The increased productivity in livestock husbandry accompanied by heightened demands on the quality of the meat produced. At the same time, the livestock industry represents an important economic activity in many countries. In large-scale farms, intensive conditions expose animals to stress, resulting in diseases and environmental decline, leading to significant economic losses. Treatment of these diseases has led to a surge in the use of veterinary drugs, fostering antimicrobial resistance and posing risks to consumers [1]. Health is closely tied to mucosal immunity and the intestinal microbiome. The intricate interactions between the host and the gut microbiome impact nutrient exchange, thereby influencing gut physiology, immunity, and morphology. On the other hand, the presence of intestinal pathogens can lead to undesirable changes in the morphology of the intestine [2]. Therefore, it is essential to promote sufficient cooperation and activation of the mucosal immune system, particularly through the production of antimicrobial proteins. Protein molecules such as lumican and olfactomedin-4 may promote intestinal homeostasis by initiating innate immune inflammatory responses that are beneficial in the early stages of enteritis and colitis [3, 4]. The mucosal surface of the intestine is formed by an epithelium, while tight junctions (TJs) exist between the epithelial cells, forming a continuous intercellular barrier, which is necessary for the separation of tissue spaces and the regulation of the selective movement of dissolved substances [5]. Both the occludin and claudin families represent TJ transmembrane proteins capable of adhesive interactions with other molecules between adhered cells, including the control of ion selectivity [6].

Consequently, there is a high demand for natural substances that have a beneficial effect on animal health, including protection against infectious diseases. Probiotic bacteria are undoubtedly among the most widely used substances of natural origin [7]. The effect of probiotics can vary depending on the properties of the specific probiotic bacteria used as well as their combination. Limosilactobacillus reuteri (L. reuteri) is among the most studied probiotic strains, which dominantly colonizes the intestines of mammals and humans. It is considered an “intestinal probiotic with prebiotic efficacy” because most of them show remarkable intestinal colonization including the secretion of bacteriocins, which also increases the expression of mucin genes, thus supporting the development and maturation of intestinal organoids and increasing the secretion of mucin itself [8]. Both animal and human studies suggest that probiotics can significantly influence the modulation of immune and inflammatory mechanisms. The use of probiotics can improve the balance of intestinal microorganisms, increase mucus secretion, and reduce the degradation of TJ proteins by reducing the presence of bacterial lipopolysaccharide (LPS) [9, 10]. Probiotic microorganisms, together with intestinal symbionts, also modulate host intestinal barrier function through their metabolites and various surface molecules [11]. In addition to this, they also promote host health by strengthening the intestinal barrier through various direct and indirect mechanisms [12]. Studying the mechanism of action for each probiotic strain is crucial. Typically, potential strains undergo microbiological testing in vitro and on two-dimensional (2D) cell lines. However, these lines lack realism in mimicking the natural reactions of the organism. Utilizing 3D animal gut models is proving to be more suitable, offering a closer simulation of the in vivo intestinal environment and facilitating a detailed study of bacteria-gut interactions. It’s important to note that live animal experiments are subject to legal restrictions in the European Union due to ethical concerns [13].

In a previous study, we demonstrated that L. reuteri B1/1 exhibited substantial adherence to non-carcinogenic porcine-derived enterocytes (CLAB) cells, especially at a concentration of 1 $\times$ 10${}^9$ CFU. This lactic acid bacteria (LAB) strain not only influenced the gene expression of pro-inflammatory cytokines and the gene expression for lumican and olfactomedin-4, but also heightened the metabolic activity of the cells after a 4 h incubation period. As the newly isolated L. reuteri B1/1 was to be included in the microorganism collection, we decided to test it further on a Transwell insert model that more closely mimics the gut. Therefore, the aim of the present study was to monitor the effect of L. reuteri B1/1 and L. fermentum CCM 7158, chosen as an indicator LAB strain, on relative gene expression of interleukins (IL-1$\beta$, IL-6, IL-8, IL-18 and IL-10), genes encoding receptors for TLR4 and TLR2, TJ proteins such as claudin-1 (CLDN1), occludin (OCLN) and important antimicrobial proteins such as lumican (LUM) and olfactomedin-4 (OLMF-4) produced by the IPEC-J2 cell line and monocyte-derived macrophages (MDMs) in a co-culture model.

