The liver with resident tissue macrophages is the site of vivid innate immunity, activated also by pathogen-associated molecular patterns (PAMPs) leaking through the intestinal barrier. As gut-derived inflammatory diseases are of outstanding importance in broiler chickens, the present study aimed to establish a proper hepatic inflammatory model by comparing the action of different PAMPs from poultry pathogens on chicken 2D and 3D primary hepatocyte—non-parenchymal cell co-cultures, the latter newly developed with a magnetic bioprinting method. The cultures were challenged by the bacterial endotoxins lipopolysaccharide (LPS) from Escherichia coli, lipoteichoic acid (LTA) from Staphylococcus aureus and by enterotoxin (ETxB) from Escherichia coli, Salmonella Typhimurium derived flagellin, phorbol myristate acetate (PMA) as a model proinflammatory agent and polyinosinic polycytidylic acid (poly I:C) for mimicking viral RNA exposure. Cellular metabolic activity was assessed with the CCK-8 test, membrane damage was monitored with the lactate dehydrogenase (LDH) leakage assay and interleukin-6 and -8 (Il-6 and -8) concentrations were measured in cell culture medium with a chicken specific ELISA. Both LPS and LTA increased the metabolic activity of the 3D cultures, concomitantly decreasing the LDH leakage, while in 2D cultures ETxB stimulated, PMA and poly I:C depressed the metabolic activity. Based on the moderately increased extracellular LDH activity, LTA seemed to diminish cell membrane integrity in 2D and poly I:C in both cell culture models. The applied endotoxins remarkably reduced the IL-8 release of 3D cultured cells, suggesting the effective metabolic adaptation and the presumably initiated anti-inflammatory mechanisms of the 3D spheroids. Notwithstanding that the IL-6 and IL-8 production of 2D cells was mostly not influenced by the endotoxins used, only the higher LTA dose was capable to evoke an IL-8 surge. Flagellin, PMA and poly I:C exerted proinflammatory action in certain concentrations in both 2D and 3D cultures, reflected by the increased cellular IL-6 release. Based on these data, LTA, flagellin, PMA and poly I:C can be considered as potent candidates to induce inflammation in chicken primary hepatic cell cultures, while LPS failed to trigger proinflammatory cytokine production, suggesting the relatively high tolerance of avian liver cells to certain bacterial endotoxins. These results substantiate that the established 3D co-cultures seemed to be proper tools for testing potential proinflammatory molecules; however, the remarkable differences between 2D and 3D models should be addressed and further studied.

Gastrointestinal inflammation leads to maldigestion and systemic diseases in poultry. To tackle the problem of the industry and to search for therapeutic candidates in vitro models are inevitable. Both immersion and air-liquid interface explant models are available, although there is limited information on the size-dependent applicability and response to different pathogen-associated molecular patterns (PAMPs) in the case of these model systems. The study aimed to compare the morphology and viability of miniature chicken gut explant cultures obtained with a biopsy punch to examine the size-dependent change over time. To verify the applicability of the model, pathogen-associated molecular patterns (PAMPs): flagellin, lipoteichoic acid (LTA) and polyinosinic polycytidylic acid (poly I:C) were applied to induce inflammation. The 2 mm diameter explants showed a decrease in metabolic activity measured by CCK-8 assay after 12 h and a significantly higher extracellular lactate dehydrogenase activity indicating cellular damage compared to the 1 mm explants, supported by histological differences after 24 h of culturing. After 12 h of incubation, the 1.5 mm explants retained columnar epithelial lining with moderate damage of the lamina propria (H&E and pan-cytokeratin staining). Exposure to 100 μg/mL poly I:C reduced the metabolic activity of the 1.5 mm explants. LTA and poly I:C increased IFN-γ concentration at both applied doses and IFN-α concentration was elevated by 50 μg/mL poly I:C treatment. Flagellin administration raised IL-2, IL-6, and RANTES levels, while higher LTA and poly I:C concentrations increased the IFN-γ/IL-10 ratio. According to the observations, the viability and integrity of the explants decreases with their size. After 12 h, the 1.5 mm diameter miniature chicken ileal explant stimulated with PAMPs can be an appropriate model to mimic diseases involving tissue damage and inflammation.

