Identification of the Effects of 5-Azacytidine on Porcine Circovirus Type 2 Replication in Porcine Kidney Cells.

Porcine circovirus type 2 (PCV2) is the main pathogen causing post-weaning multisystemic wasting syndrome (PMWS), which mainly targets the body's immune system and poses a serious threat to the global pig industry. 5-Azacytidine is a potent inhibitor of DNA methylation, which can participate in many important physiological and pathological processes, including virus-related processes, by inhibiting gene expression. However, the impact of 5-Aza on PCV2 replication in cells is not yet clear. We explored the impact of 5-Aza on PCV2 infection utilizing PK15 cells as a cellular model. Our objective was to gain insights that could potentially offer novel therapeutic strategies for PCV2. Our results showed that 5-Aza significantly enhanced the infectivity of PCV2 in PK15 cells. Transcriptome analysis revealed that PCV2 infection activated various immune-related signaling pathways. 5-Aza may activate the MAPK signaling pathway to exacerbate PCV2 infection and upregulate the expression of inflammatory and apoptotic factors.

Integrated Metabolomic and transcriptomic analyses reveal deoxycholic acid promotes transmissible gastroenteritis virus infection by inhibiting phosphorylation of NF-κB and STAT3.

BACKGROUND: Acute diarrhea, dehydration and death in piglets are all symptoms of transmissible gastroenteritis virus (TGEV), which results in significant financial losses in the pig industry. It is important to understand the pathogenesis and identify new antiviral targets by revealing the metabolic interactions between TGEV and host cells. RESULTS: We performed metabolomic and transcriptomic analyses of swine testicular cells infected with TGEV. A total of 1339 differential metabolites and 206 differentially expressed genes were detected post TEGV infection. The differentially expressed genes were significantly enriched in the HIF-1 signaling pathway and PI3K-Akt signaling. Integrated analysis of differentially expressed genes and differential metabolites indicated that they were significantly enriched in the metabolic processes such as nucleotide metabolism, biosynthesis of cofactors and purine metabolism. In addition, the results showed that most of the detected metabolites involved in the bile secretion was downregulated during TGEV infection. Furthermore, exogenous addition of key metabolite deoxycholic acid (DCA) significantly enhanced TGEV replication by NF-κB and STAT3 signal pathways. CONCLUSIONS: We identified a significant metabolite, DCA, related to TGEV replication. It added TGEV replication in host cells by inhibiting phosphorylation of NF-κB and STAT3. This study provided novel insights into the metabolomic and transcriptomic alterations related to TGEV infection and revealed potential molecular and metabolic targets for the regulation of TGEV infection.

SADS-CoV nsp1 inhibits the STAT1 phosphorylation by promoting K11/K48-linked polyubiquitination of JAK1 and blocks the STAT1 acetylation by degrading CBP.

The newly discovered zoonotic coronavirus swine acute diarrhea syndrome coronavirus (SADS-CoV) causes acute diarrhea, vomiting, dehydration, and high mortality rates in newborn piglets. Although SADS-CoV uses different strategies to evade the host's innate immune system, the specific mechanism(s) by which it blocks the interferon (IFN) response remains unidentified. In this study, the potential of SADS-CoV nonstructural proteins (nsp) to inhibit the IFN response was detected. The results determined that nsp1 was a potent antagonist of IFN response. SADS-CoV nsp1 efficiently inhibited signal transducer and activator of transcription 1 (STAT1) phosphorylation by inducing Janus kinase 1 (JAK1) degradation. Subsequent research revealed that nsp1 induced JAK1 polyubiquitination through K11 and K48 linkages, leading to JAK1 degradation via the ubiquitin-proteasome pathway. Furthermore, SADS-CoV nsp1 induced CREB-binding protein degradation to inhibit IFN-stimulated gene production and STAT1 acetylation, thereby inhibiting STAT1 dephosphorylation and blocking STAT1 transport out of the nucleus to receive antiviral signaling. In summary, the results revealed the novel mechanisms by which SADS-CoV nsp1 blocks the JAK-STAT signaling pathway via the ubiquitin-proteasome pathway. This study yielded valuable findings on the specific mechanism of coronavirus nsp1 in inhibiting the JAK-STAT signaling pathway and the strategies of SADS-CoV in evading the host's innate immune system.

