large intestine, how the highly regulated environment impacts the development of the CD4 T cell response and the T cell receptor repertoire in T. muris infection, and the importance of basophil Notch signaling for this process. A better understanding of the response to helminth infection and manipulation of Notch signaling-dependent cellular responses can lead to the discovery of novel treatments for helminth infection and allergic diseases.