In the body, the intestinal epithelium performs a number of important functions such as digesting and absorbing nutrients, providing a physical barrier, and shielding the body from the challenging conditions of the gut lumen. The epithelial barrier ensures selectivity, preventing the entry of a potentially harmful luminal contents by rejection, while facilitating the controlled absorption and secretion of significant amounts of solutes and water in a specific direction. Individual epithelial cells are linked through a network of intercellular junctions - TJs, which are of particular significance in defining the properties of the paracellular barrier and its selectivity [32]. To determine barrier strength, TEER is commonly measured in in vitro cell models and permeability to paracellular markers such as the enzyme horseradish peroxidase, inulin, or mannitol is assessed [33]. Our results showed that a basolateral challenge in the form of LPS had an effect on the reduction of TEER values already after 6 h of application. The fact that basolateral LPS challenge leads to a significant decrease in TEER was also demonstrated by Wine et al. [34], who found that the basolateral aspect of the T84 cell line by infection with invasive E. coli (O157:H7) led to a significant decrease in TEER values. This reduction was more significant after basolateral application compared to the apical compartment/application of E. coli. There is evidence that other pathogens, such as C. jejuni, enter intestinal epithelial cells through? the basolateral membrane, highlighting the importance of this finding [35].

Furthermore, it is well known that the intestinal barrier function is to some extent supported by TJ proteins [36]. Epithelial cell TJs are comprised of numerous junctional molecules, including claudins, occludins and zonula occludens, which regulate the paracellular permeability of various macromolecules, ions, and water between neighboring cells. Occludin is a key transmembrane protein in the TJs, and both occludin and claudin-1 play essential roles in maintaining the intestinal permeability and barrier function of the TJs. There is evidence that LPS-induced inflammation disrupts the integrity of intestinal epithelial cells and TJs [37]. Disruption of TJ integrity leads to immune cell activation and inflammatory processes in the affected tissues [38]. Consistent with the previous statement, in our study, basolateral application of LPS to MDMs affected the expression of the gene encoding OCLN which resulted in a significant down-regulation in IPEC-J2 cells representing the apical compartment. Similar results were reported by Zhao et al. [25], who observed a decrease in mRNA levels for the gene encoding OCLN after treating IPEC-J2 cells with bacterial LPS. Similarly, in the work of Wu et al. [37], a down-regulation of the expression of genes encoding the proteins occludin and claudin-1 was observed by applying LPS at a concentration of 1 µg/mL.

On the other hand, strong adhesion provides the initial interaction of probiotics with intestinal epithelial cells, which is key for LAB strains to exert their beneficial effects on host health. Adhesion to intestinal cells limits the presence of potential pathogens, thereby providing protection to intestinal epithelial cells (IECs) [39]. The genus Limosilactobacillus (līmōsus, Lat. - slimy), which also includes the strains L. reuteri and L. fermentum, is characterized by the ability of most strains in this genus to produce exopolysaccharides (EPS) from sucrose to promote biofilm formation on intestinal epithelia [40]. In this study, L. fermentum CCM 7158 was used as an indicator LAB strain because it is included in the Czech Collection of Microorganisms (CCM) and its properties have been extensively studied in previous studies under both in vitro and in vivo [41, 42, 43]. An interesting finding in our work is that simultaneous treatment of MDMs with LPS and treatment of IPEC-J2 with the probiotic strain L. fermentum CCM 7158 led to the down-regulation of mRNA for CLDN-1 and OCLN in intestinal cells. However, when L. reuteri B1/1 was used, the mRNA levels for the above-mentioned genes were significantly increased compared to the control group of cells. Similarly, genes encoding the antimicrobial proteins LUM and OLFM-4, which are also involved in maintaining the integrity of the intestinal epithelial layer [3], were significantly up-regulated in the IPEC-J2 cells treated with L. reuteri B1/1. In light of these results, we could suggest that L. reuteri B1/1 may act in a stimulatory manner on intestinal epithelia. Similarly, in our recent study, we observed the stimulatory effect of L. reuteri B1/1 on non-carcinogenic porcine-derived enterocytes (CLAB) at both concentrations used (10${}^7$ and 10${}^9$ CFU/mL) for mRNA transcription levels of lumican and olfactomedin-4 [30]. Likewise, in our other research under in vivo conditions, broiler chickens were orally exposed to L. reuteri B1/1 for 7 days, while also receiving a single dose of infectious Campylobacter jejuni on day 4. Specifically, in this experimental group we observed an increase in gene expression of the NLRP3 inflammasome along with other key immune molecules such as IL-1$\beta$, IL-18, and CASP-1 in the caecum. We hypothesize that this stimulation contributes to an improved ability of the innate immune system to more effectively countert Campylobacter jejuni infection in broiler chickens [44].

