Fumonisin B1 damages the barrier functions of porcine intestinal epithelial cells in vitro.

Fumonisins (Fums) are mycotoxins widely distributed in crops and feed, and ingestion of Fums-contaminated crops is harmful to animal health. The purpose of this study is to explore the effect of Fum B1 (FB1 ) on barrier functions of porcine intestinal epithelial cells, IPEC-J2, to clarify the intestinal toxicity of Fums in pigs. The results showed that the persistent treatment of FB1 significantly decreased the viability of IPEC-J2. Moreover, the expressions of Claudin 1, Occludin, Zonula Occluden-1 (ZO-1) on the messenger RNA (mRNA), and protein levels and MUC1 on the mRNA level were significantly inhibited after FB1 treatment, while the mRNA relative expression level of MUC2 was clearly increased. FB1 also enhanced the monolayer cell permeability of IPEC-J2. Importantly, FB1 promoted the expression of phosphorylated extracellular regulated protein kinase (p-ERK1/2 ). These data suggest that long-term treatment of FB1 can suppress IPEC-J2 proliferation, damage tight junctions of IPEC-J2, and regulate expression of mucins to induce the damage of barrier functions of porcine intestinal epithelial cells, which may be associated with the ERK1/2 phosphorylation pathway.

Co-infection with porcine bocavirus and porcine circovirus 2 affects inflammatory cytokine production and tight junctions of IPEC-J2 cells.

Porcine bocavirus (PBoV) has a high prevalence in both healthy and diseased swine around the world. It was recently reported that PBoV and porcine circovirus type 2 (PCV2)-which contribute to porcine diarrheal disease-have a high rate of co-infection. To clarify the pathogenesis of PBoV, we examined the co-infection rate and effects of these two pathogens in IPEC-J2 porcine intestinal enterocytes. Both single and co-infection had cytopathic effects in IPEC-J2 cells. The apoptosis and proliferation rates of cells infected with both viruses did not differ significantly from those of cells infected with either one alone. PBoV and PCV2 induced the upregulation of inflammatory cytokines and the downregulation of the tight junction proteins occludin and claudin 1 in the early stage of infection, leading to destruction of epithelial barrier integrity and enhanced cytotoxicity. These findings provide insight into the pathogenic mechanisms of PBoV and PCV2 and a basis for developing effective strategies to prevent the spread of gastrointestinal diseases in pigs and other livestock.

Identifying the location of epidermal growth factor-responsive element involved in the regulation of type IIb sodium-phosphate cotransporter expression in porcine intestinal epithelial cells.

Phosphate is an important mineral nutrient for both human and animals in growth and physiological functions; thus, much effort in the past has been made to clarify the mechanisms governing its absorption. Previous studies have found that epidermal growth factor (EGF) inhibits phosphate absorption in human intestinal cells via modulating the interaction of transcriptional factor c-myb with sodium-phosphate cotransporter (NaPi-IIb) gene promoter. This finding provoked our interest in determining the effect of EGF on NaPi-IIb gene expression in intestinal cells of pigs and the location of EGF-responsive element in the gene promoter. Using quantitative PCR, it was observed that EGF significantly reduced NaPi-IIb gene expression in porcine intestinal epithelial IPEC-J2 cells. Transfection with a series of constructs that contain different lengths of the 5'-flanking promoter region of the NaPi-IIb gene manifested that EGF-responsive element is located in the -1200 to -800 region. Further, c-myb was extracted from the cell nucleus of IPEC cells that were exposed to EGF or not via immunoprecipitation. The electrophoretic mobility shift assay showed a specific binding of transcription factor c-myb to labelled probes encompassing DNA sequence from -1092 to -1085 (-TCCAGTTG-). This protein-DNA complex was decreased with cells exposed to EGF and abrogated when c-myb was pre-incubated with excessive unlabelled competitive probes. Results from mutagenesis studies demonstrated that the c-myb-binding site is the EGF-responsive element involved in the regulation of NaPi-IIb expression. Identifying the location of EGF-responsive element contributes to understanding mechanisms underlying EGF down-regulated NaPi-IIb gene expression and provides a foundation for further investigating EGF-regulatory functions in phosphate absorption in pig intestine.

In vitro study of biological activities of anthocyanin-rich berry extracts on porcine intestinal epithelial cells.

