Pig Intestinal Epithelial Cells Research Articles

Porcine epidemic diarrhea virus causes diarrhea by activating EGFR to regulates NHE3 activity and mobility on plasma membrane.

As part of the genus Enteropathogenic Coronaviruses, Porcine Epidemic Diarrhea Virus (PEDV) is an important cause of early diarrhea and death in piglets, and one of the most difficult swine diseases to prevent and control in the pig industry. Previously, we found that PEDV can block Na+ absorption and induce diarrhea in piglets by inhibiting the activity of the sodium-hydrogen ion transporter NHE3 in pig intestinal epithelial cells, but the mechanism needs to be further explored. The epidermal growth factor receptor (EGFR) has been proved to be one of the co-receptors involved in many viral infections and a key protein involved in the regulation of NHE3 activity in response to various pathological stimuli. Based on this, our study used porcine intestinal epithelial cells (IPEC-J2) as an infection model to investigate the role of EGFR in regulating NHE3 activity after PEDV infection. The results showed that EGFR mediated viral invasion by interacting with PEDV S1, and activated EGFR regulated the downstream EGFR/ERK signaling pathway, resulting in decreased expression of NHE3 and reduced NHE3 mobility at the plasma membrane, which ultimately led to decreased NHE3 activity. The low level of NHE3 expression in intestinal epithelial cells may be a key factor leading to PEDV-induced diarrhea in newborn piglets. This study reveals the importance of EGFR in the regulation of NHE3 activity by PEDV and provides new targets and clues for the prevention and treatment of PEDV-induced diarrhea in piglets.

Porcine Circovirus 2 Increases the Frequency of Transforming Growth Factor-β 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-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.

Thioredoxin A of Streptococcus suis Serotype 2 Contributes to Virulence by Inhibiting the Expression of Pentraxin 3 to Promote Survival Within Macrophages.

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that can infect humans in contact with infected pigs or their byproducts. It can employ different types of genes to defend against oxidative stress and ensure its survival. The thioredoxin (Trx) system is a key antioxidant system that contributes adversity adaptation and pathogenicity. SS2 has been shown to encode putative thioredoxin genes, but the biological roles, coding sequence, and underlying mechanisms remains uncharacterized. Here, we demonstrated that SSU05_0237-ORF, from a clinical SS2 strain, ZJ081101, encodes a protein of 104 amino acids with a canonical CGPC active motif and an identity 70-85% similar to the thioredoxin A (TrxA) in other microorganisms. Recombinant TrxA efficiently catalyzed the thiol-disulfide oxidoreduction of insulin. The deletion of TrxA led to a significantly slow growth and markedly compromised tolerance of the pathogen to temperature stress, as well as impaired adhesion ability to pig intestinal epithelial cells (IPEC-J2). However, it was not involved in H2O2 and paraquat-induced oxidative stress. Compared with the wild-type strain, the ΔTrxA strain was more susceptible to killing by macrophages through increasing NO production. Treatment with TrxA mutant strain also significantly attenuated cytotoxic effects on RAW 264.7 cells by inhibiting inflammatory response and apoptosis. Knockdown of pentraxin 3 in RAW 264.7 cells was more vulnerable to phagocytic activity, and TrxA promoted SS2 survival in phagocytic cells depending on pentraxin 3 activity compared with the wild-type strain. Moreover, a co-inoculation experiment in mice revealed that TrxA mutant strain is far more easily cleared from the body than the wild type strain in the period from 8-24h, and exhibits significantly attenuated oxidative stress and liver injury. In summary, we reveal the important role of TrxA in the pathogenesis of SS2.