At the same time, enterocytes are an important component of the highly regulated communication network that provides protection to the intestinal mucosa, their ability to recognize microorganisms via pattern recognition receptors (PRRs) is essential [45]. The best-known of these PRRs are the Toll-like receptors (TLRs). Interaction of microorganisms with TLRs either leads to activation and triggering of the signaling pathway or, conversely, blocks its activation through the mechanism of negative regulators [46]. Structural components of the LAB cell wall, as well as the EPS, can be produced and released into the surrounding environment and are capable of interacting with TLRs in the gut [47]. In our previous study with the IPEC-J2/moDCs co-culture model, we demonstrated the ability of EPS derived from L. reuteri Biocenol™ to increase mRNA levels in dendritic cell monocultures [16]. In the present work we used live lactobacilli, where by treating apically deposited IPEC-J2 cells with L. fermentum CCM 7158, we observed an increase in gene expression for the gene encoding the TLR4 receptor not only in directly treated enterocytes but also in basolaterally deposited MDMs. Although the gene encoding TLR2 could not be captured in IPEC-J2 due to the reduced gene expression levels, the expression of this gene was increased in MDMs. This up-regulation in basolaterally deposited macrophages correlates with the detected increased expression of the pro-inflammatory cytokines studied (IL-1$\beta$, IL-6, IL-18), precisely in this experimental group. We hypothesize that this increase in genes encoding TLR receptors implies an active maintenance of the host’s vigilance against pathogens. Our results are not surprising, as it is well-known that TLR signaling triggers an initial cytokine reaction which intensifies the inflammatory response in the host while engaging other cells in the fight against infectious agents. Macrophages participate in cytokine production following their TLR9, TLR2, and TLR4 activation by releasing cytokines such as IL-12, IL-1$\beta$, IL-6, and IL-10 [48, 49, 50].

Numerous studies have demonstrated that LPS disrupts the structural integrity of the intestinal epithelia, triggers inflammation, and leads to the release of inflammatory cytokines such as IL-1$\beta$, IL-8, IL-6, and TNF-$\alpha$ [51, 52, 53]. The results obtained in our study by monitoring the mRNA transcriptional levels of pro-inflammatory cytokines (IL-8, IL-1$\beta$, IL-6, IL-18) in the LPS-induced experimental group indicate an increased release of cytokines by each cell type. Similarly, previous studies have shown that LPS is able to induce inflammation in intestinal epithelial cells, as manifested by increased levels of the pro-inflammatory cytokines monitored [54, 55, 56]. When comparing transcriptional levels of the studied pro-inflammatory cytokines in cells exposed to LPS and cells treated with L. reuteri B1/1, it is evident that the simultaneous treatment of IPEC-J2 cells with LAB and LPS challenge of MDMs significantly suppressed the inflammation induced by LPS alone without probiotic treatment. However, an interesting finding of our work was the different cellular responses of both IPEC-J2 and MDMs to the two LAB strains used. While L. reuteri B1/1 had the potential to increase the expression of genes encoding the antimicrobial peptides LUM and OLFM-4, as well as genes encoding TJ-related genes (CLDN-1 and OCLN), we did not observe these effects in the case of L. fermentum CCM 7158. On the contrary, L. fermentum CCM 7158 was able to increase the mRNA levels of pro-inflammatory cytokines (IL-18, IL-6, and IL-1$\beta$) in indirectly treated MDMs. Along with evaluating the gene expression of pro-inflammatory cytokines, IL-10 and an essential anti-inflammatory cytokine were also assessed. IL-10 plays a pivotal role in preserving gut homeostasis, the fine balance between inflammation processes triggered by pathogens, and plays a role in facilitating immunoregulatory mechanisms triggered by probiotics as well [57, 58]. In our experiment, cells from both the apical and basolateral compartments showed increased gene expression for this anti-inflammatory cytokine for both probiotic strains used and applied to IPEC-J2 cells. Although production of the anti-inflammatory cytokine IL-10 was observed in both LAB strains used in both cell cultures, we hypothesize that L. fermentum CCM 7158 has both an anti-inflammatory and immunostimulatory potential, as it was able to increase pro-inflammatory cytokines when co-induced with LPS in DMD cells. Similarly, in the study by Huang et al. [59], the strain E. faecium EF1 was found to exhibit both anti-inflammatory and immunostimulatory activities after oral administration in suckling piglets. The simultaneous anti- and pro-inflammatory properties of probiotics have also been demonstrated in several other in vitro studies [60, 61]. However, it must be taken into account that different strains of probiotics have different effects on cellular activity, cytokine release, and immunological interactions. Therefore, it is important to test bacterial strains under in vitro conditions, which allows the identification/elimination of toxic bacteria in order to reduce the number of preclinical tests performed on animals [62].