BACKGROUND: Anthocyanins, compounds that represent the major group of flavonoids in berries, are one of the most powerful natural antioxidants. The aim of this study was to evaluate biological activities and comparison of anthocyanin-rich extracts prepared from chokeberry (Aronia melanocarpa), elderberry (Sambucus nigra), bilberry (Vaccinium myrtillus) and blueberry (V. corymbosum) on the porcine intestinal epithelial IPEC-1 cell line. RESULTS: The IC50 values calculated in the antioxidant cell-based dichlorofluorescein assay (DCF assay) were 1.129 mg L(-1) for chokeberry, 1.081 mg L(-1) for elderberry, 2.561 mg L(-1) for bilberry and 2.965 mg L(-1) for blueberry, respectively. We found a significant negative correlation (P < 0.001) between cyanidin glycosides content and IC50 values. Moreover, extracts rich in cyanidin glycosides stimulated proliferation of IPEC-1 cells and did not have cytotoxic effect on cells at an equivalent in vivo concentration. CONCLUSIONS: We found that the chokeberry and elderberry extracts rich in cyanidin glycosides possess better antioxidant and anticytotoxic activities in comparison to blueberry or bilberry extracts with complex anthocyanin profiles.

Selenomethionine Alleviates Deoxynivalenol-Induced Oxidative Injury in Porcine Intestinal Epithelial Cells Independent of MAPK Pathway Regulation.

Deoxynivalenol (DON) is a prevalent contaminant in feed and food, posing a serious threat to the health of both humans and animals. The pig stands as an ideal subject for the study of DON due to its recognition as the most susceptible animal to DON. In this study, the IPEC-J2 cells were utilized as an in vitro model to explore the potential of SeMet in alleviating the intestinal toxicity and oxidative injury in intestinal epithelial cells when exposed to DON. Cells were treated either with or without 4.0 µM SeMet, in combination with or without a simultaneous treatment with 0.5 µg/mL DON, for a duration of 24 h. Then, cells or related samples were analyzed for cell proliferation, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) level, gene expressions, and protein expressions. The results showed that SeMet mitigated the cellular toxicity caused by DON, evidenced by elevated cell proliferation and the reduced LDH release of IPEC-J2 cells in the SeMet + DON group vs. the DON group. Moreover, the SeMet treatment markedly promoted antioxidant functions and decreased the oxidative injury in IPEC-J2 cell, which is indicated by the decreased ROS level and up-regulated mRNA levels of GPX1, TXNRD1, Nrf2, and GCLC in IPEC-J2 cells in the SeMet + DON group vs. the DON group. However, in both the absence and presence of exposure to DON, the SeMet treatment did not affect the protein expression of MAPK (JNK, Erk1/2, and P38) and phosphorylated MAPK (p-JNK, p-Erk1/2, and p-P38) in IPEC-J2 cells. Collectively, SeMet alleviated the DON-induced oxidative injury in porcine intestinal epithelial cells independent of the MAPK pathway regulation.

In vitro evaluation of the immunomodulatory and wakame assimilation properties of Lactiplantibacillus plantarum strains from swine milk.

The emergence and spread of antibiotic resistance threat forced to explore alternative strategies for improving the resistance to pathogens in livestock production. Probiotic lactic acid bacteria represent an alternative for this objective. In this study, seven Lactiplantibacillus plantarum strains from porcine colostrum and milk were isolated, identified and characterized in terms of their abilities to modulate immunity in porcine intestinal epithelial (PIE) cells. Then, two potential immunoregulatory strains were studied in terms of their ability to utilize and grow in wakame (Undaria pinnafida). Isolates were identified by 16S rRNA gene and evaluated by studying their interaction with PIE cells. The expressions of peptidoglycan recognition proteins (PGRPs), nucleotide-binding oligomerization domain (NODs), host defense peptides (pBD), and type I interferons (IFNs) were evaluated by RT-qPCR. The strain 4M4417 showed a remarkable capacity to differentially regulate the expression of PGRP1, PGRP3, NOD1, NOD2, and pBD1 in PIE cells. On the other hand, the strain 4M4326 was the most efficient to improve the expression of IFN-α and IFN-ß in PIE cells challenged with poly (I:C). Both L. plantarum 4M4326 and 4M4417 were characterized in terms of their ability to utilize wakame. Results demonstrated that both strains efficiently grew in wakame-based broth. Our results suggest that L. planatrum 4M4326 and 4M4417 are interesting candidates to develop immunomodulatory feeds based on wakame utilization. These new immunosynbiotic feeds could help to reduce severity of intestinal infections and improve immune health status in pigs.

