TDK conference 2014

Application of alternative growth promoters, such as probiotics and certain microbial metabolites, amongst them butyrate as feed additives is getting more abundant in the animal production. Previous studies have already described the anti-inflammatory effect of these materials on the intestinal wall, therefore presumably they possess similar effect on other organs, e.g. liver as well. In this study, we investigated the anti-inflammatory effect of butyrate and that of the spent culture supernatant (SCS) of Lactobacillus plantarum 2142 (Lp2142) on primary culture of porcine hepatocytes stimulated with bacterial lipopolysaccharides (LPS) as an in vitro inflammatory model (model 1). As Kupffer cells have a central role in establishing inflammatory response, further aim of our investigations was to create such a hepatocyte - Kupffer cell co-culture, in which the ratio of the two cell types can be set, modelling various inflammatory processes (model 2).

Hepatocytes were isolated by multi-step perfusion of processus caudatus of the liver, excised from male pigs of the Hungarian Large White breed, weighing 15 kg. Cells were seeded onto culture plates coated by collagen, and cultures became confluent in one day. In case of model 1, cells were treated with LPS and sodium butyrate or the SCS of Lp2142 for 1 hour, then after 24 hours incubation time concentrations of inflammatory cytokines IL-6 and IL-8 were determined from culture medium by using ELISA method. Ratio of Kupffer cells in the hepatic cultures was investigated by immunohistochemistry (detection of macrophage specific CD-68 antigen). In model 2, after hepatocyte isolation Kupffer cells were separated by percoll solutions of different concentrations, then both cell types were seeded onto plates in 6:1 and 2:1 ratios (hepatocyte:Kupffer cells). Confirmation of the applied ratios and the characterization of co-cultures was conducted by immunohistochemical method, similarly as in model 1.

In model 1, rate of Kupffer cells was 6.74±1.07%, in accordance with values published in scientific literature. Concentrations of IL-6 and IL-8 in culture medium of hepatocytes with physiological Kupffer cell ratio did not decrease either after butyrate or Lp2142 treatment compared to control group treated with LPS only. However, according to literature data both butyrate and Lp2142 are able to effectively decrease the amount of inflammatory cytokines in vivo. The fact that anti-inflammatory effect highly depends on the type of inflammation may provide an explanation for the differences between results of in vitro and in vivo studies. In case of chronic inflammations, rate of Kupffer cells is significantly increasing in the liver, therefore, establishing a co-culture modelling increased Kupffer cell ratio (model 2) is of special importance. Immunohistochemical investigation performed on this model has justified cell ratios set, thus it can be suitable for the investigation of further inflammatory processes.