Special Staining and Protein Expression of VEGF/EGFR and P53/NF-κB in Cryptorchid Tissue of Erhualian Pigs.

Erhualian pigs exhibit one of the highest reproductive rates globally, and cryptorchidism is a crucial factor affecting reproductive abilities of boars. This investigation focused on cryptorchid tissues from Erhualian pigs, where the histological structure of cryptorchidism was observed using specialized staining. In addition, protein expression of P53/NF-κB in cryptorchid tissues was assessed using Western blot and immunohistochemistry. In comparison to normal Erhualian testes, Masson's trichrome staining indicated a reduction in collagen fibers in the connective tissue and around the basal membrane of the seminiferous tubules in cryptorchid testes. Moreover, collagen fiber distribution was observed to be disordered. Verhoeff Van Gieson (EVG) and argyrophilic staining demonstrated brownish-black granular nucleoli organized regions in mesenchymal cells and germ cells. When compared to normal testicles, the convoluted seminiferous tubules of cryptorchids exhibited a significantly reduced number and diameter (p < 0.01). Notably, VEGF/EGFR and P53/NF-κB expression in cryptorchidism significantly differed from that in normal testes. In particular, the expression of VEGF and P53 in cryptorchid tissues was significantly higher than that in normal testes tissues, whereas the expression of EGFR in cryptorchid tissues was significantly lower than that in normal testes tissues (all p < 0.01). NF-κB expressed no difference in both conditions. The expressions of VEGF and NF-κB were observed in the cytoplasm of testicular Leydig cells and spermatogenic cells, but they were weak in the nucleus. EGFR and P53 were more positively expressed in the cytoplasm of these cells, with no positive expression in the nucleus. Conclusion: There were changes in the tissue morphology and structure of the cryptorchid testis, coupled with abnormally high expression of VEGF and P53 proteins in Erhualian pigs. We speculate that this may be an important limiting factor to fecundity during cryptorchidism.

Knockout of IRF3 and IRF7 genes by CRISPR/Cas9 technology enhances porcine virus replication in the swine testicular (ST) cell line.

Antiviral vaccines for pig diseases are essential to prevent epidemic outbreaks. However, their production is often hindered by inefficient manufacturing processes that yield lower quantities of the vaccine. To accelerate the progress of various areas of bioproduction, we have considered the necessity of enhancing viral replication efficiency by optimizing ST (swine testicular) cell lines that are commonly utilized in virus manufacturing. CRISPR/Cas9 gene-editing technology were utilized to create IRF3 or IRF7 knockout cell lines that facilitate high-titer viral stock production. Compared to the parental cell lines, the ST IRF3/7 KO cell line displayed a compromised antiviral response to a panel of viruses (Porcine epidemic diarrhea virus, Senecavirus A, Parainfluenza virus 5, and Getah virus), as evidenced by decreased expression of interferon and certain antiviral factors. The inhibition of these responses led to heightened viral replication and increased cytopathic effects, ultimately promoting apoptosis. As a result, the development of these cell lines offers a more efficient approach for biopharmaceutical companies to boost their virus production and reduce associated costs.

A High-Fat Diet Increases the Characteristics of Gut Microbial Composition and the Intestinal Damage Associated with Non-Alcoholic Fatty Liver Disease.

