Exploring the impact of the host defense peptide Pap12-6 on immune response and epithelial integrity in chicken-derived ileal explant cultures

Abstract

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.

Introduction

Due to the excessive and long-term use of antibiotics, the spread of antimicrobial resistance (AMR) has become a serious and growing concern worldwide, including in livestock farming (Li et al., 2024). AMR poses a serious challenge for combating infections, as it is often associated with higher mortality and morbidity rates, the lack of effective treatments, and higher healthcare costs (Chettri et al., 2024; Hedman et al., 2020; Li et al., 2024; Min et al., 2024). At the same time, economic and safe meat and egg production must also be maintained while ensuring proper animal health and well-being (Márton et al., 2025).

In the fight against AMR, host defense peptides (HDPs) – also known as antimicrobial peptides (AMPs) – are increasingly referred to as putative antibiotic-substitutive agents and have attracted great attention from the poultry sector. These mostly cationic peptides, containing less than 100 residues, can be found in every living organism as an integral part of innate immunity and help overcome infections (Sebők et al., 2024). The efficacy of HDPs originates in their dual role; in addition to directly attacking microbes, they can also exert remarkable beneficial effects on host cells, including immunomodulatory activity (Sebők et al., 2024). However, this property shows a significant diversity among different peptides and specific biological conditions, such as the various cell types, signaling pathways, receptors, and inflammatory environments involved (Duarte-Mata and Salinas-Carmona, 2023). Another aspect that needs to be considered is the HDPs’ peptide nature. In this context, it is worth taking into account certain limiting factors, for instance, high susceptibility to proteases, questionable pharmacokinetics, potential cytotoxicity, or expensive production on a large scale (Biswaro et al., 2018; Boullet et al., 2019; Koo and Seo, 2019). On this basis, designing small, synthetic peptides offers a great approach to counter the drawbacks mainly provided by naturally occurring HDPs (Ramesh et al., 2016).

Since the gastrointestinal (GI) tract is constantly and extremely exposed to environmental and microbial stimuli (Wickramasuriya et al., 2022), and even subclinical disorders of the digestive tract can lead to inflammation, oxidative stress, and impaired immune function, preserving gut health is essential for efficient production performance (Surai et al., 2021). According to this, it has become evident that maintaining enteral health is an unavoidable aspect of poultry farming (Ducatelle et al., 2023). The beneficial cellular effects of HDP treatment on gut health have been widely investigated in different species; however, far fewer studies focus on chicken or chicken-derived cells. According to the literature, HDPs can have a versatile impact on the intestinal homeostasis of poultry, as they can modulate immunological processes and the abundance of different cell types, support intestinal morphology and epithelial integrity, regulate the oxidative state, shape the gut microbiome, or directly enhance digestive and absorptive capacity (Márton et al., 2025). Therefore, investigating these cellular effects in chickens is crucial for gaining a deeper understanding of the effects of HDPs.

Due to continuous exposure to pathogens, the gut establishes a multifaceted barrier consisting of physical, immunological, chemical, and microbial lines of defense, composed of a large set of cell types with specific roles (Ducatelle et al., 2023; Wickramasuriya et al., 2022). This complexity of the intestines makes it challenging for researchers to model the cellular events reacting to different types of stimuli under in vitro conditions (Randall et al., 2011). To investigate these mechanisms, intestinal epithelial cell cultures are commonly used models. However, it is difficult to reproduce the intricate connections with other cell types in experimental systems made up solely of epithelial cells, as these interactions could be crucial for coordinating a proper immune response during inflammation and for reinforcing epithelial barrier function (Marks et al., 2022). Therefore, the absence of cellular variety in solely epithelial cell lines is an obvious drawback (Marks et al., 2022). As a solution, the use of intestinal tissue explant cultures is a remarkable attempt to counteract this challenge (Randall et al., 2011) and appears to be a valuable model for investigating immunomodulatory agents, considering the involvement of different cell types in their complexity (Kallapura et al., 2015). In addition, the layered and polarized structure of cells in an ex vivo explant culture also represents a significant advantage (Mátis et al., 2025). As a result, chicken-derived intestinal explant cultures have been established in recent years (Kallapura et al., 2015; Randall et al., 2011; Zhang et al., 2017).