In our current study, using the porcine intestine in vitro model by co-cultivating IPEC-J2 and MDMs, we investigated the immunomodulatory potential of a recently isolated LAB strain L. reuteri B1/1 and the indicator LAB strain L. fermentum CCM 7158 on the inflammatory response to LPS challenge. An in vitro immunoprotective potential of L. reuteri B1/1 was suggested as it was able to suppress the enhanced inflammatory response of both cells used to LPS challenge. On the contrary, L. fermentum CCM 7158 increased the mRNA levels of pro-inflammatory cytokines (IL-18, IL-6, and IL-1$\beta$) in indirectly treated MDMs when LPS was simultaneously applied to basolaterally deposited macrophages. Although the production of the anti-inflammatory cytokine IL-10 was observed in both LAB strains used in both cell cultures, we hypothesize that L. fermentum has both anti-inflammatory and immunostimulatory effects. In addition, we demonstrated the ability of L. reuteri B1/1 to up-regulate the mRNA levels of genes encoding the antimicrobial proteins LUM and OLFM-4 in directly treated IPEC-J2 cells. Moreover, L. reuteri B1/1 was able to increase the TJ-related genes CLDN1 and OCLN, which were significantly down-regulated by the LPS challenge. Our results suggest that the recently isolated LAB strain L. reuteri B1/1 could have the potential to alleviate LPS-induced epithelial disruption and influence the production of antimicrobial molecules by enterocytes. Additional research is required to enhance our understanding of how L. reuteri B1/1 influences host mucosal immunity, and the mechanism of action for potential commercial use of this new probiotic lactobacillus isolate.

All data from this study are included in the manuscript or are available on request from the first or corresponding author.

Conceptualization, ZK, VK, JS and DM; Methodology, Data curation, ZK and JS; Formal analysis, Writing - Original draft preparation, ZK and VK; Supervision, DM; The cell cultures were prepared by ZK, and JS; Writing - Review and Editing ZK and VK; The management for research activity planning and the financial support for the project leading to this publication was performed by VK and DM. All authors read and approved the final manuscript. All authors have participated sufficiently in the work to take public responsibility for appropriate portions of the content and agreed to be accountable for all aspects of the work in ensuring that questions related to its accuracy or integrity. All authors contributed to editorial changes in the manuscript.

The procedure of blood sampling was performed in accordance with the guidelines for animal welfare and was approved by the Ethics Committee for the approval of research involving animals by the legislative requirements applicable at the UVMP in Košice with permit No. EKVP/2023-04.

We are grateful to assoc. prof. Jaroslav Novotný, DVM, PhD, from the Clinic of Swine, from the University of Veterinary Medicine and Pharmacy in Košice, for providing blood samples from which the monocyte-derived macrophages were isolated.

This work was funded by the Slovak Research and Development Agency under the contract no. (APVV-21-0129) and the Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic VEGA 1/0098/22.

The authors declare no conflict of interest.