Glutamine Promotes Porcine Intestinal Epithelial Cell Proliferation through the Wnt/ß-Catenin Pathway.

Glutamine (Gln) is a critical nutrient required by neonatal mammals for intestinal growth, especially for newborn piglets. However, the mechanisms underlying the role of Gln in porcine intestinal epithelium development are not fully understood. The objective of the current study was to explore the possible signaling pathway involved in the promotion of porcine intestinal epithelial cell (IPEC-J2) proliferation by Gln. The results showed that 1 mM Gln promoted IPEC-J2 cell proliferation, and tandem mass tag proteomics revealed 973 differentially expressed proteins in Gln-treated IPEC-J2 cells, 824 of which were upregulated and 149 of which were downregulated. Moreover, gene set enrichment analysis indicated that the Wnt signaling pathway is activated by Gln treatment. Western blotting analysis further confirmed that Gln activated the Wnt/ß-catenin signaling pathway. In addition, Gln increased not only cytosolic ß-catenin but also nuclear ß-catenin protein expression. LF3 (a ß-catenin/TCF4 interaction inhibitor) assay and ß-catenin knockdown demonstrated that Gln-mediated promotion of Wnt/ß-catenin signaling and cell proliferation were blocked. Furthermore, the inhibition of TCF4 expression suppressed Gln-induced cell proliferation. These findings further confirmed that Wnt/ß-catenin signaling is involved in the promotion of IPEC-J2 cell proliferation by Gln. Collectively, these findings demonstrated that Gln positively regulated IPEC-J2 cell proliferation through the Wnt/ß-catenin pathway. These data greatly enhance the current understanding of the mechanism by which Gln regulates intestinal development.

Porcine delta coronavirus inhibits NHE3 activity of porcine intestinal epithelial cells through miR-361-3p/NHE3 regulatory axis.

Porcine deltacoronavirus (PDCoV) infection in piglets can cause small intestinal epithelial necrosis and atrophic enteritis, which leads to severe damages to host cells, and result in diarrhea. In this study, we investigated the relationship between miR-361, SLC9A3(Solute carrier family 9, subfamily A, member 3), and NHE3(sodium-hydrogen exchanger member 3) in in porcine intestinal epithelial cells (IPI-2I) cells after PDCoV infection. Our results showed that the ssc-miR-361-3p expression inhibits the mRNA level of SLC9A3 gene which lead to the descending of NHE3 protein expression, and the NHE3 activity was suppressed. NHE3 activity was suppressed via down-regulation expression of SLC9A3 mRNA by transfection with siRNA. Ssc-miR-361-3p mimics and inhibitors were used to change the expression of ssc-miR-361-3p in IPI-2I cells. Ssc-miR-361-3p overexpression reduced the mRNA level of SLC9A3 gene, the level of NHE3 protein expression and NHE3 activity in IPI-2I cells, while ssc-miR-361-3p inhibits NHE3. Furthermore, luciferase reporter assay showed that SLC9A3 gene was a direct target of ssc-miR-361-3p. Ssc-miR-361-3p inhibition restored NHE3 activity in PDCoV infected IPI-2I cells by up-regulating SLC9A3 mRNA expression and NHE3 protein expression. These results demonstrate that the PDCoV infection can inhibit NHE3 activity through miR-361-3p/SLC9A3 regulatory axis. The relevant research is reported for the first time in PDCoV, which has significance in exploring the pathogenic mechanism of PDCoV and can provide a theoretical basis for its prevention and control. suggesting that NHE3 and ssc-miR-361-3p may be potential therapeutic targets for diarrhea in infected piglets.

Porcine Circovirus 2 Increases the Frequency of Transforming Growth Factor-ß via the C35, S36 and V39 Amino Acids of the ORF4.