In the present study, a chicken-derived ileal explant culture was used to investigate the effects of Pap12-6 (PAP) on enteral immune state and intestinal barrier function. This 12-meric synthetic HDP was designed by Kim et al. to display enhanced direct and indirect antimicrobial effects compared to its parent peptide, papiliocin, a natural 37-residue HDP of swallowtail butterfly (Papilio xuthus) larvae (Kim et al., 2019). As a result, PAP was able to exert potent anti-inflammatory activities in mouse- and human-derived cell lines and in in vivo mouse models (Kim et al., 2019; Son et al., 2019). Moreover, its potent immunomodulatory and antioxidant effects on a chicken-derived primary hepatic co-culture have already been published by our research team (Márton et al., 2024). The ileal explant culture used in the present experiment has been established by our research group as a useful model for mimicking inflammatory response and testing immunomodulatory agents (Karaffová et al., 2024; Kiššová et al., 2025; Mátis et al., 2024, 2025). In the present study, PAP was applied at different concentrations (25 and 50 µg/ml), both on its own and under inflammation evoked by polyinosinic-polycytidylic acid (Poly I:C), a synthetic viral dsRNA analog. To investigate the impact of PAP on immune state, the levels of interleukin (IL)-2, IL-6, IL-8, interferon (IFN)-γ, transforming growth factor (TGF)-ß1, and “regulated on activation, normal T cells expressed and secreted” (RANTES) were aimed to be determined. The selected cytokines are essential in inflammatory response, maintaining gut homeostasis, or recruiting leukocytes to the site of injury, and it was hypothesized that PAP treatment would alter cytokine levels without compromising cell viability. Furthermore, the intracellular expression of tight junction (TJ) proteins was aimed to be examined. Occludin and claudin-3, two commonly investigated and crucial TJ proteins, were selected to gain insight into the contribution of PAP to TJ connections and, therefore, to intestinal epithelial integrity.

Materials and methods

Isolation of the explants

Ileal explants were isolated from a 3-week-old male broiler chicken, in compliance with institutional regulations, European Union animal welfare legislations, and the directives of the University of Veterinary Medicine Budapest's Local Animal Welfare Committee. The experiment was approved by the Government Office of Zala County, Plant Protection, Food Chain Safety, and Soil Conservation Directorate, Zalaegerszeg, Hungary (approval date: May 11, 2020; license number: GK-419/2020). Before the experiment, constant monitoring of chicken feeding and handling practices was carried out, in accordance with Ross Technology requirements. Unless otherwise indicated, reagents and kits mentioned hereinafter are products of Merck KGaA, Darmstadt, Germany.

Explants were isolated according to the previously developed protocol of our research group (Mátis et al., 2025). Following decapitation and removal of the abdominal feathers, the body cavity was opened, and an approximately 15 cm-long section of the ileum, 10 cm distal to the Meckel’s diverticulum, was excised. After the adipose tissue was detached from the removed intestinal section, the inner and outer parts of the obtained piece of ileum were thoroughly washed with phosphate buffered saline (PBS) + 1 % penicillin-streptomycin (Pen-Strep) solution (SKU: 15140122, Thermo Fisher Scientific Inc., Waltham, MA, USA). Next, the ileal section was longitudinally cut and washed with PBS+Pen-Strep until the absence of visible contamination. Thereafter, biopsy punches with a diameter of 1.5 mm were used to obtain the explants while the removed intestinal fragment was kept on ice and periodically irrigated with PBS+Pen-Strep. Explants were individually placed into the wells of a type IV collagen (SKU: C7661)-precoated 96-well microplate (Greiner Bio-One Hungary LLC, Mosonmagyaróvár, Hungary). In advance, the wells had been filled with 200 µl/well of cell culture medium composed of Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 Ham (SKU: D6421), supplemented with 2.5 % fetal bovine serum (FBS), 1 % glutamine, 1 % Pen-Strep, and the stock doses of the ingredients from HCM™ SingleQuots ™ Kit (SKU: CC-4182, Biocenter LLC, Szeged, Hungary). This latter consisted of amphotericin B, ascorbic acid, bovine serum albumin, gentamicin, human epidermal growth factor, hydrocortisone, insulin, and transferrin. Six treatment groups were prepared, each of them containing six replicates. Next, explants were incubated for 1 h, under 5 % CO2, at 37°C, followed by the exchange of cell culture medium to the freshly prepared treatment solutions.

Histological examination of untreated explants

After 24 h of fixation at room temperature in formaldehyde solution, some of the control, untreated explants were trimmed and dehydrated with a series of ethanol and xylene in an automatic tissue processor. The dehydrated tissue samples were embedded in paraffin blocks, and 4 µm-thick sections were cut manually and mounted onto Superfrost+ adhesion slides (Thermo Fisher Scientific Inc., Waltham, MA, USA). The unstained sections were deparaffinized and rehydrated in xylene and alcohol, respectively. Routine hematoxylin and eosin (H&E) staining (Fig. 1) was performed in an automatic staining instrument. The slides were digitized with a Pannoramic Midi slide scanner using a 20× objective (3D Histech LLC, Budapest, Hungary).

Fig. 1.

Hematoxylin and eosin (H&E) staining of a chicken ileal explant randomly selected from control, untreated cultures. The preservation of villous architecture is visible (arrows). Scale bar = 100 µm (a). Intact epithelial lining of the crypts (arrow heads), which, like the absorptive enterocytes covering the villi, exhibits a normal, columnar morphology. Scale bar = 20 µm (b).