Efficacy of TurmiZn, a Metallic Complex of Curcuminoids-Tetrahydrocurcumin and Zinc on Bioavailability, Antioxidant, and Cytokine Modulation Capability

Complexes of curcumin with metals have shown much-improved stability, solubility, antioxidant capability, and efficacy when compared to curcumin. The present research investigates the relative bioavailability, antioxidant, and ability to inhibit inflammatory cytokine production of a curcuminoid metal chelation complex of tetrahydrocurcumin-zinc-curcuminoid termed TurmiZn. In vitro uptake assay using pig intestinal epithelial cells showed that TurmiZn has an ~3-fold increase (p ≤ 0.01) in uptake compared to curcumin and a ~2-fold increase (p ≤ 0.01) over tetrahydrocurcumin (THC). In a chicken model, an oral 1-g dose of TurmiZn showed a ~2.5-fold increase of a specific metabolite peak compared to curcumin (p = 0.004) and a ~3-fold increase compared to THC (p = 0.001). Oral doses (5 g/Kg) of TurmiZn in rats also showed the presence of curcumin and THC metabolites in plasma, indicating bioavailability across cell membranes in animals. Determination of the antioxidant activity by a 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging assay indicated that TurmiZn was about 13x better (p ≤ 0.0001) than curcumin and about 4X better (p ≤ 0.0001) than THC, in reducing free radicals. In vitro experiments further showed significant (p ≤ 0.01) reductions of lipopolysaccharide (LPS)-induced proinflammatory cytokines such as interleukin (IL) IL-6, IL-8, IL-15, IL-18, and tumor necrosis factor (TNF)-alpha, while showing a significant (p ≤ 0.01) increase of granulocyte-macrophage colony-stimulating factor (GM-CSF) in dog kidney cells. In vivo cytokine modulations were also observed when TurmiZn was fed for 6 weeks to newborn chickens. TurmiZn reduced IL-1 and IL-6, but significantly reduced (p ≤ 0.01) IL-10 levels while there was a concurrent significant (p = 0.02) increase in interferon gamma compared to controls. Overall, these results indicate that TurmiZn has better bioavailability and antioxidant capability than curcumin or THC and has the ability to significantly modulate cytokine levels. Thus, TurmiZn could be an excellent candidate for a novel ingredient that can be incorporated into food and supplements to help overall health during the aging process.

Antiviral effects of Pediococcus acidilactici isolated from Tibetan mushroom and comparative genomic analysis.

Rotavirus is one of the main pathogens that cause diarrhoea in young animals, and countless animals have died of rotavirus infection worldwide. Three strains of lactic acid bacteria isolated from Tibetan mushrooms were used to study the inhibition of rotavirus in vitro and in vivo. One part was to identify and study the biochemical and probiotic characteristics of three isolated lactic acid bacteria, and the other part was to evaluate the inhibitory effect on rotavirus via in vivo and in vitro experiments. The whole genome of the lactic acid bacteria with the best antiviral effect was sequenced, and the differences between them and the standard strains were analyzed by comparative genomic analysis, so as to provide a theoretical basis for exploring the antiviral effect of lactic acid bacteria.The three strains were identified as Pediococcus acidilactici, Lactobacillus casei and Lactobacillus paracasei. Pediococcus acidilactici showed good acid tolerance, bile salt tolerance, survival in artificial intestinal fluid, survival in gastric fluid and bacteriostasis. In in vitro experiments, pig intestinal epithelial cells cocultured with Pediococcus acidilactici exhibited reduced viral infection. In the in vivo experiment, the duodenum of mice fed Pediococcus acidilactici had extremely low numbers of virus particles. The total genome size was 2,026,809 bp, the total number of genes was 1988, and the total length of genes was 1,767,273 bp. The proportion of glycoside hydrolases and glycoside transferases in CAZy was 50.6 and 29.6%, respectively. The Metabolism function in KEEG had the highest number of Global and overview maps. Among the comparative genomes, Pediococcus acidilactici had the highest homology with GCF 000146325.1, and had a good collinearity with GCF 013127755.1, without numerous gene rearrangement events such as insertion, deletion, inversion and translocation. In conclusion, Pediococcus acidilactici was a good candidate strain for antiviral probiotics.

A Novel Probiotic Bacillus subtilis Strain Confers Cytoprotection to Host Pig Intestinal Epithelial Cells during Enterotoxic Escherichia coli Infection.