Porcine circovirus 2 (PCV2) is one of the most important endemic swine pathogens, inducing immunosuppression in pigs and predisposing them to secondary bacterial or viral infections. Our previous studies show that PCV2 infection stimulated pig intestinal epithelial cells (IPEC-J2) to produce the secretory transforming growth factor-ß (TGF-ß), which, in turn, caused CD4+ T cells to differentiate into regulatory T cells (Tregs). This may be one of the key mechanisms by which PCV2 induces immunosuppression. Here, we attempt to identify the viral proteins that affect the TGF-ß secretion, as well as the key amino acids that are primarily responsible for this occurrence. The three amino acids C35, S36 and V39 of the ORF4 protein are the key sites at which PCV2 induces a large amount of TGF-ß production in IPEC-J2 and influences the frequency of Tregs. This may elucidate the regulatory effect of PCV2 on the Tregs differentiation from the perspective of virus structure and intestinal epithelial cell interaction, laying a theoretical foundation for improving the molecular mechanism of PCV2-induced intestinal mucosal immunosuppression in piglets.

Potential Toxicity and Mechanisms of T-2 and HT-2 Individually or in Combination on the Intestinal Barrier Function of Porcine Small Intestinal Epithelial Cells.

Under natural conditions, T-2 toxin can be easily metabolized to HT-2 toxin by deacetylation, and T-2 and HT-2 are usually co-contaminated in grain and feed at a high detected rate. Our previous information indicated that T-2 toxin could injure the function of the intestinal barrier, but the combined toxicity and mechanism of T-2 and HT-2 on the intestinal cells of porcines are still unknown. Therefore, we aimed to explore T-2 and HT-2 individually and combined on cellular viability, cell membrane integrity, the expression of tight junction-related proteins, and the generation of inflammatory factors in porcine intestinal epithelial cells (IPEC-J2). The results showed that T-2 and HT-2, individually or in combination, could induce a decrease in cell viability, an increase in LDH release and IL-1, IL-6, and TNF-α generation, and a decrease in the anti-inflammatory factor IL-10. Based on the analysis of immunofluorescence staining, real-time PCR, and western blotting, the tight junction protein expressions of Claudin-1, Occludin, and ZO-1 were significantly decreased in the T-2 and HT-2 individual or combination treated groups compared with the control. Furthermore, all the parameter changes in the T-2 + HT-2 combination group were much more serious than those in the individual dose groups. These results suggest that T-2 and HT-2, individually and in combination, could induce an intestinal function injury related to an inflammatory response and damage to the intestinal barrier function in porcine intestinal epithelial cells. Additionally, T-2 and HT-2 in combination showed a synergistic toxic effect, which will provide a theoretical basis to assess the risk of T-2 + HT-2 co-contamination in porcine feed.

Ligilactobacillus salivarius Strains Isolated From the Porcine Gut Modulate Innate Immune Responses in Epithelial Cells and Improve Protection Against Intestinal Viral-Bacterial Superinfection.

Previously, we constructed a library of Ligilactobacillus salivarius strains from the intestine of wakame-fed pigs and reported a strain-dependent capacity to modulate IFN-ß expression in porcine intestinal epithelial (PIE) cells. In this work, we further characterized the immunomodulatory activities of L. salivarius strains from wakame-fed pigs by evaluating their ability to modulate TLR3- and TLR4-mediated innate immune responses in PIE cells. Two strains with a remarkable immunomodulatory potential were selected: L. salivarius FFIG35 and FFIG58. Both strains improved IFN-ß, IFN-λ and antiviral factors expression in PIE cells after TLR3 activation, which correlated with an enhanced resistance to rotavirus infection. Moreover, a model of enterotoxigenic E. coli (ETEC)/rotavirus superinfection in PIE cells was developed. Cells were more susceptible to rotavirus infection when the challenge occurred in conjunction with ETEC compared to the virus alone. However, L. salivarius FFIG35 and FFIG58 maintained their ability to enhance IFN-ß, IFN-λ and antiviral factors expression in PIE cells, and to reduce rotavirus replication in the context of superinfection. We also demonstrated that FFIG35 and FFIG58 strains regulated the immune response of PIE cells to rotavirus challenge or ETEC/rotavirus superinfection through the modulation of negative regulators of the TLR signaling pathway. In vivo studies performed in mice models confirmed the ability of L. salivarius FFIG58 to beneficially modulate the innate immune response and protect against ETEC infection. The results of this work contribute to the understanding of beneficial lactobacilli interactions with epithelial cells and allow us to hypothesize that the FFIG35 or FFIG58 strains could be used for the development of highly efficient functional feed to improve immune health status and reduce the severity of intestinal infections and superinfections in weaned piglets.

