Establishment of an In Vitro Co-Culture Model of the Piglet Gut to Study Inflammatory Response and Barrier Integrity

• Abstract
• Introduction
• Results
• Discussion
• Materials and Methods
• Culture of IPEC-J2
• Isolation of PBMCs
• Cultivation media of PBMCs
• Direct gut co-culture model
• Indirect gut co-culture model
• Stimulation of indirect gut co-culture model with ConA
• Stimulation of indirect gut co-culture model with LPS and LTA
• Cytokine detection by LUMINEX analysis
• Addition of licorice extract and oregano oil
• Statistical analysis
• Contributorsʼ Statement
• References

Abstract

In intensive farming, piglets are exposed to various challenges that activate intestinal inflammatory processes, negatively affecting animal health and leading to economic losses. To study the role of the inflammatory response on epithelial barrier integrity, co-culture systems that mimic in vivo complexity are more and more preferred over cell monocultures. In this study, an in vitro gut co-culture model consisting of intestinal porcine epithelial cells and porcine peripheral blood mononuclear cells was established. The model provides an appropriate tool to study the role of the inflammatory response on epithelial barrier integrity and to screen for feed and food components, exerting beneficial effects on gut health. In the established model, inflammation-like reactions and damage of the epithelial barrier, indicated by a decrease of transepithelial electrical resistance, were elicited by activation of peripheral blood mononuclear cells via one of 3 stimuli: lipopolysaccharide, lipoteichoic acid, or concanavalin A. Two phytogenic substances that are commonly used as feed additives, licorice extract and oregano oil, have been shown to counteract the drop in transepithelial electrical resistance values in the gut co-culture model. The established co-culture model provides a powerful in vitro tool to study the role of intestinal inflammation on epithelial barrier integrity. As it consists of porcine epithelial and porcine blood cells it perfectly mimics in vivo conditions and imitates the inter-organ communication of the piglet gut. The developed model is useful to screen for nutritional components or drugs, having the potential to balance intestinal inflammation and strengthen the epithelial barrier integrity in piglets.

Introduction

In intensive pig production, animals are constantly exposed to pathogenic and nonpathogenic challenges (e.g., critical periods like weaning) that can induce a release of immunological agents, resulting in intestinal disorder, and poor animal health, and, due to reduced performance, can lead to major economic losses as a final consequence. Measures aimed at maximization of animal health and productivity require considering the effects of inflammatory processes on GI health [1], [2]. In this context, nutritional strategies balancing inflammatory reactions are intensively discussed to attenuate epithelial damage and improve GI health and animal welfare. Several studies reported that different types of feed additives (e.g., phytochemicals, probiotics, isolated natural compounds) seem to be able to improve the GI health of piglets [3], [4], [5], [6].

In addition to in vivo studies focusing on the effects of food components on the GI tract of piglets, numerous in vitro models of the intestine have been used to simulate sub-processes of gut physiology such as nutritional aspects and gut interactions [7]. In this context, co-culture systems, mimicking the in vivo complexity of intestinal inter-organ interactions, are more and more preferred over frequently used cell monocultures. This aims at closing the gap between using simplified single cell lines and the dynamic biological processes occurring under in vivo conditions [8]. However, most of these studies focus on modeling the human gut and investigate the role of food compounds and intestinal inflammation using co-cultured cell lines of human origin [9]. Merely 1 scientific group describes a co-culture system consisting of 2 porcine cell lines [10], [11] where only the negative influence of mycotoxins on the gut barrier rather than the role of inflammatory processes on the epithelial barrier has been studied.

Therefore, in this study, we established a porcine intestinal gut co-culture model, consisting of IPEC-J2 and porcine PBMCs, mimicking homeostatic and inflammatory conditions in the intestine and suitable to study the role of immune activation on GI barrier integrity in piglets. We tested different ways of co-cultivating the 2 cell types, enabling direct cell communication via cell-cell contact or indirect cell communication via soluble factors like cytokines. Three different stimuli, physiologically relevant bacterial LPS and LTA, as well as the prominent T-cell activator ConA, were tested to induce inflammation-like responses. Moreover, the potentially barrier-protective effects of 2 natural feed additives–licorice extract and oregano oil–were tested.

Results

To exclude potential effects of co-cultivation (direct or indirect) of IPEC-J2 and PBMCs on epithelial barrier integrity, TEER and FITC-dextran permeability assay were performed either with IPEC-J2 alone or in combination with PBMCs (IPEC-J2 + PBMCs) after 72 h ([Fig. 1]). Neither direct nor indirect co-cultivation of the 2 different cell types, without any further stimulation, affected TEER values or the FITC-dextran transport through the barrier compared with IPEC-J2 monocultures.

To study the role of activated immune cells on the integrity of the epithelial barrier, the indirect gut co-culture model was stimulated using the PBMC activator ConA ([Fig. 2]). In the IPEC-J2 monoculture ([Fig. 2 a]), apical, basolateral, or simultaneous apical and basolateral addition of ConA did not reduce TEER values as compared to the untreated IPEC-J2 control at the 3 measured time points (24, 48, or 72 h). After 72 h, the basolateral addition of ConA, however, significantly enhanced the TEER value (p < 0.05). In the indirect IPEC-J2/PBMC co-culture ([Fig. 2 b]), basolateral as well as apical + basolateral ConA-stimulation led to an impairment of TEER values after 24, 48, and 72 h as compared to the unstimulated control (p < 0.001, P < 0.001, and p < 0.01, respectively). The apical addition of ConA to the co-culture did not affect TEER values.

The effects of the bacterial toxins LPS and LTA on epithelial barrier integrity were assessed on IPEC-J2 monocultures as well as in the indirect co-culture model via TEER measurements after 24, 48, and 72 h of incubation.

Neither basolateral LPS nor basolateral LTA stimulation of IPEC-J2 monocultures affected TEER values ([Fig. 3]). In contrast to IPEC-J2 monocultures, basolateral LPS stimulation of the indirect co-culture reduced TEER values at all 3 time points ([Fig. 4]). The effect was most pronounced at 1000 ng/mL LPS (p < 0.001) (% of TEER reduction: 26.4, 29.5, 31 after 24, 5, and 72 h, respectively), followed by 100 ng/mL LPS (% of TEER reduction: 25.5 [p < 0.001], 26.4, and 29.1 [both p < 0.01] after 24, 48, and 72 h, respectively), and 10 ng/mL LPS (% of TEER reduction: 19.9 [p < 0.001], 17.8 [p < 0.05], 16.5 [not significant] after 24, 48, and 72 h, respectively). Basolateral treatment with 10 000 ng/mL LTA led to a reduction of TEER after 24 h (p < 0.05) (% of TEER reduction: 22.2) and 72 h (% of TEER reduction: 21), whereas the 2 lower LTA concentrations of 100 and 1000 ng/mL did not show an effect. The positive control ConA reduced TEER at all 3 time points (p < 0.001) (% of TEER reduction: 36.4, 46.1, and 46.2 after 24, 48, and 72 h, respectively).