ABSTRACTEnteric infections caused by enterotoxic Escherichia coli (ETEC) negatively impact the growth performance of piglets during weaning, resulting in significant economic losses for the producers. With the ban on antibiotic usage in livestock production, probiotics have gained a lot of attention as a potential alternative. However, strain specificity and limited knowledge on the host-specific targets limit their efficacy in preventing ETEC-related postweaning enteric infections. We recently isolated and characterized a novel probiotic Bacillus subtilis bacterium (CP9) that demonstrated antimicrobial activity. Here, we report anti-ETEC properties of CP9 and its impact on metabolic activity of swine intestinal epithelial (IPEC-J2) cells. Our results showed that pre- or coincubation with CP9 protected IPEC-J2 cells from ETEC-induced cytotoxicity. CP9 significantly attenuated ETEC-induced inflammatory response by reducing ETEC-induced nitric oxide production and relative mRNA expression of the Toll-like receptors (TLRs; TLR2, TLR4, and TLR9), proinflammatory tumor necrosis factor alpha, interleukins (ILs; IL-6 and IL-8), augmenting anti-inflammatory granulocyte-macrophage colony-stimulating factor and host defense peptide mucin 1 (MUC1) mRNA levels. We also show that CP9 significantly (P < 0.05) reduced caspase-3 activity, reinstated cell proliferation and increased relative expression of tight junction genes, claudin-1, occludin, and zona occludens-1 in ETEC-infected cells. Finally, metabolomic analysis revealed that CP9 exposure induced metabolic modulation in IPEC J2 cells with the greatest impact seen in alanine, aspartate, and glutamate metabolism; pyrimidine metabolism; nicotinate and nicotinamide metabolism; glutathione metabolism; the citrate cycle (TCA cycle); and arginine and proline metabolism. Our study shows that CP9 incubation attenuated ETEC-induced cytotoxicity in IPEC-J2 cells and offers insight into potential application of this probiotic for ETEC infection control.IMPORTANCE ETEC remains one of the leading causes of postweaning diarrhea and mortality in swine production. Due to the rising concerns with the antibiotic use in livestock, alternative interventions need to be developed. In this study, we analyzed the cytoprotective effect of a novel probiotic strain in combating ETEC infection in swine intestinal cells, along with assessing its mechanism of action. To our knowledge, this is also the first study to analyze the metabolic impact of a probiotic on intestinal cells. Results from this study should provide effective cues in developing a probiotic intervention for ameliorating ETEC infection and improving overall gut health in swine production.

Genome-wide transcriptome analysis of porcine epidemic diarrhea virus virulent or avirulent strain-infected porcine small intestinal epithelial cells.

Porcine epidemic diarrhea virus (PEDV) is the main cause of diarrhea, vomiting, and mortality in pigs, which results in devastating economic loss to the pig industry around the globe. In recent years, the advent of RNA-sequencing technologies has led to delineate host responses at late stages of PEDV infection; however, the comparative analysis of host responses to early-stage infection of virulent and avirulent PEDV strains is currently unknown. Here, using the BGI DNBSEQ RNA-sequencing, we performed global gene expression profiles of pig intestinal epithelial cells infected with virulent (GDS01) or avirulent (HX) PEDV strains for 3, 6, and 12 ​h. It was observed that over half of all significantly dysregulated genes in both infection groups exhibited a down-regulated expression pattern. Functional enrichment analyses indicated that the differentially expressed genes (DEGs) in the GDS01 group were predominantly related to autophagy and apoptosis, whereas the genes showing the differential expression in the HX group were strongly enriched in immune responses/inflammation. Among the DEGs, the functional association of TLR3 and IFIT2 genes with the HX and GDS01 strains replication was experimentally validated by TLR3 inhibition and IFIT2 overexpression systems in cultured cells. TLR3 expression was found to inhibit HX strain, but not GDS01 strain, replication by enhancing the IFIT2 expression in infected cells. In conclusion, our study highlights similarities and differences in gene expression patterns and cellular processes/pathways altered at the early-stage infection of PEDV virulent and avirulent strains. These findings may provide a foundation for establishing novel therapies to control PEDV infection.