Activation of Interleukin-1ß Release and Pyroptosis by Transmissible Gastroenteritis Virus Is Dependent on the NOD-Like Receptor Protein 3 Inflammasome in Porcine Intestinal Epithelial Cell Line.

Transmissible gastroenteritis virus (TGEV) is a coronavirus, which causes fatal severe diarrhea and leads to high mortality in newborn piglets. Inflammasomes are hub molecules that induce proinflammatory cytokine production and maturation to initiate innate immune defenses upon cellular infection. To date, the potential role of inflammasome in TGEV infection in porcine intestinal epithelial cells has not been elucidated. The present study aims to investigate the function of the inflammasome in response to TGEV infection in porcine intestinal epithelial cells. Our results revealed that TGEV infection induced the production of pro-interleukin-1ß (pro-IL-1ß) and enhanced its processing and maturation in porcine intestinal epithelial cells through caspase-1 activation. In addition, TGEV infection in porcine intestinal epithelial cells induced pyroptosis, indicated by cell death and the production and cleavage of gasdermin D (GSDMD). Meanwhile, TGEV infection sufficiently activated the expression and assembly of the NOD-like receptor protein 3 (NLRP3) inflammasome in porcine intestinal epithelial cells, and inhibition of NLRP3 blocked TGEV-induced IL-1ß release. We also found that inhibition of NLRP3 enhanced the replication of TGEV without inducing cell death. In conclusion, these data demonstrated that activation of IL-1ß release and pyroptosis is dependent on NLRP3 inflammasome, thus NLRP3 inflammasome may play a central role in the innate immune response to TGEV infection.

Comprehensive Analysis of Transcriptome-wide m6A Methylome Upon Clostridium perfringens Beta2 Toxin Exposure in Porcine Intestinal Epithelial Cells by m6A Sequencing.

Piglet diarrhea is a swine disease responsible for serious economic impacts in the pig industry. Clostridium perfringens beta2 toxin (CPB2), which is a major toxin of C. perfringens type C, may cause intestinal diseases in many domestic animals. N6-methyladenosine (m6A) RNA methylation plays critical roles in many immune and inflammatory diseases in livestock and other animals. However, the role of m6A methylation in porcine intestinal epithelial (IPEC-J2) cells exposed to CPB2 has not been studied. To address this issue, we treated IPEC-J2 cells with CPB2 toxin and then quantified methylation-related enzyme expression by RT-qPCR and assessed the m6A methylation status of the samples by colorimetric N6-methyladenosine quantification. The results showed that the methylation enzymes changed to varying degrees while the m6A methylation level increased (p < 0.01). On this basis, we performed N6-methyladenosine sequencing (m6A-seq) and RNA sequencing (RNA-seq) to examine the detailed m6A modifications and gene expression of the IPEC-J2 cells following CPB2 toxin exposure. Our results indicated that 1,448 m6A modification sites, including 437 up-regulated and 1,011 down-regulated, differed significantly between CPB2 toxin exposed cells and non-exposed cells (p < 0.05). KEGG pathway analysis results showed that m6A peaks up-regulated genes (n = 394) were mainly enriched in cancer, Cushing syndrome and Wnt signaling pathways, while m6A peaks down-regulated genes (n = 920) were mainly associated with apoptosis, small cell lung cancer, and the herpes simplex virus 1 infection signaling pathway. Furthermore, gene expression (RNA-seq data) analysis identified 1,636 differentially expressed genes (DEGs), of which 1,094 were up-regulated and 542 were down-regulated in the toxin exposed group compared with the control group. In addition, the down-regulated genes were involved in the Hippo and Wnt signaling pathways. Interestingly, the combined results of m6A-seq and RNA-seq identified genes with up-regulated m6A peaks but with down-regulated expression, here referred to as "hyper-down" genes (n = 18), which were mainly enriched in the Wnt signaling pathway. Therefore, we speculate that the genes in the Wnt signaling pathway may be modified by m6A methylation in CPB2-induced IPEC-J2 cells. These findings provide new insights enabling further exploration of the mechanisms underlying piglet diarrhea caused by CPB2 toxin.

