Immunology Core

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IMMUNOLOGY CORE – ABSTRACT Diarrheal disease is the second leading cause of death in children under five years of age. Understanding the processes involved in bacterial pathogenesis and host response to enteric pathogens is essential to improve treatment and develop effective prevention strategies. Such studies have been hindered by the lack of reliable models that fully recapitulate the complex cellular and molecular events and interactions that take place in the human gut. The Immunology Core (IC) will establish and refine co-culture models of human intestinal epithelial and innate immune cells to recreate the tissue structure, cell interaction, and coordinated responses of the human gut. This physiologically relevant system will generate new insights into the coordinated actions of epithelial and immune cells during enteric infections. The IC will also support the studies performed by the projects by providing technical expertise for implementation of immune co-cultures. The IC has two main Aims. Aim 1. As a core developmental Aim, the IC will: 1) Refine the enteroid-immune cell co-culture by seeding epithelial cell monolayers on a collagen-coated porous scaffold (i.e. Alvetex®) that will allow direct cell-to-cell contact and migration. 2) Establish a multi-cellular model consisting of epithelial cell monolayers co-cultured with different innate phagocytic cells: monocyte-derived macrophages (MF), polymorphonuclear neutrophils (PMN), and dendritic cells (DC) isolated from human peripheral blood. Phagocytic cells not only perform anti-microbial functions but also initiate adaptive immunity. We have successfully engrafted human phagocytes individually to enteroid monolayers. To better recreate individual and synergistic function of epithelial and innate immune cells, we will combine these three main phagocytic lineages with enteroid monolayers. Tissue structure and integration, cell phenotype, viability, and functionality will be assessed by histology, confocal microscopy, flow cytometry and microarray technology. Phagocytic activity, transmigration and cytokine and chemokine production will be examined to determine cell function. 3) Develop a co-culture model containing epithelial cells and TCRgd+ T cells from human peripheral blood. TCRgd+ T cells within the mucosal barrier, also known as intraepithelial lymphocytes (IEL), are rapidly mobilized and deploy diverse innate immune functions in response to pathogens. Cell phenotype will be characterized by flow cytometry, and function will be assessed by cytokine production and cell cytotoxicity. Tissue structure will be examined by histology and microscopy. Cell movement will be monitored with particle tracking system/software and confocal microscopy. Optimal conditions for the generation of immune co-cultures will be defined, and protocols produced and made available to the research projects. Aim 2. As a support service Aim, the IC will write detailed protocols to implement the methods described above and will provide training and technical assistance to facilitate the experimental work of the individual projects. Collectively, the work proposed will generate new knowledge on enteric infection and immunity that can inform prophylactic and therapeutic development efforts.