To study the potential counteraction of licorice extract (500 µg/mL) and oregano oil (12.5 µg/mL) on epithelial barrier disruption induced by inflammation-like conditions, the indirect gut co-culture model challenged with ConA was used. To exclude potential effects on epithelial barrier integrity by the test substances themselves, TEER was additionally measured after the addition of licorice extract or oregano oil without ConA stimulation ([Table 1]). Neither apical nor basolateral application of the test substances alone affected TEER values compared with the unstimulated control.

[Fig. 5] shows the influence of the addition of licorice extract and oregano oil on TEER values in the ConA stimulated co-culture model. The unstimulated cell control was tested alongside the test samples in all experiments and was higher compared to ConA treated cells after all time points. The basolateral addition of licorice extract to ConA stimulated indirect co-cultures increased TEER values after 24 and 48 h (p < 0. 05 and p < 0. 01, respectively) compared with the ConA-treated control. After 72 h, no effect was shown compared with the ConA control. The apical addition of licorice extract did not significantly affect TEER values at any time point.

The basolateral addition of oregano oil increased TEER values compared with ConA stimulated control after all 3 time points (p < 0. 01) as well. The apical addition of oregano oil did not enhance TEER values significantly at any time point.

Discussion

We established a functional co-culture model consisting of porcine epithelial and porcine immune cells, used to model inter-organ communication during exaggerated inflammatory-like conditions and to investigate the role of feed components or drugs on epithelial barrier integrity.

Two modes of co-cultivation of different cell types are conceivable, one enabling direct cell-cell interaction between the 2 cell types (direct co-culture model) [12] and the second allowing indirect cell-cell communication via soluble factors (e.g., cytokines [indirect co-culture model]) [13]. A previous study showed that the mode of co-cultivation can affect the cellular response [14]. However, the interactions strongly differ between cell types, observed parameters, and research questions. Therefore, direct and indirect gut co-culture models were established as a first step in our study to exclude potential alterations of epithelial integrity caused by hyper-responsiveness of the 2 different cell types and to imitate homeostatic conditions in the intestine. Our results show that the mere presence of the 2 porcine cell types in 1 cell culture vessel did not affect the epithelial barrier permeability, qualifying both models of co-cultivation. Subsequently, we selected the indirect co-culture model for further development of a model involving inflammatory stress and the barrier function, due to easier handling and lower susceptibility to errors of the indirect co-culture model.

As a next step, 3 different stressors were selected, the most prominent PBMC activator ConA [15], [16], as well as the physiologically relevant bacterial toxins LPS [17] and LTA, which are extensively used to study immunological questions [18], [19], to activate PBMCs and to mimic the intended inflammation status in a gut co-culture model. LPS and LTA, both prominent cell wall components of gram-positive (LTA) and gram-negative bacteria (LPS), can stimulate systemic as well as localized inflammation in vivo [20], [21]. As they play an important role in the reduction of animal performance and the suppressed growth of livestock, we selected these physiological stressors next to the jack-bean derived PBMC activator ConA.

Results demonstrate that the epithelial barrier integrity of IPEC-J2 monocultures was not affected by treatment with one of the 3 stimuli, indicated by stable TEER values. A tight monolayer of prominent epithelial cell lines (e.g., HT-29 or T84 cells) to LPS [22] or bacterial challenge [23], [24] is also known from scientific literature and might only show one side of the full picture, as other effects and interactions of those substances are described. In contrast to stimulation of the IPEC-J2 monolayer, basolateral application of ConA to the IPEC-J2/PBMC gut co-culture model effectively activated the immune cells, which was reflected in a time-dependent drop in the TEER values, indicating a disruption of the epithelial barrier integrity. A time- and dose-dependent disruption of the epithelial integrity was also observed for basolateral treatment with LPS, whereas treatment with LTA showed a dose-dependent trend of epithelial disruption but only led to a significant drop of TEER values at the highest test concentration. Results from former studies also have shown a more potent activation of PBMCs by LPS than by LTA tested in comparable concentrations [25], [26], and from in vivo studies, LPS also is known to induce a breakdown of the epithelial barrier and function [25].

The findings support our hypothesis that inflammation-like reactions in the gut co-culture model induce damage to the epithelial barrier integrity. This confirmed its suitability to study further inflammatory effects on the gut barrier model on one hand and to screen for protective substances that counteract the damaged gut barrier on the other hand. However, effects were restricted to basolateral addition of the 3 stimuli, while apical stimulation did not show any significant effects on epithelial barrier integrity. The polarized IPEC-J2 cell-layer, which has been described by Nossol et al. [27], is not negatively affected by apical application of several stimuli [22], [23], [28], and the tight layer does not allow the passage of the stimuli to the PBMCs contained in the basolateral compartment and prevent PBMC activation. These results are in line with previous findings on restricted transport of apically added LPS to the basolateral compartments through a tight Caco-2 cell monolayer [29].

Taken together, as activation of PBMCs by ConA induced the most pronounced effect on epithelial barrier integrity, it was used for continuing experiments. This result was further confirmed by the analysis of 2 cytokines IL-12 and IFN-γ, which were significantly up-regulated after stimulation with ConA but not after LPS or LTA treatment. ConA is well known from the scientific literature to activate cytokine release from PBMCs [15], [16] and affect all major T cell subsets (CD4+, CD8+, and γδ T cells), as described by Vatzia et al. [30]. Stressful life periods like weaning are known to be directly linked to exaggerated inflammatory cytokine expression in the gut [31], and with our new cell culture model, substances to counteract inflammatory cytokine expressions can be screened to further support animal health in this period. Although LPS and LTA are also known to induce a cytokine response in PBMCs, the missing or weak increase of cytokine levels could be explained by an improper incubation time with the bacterial toxins in the current experiments [32], [33]. These results make ConA a suitable stimulus to study the role of simulated inflammation on intestinal physiology in our established indirect co-culture model, and it was applied in the following experiments.

As a next step, the gut co-culture model stimulated by ConA was used to study the role of potential immunomodulators on ongoing inflammatory reactions in the intestine. ConA is known as a potent antigen-independent mitogen that functions as an inducer, leading T cells to polyclonal proliferation and cytokine induction [30], [34]. Various nutritional strategies are implemented to help piglets to cope with the post-weaning period and overcome weaning-related difficulties [35], [36], [37]. However, nutritional approaches in animal nutrition are very complex, and continuous progress to understand the mode of action is indispensable. In our study, 2 phytogenic test substances, licorice extract and oregano oil, were used to assess the effects of those immunomodulators to protect the epithelial barrier. It was hypothesized that the application of the 2 substances can counteract the observed epithelial disruption induced by ConA-activated PBMCs due to their anti-inflammatory activity [37], [38]. Our results could show that basolateral treatment of the gut co-culture model with licorice extract and oregano oil significantly protects the epithelial barrier after induced damage, proven by enhanced TEER values after all 3 measured time points. Modulation of proinflammatory cytokine levels and the overall immune status by phytogenic compounds are also known from the literature [39], [40] and support our hypothesis of anti-inflammatory activity. Moreover, the influence of phytochemicals on tight junction abundance in IPEC-J2 could be involved in barrier recovery after damage [41]. The apical application had no effects, which indicates limited availability of the active phytochemicals at the basolateral compartment containing the PBMCs, releasing the immunological active compounds. The reason could be limited stability and/or passage of the phytochemicals across the epithelial barrier.