Mitochondrial and glycolytic extracellular flux analysis optimization for isolated pig intestinal epithelial cells

Intestinal epithelial cells (IECs) are crucial to maintain intestinal function and the barrier against the outside world. To support their function they rely on energy production, and failure to produce enough energy can lead to IEC malfunction and thus decrease intestinal barrier function. However, IEC metabolic function is not often used as an outcome parameter in intervention studies, perhaps because of the lack of available methods. We therefore developed a method to isolate viable IECs, suitable to faithfully measure their metabolic function by determining extracellular glycolytic and mitochondrial flux. First, various methods were assessed to obtain viable IECs. We then adapted a previously in-house generated image-analysis algorithm to quantify the amount of seeded IECs. Correcting basal respiration data of a group of piglets using this algorithm reduced the variation, showing that this algorithm allows for more accurate analysis of metabolic function. We found that delay in metabolic analysis after IEC isolation decreases their metabolic function and should therefore be prevented. The presence of antibiotics during isolation and metabolic assessment also decreased the metabolic function of IECs. Finally, we found that primary pig IECs did not respond to Oligomycin, a drug that inhibits complex V of the electron transport chain, which may be because of the presence of drug exporters. A method was established to faithfully measure extracellular glycolytic and mitochondrial flux of pig primary IECs. This tool is suitable to gain a better understanding of how interventions affect IEC metabolic function.

Potential Probiotic Bacillus subtilis Isolated from a Novel Niche Exhibits Broad Range Antibacterial Activity and Causes Virulence and Metabolic Dysregulation in Enterotoxic E. coli.

Microbial life in extreme environments, such as deserts and deep oceans, is thought to have evolved to overcome constraints of nutrient availability, temperature, and suboptimal hygiene environments. Isolation of probiotic bacteria from such niche may provide a competitive edge over traditional probiotics. Here, we tested the survival, safety, and antimicrobial effect of a recently isolated and potential novel strain of Bacillus subtilis (CP9) from desert camel in vitro. Antimicrobial assays were performed via radial diffusion, agar spot, and co-culture assays. Cytotoxic analysis was performed using pig intestinal epithelial cells (IPEC-J2). Real time-PCR was performed for studying the effect on ETEC virulence genes and metabolomic analysis was performed using LC-MS. The results showed that CP9 cells were viable in varied bile salts and in low pH environments. CP9 showed no apparent cytotoxicity in IPEC-J2 cells. CP9 displayed significant bactericidal effect against Enterotoxic E. coli (ETEC), Salmonella Typhimurium, and Methicillin-resistant Staphylococcus aureus (MRSA) in a contact inhibitory fashion. CP9 reduced the expression of ETEC virulent genes during a 5 h co-culture. Additionally, a unique emergent metabolic signature in co-culture samples was observed by LC-MS analysis. Our findings indicate that CP9 exhibits a strong antibacterial property and reveals potential mechanisms behind.

Comparative Transcriptome Profiling of Human and Pig Intestinal Epithelial Cells after Porcine Deltacoronavirus Infection.