The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing annually, and emerging evidence suggests that the gut microbiota plays a causative role in the development of NAFLD. However, the role of gut microbiota in the development of NAFLD remains unclear and warrants further investigation. Thus, C57BL/6J mice were fed a high-fat diet (HFD), and we found that the HFD significantly induced obesity and increased the accumulation of intrahepatic lipids, along with alterations in serum biochemical parameters. Moreover, it was observed that the HFD also impaired gut barrier integrity. It was revealed via 16S rRNA gene sequencing that the HFD increased gut microbial diversity, which enriched Colidextribacter, Lachnospiraceae-NK4A136-group, Acetatifactor, and Erysipelatoclostridium. Meanwhile, it reduced the abundance of Faecalibaculum, Muribaculaceae, and Coriobacteriaceae-UCG-002. The predicted metabolic pathways suggest that HFD enhances the chemotaxis and functional activity of gut microbiota pathways associated with flagellar assembly, while also increasing the risk of intestinal pathogen colonization and inflammation. And the phosphotransferase system, streptomycin biosynthesis, and starch/sucrose metabolism exhibited decreases. These findings reveal the composition and predictive functions of the intestinal microbiome in NAFLD, further corroborating the association between gut microbiota and NAFLD while providing novel insights into its potential application in gut microbiome research for NAFLD patients.

Integrated metabolomics and transcriptomics analyses reveal metabolic responses to TGEV infection in porcine intestinal epithelial cells.

Transmissible gastroenteritis virus (TGEV) is a coronavirus that infects piglets with severe diarrhoea, vomiting, dehydration, and even death, causing huge economic losses to the pig industry. The underlying pathogenesis of TGEV infection and the effects of TGEV infection on host metabolites remain poorly understood. To investigate the critical metabolites and regulatory factors during TGEV infection in intestinal porcine epithelial cells (IPEC-J2), we performed metabolomic and transcriptomic analyses of TGEV-infected IPEC-J2 cells by LC/MS and RNA-seq techniques. A total of 87 differential metabolites and 489 differentially expressed genes were detected. A series of metabolites and candidate genes from glutathione metabolism and AMPK signalling pathway were examined through combined analysis of metabolome and transcriptome. We found glutathione peroxidase 3 (GPX3) is markedly reduced after TGEV infection, and a significant negative correlation between AMPK signalling pathway and TGEV infection. Exogenous addition of the AMPK activator COH-SR4 significantly downregulates stearoyl coenzyme A (SCD1) mRNA and inhibits TGEV replication; while exogenous GSK-690693 significantly promotes TGEV infection by inhibiting AMPK signalling pathway. In summary, our study provides insights into the key metabolites and regulators for TGEV infection from the metabolome and transcriptome perspective, which will offer promising antiviral metabolic and molecular targets and enrich the understanding of the existence of a similar mechanism in the host.

Regulation of the three-dimensional chromatin organization by transposable elements in pig spleen.

Like other mammalian species, the pig genome is abundant with transposable elements (TEs). The importance of TEs for three-dimensional (3D) chromatin organization has been observed in species like human and mouse, yet current understanding about pig TEs is absent. Here, we investigated the contribution of TEs for the 3D chromatin organization in three pig tissues, focusing on spleen which is crucial for both adaptive and innate immunity. We identified dozens of TE families overrepresented with CTCF binding sites, including LTR22_SS, LTR15_SS and LTR16_SSc which are pig-specific families of endogenous retroviruses (ERVs). Interestingly, LTR22_SS elements harbor a CTCF motif and create hundreds of CTCF binding sites that are associated with adaptive immunity. We further applied Hi-C to profile the 3D chromatin structure in spleen and found that TE-derived CTCF binding sites correlate with chromatin insulation and frequently overlap TAD borders and loop anchors. Notably, one LTR22_SS-derived CTCF binding site demarcate a TAD boundary upstream of XCL1, which is a spleen-enriched chemokine gene important for lymphocyte trafficking and inflammation. Overall, this study represents a first step toward understanding the function of TEs on 3D chromatin organization regulation in pigs and expands our understanding about the functional importance of TEs in mammals.

SADS-CoV nsp1 inhibits the IFN-ß production by preventing TBK1 phosphorylation and inducing CBP degradation.

