Correction: Trolox and Ascorbic Acid Reduce Direct and Indirect Oxidative Stress in the IPEC-J2 Cells, an In Vitro Model for the Porcine Gastrointestinal Tract.
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ABSTRACT:
SUBMITTER: PLOS ONE Staff
PROVIDER: S-EPMC4416923 | biostudies-literature | 2015
REPOSITORIES: biostudies-literature
Publications
Correction: Trolox and Ascorbic Acid Reduce Direct and Indirect Oxidative Stress in the IPEC-J2 Cells, an In Vitro Model for the Porcine Gastrointestinal Tract.
PloS one 20150501 5
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Project description:BackgroundPorcine circovirus-associated disease (PCVAD) is caused by a small pathogenic DNA virus, Porcine circovirus type 2 (PCV2), and is responsible for severe economic losses. PCV2-associated enteritis appears to be a distinct clinical manifestation of PCV2. Most studies of swine enteritis have been performed in animal infection models, but none have been conducted in vitro using cell lines of porcine intestinal origin. An in vitro system would be particularly useful for investigating microfilaments, which are likely to be involved in every stage of the viral lifecycle.MethodsWe confirmed that PCV2 infects the intestinal porcine epithelial cell line IPEC-J2 by means of indirect immunofluorescence, transmission electron microscopy, flow cytometry and qRT-PCR. PCV2 influence on microfilaments in IPEC-J2 cells was detected by fluorescence microscopy and flow cytometry. We used Cytochalasin D or Cucurbitacin E to reorganize microfilaments, and observed changes in PCV2 invasion, replication and release in IPEC-J2 cells by qRT-PCR.ResultsPCV2 infection changes the ultrastructure of IPEC-J2 cells. PCV2 copy number in IPEC-J2 cells shows a rising trend as infection proceeds. Microfilaments are polymerized at 1 h p.i., but densely packed actin stress fibres are disrupted and total F-actin increases at 24, 48 and 72 h p.i. After Cytochalasin D treatment, invasion of PCV2 is suppressed, while invasion is facilitated by Cucurbitacin E. The microfilament drugs have opposite effects on viral release.ConclusionPCV2 infects and proliferates in IPEC-J2 cells, demonstrating that IPEC-J2 cells can serve as a cell intestinal infection model for PCV2 pathogenesis. Furthermore, PCV2 rearranges IPEC-J2 microfilaments and increases the quantity of F-actin. Actin polymerization may facilitate the invasion of PCV2 in IPEC-J2 cells and the dissolution of cortical actin may promote PCV2 egress.
| S-EPMC4237784 | biostudies-literature
Project description:Rotavirus infections in nursing or post-weaning piglets are known to cause diarrhea, which can lead to commercial losses. Probiotic supplementation is used as a prophylactic or therapeutic approach to dealing with microbial infections in humans and animals. To evaluate the effect of probiotic bacteria on porcine rotavirus infections, non-transformed porcine intestinal epithelial IPEC-J2 cells were used as an in vitro model, and three different procedures were tested. When cells were exposed to seven probiotics at concentrations of 105, 106, or 107 CFU/mL for 16 h and removed before rotavirus challenge, infection reduction rates determined by flow cytometry were as follows: 15% (106) and 18% (105) for Bifidobacterium longum R0175, 15% (107) and 16% (106) for B. animalis lactis A026, and 15% (105) for Lactobacillus plantarum 299V. When cells were exposed to three selected probiotic strains for 1 h at higher concentrations, that is, 108 and 5 × 108 CFU/mL, before infection with rotavirus, no significant reduction was observed. When the probiotic bacteria were incubated with the virus before cell infection, a significant 14% decrease in the infection rate was observed for B. longum R0175. The results obtained using a cell-probiotics-virus platform combined with flow cytometry analysis suggest that probiotic bacteria can have a protective effect on IPEC-J2 cells before infection and can also prevent rotavirus infection of the cells.
| S-EPMC9402510 | biostudies-literature
Project description:The porcine small intestinal epithelial cell line, IPEC-J2, is useful to characterize the interactions of enterocytes with enteric viruses in vitro. We investigated whether IPEC-J2 cells are susceptible to porcine deltacoronavirus (PDCoV) infection. We conducted quantification of infectious virus or viral RNA, immunofluorescent (IF) staining for the detection of PDCoV antigens, and TUNEL assay in IPEC-J2 cells inoculated with the strain OH-FD22-P8 grown in LLC-PK cells, and supplemented with 10 μg/ml of trypsin in the cell culture medium. Cytopathic effects (CPE) that consisted of enlarged and rounded cells followed by cell shrinkage and detachment, were identified by the 3rd viral passage in the IPEC-J2 cells. PDCoV antigen was detected in the cells showing CPE. By double IF and TUNEL staining, most PDCoV antigen-positive IPEC-J2 cells failed to show TUNEL-positive signals, indicating that PDCoV-infected IPEC-J2 cells may not undergo apoptosis, but rather necrosis, similar to necrotic cell death of infected enterocytes in vivo. There was increased interleukin-6 in PDCoV-infected IPEC-J2 cell culture supernatants at post-inoculation hour (PIH) 48-96, as evaluated by ELISA, concurrent with increased titers of PDCoV at PIH 24-72. The susceptibility of IPEC-J2 cells to PDCoV infection supports their usefulness to characterize the interactions of enterocytes with PDCoV. We also demonstrated that IPEC-J2 cell culture-passaged PDCoV (OH-FD22-P8-I-P4) was enteropathogenic in 10-day-old gnotobiotic pigs, and induced systemic innate and pro-inflammatory cytokine responses during the acute PDCoV infection.