In addition, an immunohistochemistry (IHC) assay targeting claudin-3 was performed on the formalin-fixed, paraffin-embedded tissue sections (Fig. 2). Sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Endogenous peroxidase activity was inhibited for 5 min employing the EnVision FLEX Peroxidase-Blocking Reagent (EnVision FLEX Mini Kit, High pH, Agilent Technologies Inc., Santa Clara, CA, USA), followed by PBS rinsing. Antigen retrieval was performed in Tris/EDTA buffer (EnVision FLEX Target Retrieval Solution High pH [50×]) using microwave heating (800 Watts for 5 min, then 180 Watts for 10 min). Sections were then flushed with PBS. The primary antibody (SKU: 341700, AB_2533158, Thermo Fisher Scientific Inc., Waltham, MA, USA) was applied and incubated at a 1:100 dilution at 4°C in a wet chamber overnight. The slides were rinsed in PBS before and after the 20-min secondary antibody incubation. Immunoreactivity was visualized with diaminobenzidine chromogen (Dako Envision Flex DAB + 1 drop Chromogen + 1 mL Envision Flex Substrate Buffer) for 2–3 min. Thereafter, the sections were rinsed in PBS. The slides were counterstained with hematoxylin according to GILL II for 1 min, and bluing was performed in PBS. After dehydration in ethanol and xylene, the slides were covered with BioMount DPX media (BioGnost Ltd., Zagreb, Croatia) and coverslips. Negative controls were immunolabeled without adding the primary antibody, whereas chicken ileal and dog ileal and colonic biopsies were used as positive controls.

Fig. 2.

Immunohistochemical staining of claudin-3 in a chicken ileal explant randomly selected from control, untreated cultures. Villous enterocytes display both membranous and cytoplasmic claudin-3 immunoreactivity. Scale bar = 20 µm.

Application of substances

The application of substances was carried out according to Table 1. Explants belonging to Control only received cell culture medium. PAP (SKU: AM-290, Isca Biochemical Ltd., Exeter, Devon, UK) and Poly I:C (SKU: P9582) were reconstituted in cell culture medium, and different concentrations of the treating solutions were also created with dilution in the same medium. When explants received a combination of PAP and Poly I:C (PI:C+PAP-low and PI:C+PAP-high), the two agents were applied at the same time.

Table 1.

Treatment groups applied to ileal explant cultures of chicken origin.

Treatment group	PAP	Poly I:C
Control	—	—
PAP-low	25 µg/ml	—
PAP-high	50 µg/ml	—
Poly I:C	—	50 µg/ml
PI:C+PAP-low	25 µg/ml	50 µg/ml
PI:C+PAP-high	50 µg/ml	50 µg/ml

PAP = addition of Pap12-6 (PAP) at 25 μg/ml (PAP-low) or 50 μg/ml (PAP-high) concentrations; PI:C = addition of 50 μg/ml polyinosinic-polycytidylic acid (Poly I:C).

After treating the explants for 12 h, the culture medium of the wells was sampled. In addition, explants were lysed by intermittent sonication (3/sec) in 70 µl of M-PER™ Mammalian Protein Extraction Reagent (SKU: 78501, Thermo Fisher Scientific Inc., Waltham, MA, USA) for 20 sec/explant using a Bandelin Sonopuls HD 2200 homogenizer (Bandelin Electronic GmbH & Co. KG, Berlin, Germany). Thereafter, medium and lysate samples were stored at −80°C until further measurements.

Measurements

Cellular viability

To investigate the metabolic activity of the cells, the Cell Counting Kit-8 (CCK-8) test (SKU: 96992) was performed after sampling, in a 96-well microplate. This assay utilizes tetrazolium salt which can be reduced by dehydrogenase enzymes of the viable cells. Therefore, the amount of formazan produced is directly proportional to the number of living cells. According to the manufacturer’s instructions, CCK-8 reagent and culture medium were directly added to the microplate containing the explants, in a 1 to 10 ratio, with a total volume of 200 µl/well. This was followed by 2 h of incubation at 37°C, after which culture medium was pipetted into a new microplate, and absorbance values were determined by a Multiscan GO 3.2 reader (Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm.

Examination of cell membrane damage, another indicator of cellular viability, was performed with the Lactate Dehydrogenase Activity Assay Kit (SKU: MAK066). In this assay, lactate dehydrogenase (LDH) released into the medium after membrane damage reduces NAD to NADH, which allows a specific colorimetric detection of the latter at 450 nm. According to the protocol provided by the manufacturer, a 96-well microplate was loaded with 50 µl/well of explant culture supernatant. Thereafter, 50 µl freshly prepared Master Reaction Mix (containing LDH Substrate Mix and LDH Assay Buffer) was added into the wells, followed by a 2-min-long incubation at 37°C, protected from light. Initial absorbance values were determined at 450 nm, using a Multiscan GO 3.2 reader, and measurements were continued every 5 min (at 37°C, protected from light) until the absorbance of the most active sample became greater than that of the highest standard. Calculation and determination of the enzymatic activity were carried out according to the equation presented in the protocol.