Porcine deltacoronavirus (PDCoV) is an emerging infectious disease of swine with zoonotic potential. Phylogenetic analysis suggests that PDCoV originated recently from a host-switching event between birds and mammals. Little is known about how PDCoV interacts with its differing hosts. Human-derived cell lines are susceptible to PDCoV infection. Herein, we compare the gene expression profiles of an established host swine cells to potential emerging host human cells after infection with PDCoV. Cell lines derived from intestinal lineages were used to reproduce the primary sites of viral infection in the host. Porcine intestinal epithelial cells (IPEC-J2) and human intestinal epithelial cells (HIEC) were infected with PDCoV. RNA-sequencing was performed on total RNA extracted from infected cells. Human cells exhibited a more pronounced response to PDCoV infection in comparison to porcine cells with more differentially expressed genes (DEGs) in human, 7486, in comparison to pig cells, 1134. On the transcriptional level, the adoptive host human cells exhibited more DEGs in response to PDCoV infection in comparison to the primary pig host cells, where different types of cytokines can control PDCoV replication and virus production. Key immune-associated DEGs and signaling pathways are shared between human and pig cells during PDCoV infection. These included genes related to the NF-kappa-B transcription factor family, the interferon (IFN) family, the protein-kinase family, and signaling pathways such as the apoptosis signaling pathway, JAK-STAT signaling pathway, inflammation/cytokine–cytokine receptor signaling pathway. MAP4K4 was unique in up-regulated DEGs in humans in the apoptosis signaling pathway. While similarities exist between human and pig cells in many pathways, our research suggests that the adaptation of PDCoV to the porcine host required the ability to down-regulate many response pathways including the interferon pathway. Our findings provide an important foundation that contributes to an understanding of the mechanisms of PDCoV infection across different hosts. To our knowledge, this is the first report of transcriptome analysis of human cells infected by PDCoV.

Porcine epidemic diarrhea virus infection blocks cell cycle and induces apoptosis in pig intestinal epithelial cells

Porcine epidemic diarrhea virus (PEDV) is responsible for the acute infectious swine disease porcine epidemic diarrhea (PED). PED causes damage to the intestine, including villus atrophy and shedding, leading to serious economic losses to the pig industry worldwide. We carried out an in vitro study to investigate cell apoptosis and the cell cycle in a PEDV-infected host using transcriptomic shotgun sequencing (RNA-Seq) to study gene responses to PEDV infection. Results revealed that the PEDV infection reduced proliferation activity, blocked the cell cycle at S-phase and induced apoptosis in IPEC-J2 cells. The expression of gene levels related to ribosome proteins and oxidative phosphorylation were significantly up-regulated post-PEDV infection. Although the significantly down-regulated on PI3K/Akt signaling pathway post-PEDV infection, the regulator–related genes of mTOR signaling pathway exerted significantly up-regulated or down-regulated in IPEC-J2 cells. These results indicated that ribosome proteins and oxidative phosphorylation process were widely involved in the pathological changes and regulation of host cells caused by PEDV infection, and PI3K/AKT and mTOR signaling pathways played a vital role in antiviral regulation in IPEC-J2 cells. These data might provide new insights into the specific pathogenesis of PEDV infection and pave the way for the development of effective therapeutic strategies.

Aggregation, adhesion and efficacy studies of probiotic candidate Pediococcus acidilactici NCDC 252: a strain of dairy origin.

Aggregation and adhesion capability and survival efficacy of candidate probiotic strain Pediococcus acidilactici NCDC 252 under simulated gastric, intestinal and vaginal conditions was studied. The strain exhibited strong autoaggregation phenotype and coaggregation with other Lactic acid bacteria (LAB) and E. coli. The adhesion studies of NCDC 252 to pig's intestinal epithelial cells showed its adhesive ability. Aggregation and adhesiveness were related through cell surface proteins as removal/extraction of surface proteins resulted in altered aggregation and no adhesiveness. Cell surface proteins were analysed by SDS-PAGE and also in silico analysed from its genome. SDS-PAGE analysis of cell surface proteins of NCDC 252 revealed two potential proteins of approximately 74.3 and 53.6kDa to be involved in host-probiotic interaction. Removal of cell surface proteins by LiCl-treatment (5moll-1) resulted in loss of aggregation and adhesiveness. Further survival of NCDC 252 under simulated gastrointestinal and vaginal conditions in terms of high viable counts confirmed its efficacy for its survival under gut and urogenital conditions. These observations suggest that it can be used further in functional foods, nutraceuticals and in combating urogenital infections. As NCDC 252 was able to survive in intestinal conditions, interaction of its cell surface proteins with intestinal mucins was studied in silico by docking. Highest affinity of adhesion was observed for MUC3B. In conclucion, NCDC 252, exhibited aggregation phenotype and adhesion capability. Survivability of NCDC 252 under simulated conditions and its interaction with human mucins confirms its efficacy to be used as probiotic.