As especially young piglets are highly susceptible to inappropriate inflammatory responses due to their poorly developed mucosal immune system, which can be related to undesired alterations in gut architecture, immunomodulators helping the animals to overcome this critical period are of high interest [42], [43]. Our results showed the barrier-protecting activity of licorice extract and oregano in a complex gut co-culture model. We could show the functionality of the model to generally study the role of feed additives on the permeability of the epithelial barrier under inflammatory conditions to support animal health. Further research might shed light on the involved inflammatory and signaling pathways, and especially the cross-talk between the 2 cell types, in more detail.

In summary, the successful establishment of a gut co-culture model consisting of porcine epithelial (IPEC-J2) and porcine blood cells (PBMCs) enabled imitation of inter-organ communication and provided the possibility of studying the influence of intestinal inflammation on epithelial barrier integrity. Thereby, it could be used as an in vitro tool to investigate the role of inflammation in the development of GI disorders in pigs and possibly other species, including humans. Moreover, the gut co-culture model could be used to screen for new substances exerting beneficial effects on gut barrier integrity, and, as a next step, to evaluate their mode of action and suitability to implement them in pigletsʼ diets or medicine.

Materials and Methods

Culture of IPEC-J2

IPEC-J2 (ACC 701; Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Culture) were routinely maintained in DMEM/Hamʼs F-12 (1 : 1) without glutamine (Biochrom), supplemented with 5% FBS, 1% ITS, 2.5 mM GlutaMAX (all purchased from Gibco Life Technologies), 5 ng/mL EGF (Corning), 16 mmol/L HEPES, and 1% P/S (both purchased from Sigma-Aldrich) at 39 °C and 5% CO₂ under humidified atmosphere. Cells were grown in T-150 cell culture flasks (Starlab International GmbH) and were subcultured every 3 – 4 days after > 95% confluence was reached, for a maximum of 15 passages. Cells were checked for mycoplasma contamination every 3 mo via PCR analysis (Venor GeM Mycoplasma Detection Kit, PCR based, Minerva 116 Biolabs).

Isolation of PBMCs

Porcine whole blood was derived from a local slaughterhouse and collected from the jugular vein of 6-mo-old pigs, which were anesthetized with high electric voltage followed by exsanguination. This procedure followed the Austrian animal welfare slaughter regulation. The collected blood was filled into centrifugation tubes containing 120 mg/mL EDTA (Sigma-Aldrich) to prevent blood coagulation. Whole blood was diluted 1 : 2 using PBS (Gibco Life Technologies). The diluted blood solution was gently overlaid onto Ficoll-PaqueTM Plus (GE Healthcare), and PBMCs were isolated by density gradient centrifugation at 300 g for 30 minutes. PBMCs were carefully collected and washed (300 g for 10 minutes) with PBS for a total of 3 times.

Cultivation media of PBMCs

Freshly isolated PBMCs were suspended in 5 different cultivation media ([Table 2]) and immediately seeded at 1 × 10⁵ cells/well in 96-well round bottom plates. Cells were stimulated with 1.25 µg/mL ConA (Sigma-Aldrich) to induce cell proliferation and were incubated at 39 °C and 5% CO₂ for a total of 72 h. Control cells were incubated with different cultivation media, without the addition of ConA. Cell proliferation of PBMCs was measured using the BrdU cell proliferation assay (Roche diagnostics international AG), according to the manufacturerʼs protocol (Fig. 1S, Supporting Information). PBMC proliferation was calculated as SI by dividing the mean absorbance values of treated cells by the mean absorbance values of untreated control cells. According to the literature, a SI > 2 was considered positive [44].

Direct gut co-culture model

For the direct gut co-culture model, enabling direct cell contact between IPEC-J2 and PBMCs [12], 1.12 cm² Transwell polyester membrane inserts with 0.4 µm pores (Corning) were inverted 180 degrees and placed in a 150 mm petri dish (Corning), which was filled with approximately 20 mL PBS to ensure a humidified atmosphere ([Fig. 6]). IPEC-J2 were seeded at the bottom side of the transwell inserts at a seeding density of 1 × 10⁵ cells/insert and were incubated at 39 °C and 5% CO₂ for 24 h, to allow the cells to attach to the bottom side of the inserts. The next day, the inserts were transferred to a 12-well plate (Eppendorf) in their original orientation with the bottom facing down. Cells were allowed to differentiate for 7 days. The cultivation medium was regularly changed every 2 – 3 days. Thereafter, cryo-conserved PBMCs were recultivated and suspended in DMEM-HI ([Table 2]) and added to the apical compartments of the inserts at a density of 3 × 10⁶ cells/insert. The cultivation medium in the basolateral compartments was changed as well to DMEM-HI. IPEC-J2 without PBMCs were used as control. The co-culture was incubated at 39 °C and 5% CO₂ for 72 h, before the influence of co-cultivation on epithelial barrier integrity was analyzed via TEER measurement and FITC-dextran (average molecular weight: 3000 – 5000; Sigma-Aldrich) permeability assay. TEER was measured using the Millicell-Electrical Resistance System (Merck Millipore) as described by Springler et al. [45]. Following TEER measurements, 1 mg/mL FITC-dextran dissolved in water was added to the apical compartments of the transwell inserts, and basolateral supernatants were collected after 4.5 h incubation time. Fluorescence was measured at an excitation wavelength of 490 nm and an emission wavelength of 528 nm.

Indirect gut co-culture model

For the indirect gut co-culture model, enabling cell interactions through soluble factors [13], IPECJ2 were seeded in the apical compartments of the transwell inserts in their original orientation–with the bottom facing down and without being flipped nor inverted, at a seeding density of 1 × 10⁵ cells/insert ([Fig. 6]). Cells were differentiated for a total of 8 days at 39 °C and 5% CO₂. The cultivation medium was changed every 2 – 3 days. For indirect co-cultivation, cryo-conserved PBMCs were recultivated and suspended in DMEM-HI ([Table 2]) and added to the basolateral compartments at a seeding density of 3 × 10⁶ cells/insert. In the apical compartments, the cultivation medium was also exchanged from DMEM to DMEM-HI. IPEC-J2 without PBMCs served as control and were included in each experiment. Measurement of TEER values and FITC-dextran permeability assay was performed after 72 h of incubation.

