The Effects of Intestinal LPS Exposure on Inflammatory Responses in a Porcine Enterohepatic Co-culture System

Abstract

A porcine enterohepatic co-culture system, with primary hepatocytes as bottom layer and IPEC-J2 epithelial cells as upper layer, was developed to study the effects of lipopolysaccharides (LPS) on the gene expression profile of pro-inflammatory cytokines (interleukin-8 (IL-8) and tumor necrosis factor-α) and CYP enzymes (CYP1A1, CYP1A2, CYP3A29). The barrier integrity of IPEC-J2 cells was investigated by transepithelial electrical resistance measurements and by fluorescein isothiocyanate-dextran-based test. Basolateral IL-8 production was significantly elevated in LPS-treated IPEC-J2 and primary hepatocyte mono-cultures as well as in the co-culture system, in a dose-independent manner. The LPS-induced changes in the expression of the CYP1A2 and CYP3A29 genes in hepatocyte mono-cultures differed from those in co-culture after LPS treatment on the apical side of the IPEC-J2 cell layer. CYP1A2 was downregulated by the LPS treatment in mono-cultures but upregulated at 10 μg/ml LPS in co-culture; gene expression of CYP3A29 showed no significant LPS-induced change in the hepatocyte mono-culture but was significantly downregulated in co-culture. The newly established co-culture system capable of mimicking enterohepatic interplay in LPS-induced inflammatory responses in vitro can be used in the future for reliable screening of potential anti-inflammatory compounds.

Porcine hepatocyte–Kupffer cell co‐culture as an in vitro model for testing the efficacy of anti‐inflammatory substances

“…Hepatocyte isolation was performed from male pigs of the Hungarian Large White breed weighing approx. 15 skg (obtained from Dunahyb Kft, Fadd, Hungary), as described previously by us (Pászti‐Gere et al., ). Briefly, freshly isolated cells were obtained by a multistep ex vivo perfusion of the liver's caudate lobe, disintegrating the extracellular matrix by enzymatic digestion with collagenase.…”

“…Briefly, freshly isolated cells were obtained by a multistep ex vivo perfusion of the liver's caudate lobe, disintegrating the extracellular matrix by enzymatic digestion with collagenase. Hepatocyte‐enriched fractions were purified with three subsequent centrifugations (50 g , 75 s) and resuspendations (Pászti‐Gere et al., ;). KCs were isolated from supernatants gained during purification of hepatocytes by centrifuging cell suspensions at 500 g for 5 min, followed by further separation of KCs with a mixture of two different concentrations of Percoll solution (15 ml of 50% and 20 ml of 25%; spinning at 500 g , 20 min); KCs were found between the two phases.…”

“…All cell cultures were incubated at 37 °C in humid atmosphere with 10% CO 2 , and culture medium was changed 4 h after seeding. Culture media contained foetal bovine serum (10%) during the first 24 h of culturing and were prepared as described earlier for hepatocyte cultivation (Pászti‐Gere et al., ).…”

“…As enteral bacterial infections are highly relevant in farm animals by causing severe economic losses and being important from food safety point of view, studying the systemic inflammatory response induced by LPS from Gram‐negative enteral pathogens is an important field of veterinary research. As the integrity of gut barrier function is diminished by bacterial endotoxins (Pászti‐Gere et al., ), increased enteral LPS release could directly affect the liver and induce hepatic inflammation by activating the KCs. Further, KCs are involved in the regulation of metabolic processes as well, being potent effectors of insulin sensitivity, carbohydrate and lipid metabolism serving as a key link between inflammation and metabolism (Baffy, ).…”

Crosstalk Between Nuclear Glucose-Regulated Protein 78 and Tumor Protein 53 Contributes to the Lipopolysaccharide Aggravated Apoptosis of Endoplasmic Reticulum Stress-Responsive Porcine Intestinal Epithelial Cells

“…Lipopolysaccharide (LPS) is a component in the outer membrane of Gram-negative bacteria and is recognized by epithelial toll-like receptor-4 [18]. Treatment with LPS results in the activation of nuclear factor (NF)-κB and subsequent upregulation of proinflammatory cytokines including interleukin (IL)-1α, IL-1β, and IL-6 [19]. Previous studies using the IPEC-J2 cell line have established a model of inflammation using Salmonella enterica-derived LPS [18,20].…”