Swine acute diarrhea syndrome (SADS) is first reported in January 2017 in Southern China. It subsequently causes widespread outbreaks in multiple pig farms, leading to economic losses. Therefore, it is an urgent to understand the molecular mechanisms underlying the pathogenesis and immune evasion of Swine acute diarrhea syndrome coronavirus (SADS-CoV). Our research discovered that SADS-CoV inhibited the production of interferon-ß (IFN-ß) during viral infection. The nonstructural protein 1 (nsp1) prevented the phosphorylation of TBK1 by obstructing the interaction between TBK1 and Ub protein. Moreover, nsp1 induced the degradation of CREB-binding protein (CBP) through the proteasome-dependent pathway, thereby disrupting the IFN-ß enhancer and inhibiting IFN transcription. Finally, we identified nsp1-Phe39 as the critical amino acid that downregulated IFN production. In conclusion, our findings described two mechanisms in nsp1 that inhibited IFN production and provided new insights into the evasion strategy adopted by SADS-CoV to evade host antiviral immunity.

Integrated of multi-omics and molecular docking reveal PHGDH, PSAT1 and PSPH in the serine synthetic pathway as potential targets of T-2 toxin exposure in pig intestinal tract.

T-2 toxin (T-2) with a molecular weight of 466.52 g/mol is an inevitable mycotoxin in food products and feeds, posing a significant threat to human and animal health. However, the underlying molecular mechanisms of the cytotoxic effects of T-2 exposure on porcine intestinal epithelial cells (IPEC-J2) remain unclear. Here, we investigated the cytotoxic effects of T-2 exposure on IPEC-J2 through the detection of cell viability, cell morphology, mitochondrial membrane potential, ROS, apoptosis and autophagy. Further transcriptomic and proteomic analyses of IPEC-J2 upon T-2 exposure were performed by using RNA-seq and TMT techniques. A total of 546 differential expressed genes (DEGs) and 269 differentially expressed proteins (DEPs) were detected. Among these, 24 common DEGs/DEPs were involved in IPEC-J2 upon T-2 exposure. Interestingly, molecular docking analysis revealed potential interactions between T-2 and three key enzymes (PHGDP, PSAT1, and PSPH) in the serine biosynthesis pathway. Besides, further experimental showed that PSAT1 knockdown exacerbated T-2-induced oxidative damage. Together, our findings indicated that the serine biosynthesis pathway including PHGDP, PSAT1, PSPH genes probably acts critical roles in the regulation of T-2-induced cell damage. This study provided new insights into the global molecular effects of T-2 exposure and identified the serine biosynthesis pathway as molecular targets and potential treatment strategies against T-2.

Hypomethylated interferon regulatory factor 8 recruits activating protein-2α to attenuate porcine epidemic diarrhea virus infection in porcine jejunum.

Interferon regulatory factor 8 (IRF8) is a key regulator of innate immune receptor signaling that resists pathogen invasion by regulating cell growth and differentiation. Porcine epidemic diarrhea virus (PEDV) targets the intestine and damages the mucosal barrier. However, whether IRF8 regulates PEDV replication remains unclear. We revealed that PEDV infection activated IRF8 expression. Moreover, IRF8 deletion drastically promoted PEDV replication and invasion, increasing the virus copies and titers. Hypomethylation enrichment of activating protein (AP)-2α was significantly negatively correlated with high IRF8 expression, and AP-2α directly targeted the IRF8 promoter to regulate PEDV replication. Furthermore, IRF8 overexpression decreased the cellular reactive oxygen species levels and mitochondrial membrane potential and increased the antioxidant enzyme activities to alleviate PEDV-induced oxidative damage. IRF8 overexpression suppressed apoptotic gene expression, thereby inhibiting apoptosis in response to PEDV stimulation. Taken together, this study demonstrates that AP-2α is involved in PEDV-induced epigenetic modification of IRF8 to reduce cell apoptosis and oxidative stress and facilitate host resistance to PEDV in the intestinal epithelium.