Stimulation of indirect gut co-culture model with ConA

To mimic inflammation in the intestine, the established indirect gut co-culture model was used to activate PBMCs via ConA stimulation. Monocultures were exposed to 1.25 µg/mL ConA, which was added to the basolateral compartments, the apical compartments, or simultaneously to the apical and basolateral compartments for a total of 72 h. As a next step, PBMCs were co-cultured beneath 8-day differentiated IPEC-J2 for 24 h, allowing PBMCs to settle on the bottom side of the wells. On the next day, PBMCs were either stimulated by adding 1.25 µg/mL ConA to the basolateral compartments, the apical compartments, or simultaneously to the apical and basolateral compartments. To assess the integrity of the epithelial barrier, TEER measurements were performed after 24, 48, and 72 h. After the final TEER measurement, supernatants from apical and basolateral compartments were collected and stored at − 80 °C for cytokine analysis.

Stimulation of indirect gut co-culture model with LPS and LTA

LPS derived from Escherichia coli O55:B5 purified by phenol extraction (Sigma-Aldrich) at 10, 100, and 1000 ng/mL and LTA from Staphylococcus aureus (Sigma-Aldrich) at 100, 1000, and 10 000 ng/mL were further used to activate PBMCs and induce inflammation-like reactions in the indirect gut co-culture model. LPS and LTA stock solutions [1 mg/mL] were prepared in endotoxin-free glass tubes using PBS under agitation and stored at − 20 °C until further usage. Stock solutions were diluted in DMEM-HI to reach the desired test concentrations directly before each experiment. To assess a possible direct influence of LPS or LTA on IPEC-J2 monocultures, monocultures were basolaterally exposed to LPS or LTA at the defined concentrations for 72 h. As a next step, LPS or LTA at the defined concentrations was added to PBMCs contained in the basolateral compartments of co-cultivated cells for 72 h, 24 h after the previous co-cultivation. Unchallenged co-cultures and basolaterally stimulated PBMCs with 1.25 µg/mL ConA served as controls. The impact on epithelial barrier integrity was analyzed via TEER measurements after 24, 48, and 72 h. After the final TEER measurement, supernatants from apical and basolateral compartments were collected and stored at − 80 °C for subsequent cytokine analysis. Moreover, IPEC-J2 were separated from PBMCs by transferring transwell inserts to new 12-well plates, and the neutral red (NR) cell viability assay (Aniara Diagnostica) was performed, according to the manufacturerʼs protocol. Cell viability was calculated as %, and values < 80% cell viability were considered cytotoxic (Fig. 2S and Fig. 3S, Supporting Information).

Cytokine detection by LUMINEX analysis

Collected PBMC supernatants were analyzed for interleukin-12p40 (IL-12p40) and IFN-γ using a multiplex fluorescent microsphere immunoassay (FMIA) as described by Ladinig et al. [46] (Fig. 4S, Supporting Information). Differing from the described protocol, the following reagents were used for IFN-γ detection: IFN-γ capture and detection antibody pair (Thermo Fisher catalog numbers MP700 and MP701B) and IFN-γ standard CSC4033 part SD066 (Thermo Fisher).

Addition of licorice extract and oregano oil

The potential of 2 natural substances, licorice extract and oregano oil, ([Table 3]; both obtained from BIOMIN Phytogenics GmbH), to counteract the damage of the epithelial barrier in the indirect gut co-culture model stimulated with ConA was analyzed. Ethanolic extracts of licorice extract (500 µg/mL) or oregano oil (12.5 µg/mL) have been prepared. Therefore, EtOH 70% (Merck KGaA) was added to the test substances, and the mixtures were shaken for 1 hour on a microplate shaker at room temperature. The prepared extracts were added simultaneously with basolaterally added ConA to the apical or basolateral compartments. The effects of 70% EtOH itself have been excluded before experiments (data not shown). Unchallenged co-culture, cells apically or basolaterally exposed to licorice extract or oregano oil but not to ConA, and cells basolaterally stimulated only with 1.25 µg/mL ConA were included as controls. The impact on epithelial barrier integrity was analyzed via TEER measurements after 24, 48, and 72 h.

Statistical analysis

Statistical analysis was performed with GraphPad Prism 8 (Version 8.0, GraphPad Software Inc). All data were tested for normal distribution using the Kolmogorov-Smirnov normality test. The normally distributed data obtained from the BrdU assay of cryo-conserved PBMCs and TEER or FITC analysis of unstimulated co-culture models were further analyzed by unpaired, 2-tailed t-test. Normally distributed data of all other experiments were analyzed by ANOVA using Dunnettʼs test as post hoc test, or, in cases where they were not normally distributed, by the non-parametric Kruskal Wallis test. Differences were considered statistically significant if their P-values were < 0.05. Diagrams were created using the GrapdPad Prism 8 software.

Contributorsʼ Statement

Design of experiments: E. Mayer, K. Teichmann, N. Reisinger, and T. Schott; conduction of experiments: T. Schott; LUMINEX analysis: A. Ladinig; statistical analysis: T. Schott and N. Reisinger; drafting the manuscript: T. Schott and E. Mayer; critical revision of the manuscript: A. Ladinig, E. Mayer, J. König, K. Teichmann, and N. Reisinger.

Conflict of Interest

BIOMIN Holding GmbH operates the BIOMIN Research Center and is a producer and trader of animal feed additives. This, however, did not influence the design of the experimental study or bias the presentation and interpretation of results. All authors declare that they have no conflict of interest.

Acknowledgements

The authors thank Dominik Wendner and Valentina Rainer for their excellent technical assistance.

