Characterization of porcine intestinal enteroid cultures under a lipopolysaccharide challenge1

Abstract

Lipopolysaccharides (LPS) located in the outer membrane of Gram-negative bacteria are recognized by Toll-like receptor 4 (TLR4). This interaction initiates a proinflammatory response that antagonizes pig performance and increases morbidity and mortality. Understanding this signaling cascade in the gastrointestinal tract of pigs has relied heavily on cell lines isolated several decades ago. Therefore, we produced and characterized a porcine intestinal enteriod model to study the impact a LPS challenge has on TLR4 localization. Modifying protocols used to isolate porcine, murine, and human intestinal epithelial stem cells (IESC), we were able to isolate porcine IESC. After 7 to 10 d of culturing the IESC, 3-dimensional intestinal enteroid structures were identified. These intestinal enteroids were characterized via immunofluorescent markers (villin, chromogranin A, and claudin 4) and were shown to contain enterocytes and enteroendocrine cells and form tight junctions. After 14 to 16 d of culture, porcine intestinal enteroids underwent a 24-h LPS challenge (10 μg/mL) to study the localization of TLR4. Control intestinal enteroids showed TLR4 disbursed throughout the cells, whereas localization was more punctate in LPS-treated intestinal enteroids. Co-staining with the early endosome marker early endosome antigen 1 showed increased internalization and colocalization of TLR4 with early endosome antigen 1 after LPS exposure (P = 0.03). In conclusion, porcine intestinal enteroids were responsive to a LPS challenge, which resulted in the internalization of TLR4 to the early endosome, and these enteroids cultures may be a viable model for understanding intestinal inflammation and receptor localization.