Evaluation of the protective effects of zinc butyrate in IPEC-J2 cells and grower pigs under heat stress.

Heat stress (HS) is a major environmental stressor primarily affecting swine performance through negative effects on intestinal health. Zinc and butyric acid supplementation help maintain intestinal integrity and barrier function, and has been shown to be beneficial to swine during stress conditions. We tested a novel formulation of zinc butyrate (ZnB) to study whether it has protective effects toward swine using pig intestinal epithelial cells (IPEC-J2) and in a grower swine HS trial. IPEC-J2 cells were grown either under an inflammatory challenge (Escherichia coli lipopolysaccharide) or HS (41.5 °C for 48 h) using Transwell plates. The tight junction integrity of the cells under various treatments, including ZnB, zinc sulfate, and calcium butyrate, was followed over a period of 36 to 48 h by measuring transepithelial electrical resistance (TER). During inflammatory challenge, ZnB-treated cells had the greatest TER (P < 0.05) at 36 h. When the cells were exposed to HS at 41.5 °C, ZnB-treated cells had similar TER to the cells incubated at 37.0 °C, indicating significant protection against HS. In the swine trial (two dietary treatments, control and an encapsulated form of 40% zinc butyrate [E-ZnB] in hydrogenated palm oil pearls, 12 pigs per treatment), grower gilts (35 ± 1 kg) were supplemented with E-ZnB for 24 d before being subjected to biphasic HS for 7 d, 30 to 32 °C for 8 h and 28 °C for 16 h, for a total duration of 56 h of HS. At the end of the HS phase, half the pigs were euthanized from each treatment (n = 6 per treatment), and growth performance was calculated. During the HS phase, average daily gain (ADG; 0.53 vs. 0.79 kg) and gain-to-feed ratio (G:F; 0.33 vs. 0.43) were greater in the E-ZnB group (P < 0.05). Although in vivo intestinal permeability increased during the HS phase (P < 0.05), no differences were observed in the present study for the intestinal health parameters measured including TER, villus height:crypt depth ratio, and in vivo and ex vivo intestinal permeability between the two treatment groups. In conclusion, results presented here demonstrate that E-ZnB supplementation during HS improves ADG and G:F in grower pigs. Although we could not measure any differences, the mode of action of butyric acid and zinc suggests that the performance improvements are related to improved intestinal health.

Efficient Production of Recombinant Protegrin-1 From Pichia pastoris, and Its Antimicrobial and in vitro Cell Migration Activity.

Protegrin (PG) belongs to the antimicrobial peptide cathelicidin family. To date, five protegrin sequences have been identified in pigs, PG-1 to PG-5. Of these, PG-1 exhibits potent antimicrobial activity against a broad range of antibiotic-resistant microorganisms as well as viruses. However, the other potential role(s) of PG beyond antimicrobial has largely been unexplored. The aim of this study was to use nonpathogenic yeast Pichia pastoris to express antimicrobially active recombinant protegrin (rPG-1). Additionally, the effect of PG-1 on cell migration and proliferation was also examined in vitro using pig intestinal epithelial cells as a model. Highest level of rPG-1 (104 ± 11 μg/mL) was detected at 24 h in fermentation culture medium. Similar to rPG-1, 0.8 ± 0.10 g/L of proform PG-1 (rProPG-1) and 0.2 ± 0.02 g/L of the PG-1 cathelin domain (rCath) was detected in fermentation culture medium. Resulting recombinant PG-1 and cleaved rProPG-1 exerted antimicrobial activity against Escherichia coli DH5α at the same level as chemically synthesized PG-1. Enhanced cell migration was observed (p < 0.05) in groups treated with rProPG-1, rCath, and rPG-1 compared to the control. Furthermore, rPG-1 was stable at temperatures ranging from 25°C to 80°C. In summary, biologically active recombinant protegrin in its pro-, cathelin-, and mature- forms were successfully expressed in P. pastoris suggesting potential feasibility for future therapeutic applications.