• References

• 1 Liu Y. Fatty acids, inflammation and intestinal health in pigs. J Anim Sci Biotechnol 2015; 6: 1-9
  CrossrefPubMedSearch in Google Scholar
• 2 Shin D, Chang SY, Bogere P, Won KH, Choi JY, Choi YJ, Lee HK, Hur J, Park BY, Kim Y, Heo J. Beneficial roles of probiotics on the modulation of gut microbiota and immune response in pigs. PLoS One 2019; 14: 1-23
  CrossrefPubMedSearch in Google Scholar
• 3 Valenzuela-Grijalva NV, Pinelli-Saavedra A, Muhlia-Almazan A, Domínguez-Díaz D, González-Ríos H. Dietary inclusion effects of phytochemicals as growth promoters in animal production. J Anim Sci Technol 2017; 59: 1-17
  CrossrefPubMedSearch in Google Scholar
• 4 Wang T, Yao W, Li J, Shao Y, He Q, Xia J, Huang F. Dietary garcinol supplementation improves diarrhea and intestinal barrier function associated with its modulation of gut microbiota in weaned piglets. J Anim Sci Biotechnol 2020; 11: 1-13
  CrossrefPubMedSearch in Google Scholar
• 5 Wang K, Chen G, Cao G, Xu Y, Wang Y, Yang C. Effects of Clostridium butyricum and Enterococcus faecalis on growth performance, intestinal structure, and inflammation in lipopolysaccharide-challenged weaned piglets. J Anim Sci 2019; 97: 4140-4151
  CrossrefPubMedSearch in Google Scholar
• 6 Chen J, Yu B, Chen D, Huang Z, Mao X, Zheng P, Yu J, Luo J, He J. Chlorogenic acid improves intestinal barrier functions by suppressing mucosa inflammation and improving antioxidant capacity in weaned pigs. J Nutr Biochem 2018; 59: 84-92
  CrossrefPubMedSearch in Google Scholar
• 7 Cencič A, Langerholc T. Functional cell models of the gut and their applications in food microbiology–a review. Int J Food Microbiol 2010; 141: S4
  CrossrefPubMedSearch in Google Scholar
• 8 Duell BL, Cripps AW, Schembri MA, Ulett GC. Epithelial cell coculture models for studying infectious diseases: benefits and limitations. J Biomed Biotechnol 2011; 2011: 852419
  CrossrefPubMedSearch in Google Scholar
• 9 Ponce de León-Rodríguez MC, Guyot JP, Laurent-Babot C. Intestinal in vitro cell culture models and their potential to study the effect of food components on intestinal inflammation. Crit Rev Food Sci Nutr 2019; 59: 3648-3666
  CrossrefPubMedSearch in Google Scholar
• 10 Gu MJ, Song SK, Lee IK, Ko S, Han SE, Bae S, Ji SY, Park BC, Song KD, Lee HK, Han SH, Yun CH. Barrier protection via Toll-like receptor 2 signaling in porcine intestinal epithelial cells damaged by deoxynivalnol. Vet Res 2016; 47: 1-11
  CrossrefPubMedSearch in Google Scholar
• 11 Gu MJ, Han SE, Hwang K, Mayer E, Reisinger N, Schatzmayr D, Park BC, Han SH, Yun CH. Hydrolyzed fumonisin B 1 induces less inflammatory responses than fumonisin B 1 in the co-culture model of porcine intestinal epithelial and immune cells. Toxicol Lett 2019; 305: 110-116
  CrossrefPubMedSearch in Google Scholar
• 12 Dehlink E, Domig KJ, Loibichler C, Kampl E, Eiwegger T, Georgopoulos A, Kneifel W, Urbanek R, Szépfalusi Z. Heat- and formalin-inactivated probiotic bacteria induce comparable cytokine patterns in intestinal epithelial cell-leucocyte cocultures. J Food Prot 2007; 70: 2417-2421
  CrossrefPubMedSearch in Google Scholar
• 13 Haller D, Bode C, Hammes WP, Pfeifer AMA, Schiffrin EJ, Blum S. Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures. Gut 2000; 47: 79-87
  CrossrefPubMedSearch in Google Scholar
• 14 Loss H, Aschenbach JR, Ebner F, Tedin K, Lodemann U. Inflammatory responses of porcine MoDC and intestinal epithelial cells in a direct-contact co-culture system following a bacterial challenge. Inflammation 2020; 43: 552-567
  CrossrefPubMedSearch in Google Scholar
• 15 Katial RK, Sachanandani D, Pinney C, Lieberman MM. Cytokine production in cell culture by peripheral blood mononuclear cells from immunocompetent hosts. Clin Diagn Lab Immunol 1998; 5: 78-81
  CrossrefPubMedSearch in Google Scholar
• 16 Verfaillie T, Cox E, To LT, Vanrompay D, Bouchaut H, Buys N, Goddeeris BM. Comparative analysis of porcine cytokine production by mRNA and protein detection. Vet Immunol Immunopathol 2001; 81: 97-112
  CrossrefPubMedSearch in Google Scholar
• 17 Guo S, Al-Sadi R, Said HM, Ma TY. Lipopolysaccharide causes an increase in intestinal tight junction permeability in vitro and in vivo by inducing enterocyte membrane expression and localization of TLR-4 and CD14. Am J Pathol 2013; 182: 375-387
  CrossrefPubMedSearch in Google Scholar
• 18 Liu CH, Chaung HC, Chang HL, Peng YT, Chung WB. Expression of Toll-like receptor mRNA and cytokines in pigs infected with porcine reproductive and respiratory syndrome virus. Vet Microbiol 2009; 136: 266-276
  CrossrefPubMedSearch in Google Scholar
• 19 Cinar MU, Islam MA, Pröll M, Kocamis H, Tholen E, Tesfaye D, Looft C, Schellander K, Uddin MJ. Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA. BMC Res Notes 2013; 6: 1
  CrossrefPubMedSearch in Google Scholar
• 20 Mani V, Weber TE, Baumgard LH, Gabler NK. Growth and development symposium: Endotoxin, inflammation, and intestinal function in livestock. J Anim Sci 2012; 90: 1452-1465
  CrossrefPubMedSearch in Google Scholar
• 21 Islam MA, Pröll M, Hölker M, Tholen E, Tesfaye D, Looft C, Schellander K, Cinar MU. Alveolar macrophage phagocytic activity is enhanced with LPS priming, and combined stimulation of LPS and lipoteichoic acid synergistically induce pro-inflammatory cytokines in pigs. Innate Immun 2013; 19: 631-643
  CrossrefPubMedSearch in Google Scholar
• 22 Leonard F, Collnot EM, Lehr CM. A three-dimensional coculture of enterocytes, monocytes and dendritic cells to model inflamed intestinal mucosa in vitro . Mol Pharm 2010; 7: 2103-2119
  CrossrefPubMedSearch in Google Scholar
• 23 Ou G, Baranov V, Lundmark E, Hammarström S, Hammarström ML. Contribution of intestinal epithelial cells to innate immunity of the human gut–studies on polarized monolayers of colon carcinoma cells. Scand J Immunol 2009; 69: 150-161
  CrossrefPubMedSearch in Google Scholar