Eugenol attenuates inflammatory response and enhances barrier function during lipopolysaccharide-induced inflammation in the porcine intestinal epithelial cells.

Eugenol (4-allyl-2-methoxyphenol) is an essential oil component, possessing antimicrobial, anti-inflammatory, and antioxidative properties; however, the effect of eugenol on porcine gut inflammation has not yet been investigated. In this study, an in vitro lipopolysaccharide (LPS)-induced inflammation model in porcine intestinal epithelial cells (IPEC-J2) has been set up. Cells were pretreated with 100 µM (16.42 mg/L) eugenol for 2 h followed by 10 µg/mL LPS stimulation for 6 h. Proinflammatory cytokine secretion; reactive oxygen species; gene expression of proinflammatory cytokines, tight junction proteins, and nutrient transporters; the expression and distribution of zonula occludens-1 (ZO-1); transepithelial electrical resistance (TEER); and cell permeability were measured to investigate the effect of eugenol on inflammatory responses and gut barrier function. The results showed that eugenol pretreatment significantly suppressed the LPS-stimulated interleukin-8 level and the mRNA abundance of tumor necrosis factor-α and restored the LPS-stimulated decrease of the mRNA abundance of tight junction proteins, such as ZO-1 and occludin, and the mRNA abundance of nutrient transporters, such as B0 1 system ASC sodium-dependent neutral amino acid exchanger 2, sodium-dependent glucose transporter 1, excitatory amino acid transporter 1, and peptide transporter 1. In addition, eugenol improved the expression and even redistribution of ZO-1 and tended to increase TEER value and maintained the barrier integrity. In conclusion, a low dose of eugenol attenuated inflammatory responses and enhanced selectively permeable barrier function during LPS-induced inflammation in the IPEC-J2 cell line.

Effect of berberine on LPS-induced expression of NF-κB/MAPK signalling pathway and related inflammatory cytokines in porcine intestinal epithelial cells.

Berberine is an alkaloid extracted from medicinal plants such as Coptis chinensis and Phellodendron chinense. It possesses anti-inflammatory, anti-tumour and anti-oxidation properties, and regulates Glc and lipid metabolism. This study explored the mechanisms of the protective effects of berberine on barrier function and inflammatory damage in porcine intestinal epithelial cells (IPEC-J2) induced by LPS. We first evaluated the effects of berberine and LPS on cell viability. IPEC-J2 cells were treated with 5 µg/ml LPS for 1 h to establish an inflammatory model, and 75, 150 and 250 µg/ml berberine were used in further experiments. The expression of IL-1ß, IL-6 and TNF-α was measured by RT-PCR. The key proteins of the NF-κB/MAPK signalling pathway (IκBα, p-IκBα, p65, p-p65, c-Jun N-terminal kinase (JNK), p-JNK, p38, p-p38, ERK1/2 and p-ERK1/2) were detected by Western blot. Upon exposure to LPS, IL-1ß, IL-6 and TNF-α mRNA levels and p-IκBα p-p65 protein levels were significantly enhanced. Pre-treatment with berberine reduced the expression of inflammatory factors and was positively correlated with its concentration, and dose dependently inhibited the expression of IκBα, p-IκBα, p-p65, p-p38 and JNK. These results demonstrated that pre-treating intestinal epithelial cells with berberine was useful in preventing and treating diarrhoea induced by Escherichia coli in weaned pigs.

Selection of Immunobiotic Ligilactobacillus salivarius Strains from the Intestinal Tract of Wakame-Fed Pigs: Functional and Genomic Studies.