The abundance of inflammation-related proteins

To investigate the effects of PAP on the immune state, concentrations of IL-2, IL-6, IFN-γ, and RANTES were assessed with Luminex xMAP technology, using MILLIPLEX® Chicken Cytokine/Chemokine Panel 1 – Immunology Multiplex Assay kit (SKU: GCYT1-16 K). Adhering with the protocol of the manufacturer, 96-well plates provided with the kit were filled with 25 µl of supernatant samples, standards, controls, and assay buffer. This was followed by the addition of four sets of antibody-coated beads with distinct colors. After overnight incubation and washing, each well of the plate was filled with 25 µl of Detection Antibodies Solution. After incubating 1 h, 25 µl/well of Streptavidin-Phycoerythrin solution was added, followed by a 30-min-long incubation and washing. Thereafter, 150 µl of Drive Fluid was pipetted into each well, and the beads were reconstituted on a plate shaker. Reading was carried out with a Luminex MAGPIX® instrument, and data were collected with xPonent software. Standard curves were depicted with Milliplex Belysa 1.1 software, using bead median fluorescence intensity (MFI).

The levels of IL-8 and TGF-ß1 were determined with chicken-specific sandwich ELISA assay (SKUs: MBS289628 and MBS261515, MyBioSource Inc., San Diego, CA, USA) according to the manufacturer’s protocols. After the required steps were completed, absorbance values were immediately determined at 450 nm, using a Multiscan GO 3.2 reader.

The abundance of TJ proteins

When examining the TJ proteins in the explants, occludin levels were assayed with a chicken-specific competitive ELISA kit, and claudin-3 levels were assessed with a chicken-specific sandwich ELISA technique (SKUs: MBS738326 and MBS085865, MyBioSource Inc., San Diego, CA, USA), using lysate samples. Following the manufacturer’s instructions, absorbance was measured at 450 nm using a Multiscan GO 3.2 reader after applying the required reagents.

Statistical analysis

R version 4.0.3 program was employed for statistical analyses of data. As Shapiro-Wilk tests indicated non-normal distribution in several treatment groups, the Wilcoxon signed-rank test was used for pairwise comparisons. The difference was considered significant if the resulting p-value turned out to be lower than 0.05. Treatment groups PAP-low, PAP-high, and Poly I:C were compared to Control, whereas the groups PI:C+PAP-low and PI:C+PAP-high were compared to both Control and Poly I:C. Results were visualized with Prism 9 (GraphPad Software Inc., San Diego, CA, USA).

Results

Cellular viability

Neither metabolic activity nor LDH activity was influenced by the treatments, except that Poly I:C significantly decreased the metabolic activity of the explants (p = 0.030) (Fig. 3).

Fig. 3.

Bar graphs of cellular metabolic activity (a) and extracellular lactate dehydrogenase (LDH) activity (b). Chicken ileal explant cultures were treated with different concentrations of Pap12-6 (PAP), both alone and in combination with polyinosinic-polycytidylic acid (Poly I:C). Columns represent means ± SEM (n = 6 / treatment group). PAP-low = 25 μg/ml PAP, PAP-high = 50 μg/ml PAP, Poly I:C = 50 μg/ml Poly I:C, PI:C+PAP-low = 25 μg/ml PAP + 50 μg/ml Poly I:C, PI:C+PAP-high = 50 μg/ml PAP + 50 μg/ml Poly I:C. Cells receiving none of the treatments are considered Control. The groups PAP-low, PAP-high, and Poly I:C were compared to Control. Combinations of Poly I:C and PAP (PI:C+PAP-low and PI:C+PAP-high) were compared to both Control and Poly I:C. Asterisks indicate significant differences between the above-mentioned treatment groups. *p < 0.05.

The abundance of inflammation-related proteins

Regarding IL-2, significantly elevated levels were observed in treatment groups PAP-low (p = 0.027) and PAP-high (p = 0.013), compared to Control. Additionally, PI:C+PAP-low significantly enhanced IL-2 production compared to both Control (p = 0.048) and Poly I:C (p = 0.028) (Fig. 4a). In the case of IFN-γ, Poly I:C caused a significant increase (p = 0.005) compared to Control, which was significantly alleviated by PI:C+PAP-high (p = 0.005), compared to the cells receiving only Poly I:C treatment. In comparison with Control, significantly higher levels of IFN-γ were observed in treatment groups PI:C+PAP-low (p = 0.004) and PI:C+PAP-high (p = 0.005) (Fig. 4b). Regarding IL-8, the concentration of the cytokine was significantly raised after applying the combinatory exposure of PI:C+PAP-low (p = 0.022), compared to the cultures solely treated with Poly I:C (Fig. 4c). When investigating the level of RANTES, Poly I:C and PI:C+PAP-low caused a significant elevation compared to Control (p = 0.030 and p = 0.017, respectively) (Fig. 4d). Concentrations of IL-6 and TGF-ß1 were not significantly influenced by any of the treatments (Fig. 4e and Fig. 4f).