• 24 Parlesak A, Haller D, Brinz S, Baeuerlein A, Bode C. Modulation of cytokine release by differentiated CACO-2 cells in a compartmentalized coculture model with mononuclear leucocytes and nonpathogenic bacteria. Scand J Immunol 2004; 60: 477-485
  CrossrefPubMedSearch in Google Scholar
• 25 Liu Y, Chen F, Odle J, Lin X, Jacobi SK, Zhu H, Wu Z, Hou Y. Fish oil enhances intestinal integrity and inhibits TLR4 and NOD2 signaling pathways in weaned pigs after LPS challenge. J Nutr 2012; 142: 2017-2024
  CrossrefPubMedSearch in Google Scholar
• 26 Bruserud Ø, Wendelbo Ø, Paulsen K. Lipoteichoic acid derived from Enterococcus faecalis modulates the functional characteristics of both normal peripheral blood leukocytes and native human acute myelogenous leukemia blasts. Eur J Haematol 2004; 73: 340-350
  CrossrefPubMedSearch in Google Scholar
• 27 Nossol C, Diesing AK, Walk N, Faber-Zuschratter H, Hartig R, Post A, Kluess J, Rothkötter HJ, Kahlert S. Air-liquid interface cultures enhance the oxygen supply and trigger the structural and functional differentiation of intestinal porcine epithelial cells (IPEC). Histochem Cell Biol 2011; 136: 103-115
  CrossrefPubMedSearch in Google Scholar
• 28 Jung HC, Eckmann L, Yang SK, Panja A, Fierer J, Morzycka-Wroblewsksa E, Kagnoff MF. A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion. J Clinical Investig 1993; 95: 55-65
  CrossrefPubMedSearch in Google Scholar
• 29 Kämpfer AAM, Urbán P, Gioria S, Kanase N, Stone V, Kinsner-Ovaskainen A. Development of an in vitro co-culture model to mimic the human intestine in healthy and diseased state. Toxicol Vitr 2017; 45: 31-43
  CrossrefPubMedSearch in Google Scholar
• 30 Vatzia E, Pierron A, Saalmüller A, Mayer E, Gerner W. Deoxynivalenol affects proliferation and expression of activation-related molecules in major porcine T-cell subsets. Toxins (Basel) 2019; 11: 644
  CrossrefPubMedSearch in Google Scholar
• 31 Pié S, Lallès JP, Blazy F, Laffitte J, Sève B, Oswald IP. Weaning is associated with an upregulation of expression of inflammatory cytokines in the intestine of piglets. J Nutr 2004; 134: 641-647
  CrossrefPubMedSearch in Google Scholar
• 32 Koch L, Frommhold D, Buschmann K, Kuss N, Poeschl J, Ruef P. LPS- and LTA-induced expression of IL-6 and TNF- in neonatal and adult blood: role of MAPKs and NF-B. Mediators Inflamm 2014; 2014: 283126
  CrossrefPubMedSearch in Google Scholar
• 33 Liu X, Hu X, Zhang X, Li Z, Lu H. Role of rheum polysaccharide in the cytokines produced by peripheral blood monocytes in TLR4 mediated HLA-B27 associated AAU. Biomed Res Int 2013; 2013: 431232
  CrossrefPubMedSearch in Google Scholar
• 34 Vatzia E, Pierron A, Hoog AM, Saalmüller A, Mayer E, Gerner W. Deoxynivalenol Has the capacity to increase transcription factor expression and cytokine production in porcine T cells. Front Immunol 2020; 11: 1-17
  CrossrefPubMedSearch in Google Scholar
• 35 Windisch W, Schedle K, Plitzner C, Kroismayr A. Use of phytogenic products as feed additives for swine and poultry. J Anim Sci 2008; 86: E140-E148
  CrossrefPubMedSearch in Google Scholar
• 36 Lallès JP, Bosi P, Smidt H, Stokes CR. Nutritional management of gut health in pigs around weaning. Proc Nutr Soc 2007; 66: 260-268
  CrossrefPubMedSearch in Google Scholar
• 37 Dong GZ, Pluske JR. The low feed intake in newly-weaned pigs: problems and possible solutions. Asian-Australasian J Anim Sci 2007; 20: 440-452
  CrossrefPubMedSearch in Google Scholar
• 38 Ayrle H, Mevissen M, Kaske M, Nathues H, Gruetzner N, Melzig M, Walkenhorst M. Medicinal plants–prophylactic and therapeutic options for gastrointestinal and respiratory diseases in calves and piglets? A systematic review. BMC Vet Res 2016; 12: 89
  CrossrefPubMedSearch in Google Scholar
• 39 Kaschubek T, Mayer E, Rzesnik S, Grenier B, Bachinger D, Schieder C, König J, Teichmann K. Effects of phytogenic feed additives on cellular oxidative stress and inflammatory reactions in intestinal porcine epithelial cells. J Anim Sci 2018; 96: 3657-3669
  CrossrefPubMedSearch in Google Scholar
• 40 Pu J, Chen D, Tian G, He J, Zheng P, Mao X, Yu J, Huang Z, Zhu L, Luo J, Luo Y, Yu B. Protective effects of benzoic acid, bacillus coagulans, and oregano oil on intestinal injury caused by enterotoxigenic escherichia coli in weaned piglets. Biomed Res Int 2018; 2018: 1829632
  CrossrefPubMedSearch in Google Scholar
• 41 Zou Y, Xiang Q, Wang J, Peng J, Wei H. Oregano Essential oil improves intestinal morphology and expression of tight junction proteins associated with modulation of selected intestinal bacteria and immune status in a pig model. Biomed Res Int 2016; 2016: 5436738
  CrossrefPubMedSearch in Google Scholar
• 42 Bachinger D, Mayer E, Kaschubek T, Schieder C, König J, Teichmann K. Influence of phytogenics on recovery of the barrier function of intestinal porcine epithelial cells after a calcium switch. J Anim Physiol Anim Nutr (Berl) 2019; 103: 210-220
  CrossrefPubMedSearch in Google Scholar
• 43 Gallois M, Rothkötter HJ, Bailey M, Stokes CR, Oswald IP. Natural alternatives to in-feed antibiotics in pig production: can immunomodulators play a role?. Animal 2009; 3: 1644-1661
  CrossrefPubMedSearch in Google Scholar
• 44 Szépfalusi Z, Nentwich I, Gerstmayr M, Jost E, Todoran L, Gratzl R, Herkner K, Urbanek R. Prenatal allergen contact with milk proteins. Clin Exp Allergy 1997; 27: 28-35
  CrossrefPubMedSearch in Google Scholar
• 45 Springler A, Hessenberger S, Schatzmayr G, Mayer E. Early activation of MAPK p44/42 is partially involved in DON-induced disruption of the intestinal barrier function and tight junction network. Toxins (Basel) 2016; 8: 264
  CrossrefPubMedSearch in Google Scholar
• 46 Ladinig A, Lunney JK, Souza CJH, Ashley C, Plastow G, Harding JCS. Cytokine profiles in pregnant gilts experimentally infected with porcine reproductive and respiratory syndrome virus and relationships with viral load and fetal outcome. Vet Res 2014; 45: 1-10
  CrossrefPubMedSearch in Google Scholar