Virus induced damage triggered by excessive inflammation and free radical production is a major threat in the poultry industry, leading to low productivity even in vaccinated flocks. The purpose of the study was to induce inflammation with the viral double-stranded RNA analog polyinosinic-polycytidylic acid (poly I:C) on chicken primary hepatocyte - non-parenchymal cell co-cultures to investigate the immunomodulatory and cell protectant effects of chicoric acid (CA) in comparison to N-acetylcysteine (NAC). Poly I:C significantly elevated the activity of the cell damage marker, lactate dehydrogenase (LDH) and the concentration of inflammatory cytokines (IL-6, IL-8, IFN-α, IFN-γ and M-CSF) in the culture medium and decreased cellular metabolic activity. CA significantly reduced the elevated LDH and cytokine levels in a dose-dependent manner, moreover, the higher (100 µg/mL) concentration of CA even elevated the level of metabolic activity. In contrast, 10 µg/mL NAC treatment decreased the level of each inflammatory cytokine but did not rectify cell damage or metabolic depression. The results indicate, that CA, present in common plants of the Asteraceae family, proves to be a beneficial hepatoprotective, and along with NAC, an immunomodulatory supplement in vitro under a stimulus mimicking viral infection.

In this study, we observed the effect of the newly isolated probiotic strain Limosilactobacillus reuteri B1/1 on the relative gene expression of selected cytokines (interleukin-15, transforming growth factor-β4), tight junction proteins (E-cadherin, occludin), biomarker active intestinal stem cells - LGR5 (leucine-rich repeat containing G protein-coupled receptor), markers of mucosal intestinal immunity (mucin-2, immunoglobulin A), as well as the creation of a new biomarker of inflammation in the intestine - calprotectin on an ex vivo model of chicken ileal explant in the prevention of Salmonella Enteritidis PT4 infection. The ability of L. reuteri B1/1 to effectively modulate the mucosal immune response under pretreatment conditions in S. Enteritidis PT4 infection in a chicken ileal explant model was confirmed. In addition, our obtained results point to the fact that the new chicken ileum explant model could be a suitable model to investigate or test the influence of natural substances such as probiotic bacteria in the interaction with the intestine as well as pathogenic microorganisms. In addition, the results of our study may contribute to a deeper understanding of the action of newly isolated probiotic bacteria at the intestinal level using ex vivo models such as chicken ileum explant, which are able to mimic in vivo conditions sufficiently.

As the threat posed by antimicrobial resistance grows more crucial, the development of compounds that can replace antibiotics becomes increasingly vital. Chicken cathelicidin-2 (Cath-2) belongs to the group of Host Defense Peptides (HDPs), which could provide a feasible solution for the treatment of gastrointestinal infections in poultry. It is a small peptide produced by the heterophil granulocytes of chickens as part of the innate immune response, and its immunomodulatory activity has already been demonstrated in several cell types. In this study, the effects of Cath-2 on the intestinal immune response were examined using ileal explant cultures isolated from chicken. Regarding our results, Cath-2 displayed a potent anti-inflammatory effect as it alleviated the LTA-caused elevation of interleukin (IL)-6 and IL-2 concentrations, and that of the IFN-γ/IL-10 ratio, furthermore, it increased the concentration of IL-10, alleviating the LTA-evoked decreased level of the anti-inflammatory cytokine. Moreover, when applied alone, it elevated the concentrations of IL-6, CXCLi2, and IL-2, providing evidence of its complex immunomodulatory mechanisms. In summary, Cath-2 was able to modulate the immune response of the intestinal wall not only by reducing pro-inflammatory cytokine release, but also through immune stimulation, demonstrating that it has the ability to improve innate immunity via a complex mechanism that may make it a suitable candidate for the control of intestinal infections.

The use of natural feed supplements is an alternative tool to diminish the damage caused by certain bacteria, improving animal health and productivity. The present research aimed to investigate the proinflammatory effect of flagellin released from the bacterial flagellum of Salmonella enterica serovar Typhimurium and to attenuate the induced inflammation with luteolin as a plant-derived flavonoid on a chicken primary hepatocyte–non-parenchymal cell co-culture. Cells were cultured in a medium supplemented with 250 ng/mL flagellin and 4 or 16 µg/mL luteolin for 24 h. Cellular metabolic activity, lactate dehydrogenase (LDH) activity, interleukin-6, 8, 10 (IL-6, IL-8, IL-10), interferon-α, γ (IFN-α, IFN-γ), hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) concentrations were determined. Flagellin significantly increased the concentration of the proinflammatory cytokine IL-8 and the ratio of IFN-γ/IL-10, while it decreased the level of IL-10, indicating that the model served adequate to study inflammation in vitro. Luteolin treatment at 4 µg/mL did not prove to be cytotoxic, as reflected by metabolic activity and extracellular LDH activity, and significantly reduced the flagellin-triggered IL-8 release of the cultured cells. Further, it had a diminishing effect on the concentration of IFN-α, H₂O₂ and MDA and restored the level of IL-10 and the ratio of IFN-γ/IL-10 when applied in combination with flagellin. These results suggest that luteolin at lower concentrations may protect hepatic cells from an excessive inflammatory response and act as an antioxidant to attenuate oxidative damage.