LGR5 and BMI1 Increase Pig Intestinal Epithelial Cell Proliferation by Stimulating WNT/β-Catenin Signaling.

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) and B-cell-specific Moloney murine leukemia virus insertion site 1 (BMI1) are markers of fast-cycling and quiescent intestinal stem cells, respectively. To determine the functions of these proteins in large animals, we investigated their effects on the proliferation of intestinal epithelial cells from pigs. Our results indicated that LGR5 and BMI1 are highly conserved proteins and that the pig proteins have greater homology with the human proteins than do mouse proteins. Overexpression of either LGR5 or BMI1 promoted cell proliferation and WNT/β-catenin signaling in pig intestinal epithelial cells (IPEC-J2). Moreover, the activation of WNT/β-catenin signaling by recombinant human WNT3A protein increased cell proliferation and LGR5 and BMI1 protein levels. Conversely, inhibition of WNT/β-catenin signaling using XAV939 reduced cell proliferation and LGR5 and BMI1 protein levels. This is the first report that LGR5 and BMI1 can increase proliferation of pig intestinal epithelial cells by activating WNT/β-catenin signaling.

CDX2 increases SLC7A7 expression and proliferation of pig intestinal epithelial cells.

Nutrient absorption mediated by nutrient transporters expressed in the intestinal epithelium supplies substrates to support intestinal processes, including epithelial cell proliferation. We evaluated the role of Caudal type homeobox 2 (CDX2), an intestine-specific transcription factor, in the proliferation of pig intestinal epithelial cells (IPEC-1) and searched for novel intestinal nutrient transporter genes activated by CDX2. Our cloned pig CDX2 cDNA contains a “homeobox” DNA binding motif, suggesting it is a transcriptional activator. CDX2 overexpression in IPEC-1 cells increased cell proliferation, the percentage of cells in S/G2 phase, and the abundance of transcripts of the cell cycle-related genes Cyclin A2; Cyclin B; Cyclin D2; proliferating cell nuclear antigen; and cell cycle cyclin-dependent kinases 1, 2 and 4, as well as the predicted CDX2 target genes SLC1A1, SLC5A1 and SLC7A7. In addition, luciferase reporter and chromatin immunoprecipitation assays revealed that CDX2 binds directly to the SLC7A7 promoter. This is the first report of CDX2 function in pig intestinal epithelial cells and identifies SLC7A7 as a novel CDX2 target gene. Our findings show that nutrient transporters are activated during CDX2-induced proliferation of normal intestinal epithelial cells.

L-Glutamate deficiency can trigger proliferation inhibition via down regulation of the mTOR/S6K1 pathway in pig intestinal epithelial cells.

The objective of this study was to investigate the effects of L-glutamate (Glu) deficiency or L-trans pyrrolidine-2,4-dicarboxylic acid (PDC) supplementation on the proliferation of pig intestinal epithelial cells (IPEC-1). First, IPEC-1 cells were cultured in normal growing medium supplemented with 0 (Control), 50, 100, or 200 µmol/L PDC to determine an appropriate concentration of PDC supplementation. Second, IPEC-1 cells were cultured in Glu-deficient medium supplemented with 0 µmol/L Glu (Glu deficiency), 50 µmol/L Glu (Control), or 50 µmol/L Glu plus 100 µmol/L PDC (PDC supplementation). Cell proliferation ( = 24), cell cycle distribution ( = 6), cell apoptosis ( = 6), and expression levels of proteins of interest ( = 4) were determined by MTT assay, flow cytometry, or western blot. The results showed that cell proliferation was inhibited ( < 0.05) by 50, 100, and 200 µmol/L PDC supplementation at 24 and 48 h after treatment. Variance analysis was performed using the GLM procedure, and the results demonstrated that Glu deficiency or PDC supplementation led to the inhibition ( < 0.05) of cell proliferation, a greater ( < 0.05) percentage of cells in the G1 phase, and a lower ( < 0.05) percentage of cells in the S phase. Moreover, Glu deficiency or PDC supplementation reduced ( < 0.05) the expression levels of excitatory AA transporter 3 (EAAT3), phosphor-mammalian target of rapamycin (p-mTOR; Ser2448), p-ribosomal protein S6 kinase 1 (S6K1; Thr389), and p-S6 (Ser235/236). This study demonstrates that Glu deficiency or PDC supplementation inhibits proliferation of IPEC-1 cells via downregulation of the mTOR/S6K1 pathway and EAAT3 expression indicating that Glu deficiency may lead to the disturbances of intestinal epithelial renewal in pigs, particularly in neonates.