Correspondence

Publication History

Received: 24 February 2021

Accepted after revision: 14 May 2021

Article published online:
18 June 2021

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

• 1 Liu Y. Fatty acids, inflammation and intestinal health in pigs. J Anim Sci Biotechnol 2015; 6: 1-9
  CrossrefPubMedSearch in Google Scholar
• 2 Shin D, Chang SY, Bogere P, Won KH, Choi JY, Choi YJ, Lee HK, Hur J, Park BY, Kim Y, Heo J. Beneficial roles of probiotics on the modulation of gut microbiota and immune response in pigs. PLoS One 2019; 14: 1-23
  CrossrefPubMedSearch in Google Scholar
• 3 Valenzuela-Grijalva NV, Pinelli-Saavedra A, Muhlia-Almazan A, Domínguez-Díaz D, González-Ríos H. Dietary inclusion effects of phytochemicals as growth promoters in animal production. J Anim Sci Technol 2017; 59: 1-17
  CrossrefPubMedSearch in Google Scholar
• 4 Wang T, Yao W, Li J, Shao Y, He Q, Xia J, Huang F. Dietary garcinol supplementation improves diarrhea and intestinal barrier function associated with its modulation of gut microbiota in weaned piglets. J Anim Sci Biotechnol 2020; 11: 1-13
  CrossrefPubMedSearch in Google Scholar
• 5 Wang K, Chen G, Cao G, Xu Y, Wang Y, Yang C. Effects of Clostridium butyricum and Enterococcus faecalis on growth performance, intestinal structure, and inflammation in lipopolysaccharide-challenged weaned piglets. J Anim Sci 2019; 97: 4140-4151
  CrossrefPubMedSearch in Google Scholar
• 6 Chen J, Yu B, Chen D, Huang Z, Mao X, Zheng P, Yu J, Luo J, He J. Chlorogenic acid improves intestinal barrier functions by suppressing mucosa inflammation and improving antioxidant capacity in weaned pigs. J Nutr Biochem 2018; 59: 84-92
  CrossrefPubMedSearch in Google Scholar
• 7 Cencič A, Langerholc T. Functional cell models of the gut and their applications in food microbiology–a review. Int J Food Microbiol 2010; 141: S4
  CrossrefPubMedSearch in Google Scholar
• 8 Duell BL, Cripps AW, Schembri MA, Ulett GC. Epithelial cell coculture models for studying infectious diseases: benefits and limitations. J Biomed Biotechnol 2011; 2011: 852419
  CrossrefPubMedSearch in Google Scholar
• 9 Ponce de León-Rodríguez MC, Guyot JP, Laurent-Babot C. Intestinal in vitro cell culture models and their potential to study the effect of food components on intestinal inflammation. Crit Rev Food Sci Nutr 2019; 59: 3648-3666
  CrossrefPubMedSearch in Google Scholar
• 10 Gu MJ, Song SK, Lee IK, Ko S, Han SE, Bae S, Ji SY, Park BC, Song KD, Lee HK, Han SH, Yun CH. Barrier protection via Toll-like receptor 2 signaling in porcine intestinal epithelial cells damaged by deoxynivalnol. Vet Res 2016; 47: 1-11
  CrossrefPubMedSearch in Google Scholar
• 11 Gu MJ, Han SE, Hwang K, Mayer E, Reisinger N, Schatzmayr D, Park BC, Han SH, Yun CH. Hydrolyzed fumonisin B 1 induces less inflammatory responses than fumonisin B 1 in the co-culture model of porcine intestinal epithelial and immune cells. Toxicol Lett 2019; 305: 110-116
  CrossrefPubMedSearch in Google Scholar
• 12 Dehlink E, Domig KJ, Loibichler C, Kampl E, Eiwegger T, Georgopoulos A, Kneifel W, Urbanek R, Szépfalusi Z. Heat- and formalin-inactivated probiotic bacteria induce comparable cytokine patterns in intestinal epithelial cell-leucocyte cocultures. J Food Prot 2007; 70: 2417-2421
  CrossrefPubMedSearch in Google Scholar
• 13 Haller D, Bode C, Hammes WP, Pfeifer AMA, Schiffrin EJ, Blum S. Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures. Gut 2000; 47: 79-87
  CrossrefPubMedSearch in Google Scholar
• 14 Loss H, Aschenbach JR, Ebner F, Tedin K, Lodemann U. Inflammatory responses of porcine MoDC and intestinal epithelial cells in a direct-contact co-culture system following a bacterial challenge. Inflammation 2020; 43: 552-567
  CrossrefPubMedSearch in Google Scholar
• 15 Katial RK, Sachanandani D, Pinney C, Lieberman MM. Cytokine production in cell culture by peripheral blood mononuclear cells from immunocompetent hosts. Clin Diagn Lab Immunol 1998; 5: 78-81
  CrossrefPubMedSearch in Google Scholar
• 16 Verfaillie T, Cox E, To LT, Vanrompay D, Bouchaut H, Buys N, Goddeeris BM. Comparative analysis of porcine cytokine production by mRNA and protein detection. Vet Immunol Immunopathol 2001; 81: 97-112
  CrossrefPubMedSearch in Google Scholar
• 17 Guo S, Al-Sadi R, Said HM, Ma TY. Lipopolysaccharide causes an increase in intestinal tight junction permeability in vitro and in vivo by inducing enterocyte membrane expression and localization of TLR-4 and CD14. Am J Pathol 2013; 182: 375-387
  CrossrefPubMedSearch in Google Scholar
• 18 Liu CH, Chaung HC, Chang HL, Peng YT, Chung WB. Expression of Toll-like receptor mRNA and cytokines in pigs infected with porcine reproductive and respiratory syndrome virus. Vet Microbiol 2009; 136: 266-276
  CrossrefPubMedSearch in Google Scholar
• 19 Cinar MU, Islam MA, Pröll M, Kocamis H, Tholen E, Tesfaye D, Looft C, Schellander K, Uddin MJ. Evaluation of suitable reference genes for gene expression studies in porcine PBMCs in response to LPS and LTA. BMC Res Notes 2013; 6: 1
  CrossrefPubMedSearch in Google Scholar
• 20 Mani V, Weber TE, Baumgard LH, Gabler NK. Growth and development symposium: Endotoxin, inflammation, and intestinal function in livestock. J Anim Sci 2012; 90: 1452-1465
  CrossrefPubMedSearch in Google Scholar
• 21 Islam MA, Pröll M, Hölker M, Tholen E, Tesfaye D, Looft C, Schellander K, Cinar MU. Alveolar macrophage phagocytic activity is enhanced with LPS priming, and combined stimulation of LPS and lipoteichoic acid synergistically induce pro-inflammatory cytokines in pigs. Innate Immun 2013; 19: 631-643
  CrossrefPubMedSearch in Google Scholar
• 22 Leonard F, Collnot EM, Lehr CM. A three-dimensional coculture of enterocytes, monocytes and dendritic cells to model inflamed intestinal mucosa in vitro . Mol Pharm 2010; 7: 2103-2119
  CrossrefPubMedSearch in Google Scholar
• 23 Ou G, Baranov V, Lundmark E, Hammarström S, Hammarström ML. Contribution of intestinal epithelial cells to innate immunity of the human gut–studies on polarized monolayers of colon carcinoma cells. Scand J Immunol 2009; 69: 150-161