Fig. 4.

Bar graphs of interleukin (IL)-2 (a), interferon (IFN)-γ (b), IL-8 (c), regulated on activation, normal T cells expressed and secreted (RANTES) (d), IL-6 (e), and transforming growth factor (TGF)-ß1 (f) concentrations. Chicken ileal explant cultures were treated with different concentrations of Pap12-6 (PAP), both alone and in combination with polyinosinic-polycytidylic acid (Poly I:C). Columns represent means ± SEM (n = 6 / treatment group). PAP-low = 25 μg/ml PAP, PAP-high = 50 μg/ml PAP, Poly I:C = 50 μg/ml Poly I:C, PI:C+PAP-low = 25 μg/ml PAP + 50 μg/ml Poly I:C, PI:C+PAP-high = 50 μg/ml PAP + 50 μg/ml Poly I:C. Cells receiving none of the treatments are considered Control. The groups PAP-low, PAP-high, and Poly I:C were compared to Control. Combinations of Poly I:C and PAP (PI:C+PAP-low and PI:C+PAP-high) were compared to both Control and Poly I:C. Asterisks indicate significant differences between the above-mentioned treatment groups. *p < 0.05, **p < 0.01.

The abundance of TJ proteins

In the case of occludin, PAP-low contributed to a significant increase in the level of the TJ protein (p = 0.010) (Fig. 5a), whereas treatment with PAP-high resulted in a significant elevation of claudin-3 production (p = 0.009) (Fig. 5b).

Fig. 5.

Bar graphs of occludin (a) and claudin-3 (b) concentrations. Chicken ileal explant cultures were treated with different concentrations of Pap12-6 (PAP), both alone and in combination with polyinosinic-polycytidylic acid (Poly I:C). Columns represent means ± SEM (n = 6 / treatment group). PAP-low = 25 μg/ml PAP, PAP-high = 50 μg/ml PAP, Poly I:C = 50 μg/ml Poly I:C, PI:C+PAP-low = 25 μg/ml PAP + 50 μg/ml Poly I:C, PI:C+PAP-high = 50 μg/ml PAP + 50 μg/ml Poly I:C. Cells receiving none of the treatments are considered Control. The groups PAP-low, PAP-high, and Poly I:C were compared to Control. Combinations of Poly I:C and PAP (PI:C+PAP-low and PI:C+PAP-high) were compared to both Control and Poly I:C. Asterisks indicate significant differences between the above-mentioned treatment groups. **p < 0.01.

Discussion

After being explored as putative antibiotic substitutive agents, HDPs and their versatile immunomodulatory effects have been increasingly investigated. However, their extremely multifaceted impact on the immune system still makes it difficult to predict the HDPs’ in vivo effects, as various biological factors and the parameters of the particular peptide can influence the immunomodulatory activity (Duarte-Mata and Salinas-Carmona, 2023; Peng et al., 2020). In poultry – despite the relevance of finding new alternatives to antibiotics –, there is limited data available on the cellular effects of HDPs, making it essential to investigate their influence on host cells. In this regard, the impact of HDPs on enteral homeostasis represents a relevant research field, given the outstanding role of gut health in the defense against pathogens. In the present experiment, ileal explant cultures of chicken origin were treated with PAP, a synthetic HDP, without inflammation, as well as in inflammatory conditions evoked by Poly I:C.

To ensure the safe application of HDPs in the future, their potential adverse effects on the cellular viability of the host cells need to be thoroughly investigated. In the present study, neither of the applied concentrations of PAP influenced the metabolic activity or cell membrane integrity, hence the viability of the explants, suggesting the harmless nature of the peptide. This observation is in accordance with the results of our previous experiment, in which PAP displayed no cytotoxic effects on a primary hepatocyte–non-parenchymal cell co-culture of chicken origin (Márton et al., 2024). Moreover, our findings are consistent with other studies conducted on cell cultures, as PAP exerted no harmful effects on the RAW 264.7 mouse macrophage cell line, the HaCaT human keratinocyte cell line (Kim et al., 2019; Son et al., 2019), and the HEK-293 human embryonic kidney cell line (Kim et al., 2019). In the present study, it can also be observed that Poly I:C slightly reduced metabolic activity, which, while not considered a cytotoxic effect due to the unchanged LDH activity, indicates a mild metabolic depression in the cells.