| S-EPMC7117386 | biostudies-literature
Project description:Cell lines matching the source epithelium are indispensable for investigating porcine intestinal transport and barrier properties on a subcellular or molecular level and furthermore help to reduce animal usage. The porcine jejunal cell line IPEC-J2 is established as an in vitro model for porcine infection studies but exhibits atypically high transepithelial resistances (TER) and only low active transport rates so that the effect of nutritional factors cannot be reliably investigated. This study aimed to properly remodel IPEC-J2 and then to re-characterize these cells regarding epithelial architecture, expression of barrier-relevant tight junction (TJ) proteins, adequate TER and transport function, and reaction to secretagogues. For this, IPEC-J2 monolayers were cultured on permeable supports, either under conventional (fetal bovine serum, FBS) or species-specific (porcine serum, PS) conditions. Porcine jejunal mucosa was analyzed for comparison. Main results were that under PS conditions (IPEC-J2/PS), compared to conventional FBS culture (IPEC-J2/FBS), the cell height increased 6-fold while the cell diameter was reduced by 50%. The apical cell membrane of IPEC-J2/PS exhibited typical microvilli. Most importantly, PS caused a one order of magnitude reduction of TER and of trans- and paracellular resistance, and a 2-fold increase in secretory response to forskolin when compared to FBS condition. TJ ultrastructure and appearance of TJ proteins changed dramatically in IPEC-J2/PS. Most parameters measured under PS conditions were much closer to those of typical pig jejunocytes than ever reported since the cell line's initial establishment in 1989. In conclusion, IPEC-J2, if cultured under defined species-specific conditions, forms a suitable model for investigating porcine paracellular intestinal barrier function.
| S-EPMC3829867 | biostudies-literature
Project description:The essential requirement of the lymphotoxin beta receptor (LTβR) in the development and maintenance of peripheral lymphoid organs is well recognized. Evidence shows that LTβR is involved in various cellular processes; however, whether it plays a role in maintaining the cellular function of intestinal porcine enterocytes (IPEC-J2), specifically during porcine epidemic diarrhea virus (PEDV) infection, remains unknown. In this study, we generated LTβR null IPEC-J2 cells using CRISPR/Cas9 to examine the importance of LTβR in cell proliferation, apoptosis, and the response to PEDV infection. Our results showed that the lack of LTβR leads to significantly decreased cell proliferation, potentially due to S phase arrest in LTβR-/- IPEC-J2 cells. Label-free digital holographic microscopy was used to record the three-dimensional morphology of both cell types for up to 72 hours and revealed significantly increased numbers of LTβR-/- cells undergoing apoptosis. Furthermore, we found that PEDV-infected LTβR-/- null IPEC-J2 cells exhibited significant suppression of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) target genes (interleukin (IL)-6 and IL-8) and mucosal barrier integrity-related genes (vascular cell adhesion molecule 1 (VCAM1) and IL-22), which may explain why LTβR-/- cells are more susceptible to PEDV infection. Collectively, our data not only demonstrate the key role of LTβR in intestinal porcine enterocytes, but also provide data for the improved understanding of the cellular response to PEDV infection.
| S-EPMC6262443 | biostudies-literature
Project description:Trefoil factors (TFFs) are regulatory peptides playing critical roles in mucosal repair and protection against a variety of insults within the gastrointestinal tract. This work aimed to explore the effects of deoxynivalenol (DON) on intestinal TFFs expression using in vivo and in vitro models. In an animal trial, twenty-four 28-d-old barrows (Duroc × Landrace × Large White; initial body weight = 7.6 ± 0.7 kg) were randomly divided into three treatments for 28 days, including a control diet (0.61 mg DON/kg feed), and two levels of DON-contaminated diets containing 1.28 and 2.89 mg DON/kg feed, respectively. Piglets exposed to DON had lower mRNA expression of TFF1, TFF2, TFF3, as well as Claudin-4 in the intestine (P < 0.05). Dietary DON exposure decreased the protein levels of TFF2 and TFF3 in the jejunum as demonstrated by western blot and immunohistochemistry. In intestinal porcine epithelial cells (IPEC-J2), DON depressed the mRNA expression of TFF2, TFF3, and Claudin-4. Overexpression of sterile alpha motif (SAM) pointed domain E26 transformation-specific (ETS) factor (SPDEF) was found to attenuate DON-induced suppression of TFFs in IPEC-J2 cells. Altogether, our work shows, for the first time, that dietary DON exposure depresses the expression of intestinal TFFs in piglets. Given the fundamental role of TFFs in intestinal mucosal homeostasis, our observations indicate that the DON content in animal feed should be strictly controlled based on the existing regulation for DON.