In this article, Ligilactobacillus salivarius FFIG strains, isolated from the intestinal tract of wakame-fed pigs, are characterized according to their potential probiotic properties. Strains were evaluated by studying their interaction with porcine intestinal epithelial (PIE) cells in terms of their ability to regulate toll-like receptor (TLR)-3- or TLR4-mediated innate immune responses, as well as by assessing their adhesion capabilities to porcine epithelial cells and mucins. These functional studies were complemented with comparative genomic evaluations using the complete genome sequences of porcine L. salivarius strains selected from subgroups that demonstrated different "immune" and "adhesion" phenotypes. We found that their immunomodulatory and adhesion capabilities are a strain-dependent characteristic. Our analysis indicated that the differential immunomodulatory and adhesive activities of FFIG strains would be dependent on the combination of several surface structures acting simultaneously, which include peptidoglycan, exopolysaccharides, lipoteichoic acid, and adhesins. Of note, our results indicate that there is no correlation between the immunomodulatory capacity of the strains with their adhesion ability to mucins and epithelial cells. Therefore, in the selection of strains destined to colonize the intestinal mucosa and modulate the immunity of the host, both properties must be adequately evaluated. Interestingly, we showed that L. salivarius FFIG58 functionally modulated the innate immune responses triggered by TLR3 and TLR4 activation in PIE cells and efficiently adhered to these cells. Moreover, the FFIG58 strain was capable of reducing rotavirus replication in PIE cells. Therefore, L. salivarius FFIG58 is a good candidate for further in vivo studying the protective effect of lactobacilli against intestinal infections in the porcine host. We also reported and analyzed, for the first time, the complete genome of several L. salivarius strains that were isolated from the intestine of pigs after the selective pressure of feeding the animals with wakame. Further genomic analysis could be of value to reveal the metabolic characteristics and potential of the FFIG strains in general and of the FFIG58 strain, in particular, relating to wakame by-products assimilation.

Efficient Selection of New Immunobiotic Strains With Antiviral Effects in Local and Distal Mucosal Sites by Using Porcine Intestinal Epitheliocytes.

Previously, we evaluated the effect of the immunobiotic strain Lactobacillus rhamnosus CRL1505 on the transcriptomic response of porcine intestinal epithelial (PIE) cells triggered by the challenge with the Toll-like receptor 3 (TLR-3) agonist poly(I:C) and successfully identified a group of genes that can be used as prospective biomarkers for the screening of new antiviral immunobiotics. In this work, several strains of lactobacilli were evaluated according to their ability to modulate the expression of IFNα, IFNß, RIG1, TLR3, OAS1, RNASEL, MX2, A20, CXCL5, CCL4, IL-15, SELL, SELE, EPCAM, PTGS2, PTEGES, and PTGER4 in PIE cells after the stimulation with poly(I:C). Comparative analysis of transcripts variations revealed that one of the studied bacteria, Lactobacillus plantarum MPL16, clustered together with the CRL1505 strain, indicating a similar immunomodulatory potential. Two sets of in vivo experiments in Balb/c mice were performed to evaluate L. plantarum MPL16 immunomodulatory activities. Orally administered MPL16 prior intraperitoneal injection of poly(I:C) significantly reduced the levels of the proinflammatory mediators tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and IL-15 in the intestinal mucosa. In addition, orally administered L. plantarum MPL16 prior nasal stimulation with poly(I:C) or respiratory syncytial virus infection significantly decreased the levels of the biochemical markers of lung tissue damage. In addition, reduced levels of the proinflammatory mediators TNF-α, IL-6, and IL-8 were found in MPL16-treated mice. Improved levels of IFN-ß and IFN-γ in the respiratory mucosa were observed in mice treated with L. plantarum MPL16 when compared to control mice. The immunological changes induced by L. plantarum MPL16 were not different from those previously reported for the CRL1505 strain in in vitro and in vivo studies. The results of this work confirm that new immunobiotic strains with the ability of stimulating both local and distal antiviral immune responses can be efficiently selected by evaluating the expression of biomarkers in PIE cells.

Protective Effects of Fucoidan against Hydrogen Peroxide-Induced Oxidative Damage in Porcine Intestinal Epithelial Cells.

