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

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Kupffer cells (KCs) are playing predominant role in the regulation and mediation of hepatic and systemic inflammatory response (Racanelli and Rehermann, 2006). As resident liver macrophages, they recognize pathogen‐associated molecular patterns (PAMP), mostly originated from the gastrointestinal tract, or bind endogenously derived damage‐associated molecular patterns (DAMP), such as apoptotic or injured cells (Bianchi, 2007). KCs are being activated, and their function is regulated predominantly by the Toll‐like receptor 4 (TLR‐4) signalling pathway (Seki and Brenner, 2008). Among others, lipopolysaccharide (LPS)‐type endotoxins, cell wall components of Gram‐negative bacteria, can trigger inflammation via the mediation of activated KCs producing high amount of pro‐inflammatory cytokines (e.g. interleukins) and chemokines (Racanelli and Rehermann, 2006; Seki and Brenner, 2008). 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., 2014), 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, 2009). Therefore, monitoring the pivotal role of KCs in the complex regulation of inflammatory and metabolic processes could highlight new ways of improving animal health and productivity.
Hepatic cell culture models are considered as relevant and practical in vitro systems for studying the inflammatory response of the organ. Isolated and cultured hepatic cells contain all metabolizing enzymes and transport proteins, mimicking physiological and pathological processes that can occur in the liver in vivo (Herédi‐Szabó et al., 2009). Notwithstanding that primary cultures of hepatocytes also contain a distinct amount of non‐parenchymal cells, hepatocytes should be co‐cultured with KCs in appropriate ratio for proper investigations on hepatic inflammation. This functional cell culture system has been already established with cells of rat (Bhatia et al., 1998) and pig origin (Hoebe et al., 2000), serving as a model for human diseases and as a research tool in veterinary medicine. In the latter case, hepatocytes were co‐cultured with KCs in 1:1 ratio to study the hepatotoxicity of certain compounds (Hoebe et al., 2000).
The main goal of the present study was to improve and characterize a porcine primary hepatocyte–KC co‐culture to serve as an in vitro inflammatory model by applying different ratios of cell types to mimic milder and severe inflammation. These cell cultures might be useful to assess some aspects of enteral bacterial infections in swine as investigating the role of KCs in different forms of LPS‐induced inflammation. Further, the efficacy of potential anti‐inflammatory substances, such as the short‐chain fatty acid butyrate and terpinen‐4‐ol, the active component of the tea tree oil (derived from Melaleuca alternifolia) and further plants (such as Illicium verum and Myristica fragrans), was aimed to be tested on the co‐cultures. As both butyrate and terpinen‐4‐ol can be considered as potent natural anti‐inflammatory agents based on numerous in vitro and in vivo studies (Carson et al., 2006; Vieira, 2012; Farkas et al., 2014), they can be proper candidates to demonstrate new application possibilities of the obtained in vitro model.
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