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.

During a microbial infection, cytokine and interferon communication downstream of JAK-STAT signaling molecules are critical for the activation of pro- and anti-inflammatory gene programs. The prevailing model relies on JAKs (Janus Kinases) activation upon cytokine or interferon docking with its receptor at the membrane, which phosphorylates STATs (signal transducers and activators of transcription proteins) and leads to their translocation into the nucleus to either activate or repress the transcription of genes. In this study, we have identified the role of an endocytic pathway responsible for JAK-STAT activation upon interactions between the IL-10 family proteins and the cytokine receptor. Using skin epithelial cell lines, I have performed experiments to show that clathrin-mediated endocytosis is required for the signaling of cytokines that use the IL-10R2 receptor. According to my preliminary results, the inhibition of clathrin-mediated endocytosis using chemical inhibitor dynasore, prior to treatment with IL-26, results in complete shutdown of pSTAT3, pSTAT1, and pErk1/2. These downstream molecules are required for activation of inflammatory gene programming. We now propose to investigate the roles of other inhibitors within the endocytic pathway including chloroquine, bafilomycin A1, and a Rab7a inhibitor on subsequent JAK-STAT signaling transduction. In order to visualize these results, confocal microscopy studies will be used to investigate trafficking in these vacuoles, followed by colocalization studies of JAK proteins with receptors in early endosomes. Our studies suggest that a change in receptor mobilization controlled through the endosomal trafficking mechanism modulates JAK-STAT signaling and downstream gene expression. This proposal is a paradigm shift which challenges the prevailing dogma that IL-10R2 dependent cytokines can activate JAK-STATs at the cell membrane to induce inflammatory responses. Furthermore, this new signaling model has the potential to impact pharmaceutical interventions as the IL-10 family plays a critical role in inflammatory bowel diseases as well as fungal and bacterial infections.

In the lungs, conventional dendritic cells (cDCs) survey the tissue for foreign antigens and initiate immune responses. A subset of lung cDCs, known as cDC1s, are phenotypically distinct from cDCs and critical for T cell activation in diseases such as lung cancer and respiratory viral infection. Lung-specific cDC1 generation in vitro has not yet been achieved, and performing mechanistic studies in vivo remains difficult because cDC1s exist in small numbers in the lung. I aim to identify an in vitro culture system to develop sufficient numbers of murine (mouse) lung-specific cDC1s. I examined how the addition of transforming growth factor-beta (TGF-β) to bone marrow from mice affects cDC1 differentiation. TGF-β is a factor of interest because it is an abundant lung cytokine during development that upregulates genes associated with DC development, but its effect on cDC1 differentiation has not been documented. I first harvested mouse bone marrow, cultured these cells in FMS-like tyrosine kinase 3 ligand (FLT3L), and subsequently added either recombinant granulocyte-macrophage stimulating factor (rGM-CSF) or recombinant TGF-β (rTGF-β) individually or together. I then used flow cytometry to assess the phenotype of generated cDCs in these cultures. Preliminary results indicate a combination of FLT3L, rGM-CSF, and rTGF-β increases the expression of the epithelial adhesion molecule (EpCAM), which our lab has newly identified to be expressed in subsets of lung cDCs. Additionally, GM-CSF suppresses XCR1 expression, another marker of cDC1s, while FLT3L induces XCR1 expression. Based on these findings, I will continue to improve the culture conditions by tracking the expression of preDC markers in only FLT3L to determine the best day to add GM-CSF and TGF-β. Understanding the development of in vitro murine cDCs would allow us to replicate similar in vitro studies for human bone marrow-derived lung cDC1s and use them for developing cell-based therapies against lung diseases.