Genome-wide analysis of circRNA regulation during spleen development of Chinese indigenous breed Meishan pigs.

BACKGROUND: Numerous circular RNAs (circRNAs) have been recently identified in porcine tissues and cell types. Nevertheless, their significance in porcine spleen development is yet unelucidated. Herein, we reported an extensive overlook of circRNA expression profile during spleen development in Meishan pigs. RESULTS: Overall, 39,641 circRNAs were identified from 6,914 host genes. Among them, many circRNAs are up- or down-regulated at different time points of pig spleen development. Using WGCNA analysis, we revealed two essential modules for protein-coding genes and circRNAs. Subsequent correlation analysis revealed 67 circRNAs/co-expressed genes that participated in immnue-associated networks. Furthermore, a competing endogenous RNA (ceRNA) network analysis of circRNAs revealed that 12 circRNAs modulated CD226, MBD2, SAMD3, SIT1, SRP14, SYTL3 gene expressions via acting as miRNA sponges. Moreover, the circRNA_21767/miR-202-3p axis regulated SIT1 expression in a ceRNA manner, which is critical for the immune-based regulation of spleen development in Meishan pigs. CONCLUSIONS: Overall, our results demonstrated that the circRNAs were differentially expressed during different stages of porcine spleen development, meanwhile the circRNAs interacted with immune-related genes in a ceRNA-based fashion. Moreover, we presented biomedical researchers with RNAseqTools, a user-friendly and powerful software for the visualization of transcriptome profile data.

Porcine Deltacoronavirus Infection Disrupts the Intestinal Mucosal Barrier and Inhibits Intestinal Stem Cell Differentiation to Goblet Cells via the Notch Signaling Pathway.

Goblet cells and their secreted mucus are important elements of the intestinal mucosal barrier, which allows host cells to resist invasion by intestinal pathogens. Porcine deltacoronavirus (PDCoV) is an emerging swine enteric virus that causes severe diarrhea in pigs and causes large economic losses to pork producers worldwide. To date, the molecular mechanisms by which PDCoV regulates the function and differentiation of goblet cells and disrupts the intestinal mucosal barrier remain to be determined. Here, we report that in newborn piglets, PDCoV infection disrupts the intestinal barrier: specifically, there is intestinal villus atrophy, crypt depth increases, and tight junctions are disrupted. There is also a significant reduction in the number of goblet cells and the expression of MUC-2. In vitro, using intestinal monolayer organoids, we found that PDCoV infection activates the Notch signaling pathway, resulting in upregulated expression of HES-1 and downregulated expression of ATOH-1 and thereby inhibiting the differentiation of intestinal stem cells into goblet cells. Our study shows that PDCoV infection activates the Notch signaling pathway to inhibit the differentiation of goblet cells and their mucus secretion, resulting in disruption of the intestinal mucosal barrier. IMPORTANCE The intestinal mucosal barrier, mainly secreted by the intestinal goblet cells, is a crucial first line of defense against pathogenic microorganisms. PDCoV regulates the function and differentiation of goblet cells, thereby disrupting the mucosal barrier; however, the mechanism by which PDCoV disrupts the barrier is not known. Here, we report that in vivo, PDCoV infection decreases villus length, increases crypt depth, and disrupts tight junctions. Moreover, PDCoV activates the Notch signaling pathway, inhibiting goblet cell differentiation and mucus secretion in vivo and in vitro. Thus, our results provide a novel insight into the mechanism underlying intestinal mucosal barrier dysfunction caused by coronavirus infection.

Evaluation of the genotype I Japanese encephalitis virus as a stable viral vector for foreign gene expression.

