Pap12-6: A host defense peptide with potent immunomodulatory activity in a chicken hepatic cell culture.

In the fight against antimicrobial resistance, host defense peptides (HDPs) are increasingly referred to as promising molecules for the design of new antimicrobial agents. In terms of their future clinical use, particularly small, synthetic HDPs offer several advantages, based on which their application as feed additives has aroused great interest in the poultry sector. However, given their complex mechanism of action and the limited data about the cellular effects in production animals, their investigation is of great importance in these species. The present study aimed to examine the immunomodulatory activity of the synthetic HDP Pap12-6 (PAP) solely and in inflammatory environments evoked by lipoteichoic acid (LTA) and polyinosinic-polycytidylic acid (Poly I:C), in a primary chicken hepatocyte-non-parenchymal cell co-culture. Based on the investigation of the extracellular lactate dehydrogenase (LDH) activity, PAP seemed to exert no cytotoxicity on hepatic cells, suggesting its safe application. Moreover, PAP was able to influence the immune response, reflected by the decreased production of interleukin (IL)-6, IL-8, and "regulated on activation, normal T cell expressed and secreted"(RANTES), as well as the reduced IL-6/IL-10 ratio in Poly I:C-induced inflammation. PAP also diminished the levels of extracellular H2O2 and nuclear factor erythroid 2-related factor 2 (Nrf2) when applied together with Poly I:C and in both inflammatory conditions, respectively. Consequently, PAP appeared to display potent immunomodulatory activity, preferring to act towards the cellular anti-inflammatory and antioxidant processes. These findings confirm that PAP might be a promising alternative for designing novel antimicrobial immunomodulatory agents for chickens, thereby contributing to the reduction of the use of conventional antibiotics.

Cecropin A: investigation of a host defense peptide with multifaceted immunomodulatory activity in a chicken hepatic cell culture.

Introduction: Host defense peptides (HDPs) are increasingly referred to as promising candidates for the reduction of the use of conventional antibiotics, thereby combating antibiotic resistance. As HDPs have been described to exert various immunomodulatory effects, cecropin A (CecA) appears to be a potent agent to influence the host inflammatory response. Methods: In the present study, a chicken primary hepatocyte-non-parenchymal cell co-culture was used to investigate the putative immunomodulatory effects of CecA alone and in inflammatory conditions evoked by polyinosinic-polycytidylic acid (Poly I:C). To examine the viability of the cells, the extracellular lactate dehydrogenase (LDH) activity was determined by colorimetric assay. Inflammatory markers interleukin (IL)-8 and transforming growth factor-ß1 (TGF-ß1) were investigated using the ELISA method, whereas concentrations of IL-6, IL-10, and interferon-γ (IFN-γ) were assayed by Luminex xMAP technology. Extracellular H2O2 and malondialdehyde levels were measured by fluorometric and colorimetric methods, respectively. Results: Results of the lower concentrations suggested the safe application of CecA; however, it might contribute to hepatic cell membrane damage at its higher concentrations. We also found that the peptide alleviated the inflammatory response, reflected by the decreased production of the pro-inflammatory IL-6, IL-8, and IFN-γ. In addition, CecA diminished the levels of anti-inflammatory IL-10 and TGF-ß1. The oxidative markers measured remained unchanged in most cases of CecA exposure. Discussion: CecA displayed a multifaceted immunomodulatory but not purely anti-inflammatory activity on the hepatic cells, and might be suggested to maintain the hepatic inflammatory homeostasis in Poly I:C-triggered immune response. To conclude, our study suggests that CecA might be a promising molecule for the development of new immunomodulatory antibiotic-substitutive agents in poultry medicine; however, there is still a lot to clarify regarding its cellular effects.

Investigation of the effects of T-2 toxin in chicken-derived three-dimensional hepatic cell cultures.