This study was conducted to evaluate the effectiveness of fucoidan in ameliorating hydrogen peroxide (H2O2)-induced oxidative stress to porcine intestinal epithelial cell line (IPEC-1). The cell viability test was initially performed to screen out appropriate concentrations of H2O2 and fucoidan. After that, cells were exposed to H2O2 in the presence or absence of pre-incubation with fucoidan. Hydrogen peroxide increased the apoptotic and necrotic rate, boosted reactive oxygen species (ROS) generation, and disturbed the transcriptional expression of genes associated with antioxidant defense and apoptosis in IPEC-1 cells. Pre-incubation with fucoidan inhibited the increases in necrosis and ROS accumulation induced by H2O2. Consistently, in the H2O2-treated IPEC-1 cells, fucoidan normalized the content of reduced glutathione as well as the mRNA abundance of NAD(P)H quinone dehydrogenase 1 and superoxide dismutase 1 while it prevented the overproduction of malondialdehyde. Moreover, H2O2 stimulated the translocation of nuclear factor-erythroid 2-related factor-2 to the nucleus of IPEC-1 cells, but this increase was further promoted by fucoidan pre-treatment. The results suggest that fucoidan is effective in protecting IPEC-1 cells against oxidative damage induced by H2O2, which may help in developing appropriate strategies for maintaining the intestinal health of young piglets.

Exopolysaccharides from Lactobacillus delbrueckii OLL1073R-1 modulate innate antiviral immune response in porcine intestinal epithelial cells.

Previous studies demonstrated that the extracellular polysaccharides (EPSs) produced by Lactobacillus delbrueckii OLL1073R-1 (LDR-1) improve antiviral immunity, especially in the systemic and respiratory compartments. However, it was not studied before whether those EPSs are able to beneficially modulate intestinal antiviral immunity. In addition, LDR-1-host interaction has been evaluated mainly with immune cells while its interaction with intestinal epithelial cells (IECs) was not addressed before. In this work, we investigated the capacity of EPSs from LDR-1 to modulate the response of porcine IECs (PIE cells) to the stimulation with the Toll-like receptor (TLR)-3 agonist poly(I:C) and the role of TLR2, TLR4, and TLR negative regulators in the immunoregulatory effect. We showed that innate immune response triggered by TLR3 activation in porcine IECs was differentially modulated by EPS from LDR-1. EPSs treatment induced an increment in the expression of interferon (IFN)-α and IFN-ß in PIE cells after the stimulation with poly(I:C) as well as the expression of the antiviral factors MxA and RNase L. Those effects were related to the reduced expression of A20 in EPS-treated PIE cells. EPS from LDR-1 was also able to reduce the expression of IL-6 and proinflammatory chemokines. Although further in vivo studies are needed, our results suggest that these EPSs or a yogurt fermented with LDR-1 have potential to improve intestinal innate antiviral response and protect against intestinal viruses.

Genomic Characterization of Lactobacillus delbrueckii TUA4408L and Evaluation of the Antiviral Activities of its Extracellular Polysaccharides in Porcine Intestinal Epithelial Cells.

In lactic acid bacteria, the synthesis of exopolysaccharides (EPS) has been associated with some favorable technological properties as well as health-promoting benefits. Research works have shown the potential of EPS produced by lactobacilli to differentially modulate immune responses. However, most studies were performed in immune cells and few works have concentrated in the immunomodulatory activities of EPS in non-immune cells such as intestinal epithelial cells. In addition, the cellular and molecular mechanisms involved in the immunoregulatory effects of EPS have not been studied in detail. In this work, we have performed a genomic characterization of Lactobacillus delbrueckii subsp. delbrueckii TUA4408L and evaluated the immunomodulatory and antiviral properties of its acidic (APS) and neutral (NPS) EPS in porcine intestinal epithelial (PIE) cells. Whole genome sequencing allowed the analysis of the general features of L. delbrueckii TUA4408L genome as well as the characterization of its EPS genes. A typical EPS gene cluster was found in the TUA4408L genome consisting in five highly conserved genes epsA-E, and a variable region, which includes the genes for the polymerase wzy, the flippase wzx, and seven glycosyltransferases. In addition, we demonstrated here for the first time that L. delbrueckii TUA4408L and its EPS are able to improve the resistance of PIE cells against rotavirus infection by reducing viral replication and regulating inflammatory response. Moreover, studies in PIE cells demonstrated that the TUA4408L strain and its EPS differentially modulate the antiviral innate immune response triggered by the activation of Toll-like receptor 3 (TLR3). L. delbrueckii TUA4408L and its EPS are capable of increasing the activation of interferon regulatory factor (IRF)-3 and nuclear factor κB (NF-κB) signaling pathways leading to an improved expression of the antiviral factors interferon (IFN)-ß, Myxovirus resistance gene A (MxA) and RNaseL.