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 establishment of a consistent ex vivo model of poultry gut tissue for the study of intestine-microbiome interactions remains still a significant challenge. In this study a pro-inflammatory response of chicken ileal explant cultures was observed after Salmonella enterica serotype Enteritidis infection reflected by up-regulation of IL-18, TNF-α, IL-1β mRNA expression and the levelof serum amyloid A (SAA) protein. In contrast, pre-treatment of ileal explants with probiotic strain Limosilactobacillus reuteri CCM 9425 was able to suppress the infection-induced up-regulation of IL-1β, IL-18 cytokines and the SAA protein. Moreover, the applied probiotics elevated the RNA level of the gene encoding the anti-inflammatory cytokine IL-10 in the probiotic group and the pre-treatment group. Using ileal explant cultures isolated from chicken offers a reliable model of the gut, for studiing the effects of microorganisms at the level of histological tissue structure, gene expression of selected markers and protein production. In summary, regarding our results the miniature chicken ileal explants exhibited appropriate innate immune responses following exposure to bacterial infection with Salmonella Enteritidis and Limosilactobacillus reuteri, furthermore, represents a suitable model for the study of host-pathogen interactions under ex vivo conditions.

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.

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 H₂O₂and 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.

Due to the spread of antimicrobial resistance, there is an urgent need to search for novel antimicrobial agents. In poultry farming, animals are constantly exposed to enteral pathogens and are simultaneously required to maintain high production performance, making it challenging to preserve enteral homeostasis. Host defense peptides (HDP), regarded as putative antibiotic substitutes, have been considered beneficial for poultry gut health; however, many peptides have yet to be investigated from this perspective. In the present study, chicken ileal explant cultures were used to examine the effects of the HDP Pap12-6 (PAP) on enteral immune state and tight junction (TJ) protein abundance. The peptide was applied at 25 and 50 µg/ml concentrations alone (PAP-low and PAP-high, respectively) and in inflammatory conditions evoked by polyinosinic-polycytidylic acid (Poly I:C, 50 µg/ml), at the same doses (PI:C+PAP-low and PI:C+PAP-high, respectively). According to the results, PAP did not influence metabolic activity and extracellular lactate dehydrogenase activity, hence the viability of the explants. Regarding the immune state, the level of interleukin (IL)-2 was increased by PAP-low, PAP-high, and PI:C+PAP-low, whereas IL-8 concentration was raised by PI:C+PAP-low treatment. Furthermore, PI:C+PAP-high decreased the elevated interferon (IFN)-γ production caused by Poly I:C. In the case of TJ proteins, PAP-low elevated the expression of occludin, whereas PAP-high increased the abundance of claudin-3. Conclusively, PAP displayed immunomodulatory effects and may have contributed to epithelial integrity without being cytotoxic, suggesting its potential as an antibiotic alternative in poultry farming. However, the detailed mechanism of action and concentration-dependent activity of PAP must be elucidated in the future.

Background: Chronic inflammatory enteropathies (CIEs) in dogs are multifactorial disorders characterized by mucosal immune dysregulation, compromised epithelial barrier function, and increased exposure to microbial components such as lipopolysaccharide (LPS). The resulting oxidative stress and inflammation contribute to local and systemic pathology. Objective: This study aimed to evaluate the immunomodulatory and antioxidant effects of three naturally occurring flavonoids—quercetin, luteolin, and grape seed extract oligomeric proanthocyanidins (GSOPs)—in LPS-stimulated canine duodenal explants. Methods: Duodenal tissue samples were cultured in vitro and challenged with LPS derived from Escherichia coli and Salmonella enterica. Explants were co-incubated with flavonoid compounds, and endpoints included evaluation of histological architecture, inflammatory cytokine production, and reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation. Results: All three flavonoids attenuated LPS-induced mucosal inflammation and ROS production to varying degrees. In addition, GSOPs significantly reduced RNS levels under both basal and LPS-stimulated conditions. Quercetin and luteolin demonstrated pronounced downregulation of TNF-α, while both compounds also reduced IL-6 concentrations under non-stimulated conditions. These effects support the hypothesis that flavonoids can mitigate both inflammatory and oxidative responses under conditions relevant to CIE. Conclusion: Quercetin, luteolin, and GSOPs show promising in vitro efficacy in modulating key mechanisms implicated in canine CIE. Their multimodal actions highlight their potential as adjunctive nutraceuticals for the management of CIE in dogs. However, further in vivo validation is warranted.