Despite being one of the most common contaminants of poultry feed, the molecular effects of T-2 toxin on the liver of the exposed animals are still not fully elucidated. To gain more accurate understanding, the effects of T-2 toxin were investigated in the present study in chicken-derived three-dimensional (3D) primary hepatic cell cultures. 3D spheroids were treated with three concentrations (100, 500, 1000 nM) of T-2 toxin for 24 h. Cellular metabolic activity declined in all treated groups as reflected by the Cell Counting Kit-8 assay, while extracellular lactate dehydrogenase activity was increased after 500 nM T-2 toxin exposure. The levels of oxidative stress markers malondialdehyde and protein carbonyl were reduced by the toxin, suggesting effective antioxidant compensatory mechanisms of the liver. Concerning the pro-inflammatory cytokines, IL-6 concentration was decreased, while IL-8 concentration was increased by 100 nM T-2 toxin exposure, indicating the multifaceted immunomodulatory action of the toxin. Further, the metabolic profile of hepatic spheroids was also modulated, confirming the altered lipid and amino acid metabolism of toxin-exposed liver cells. Based on these results, T-2 toxin affected cell viability, hepatocellular metabolism and inflammatory response, likely carried out its toxic effects by affecting the oxidative homeostasis of the cells.

Modulation of the immune response by the host defense peptide IDR-1002 in chicken hepatic cell culture.

IDR-1002, a synthetic host defense peptide (HDP), appears to be a potential candidate for the treatment of bacterial infections and the consequent inflammatory response due to its potent immunomodulatory activity. This is of relevance to the emerging issue of antimicrobial resistance in the farming sector. In this study, the effects of IDR-1002 were investigated on a chicken hepatocyte‒non-parenchymal cell co-culture, and the results revealed that IDR-1002 had complex effects on the regulation of the hepatic innate immunity. IDR-1002 increased the levels of both RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) and Macrophage colony stimulating factor (M-CSF), suggesting the peptide plays a role in the modulation of macrophage differentiation, also reflected by the reduced concentrations of interleukin (IL)-6 and IL-10. The pro-inflammatory cytokine release triggered by the bacterial cell wall component lipoteichoic acid (LTA) was ameliorated by the concomitantly applied IDR-1002 based on the levels of IL-6, chicken chemotactic and angiogenic factor (CXCLi2) and interferon (IFN)-γ. Moreover, the production of nuclear factor erythroid 2-related factor 2 (Nrf2), an essential transcription factor in the antioxidant defense pathway, was increased after IDR-1002 exposure, while protein carbonyl (PC) levels were also elevated. These findings suggest that IDR-1002 affects the interplay of the cellular immune response and redox homeostasis, thus the peptide represents a promising tool in the treatment of bacterially induced inflammation in chickens.

Detrimental consequences of tebuconazole on redox homeostasis and fatty acid profile of honeybee brain.

Excessive use of azole fungicides in agriculture poses a potential threat to honeybees and other pollinator insects; however, the detailed effects of these molecules remain largely unclear. Hence, in the present study it was aimed to investigate the acute sublethal effects of tebuconazole on the redox homeostasis and fatty acid composition in the brain of honeybees. Our findings demonstrate that tebuconazole decreased total antioxidant capacity, the ratio of reduced to oxidized glutathione and disturbed the function of key antioxidant defense enzymes along with the induction of lipid peroxidation indicated by increased malondialdehyde levels, while it also altered the fatty acid profile of the brain. The present study highlights the negative impact of tebuconazole on honeybees and contributes to the understanding of potential consequences related to azole exposure on pollinator insects' health, such as the occurrence of colony collapse disorder.

Luteolin: A Phytochemical to Mitigate S. Typhimurium Flagellin-Induced Inflammation in a Chicken In Vitro Hepatic Model.

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.

Feline Uroepithelial Cell Culture as a Novel Model of Idiopathic Cystitis: Investigations on the Effects of Norepinephrine on Inflammatory Response, Oxidative Stress, and Barrier Function.

