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.

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/β-catenin signaling pathway. In addition, Gln increased not only cytosolic β-catenin but also nuclear β-catenin protein expression. LF3 (a β-catenin/TCF4 interaction inhibitor) assay and β-catenin knockdown demonstrated that Gln-mediated promotion of Wnt/β-catenin signaling and cell proliferation were blocked. Furthermore, the inhibition of TCF4 expression suppressed Gln-induced cell proliferation. These findings further confirmed that Wnt/β-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/β-catenin pathway. These data greatly enhance the current understanding of the mechanism by which Gln regulates intestinal development.

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-β 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.

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.

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.

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-β (TGF-β), 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-β 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-β 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.

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.

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-β 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-β, 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-β, 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.

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-1β (pro-IL-1β) 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-1β 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-1β release and pyroptosis is dependent on NLRP3 inflammasome, thus NLRP3 inflammasome may play a central role in the innate immune response to TGEV infection.

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.