Proinflammatory cytokines suppress stemness-related properties and expression of tight junction in canine intestinal organoids

Abstract Recent advancements in canine intestinal organoid research have paved the way for the development of enhanced in vitro models, crucial for exploring intestinal physiology and diseases. Despite these strides, there is a notable gap in creating specific in vitro models that focus on intestinal inflammation. Our study aims to bridge this gap by investigating the impact of proinflammatory cytokines on canine intestinal epithelial cells (IECs) within the context of organoid models. Canine intestinal organoids were treated with proinflammatory cytokines TNF-α, IFN-γ, and IL-1β. The expression of stem cell markers Lgr5 , Sox9 , Hopx , and Olfm4 was evaluated through RT-qPCR, while membrane integrity was assessed using immunofluorescence staining for tight junction proteins and transport assays for permeability. IFN-γ significantly decreased Lgr5 expression, a key intestinal stem cell marker, at both 24 and 48 h post-treatment ( p =0.030 and p =0.002, respectively). Conversely, TNF-α increased Olfm4 expression during the same intervals ( p =0.018 and p =0.011, respectively). A reduction in EdU-positive cells, indicative of decreased cell proliferation, was observed following IFN-γ treatment. Additionally, a decrease in tight junction proteins E-cadherin and ZO-1 ( p <0.001 and p =0.003, respectively) and increased permeability in IECs ( p =0.012) were noted, particularly following treatment with IFN-γ. The study highlights the profound impact of proinflammatory cytokines on canine IECs, influencing both stem cell dynamics and membrane integrity. These insights shed light on the intricate cellular processes underlying inflammation in the gut and open avenues for more in-depth research into the long-term effects of inflammation on intestinal health.