Enteral homeostasis is a key element of normal immune function and production efficiency in poultry. Therefore, the gut must be able to effectively respond to infections, and its steady state must also be maintained. Pathogen-associated molecular patterns (PAMPs) are detected by pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), different types of which can detect different microbial components and activate molecules crucial for inflammation (Broom, 2019; Dai et al., 2022; Keestra et al., 2013). Finally, the cascade regulates the production of different cytokines and chemokines to provide an effective immune response (Keestra et al., 2013). To investigate the impact of PAP on enteral immune processes, the levels of IL-2, IL-6, IFN-γ, TGF-ß1, IL-8, and RANTES were determined after applying the peptide alone and in Poly I:C-induced inflammation. Poly I:C, a synthetic molecule similar to viral dsRNA, serves as a ligand for TLR3 and is a suitable agent to create inflammatory conditions in intestinal explant cultures (Mátis et al., 2025).

IL-2, a pleiotropic cytokine, has a key role in mediating T cell-dependent immune response, it is essential for the growth and antibody production of B cells, and it can also contribute to the proliferation and differentiation of natural killer (NK) cells (Akdis et al., 2011; Zhou et al., 2019). Therefore, IL-2 is crucial for maintaining GI homeostasis and preventing chronic enteral inflammation; moreover, it can augment tolerance to dietary antigens (Zhou et al., 2019). In the present study, PAP increased the production of IL-2 both alone and in Poly I:C-induced inflammation, suggesting the peptide’s protective and immunostimulatory effects by regulating cell-mediated immunity. In agreement with this finding, melittin, another HDP, also exerted the same effect on intestinal IL-2 mRNA expression in an in vivo experiment on laying quails (Li et al., 2023). In addition, HDP chicken cathelicidin-2 also elevated IL-2 levels of chicken-derived ileal explant cultures, in a former experiment of our research team (Mátis et al., 2024). However, in the literature, the immunomodulatory effects of HDPs on the poultry intestine have also shown opposing outcomes concerning IL-2 production. Namely, in broiler chickens, camel lactoferrin 36 (Daneshmand et al., 2019) and chicken NK-lysin expressed by Bacillus subtilis (Wickramasuriya et al., 2023) contributed to an enteral IL-2 decrease. Furthermore, melittin, when tested at a different concentration in another section of the small intestine (Li et al., 2023), and chicken cathelicidin-2, in lipoteichoic acid (LTA)-induced inflammation (Mátis et al., 2024), displayed similar IL-2-reducing effects, despite demonstrating opposing activities under previously described conditions. Based on these observations, it is evident that HDPs can exhibit highly diverse effects, even in the case of the same peptide, making it challenging to evaluate their overall contribution to the immune state of the host. Therefore, it is crucial to investigate their immunomodulatory activity across a wide range of experimental setups and with regard to different inflammatory mediators.

Another crucial participant in enteral immune processes is IL-6. Besides its main effect being proinflammatory, IL-6 displays a versatile impact on gut health, with a major influence ranging from acting on physiological cellular events to pathological phenomena (Alhendi and Naser, 2023). IL-6 has a role in the activation of immune cells, both in innate and adaptive immunity, the survival of neutrophils, the proliferation of intestinal epithelial cells, as well as the switch from acute to chronic inflammation (Alhendi and Naser, 2023; Shahini and Shahini, 2023). Therefore, the production of IL-6 is strictly controlled by a complex network of upstream molecular factors, allowing a balanced regulation of IL-6 signaling. By contributing to a shift from maintaining gut homeostasis to exaggerating inflammation, overproduction of the cytokine has been linked to different intestinal pathologies, such as inflammatory bowel disease (IBD) (Alhendi and Naser, 2023). It was described that many HDPs were able to influence intestinal IL-6 expression in poultry, providing examples of both increasing (Daneshmand et al., 2019; Kogut et al., 2013; Mátis et al., 2024) and decreasing effects (Lee et al., 2023a; Zhang et al., 2021; Zhu et al., 2022). Even though in previous studies, PAP was able to alleviate IL-6 release from the RAW 264.7 murine macrophage cell line (Kim et al., 2019; Son et al., 2019) and a primary chicken-derived hepatic co-culture (Márton et al., 2024), the present study failed to present similar results. Nevertheless, the immunomodulatory effects of PAP on other intestinal cell cultures have not been examined before, leaving open the possibility that the absence of PAP’s IL-6-altering effect observed might be because the same concentrations of the peptide acted differently in a different tissue type. However, to gain a deeper understanding of the above, the investigation of additional concentrations of PAP and further studies involving chicken-derived intestinal cells are a must, and their absence represents a limitation in the present study.

IFN-γ, a type II interferon, has a paramount role in immune cell activation, intestinal HDP and mucus production, promotion of the cells’ cytotoxic effects, and apoptosis of mucosal epithelial cells. Primarily produced in viral infection (Xie et al., 2020), IFN-γ is a crucial contributor to, and exaggerator of, intestinal inflammation (Cardoso Dal Pont et al., 2023; Li et al., 2023). In the present study, PI:C+PAP-high exerted a decreasing effect on IFN-γ concentration compared to the increase caused by Poly I:C, suggesting that the peptide was able to alleviate inflammation – however, the levels remained higher than those of Control. In contrast, when PAP was previously applied to chicken hepatic co-cultures in a former study of our research team, the same concentrations of the peptide were not able to significantly influence IFN-γ level in Poly I:C-triggered inflammation (Márton et al., 2024). This could indicate the previously mentioned tissue-specific immunomodulatory effects of PAP. At the same time, it is worth mentioning that in the chicken intestine, HDP chicken NK-lysin was able to exert the same reducing effect on IFN-γ gene expression when administered to enhance vaccination efficacy in Eimeria infection (Lee et al., 2022, 2023b). On the other hand, the opposite effect was also documented after treating broiler chickens with Brevibacillus texaporus-derived HDPs (BT peptides) in Salmonella enterica serovar Enteritidis-induced inflammation (Kogut et al., 2013).