Evaluation of preparation methods for MS-based analysis of intestinal epithelial cell proteomes.

The gut epithelium formed between an organism and the environment plays an essential role in host-microbe interactions, yet remains one of the least characterized mammalian tissues. Especially the membrane proteins, which are critical to bacterial adhesion, are understudied, because these proteins are low in abundance, and large amounts of sample is needed for their preparation and for undertaking MS-based analysis. The aim of this study was to evaluate three different methods for isolation and preparation of pig intestinal epithelial cells for MS-based analysis of the proteome. Samples were analyzed by LC and electrospray QTOF-MS. The methods were evaluated according to efficiency, purity, transmembrane protein recovery, as well as for suitability to large-scale preparations. Our data clearly demonstrate that mucosal shaving is by far the best-suited method for in-depth MS analysis in terms of ease and speed of sample preparation, as well as protein recovery. In comparison, more gentle methods where intestinal epithelial cells are harvested by shaking are more time consuming, result in lower protein yield, and are prone to increased technical variation due to multiple steps involved.

Peptidoglycan Recognition Proteins Are Differentially Regulated by Time and Long‐Chain Polyunsaturated Fatty Acids In Neonatal Pig Intestinal Epithelial Cells, IPECJ2

Peptidoglycan recognition proteins (PGlyRP) are a family of highly conserved pattern recognition receptors of the innate immune system. PGlyRP1, 3, and 4 have direct bactericidal activity, while PGlyRP2 is bound and secreted from cells and hydrolyzes peptidoglycan through amidase activity. PGlyRP3 is expressed in human colon‐like CaCO‐2 cells and is known to enhance anti‐inflammatory responses post‐bacterial challenge. This study hypothesized that PGlyRP expression in IPECJ2 cells would be regulated by time post‐confluence and by long chain polyunsaturated fatty acid (LC‐PUFA) treatment. IPECJ2 cells were grown to confluence and mRNA abundance was measured by RT‐PCR. PGlyRP 2 and 4 were expressed in IPECJ2 cells and differentially regulated over time (P &lt; 0.001). PGlyRP2 mRNA abundance decreased over 16d (P &lt; 0.001), whereas PGlyRP4 mRNA abundance increased over time. In addition, LC‐PUFA (n‐3 and n‐6) dose‐dependently increased expression of PGlyRP2 mRNA by 100‐175% within 24h compared to vehicle‐treated cells (P&lt; 0.05). PGlyRP4 mRNA increased 65 % when treated with 100 μM eicosapentaenoic acid (n‐3) compared to vehicle treated cells (P&lt;0.05). In conclusion, IPECJ2 cells express PGlyRP2 and 4, have differential patterns of expression over time post‐confluence and are acutely regulated by LC‐PUFA. These data implicate a potential mechanism for dietary LC‐PUFA to modulate intestinal health by altering mucosal signaling mechanisms between luminal microbiotia, enterocytes and the mucosal immune system. Funded by Undergraduate Research Grant.

Ferrous bisglycinate increased iron transportation through DMT1 and PepT1 in pig intestinal epithelial cells compared with ferrous sulphate

Ferrous bisglycinate increased iron transportation through DMT1 and PepT1 in pig intestinal epithelial cells compared with ferrous sulphate