  CrossrefPubMedSearch in Google Scholar
• 24 Parlesak A, Haller D, Brinz S, Baeuerlein A, Bode C. Modulation of cytokine release by differentiated CACO-2 cells in a compartmentalized coculture model with mononuclear leucocytes and nonpathogenic bacteria. Scand J Immunol 2004; 60: 477-485
  CrossrefPubMedSearch in Google Scholar
• 25 Liu Y, Chen F, Odle J, Lin X, Jacobi SK, Zhu H, Wu Z, Hou Y. Fish oil enhances intestinal integrity and inhibits TLR4 and NOD2 signaling pathways in weaned pigs after LPS challenge. J Nutr 2012; 142: 2017-2024
  CrossrefPubMedSearch in Google Scholar
• 26 Bruserud Ø, Wendelbo Ø, Paulsen K. Lipoteichoic acid derived from Enterococcus faecalis modulates the functional characteristics of both normal peripheral blood leukocytes and native human acute myelogenous leukemia blasts. Eur J Haematol 2004; 73: 340-350
  CrossrefPubMedSearch in Google Scholar
• 27 Nossol C, Diesing AK, Walk N, Faber-Zuschratter H, Hartig R, Post A, Kluess J, Rothkötter HJ, Kahlert S. Air-liquid interface cultures enhance the oxygen supply and trigger the structural and functional differentiation of intestinal porcine epithelial cells (IPEC). Histochem Cell Biol 2011; 136: 103-115
  CrossrefPubMedSearch in Google Scholar
• 28 Jung HC, Eckmann L, Yang SK, Panja A, Fierer J, Morzycka-Wroblewsksa E, Kagnoff MF. A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion. J Clinical Investig 1993; 95: 55-65
  CrossrefPubMedSearch in Google Scholar
• 29 Kämpfer AAM, Urbán P, Gioria S, Kanase N, Stone V, Kinsner-Ovaskainen A. Development of an in vitro co-culture model to mimic the human intestine in healthy and diseased state. Toxicol Vitr 2017; 45: 31-43
  CrossrefPubMedSearch in Google Scholar
• 30 Vatzia E, Pierron A, Saalmüller A, Mayer E, Gerner W. Deoxynivalenol affects proliferation and expression of activation-related molecules in major porcine T-cell subsets. Toxins (Basel) 2019; 11: 644
  CrossrefPubMedSearch in Google Scholar
• 31 Pié S, Lallès JP, Blazy F, Laffitte J, Sève B, Oswald IP. Weaning is associated with an upregulation of expression of inflammatory cytokines in the intestine of piglets. J Nutr 2004; 134: 641-647
  CrossrefPubMedSearch in Google Scholar
• 32 Koch L, Frommhold D, Buschmann K, Kuss N, Poeschl J, Ruef P. LPS- and LTA-induced expression of IL-6 and TNF- in neonatal and adult blood: role of MAPKs and NF-B. Mediators Inflamm 2014; 2014: 283126
  CrossrefPubMedSearch in Google Scholar
• 33 Liu X, Hu X, Zhang X, Li Z, Lu H. Role of rheum polysaccharide in the cytokines produced by peripheral blood monocytes in TLR4 mediated HLA-B27 associated AAU. Biomed Res Int 2013; 2013: 431232
  CrossrefPubMedSearch in Google Scholar
• 34 Vatzia E, Pierron A, Hoog AM, Saalmüller A, Mayer E, Gerner W. Deoxynivalenol Has the capacity to increase transcription factor expression and cytokine production in porcine T cells. Front Immunol 2020; 11: 1-17
  CrossrefPubMedSearch in Google Scholar
• 35 Windisch W, Schedle K, Plitzner C, Kroismayr A. Use of phytogenic products as feed additives for swine and poultry. J Anim Sci 2008; 86: E140-E148
  CrossrefPubMedSearch in Google Scholar
• 36 Lallès JP, Bosi P, Smidt H, Stokes CR. Nutritional management of gut health in pigs around weaning. Proc Nutr Soc 2007; 66: 260-268
  CrossrefPubMedSearch in Google Scholar
• 37 Dong GZ, Pluske JR. The low feed intake in newly-weaned pigs: problems and possible solutions. Asian-Australasian J Anim Sci 2007; 20: 440-452
  CrossrefPubMedSearch in Google Scholar
• 38 Ayrle H, Mevissen M, Kaske M, Nathues H, Gruetzner N, Melzig M, Walkenhorst M. Medicinal plants–prophylactic and therapeutic options for gastrointestinal and respiratory diseases in calves and piglets? A systematic review. BMC Vet Res 2016; 12: 89
  CrossrefPubMedSearch in Google Scholar
• 39 Kaschubek T, Mayer E, Rzesnik S, Grenier B, Bachinger D, Schieder C, König J, Teichmann K. Effects of phytogenic feed additives on cellular oxidative stress and inflammatory reactions in intestinal porcine epithelial cells. J Anim Sci 2018; 96: 3657-3669
  CrossrefPubMedSearch in Google Scholar
• 40 Pu J, Chen D, Tian G, He J, Zheng P, Mao X, Yu J, Huang Z, Zhu L, Luo J, Luo Y, Yu B. Protective effects of benzoic acid, bacillus coagulans, and oregano oil on intestinal injury caused by enterotoxigenic escherichia coli in weaned piglets. Biomed Res Int 2018; 2018: 1829632
  CrossrefPubMedSearch in Google Scholar
• 41 Zou Y, Xiang Q, Wang J, Peng J, Wei H. Oregano Essential oil improves intestinal morphology and expression of tight junction proteins associated with modulation of selected intestinal bacteria and immune status in a pig model. Biomed Res Int 2016; 2016: 5436738
  CrossrefPubMedSearch in Google Scholar
• 42 Bachinger D, Mayer E, Kaschubek T, Schieder C, König J, Teichmann K. Influence of phytogenics on recovery of the barrier function of intestinal porcine epithelial cells after a calcium switch. J Anim Physiol Anim Nutr (Berl) 2019; 103: 210-220
  CrossrefPubMedSearch in Google Scholar
• 43 Gallois M, Rothkötter HJ, Bailey M, Stokes CR, Oswald IP. Natural alternatives to in-feed antibiotics in pig production: can immunomodulators play a role?. Animal 2009; 3: 1644-1661
  CrossrefPubMedSearch in Google Scholar
• 44 Szépfalusi Z, Nentwich I, Gerstmayr M, Jost E, Todoran L, Gratzl R, Herkner K, Urbanek R. Prenatal allergen contact with milk proteins. Clin Exp Allergy 1997; 27: 28-35
  CrossrefPubMedSearch in Google Scholar
• 45 Springler A, Hessenberger S, Schatzmayr G, Mayer E. Early activation of MAPK p44/42 is partially involved in DON-induced disruption of the intestinal barrier function and tight junction network. Toxins (Basel) 2016; 8: 264
  CrossrefPubMedSearch in Google Scholar
• 46 Ladinig A, Lunney JK, Souza CJH, Ashley C, Plastow G, Harding JCS. Cytokine profiles in pregnant gilts experimentally infected with porcine reproductive and respiratory syndrome virus and relationships with viral load and fetal outcome. Vet Res 2014; 45: 1-10
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material