Another important contributor to gut homeostasis is TGF-ß, the most abundant intestinal isoform of which is TGF-ß1 (Tanigawa et al., 2005). This multifunctional cytokine regulates the proliferation, apoptosis, and differentiation of intestinal cells, and it promotes angiogenesis, extracellular matrix formation, wound healing, and mucosal barrier function (Howe et al., 2005; Ihara et al., 2017; Tanigawa et al., 2005). Moreover, it serves as a key suppressor of the immune response (Howe et al., 2005; Ihara et al., 2017). In the present experiment, PAP was not able to influence the concentration of TGF-ß1, suggesting that the immune state was not altered through the modulation of this cytokine. Even though the authors are not aware of any other experiments that have investigated the impact of PAP or further papiliocins on TGF-ß1 production, it was described that few other HDPs regulated the intestinal gene expression of the cytokine in chicken. While soybean bioactive peptides reduced the jejunal expression in Eimeria-challenged broiler chickens (Osho et al., 2019), avian ß-defensin-2 and plectasin combined with plant oils elevated TGF-ß1 production of the intestines in healthy chickens (Shreeya et al., 2023; Xie et al., 2020).

In the immune response, chemokines have an essential role in recruiting leukocytes to the site of infection; and based on the subfamily of interest, they exert their chemoattractant effect on specific immune cells (Ajuebor et al., 2001). In this term, IL-8 (often referred to as CXCL8, and in chicken: CXCLi2), a chemokine produced by various GI cells, is primarily responsible for attracting and activating neutrophils (Adumitrăchioaiei et al., 2024). Therefore, IL-8 contributes to an acute neutrophil response, degranulation of polymorphonuclear cells, and respiratory burst, thereby supporting the antimicrobial defense of the host (Cotton et al., 2016). Based on this proinflammatory action, it was described that many GI diseases, such as IBD (Cotton et al., 2016; Williams et al., 2000), gastric and colonic carcinomas (Maheshwari et al., 2002), or ulcerative colitis (Williams et al., 2000), were accompanied by elevated intestinal IL-8 production. In the present study, PI:C+PAP-low raised the level of IL-8, suggesting that the peptide stimulated the immune response in inflammatory conditions. This finding is contrary to the reducing effect exerted by PAP on chicken hepatic co-cultures in our previous study (Márton et al., 2024). Given these conflicting results, the putative tissue-specific activity of PAP is to be considered again and further examined. Nevertheless, BT peptides also contributed to a local IL-8 increase in broiler chickens after Salmonella enterica serovar Enteritidis challenge (Kogut et al., 2013). According to the observations in that experiment, Kogut et al. suggested that BT peptides might contribute to a so-called priming effect, thereby strengthening the activation of phagocytes and enhancing the sensitivity of the tissues to microbial stimulus (Kogut et al., 2013). Moreover, in addition to the proinflammatory role of IL-8, the beneficial effects of the chemokine in the GI tract — namely, its involvement in GI tissue remodeling, angiogenesis, and epithelialization — have also been described (Maheshwari et al., 2002). Based on this, the increased level of IL-8 caused by PAP may suggest a gut-protective function of the peptide. However, as the investigation of the immunomodulatory effects of HDPs in the poultry gut is still in its infancy, more research is required to fully understand the underlying mechanisms and consequences of the altered IL-8 level.

RANTES, another chemokine less frequently examined related to HDPs, is mainly produced by macrophages, T lymphocytes, and endothelial cells of the inflamed gut tissue (Grimm and Doe, 1996). As a response to inflammation, the secretion of RANTES mainly contributes to the recruitment and activation of monocytes, eosinophils, T lymphocytes, and mast cells (Ajuebor et al., 2001; Ajuebor and Swain, 2002). Even though the exact action of RANTES in the gut is still under investigation, it was confirmed that RANTES played a role in the development of IBD (Grimm and Doe, 1996), irritable bowel syndrome (IBS) (Berg et al., 2020), enteral ischemia reperfusion injury (IRI) (Chen et al., 2004), allergic reactions to food components (Lee et al., 2004), and chronic colitis (Ajuebor et al., 2001). In the present experiment, Poly I:C induced the production of RANTES, but this elevation was not significantly influenced by PAP (the significant effect of PAP was observed only in comparison to Control). This observation is contrary to the decreasing effect exerted by PAP in our former study conducted on chicken hepatic co-cultures (Márton et al., 2024). Despite RANTES being relatively rarely investigated in the context of HDPs, in the literature, certain peptides influenced its gene expression or concentration in various ways, providing results of both increasing (Sebők et al., 2023; van der Does et al., 2010) and decreasing activities (Anupa et al., 2015; Tripathi et al., 2017; Yan et al., 2018).