Feline idiopathic cystitis (FIC) is one of the most common urinary tract disorders in domestic cats. As stress is suggested to play a key role in the pathogenesis of FIC, the effects of norepinephrine (NE) as a stress mediator were investigated on a novel feline primary uroepithelial cell culture, serving as an in vitro model of the disease. The uroepithelial cells gained from the mucosa of the bladder of a euthanized cat were cultured for 6 days and were acutely exposed to NE (10, 100, and 1000 µM) for 1 h. NE increased the metabolic activity of the cultured cells and elevated the extracellular concentrations of the pro-inflammatory mediators interleukin-6 (IL-6) and stromal cell derived factor 1 (SDF-1), confirming that NE can trigger an inflammatory response in the uroepithelium. Cellular protein carbonyl levels were increased by NE exposure, while malondialdehyde and glucose regulated protein 78 concentrations remained unchanged, indicating that NE may provoke the oxidative damage of proteins without inducing lipid peroxidation or endoplasmic reticulum stress. Further, it can be strongly suggested that an acute NE challenge might diminish the barrier function of uroepithelial cells, as reflected by the decreased glycosaminoglycan concentration, claudin-4 protein expression, and reduced TER values of the NE-treated cell cultures. Based on these results, short-term NE exposure mimicking acute stress can provoke an inflammatory response and decrease the barrier integrity of cultured feline uroepithelial cells. Hence, it is highly expected that stress-associated NE release may play an important mediatory role in the pathogenesis of FIC.

Immunomodulatory effects of chicken cathelicidin-2 on a primary hepatic cell co-culture model.

Cathelicidin-2 is an antimicrobial peptide (AMP) produced as part of the innate immune system of chickens and might be a new candidate to combat infection and inflammation within the gut-liver axis. Studying the hepatic immune response is of high importance as the liver is primarily exposed to gut-derived pathogen-associated molecular patterns. The aim of the present study was to assess the effects of chicken cathelicidin-2 alone or combined with lipoteichoic acid (LTA) or phorbol myristate acetate (PMA) on cell viability, immune response and redox homeostasis in a primary hepatocyte-non-parenchymal cell co-culture of chicken origin. Both concentrations of cathelicidin-2 decreased the cellular metabolic activity and increased the extracellular lactate dehydrogenase (LDH) activity reflecting reduced membrane integrity. Neither LTA nor PMA affected these parameters, and when combined with LTA, cathelicidin-2 could not influence the LDH activity. Cathelicidin-2 had an increasing effect on the concentration of the proinflammatory CXCLi2 and interferon- (IFN-)γ, and on that of the anti-inflammatory IL-10. Meanwhile, macrophage colony stimulating factor (M-CSF), playing a complex role in inflammation, was diminished by the AMP. LTA elevated IFN-γ and decreased M-CSF levels, while PMA only increased the concentration of M-CSF. Both concentrations of cathelicidin-2 increased the H2O2 release of the cells, but the concentration of malondialdehyde as a lipid peroxidation marker was not affected. Our findings give evidence that cathelicidin-2 can also possess anti-inflammatory effects, reflected by the alleviation of the LTA-triggered IFN-γ elevation, and by reducing the M-CSF production induced by PMA. Based on the present results, cathelicidin-2 plays a substantial role in modulating the hepatic immune response with a multifaceted mode of action. It was found to have dose-dependent effects on metabolic activity, membrane integrity, and reactive oxygen species production, indicating that using it in excessively high concentrations can contribute to cell damage. In conclusion, cathelicidin-2 seems to be a promising candidate for future immunomodulating drug development with an attempt to reduce the application of antibiotics.

Protective effects of chicoric acid on polyinosinic-polycytidylic acid exposed chicken hepatic cell culture mimicking viral 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.

Two Sides to Every Question: Attempts to Activate Chicken Innate Immunity in 2D and 3D Hepatic Cell Cultures.

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.