Feline Chronic Gingivostomatitis (FCGS) is a common oral inflammatory disease of cats. FCGS is associated with chronic viral infections, such as feline calicivirus (FCV) and feline foamy virus (FFV) and can be treated by full mouth extraction (FME) or mesenchymal stromal cell (MSC) therapy in cases where FME fails. We hypothesized that FCGS is characterized by a dysfunctional, exhausted, CD8+ T cells state that is characterized by decreased cytokine production, decreased proliferative potential, decreased cytotoxic activity and increased expression of inhibitory receptors. We further hypothesized that MSC therapy helps to reinvigorate exhausted CD8+ T cells. Transcriptomic analysis of peripheral blood CD8+ T cells indicate upregulation of genes and pathways associated with exhaustion in peripheral blood CD8+ T cells in cats with FCGS. Moreover, confocal immunofluorescence indicated a marked expansion of CTLA-4 positive T cells in the mandibular lymph nodes of cats with FCGS. Finally, MSC therapy suppressed CD8+ T cell activation and proliferation. By comparing the pre-treatment pathways of CD8+ T cell effector activity in successful and failed FME therapy, we identified IL12R, IL18R, and AKT1S1 as crucial for successful FME therapy, potentially linked with the Ras-MEK-ERK pathway, reflecting enhanced T cell signaling pathway activation. In summary, our study found CD8 T cell exhaustion in FCGS and identifies key molecular targets for FME and MSCs treatment.

The intestinal barrier separates the gut lumen from the internal tissues of the body. It is comprised of a layer of mucus, a monolayer of intestinal epithelial cells adjoined and maintained by junctional protein complexes, and a lamina propria which contains specialized immune cells. This barrier is responsible for the absorption of nutrients, preventing the entry of harmful pathogens and substances, and is a critical component of the innate immune system. This dissertation presents a comprehensive review of the role of the intestinal barrier and innate immune response to the apicomplexan parasite, Cryptosporidium parvum, and the use of intestinal organoids as a model of study (Chapter 1) and the effects of both inflammation and Diabetes mellitus on the function of the intestinal epithelium (Chapter 2). It shows that inflammatory cytokines directly increase the permeability of the bovine intestinal epithelium while altering tight junction morphology and reducing cellular turnover, using bovine intestinal organoids as a model (Chapter 3). Finally, this work uses feline intestinal organoids to show that hyperglycemia-like conditions directly increase the permeability of feline intestinal epithelium while altering tight junctional morphology, as mediated through the activation of protein kinase C-α (PKCα); the drug fenofibrate prevents hyperglycemia-induced barrier dysfunction and restores PKCα activity to baseline levels (Chapter 4).

Conventional in-vitro models of intestinal epithelium such as human intestinal/colonic cancer cell lines lack essential features of the canine intestine, limiting their use to study canine intestinal barrier function, cellular metabolism, or host-microbe interactions. Intestinal organoids, also known as enteroids, represent a recent primary cell culture technology that has revolutionized the field of intestinal disease and physiology modeling. Enteroids are 3D structures that are composed of a single layer epithelium arranged in crypt- and villi-like domains with its apical and basal membranes facing the luminal and external sides of the enteroid, respectively. The conversion of the 3D organization of enteroids into a 2D monolayer enables accessibility to intestinal cells' apical membrane for experimental infections or drug treatments. This study aimed to develop a canine enteroid-derived monolayer model to study gut barrier structure and function. Jejunal segments were obtained from three donor dogs during a resection and anastomosis surgery and used to isolate the intestinal crypts by EDTA-based tissue dissociation. Then, the intestinal crypts were resuspended in growth factor reduced Matrigel and supplemented with the commercial IntestiCult™ Human Organoid Growth Medium to generate 3D enteroids. Canine enteroids were mechanically and enzymatically disrupted into single cells and cultured on Matrigel-coated porous membranes of transwell culture systems, reaching 100% confluency around day four. We performed immunofluorescence, histochemistry, and transmission electron microscopy (TEM) to study intestinal epithelium morphology and structure. Intestinal epithelium barrier function was analyzed by Fluorescein Isothiocyanate-Dextran 4kD (FITC-D4) intestinal permeability Assay and Trans-Epithelial Electrical Resistance (TEER) measurement. Canine enteroids and 2D monolayers showed positive staining for ZO-1, Ki67, and Periodic Acid of Schiff (PAS), indicating epithelial cell heterogeneity and polarization. A functional intestinal barrier was confirmed by FITC-D4 assay and TEER measurement. This protocol provides a canine-specific, complex, and long-term ex-vivo model to study intestinal epithelial function.