Alongside the influence of PAP on cellular viability and immune state, the effects of the peptide on another key component of gut health were also investigated. Intestinal epithelial integrity, contributing to the physical line of defense, is primarily established by TJ connections constituted by TJ proteins. These cellular connections are localized along the lateral sides of the enterocytes, close to their apical surfaces. The different TJ proteins identified can be divided into two major groups: transmembrane proteins and intracellular plaque proteins. Transmembrane proteins, such as occludin and claudins, are responsible for connecting the adjacent cells via binding to the adjoining transmembrane protein (González-Mariscal et al., 2003; Suzuki, 2020). Maintaining proper intestinal barrier integrity is of paramount importance in preventing the inflow of pathogens and pathogenic metabolites into the circulation and in supporting the absorptive function of the gut (González-Mariscal et al., 2003). Therefore, impairment of this integrity results in inflammation and damage to other tissues and organs, as well (Suzuki, 2020). It has been demonstrated that gene expressions or abundance of TJ proteins may be reduced in various enteric infections of chickens, which can be significantly alleviated or improved by the application of HDPs (Daneshmand et al., 2019, 2020; Lee et al., 2023a; Li et al., 2023; Wickramasuriya et al., 2021, 2023). In addition, their beneficial effects on TJ proteins can also be remarkable in healthy poultry without existing infection, which, due to continuous exposure to pathogens, can also significantly contribute to gut health (Dai et al., 2022; Xie et al., 2020; Zhang et al., 2021; Zhu et al., 2022). In the present study, the intracellular level of occludin was elevated by PAP-low, and the expression of claudin-3 was also augmented by PAP-high, suggesting that the peptide contributed to enhanced TJ connectivity and barrier integrity. To the best of the authors’ knowledge, the effects of PAP or papiliocins on gut barrier function have never been investigated; however, our findings are consistent with other HDPs applied in poultry. To name some regarding occludin, camel lactoferrins (Daneshmand et al., 2019, 2020), chicken NK-lysin (Wickramasuriya et al., 2021), microcin C7 (Dai et al., 2022), and mastoparan X (Zhu et al., 2022) were able to increase the gene expression or concentration of the TJ protein in broiler chickens, whereas melittin contributed to a similar effect in laying quails (Li et al., 2023). In addition, it was also described that HDPs melittin (Li et al., 2023), plectasin (Zhang et al., 2021), camel lactoferrins (Daneshmand et al., 2019, 2020), and mastoparan X (Zhu et al., 2022) stimulated the expression of different claudins in in vivo poultry experiments.

Overall, the present study’s results support the growing body of evidence regarding the versatile effects of HDPs on the poultry intestinal cells and may contribute to a deeper understanding in this research field. Nevertheless, it is important to note that our results were obtained under experimental conditions that differ in certain aspects (such as the type of inflammatory stimulus or the specific HDP used) from those described in the cited literature. Therefore, these differences limit the extent to which our findings should be directly compared with the results of the referenced studies, representing a limitation. Furthermore, the absence of investigating the underlying mechanism of action using a broader range of measurement techniques, as well as the direct assessment of barrier integrity and TJ connections, should also be considered as limitations of the present study and need to be addressed in the future.

Conclusions

The present study aimed to investigate the effects of PAP on the immune state and intestinal epithelial integrity of a chicken ileal explant culture after applying the peptide both individually and under inflammatory conditions evoked by Poly I:C. Based on our results, PAP appeared to exert no cytotoxic effects on ileal cells, indicating its safe application in poultry. Furthermore, PAP displayed an immunomodulatory activity, as it was able to increase the levels of IL-2 and IL-8 while decreasing the Poly I:C-triggered elevated concentration of IFN-γ. In addition, the peptide may have contributed to enhanced TJ connectivity of the explants, reflected by the augmented abundance of occludin and claudin-3. Therefore, PAP is suggested to exert beneficial effects on chicken gut homeostasis to promote the host’s immune function. However, it is important to highlight that the complex effects of these examined parameters, and especially the intricate and not-fully-understood influence of HDPs on intestinal cellular processes, make it challenging to evaluate the results unequivocally. Additionally, the complex mechanism of action of the peptide, as well as the exact implications of its impact, require further detailed investigation. In conclusion, the present study aimed to contribute to a broader understanding of HDPs in relation to poultry health, and based on the results, PAP may be a potential candidate for developing novel antibiotic-substitutive agents to combat AMR in livestock farming.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.