Effects of Acute Heat Stress on a Newly Established Chicken Hepatocyte—Nonparenchymal Cell Co-Culture Model

Abstract

Heat stress is one of the most important issues in broiler flocks impairing animal health and productivity. On a cellular level, excess heat exposure can trigger heat shock response acting for the restoration of cell homeostasis by several mechanisms, such as affecting heat shock protein synthesis, redox homeostasis and pro-inflammatory cytokine production. The major aim of this study was to establish a novel avian hepatocyte—nonparenchymal cell co-culture as a model for investigating the cellular effects of heat stress and its interaction with inflammation in chicken liver. Cell fractions were isolated by differential centrifugation from a freshly perfused chicken liver, and hepatocyte mono-cultures as well as hepatocyte–nonparenchymal cell co-cultures (with cell ratio 6:1, hepatocytes to nonparenchymal cells, mimicking a milder hepatic inflammation) were prepared. Isolated and cultured cells were characterized by flow cytometry and immunocytochemistry applying hepatocyte- and macrophage-specific antibodies. Confluent cell cultures were exposed to 43 °C temperature for 1 or 2 h, while controls were cultured at 38.5 °C. The metabolic activity, LDH enzyme activity, reactive oxygen species (H2O2) production, extracellular concentration of heat shock protein 70 (HSP70), and that of the pro-inflammatory cytokines interleukin (IL-)6 and IL-8 were assessed. Shorter heat stress applied for 1 h could strongly influence liver cell function by significantly increasing catabolic metabolism and extracellular H2O2 release, and by significantly decreasing HSP70, IL-6, and IL-8 production on both cell culture models. However, all these alterations were restored after 2 h heat exposure, indicating a fast recovery of liver cells. Hepatocyte mono-cultures and hepatocyte—nonparenchymal cell co-cultures responded to heat stress in a similar manner, but the higher metabolic rate of co-cultured cells may have contributed to a better capability of inflamed liver cells for accommodation to stress conditions. In conclusion, the established new primary cell culture models provide suitable tools for studying the hepatic inflammatory and stress response. The results of this study highlight the impact of short-term heat stress on the liver in chickens, underline the mediatory role of oxidative stress in acute stress response, and suggest a fast cellular adaptation potential in liver cells.

Acute Heat Stress Induces the Differential Expression of Heat Shock Proteins in Different Sections of the Small Intestine of Chickens Based on Exposure Duration

“…Therefore, proteins and genes did not show the same expression level in this study, although their expression trend was almost the same. This trend of expression indicates that short-term heat stress induces the production of HSPs that contribute to the restoration of normal cell function, which is reflected by the reduction of the gene expression of HSPs in the later stages of acute heat stress [52].…”

Cellular Effects of T-2 Toxin on Primary Hepatic Cell Culture Models of Chickens

“…Different chicken-derived primary cell culture models of hepatic origin were successfully established and characterized by our research group previously [24]. In contrast to mammals, no such primary hepatic cell culture model exists.…”

The Effects of Matriptase Inhibition on the Inflammatory and Redox Homeostasis of Chicken Hepatic Cell Culture Models

“…Liver cells were isolated and cell cultures were prepared as described in a previous study of our research group [ 23 ], using three-week-old male Ross-308 type broiler chickens, obtained from Gallus Ltd. (Devecser, Hungary). Cell isolation was carried out in accordance with national and EU laws and institutional guidelines, also confirmed by the Local Animal Welfare Committee of the University of Veterinary Medicine, Budapest.…”

“…After disrupting the capsule of the liver, the gained cells were suspended in 2.5% bovine serum albumin (BSA) containing HBSS and were filtered through three layers of sterile gauze, followed by a 45 min incubation on ice. Thereafter, hepatocytes and NP cells were isolated by a multistep differential centrifugation as previously described [ 23 ]. Both cell fractions were resuspended in Williams’ Medium E, supplemented with 0.22% NaHCO 3 , 50 mg/L gentamycin, 2 mM glutamine, 4 µg/L dexamethasone, 20 IU/L insulin, and 5% foetal bovine serum (FBS).…”

“…The viability of hepatocytes and NP cells was examined by the trypan blue exclusion test, and cell suspensions were diluted after cell counting in a Bürker chamber to the appropriate seeding concentrations (hepatocyte mono-cultures: 10 6 cells/mL; co-cultures: 8.5 × 10 5 cells/mL hepatocytes, 1.5 × 10 5 cells/mL NP cells). Both hepatocyte and NP cell enriched fractions have been previously characterized by flow cytometry and immunofluorescent detection of specific markers for hepatocytes and macrophages [ 23 ].…”