| S-EPMC6891430 | biostudies-literature
Project description:in vitro microarray study of transcriptional changes of jejunal cells
2018-11-14 | GSE111184 | GEO
Project description:Autophagy is an important cellular response against intracellular pathogens. However, some viruses have evolved mechanisms to hijack this defensive process to provide favorable conditions for virus replication in host cells. The porcine epidemic diarrhea virus (PEDV) has been shown to alter autophagy pathways; however, it is still unknown through which receptors PEDV induces autophagy in IPEC-J2 cells, whether autophagy facilitates PEDV replication, and which functional domains of PEDV proteins are primarily responsible for inducing autophagy. Here, we found that PEDV infection induces autophagy in host cells via distinct and uncoupled molecular pathways. RNA-seq technology was used to analyze the expression patterns of intracellular genes in PEDV-infected IPEC-J2 cells using transcriptomics. The results demonstrate that PEDV triggers autophagy via the cellular pathogen receptor TLR4 and the AKT-mTOR pathway. As evidenced by autophagosome detection, PEDV infection increases autophagosomes and light chain 3 (LC3)-II as well as downregulated AKT-mTOR phosphorylation. Our study revealed that the binding of the viral protein NSP61-2C (56-151aa) to TLR4 triggers autophagy and inactivates the AKT-mTOR pathway, both of which are critical for facilitating PEDV infection. Through screening and analysis, TLR4 was found to be a key gene involved in PEDV-induced autophagy. The screening of the key functional domains of NSP6 (56-151aa) for their ability to induce autophagy in IPEC-J2 cells provided a basis for the in-depth analysis of the pathogenic mechanism of PEDV infection-induced autophagy and promotion of self-replication and also provided an important target for the study of PEDV antiviral drugs. In conclusion, we elucidated that the PEDV infection of IPEC-J2 cells could induce autophagy and found that PEDV could use autophagy to promote its own replication.
| S-EPMC11598845 | biostudies-literature
Project description:The in vitro anti-inflammatory effect of apigenin and its trimethylated analogue (apigenin-trimethylether) has been investigated in order to evaluate whether these flavonoids could attenuate LPS-induced inflammation in IPEC-J2 non-transformed intestinal epithelial cells. Levels of IL-6, IL-8, TNF-α, and COX-2 mRNA were measured as a marker of inflammatory response. The extracellular H2O2 level in IPEC-J2 cells was also monitored by Amplex Red assay. Our data revealed that both compounds had significant lowering effect on the inflammatory response. Apigenin (at 25 μM) significantly decreased gene expression of IL-6 in LPS-treated cells, while apigenin-trimethylether in the same concentration did not influence IL-6 mRNA level. Both apigenin and apigenin-trimethylether reduced IL-8 gene expression significantly. TNF-α mRNA level was decreased by apigenin-trimethylether, which was not influenced by apigenin. Treatment with both flavonoids caused significant reduction in the mRNA level of COX-2, but the anti-inflammatory effect of the methylated analogue was more effective than the unmethylated one. Furthermore, both flavonoids reduced significantly the level of extracellular H2O2 compared to the control cells. In conclusion, the methylated apigenin analogue could avoid LPS-induced intestinal inflammation and it could be applied in the future as an effective anti-inflammatory compound.
| S-EPMC4477253 | biostudies-literature
Project description:Porcine circovirus 2 (PCV2) causes immunosuppression. Piglets infected with PCV2 can develop enteritis. Given that the gut is the largest immune organ, however, the response of the gut's immune system to PCV2 is still unclear. Here, IPEC-J2 cells with different treatments were co-cultured with PBMC or CD4+ T cells (Transwell). Flow cytometry and Western blotting revealed that PCV2-infected IPEC-J2 increased the frequency of CD4+ T cells among piglets' peripheral blood mononuclear cells (PBMCs) and caused CD4+ T cells to undergo a transformation into Foxp3+ regulatory T cells (Treg cells) via activating CD4+ T ERK. Cytokines production and an inhibitor assay showed that the induction of Tregs by PCV2-infected IPEC-J2 was dependent on TGF-β induced by PCV2 in IPEC-J2, which was associated with the activation of NF-κB. Taken together, PCV2-infected IPEC-J2 activated NF-κB to stimulate the synthesis of TGF-β, which enhanced the differentiation of CD4+ T cells into Treg cells through the activation of ERK in CD4+ T cells. This information sheds light on PCV2's function in the intestinal immune system and suggests a potential immunosuppressive mechanism for PCV2 infection.
| S-EPMC9698303 | biostudies-literature