Manipulation of the flavivirus genome to accommodate and express a heterologous gene of interest has become an attractive approach for gene delivery and the development of viral-vectored vaccines. However, due to the inherent genetic instability of the flavivirus genomes, the construction of recombinant viruses carrying a foreign gene could be problematic and heavily resistant. In this study, the possibility of the Japanese encephalitis virus (JEV) as a stable flavivirus vector for the expression of a foreign gene was assessed using reverse genetics. The full-length cDNA genome of genotype I (GI) JEV inherently possessed excellent stability and manipulability in a bacterial host, while mutations and deletions accumulated in the cDNA genomes of genotype Ⅲ (GⅢ) JEV strains. Using the GI JEV as backbones, we generate a panel of recombinant viruses expressing various foreign genes. All recombinant viruses exhibited excellent genetic stability and efficiently express foreign genes for at least ten serial passages in vitro. In application, a convenient, rapid and reliable image-based assay for neutralizing antibody testing and antiviral drug discovery was established with a mCherry-reporter recombinant virus (rBJ-mCherry). Meanwhile, the recombinant viruses expressing the antigens of the African swine fever virus (ASFV) or Classical swine fever virus (CSFV) could effectively induce antibody responses to the JEV vector and foreign antigens in a mouse vaccination model. Therefore, GI JEV strains could serve as viral vectors accommodating the expression of large foreign genes.

Sodium Butyrate Ameliorates Deoxynivalenol-Induced Oxidative Stress and Inflammation in the Porcine Liver via NR4A2-Mediated Histone Acetylation.

Mycotoxin-induced liver injury is often accompanied by oxidative stress (OS) and inflammation. This research aimed to explore the potential mechanism of sodium butyrate (NaBu) in modulating hepatic anti-oxidation and anti-inflammation pathways in deoxynivalenol (DON)-exposed piglets. The results show that DON induced liver injury, increased mononuclear cell infiltration, and decreased serum total protein and albumin concentrations. Transcriptomic analysis revealed that reactive oxygen species (ROS) and TNF-α pathways were highly activated upon DON exposure. This is associated with disturbed antioxidant enzymes and increased inflammatory cytokines secretion. Importantly, NaBu effectively reversed the alterations caused by DON. Mechanistically, the ChIP-seq result revealed that NaBu strongly depressed DON-increased enrichment of histone mark H3K27ac at the genes involved in ROS and TNF-α-mediated pathways. Notably, we demonstrated that nuclear receptor NR4A2 was activated by DON and remarkably recovered with the treatment of NaBu. In addition, the enhanced NR4A2 transcriptional binding enrichments at the promoter regions of OS and inflammatory genes were hindered by NaBu in DON-exposed livers. Consistently, elevated H3K9ac and H3K27ac occupancies were also observed at the NR4A2 binding regions. Taken together, our results indicated that a natural antimycotic additive, NaBu, could mitigate hepatic OS and inflammatory responses, possibly via NR4A2-mediated histone acetylation.

Lactoferrin Relieves Deoxynivalenol-Induced Oxidative Stress and Inflammatory Response by Modulating the Nrf2/MAPK Pathways in the Liver.

Deoxynivalenol (DON), one of the most common mycotoxins contaminating food and feed, has been shown to induce hepatotoxicity. Lactoferrin (LF) enriched in human milk is a critical functional food component and performs the hepatoprotection function. Here, we aimed to explore whether dietary LF supplementation can protect from DON-induced hepatotoxicity and uncover the underlying mechanism in mice and alpha mouse liver 12 (AML12) hepatocytes. In vivo results revealed that LF alleviated DON-induced liver injury, reflected by repairing the hepatic histomorphology and decreasing the plasma alanine aminotransferase (ALT) level and the number of blood white blood cells (WBC) and neutrophils (Neu). Moreover, LF decreased the hepatic reactive oxygen species (ROS) and malondialdehyde (MDA) accumulation and enhanced the hepatic GSH-px activity and protein expression of Nrf2 and GPX4 to reverse the DON-induced hepatic oxidative stress. Furthermore, LF downregulated the pro-inflammatory-response-related gene expressions (IL1ß, TNFα, and Tlr4) and the phosphorylation levels of IKK, IκBα, and p38 in the liver of DON-exposed mice. Additionally, in vitro studies confirmed that LF ameliorated the DON-induced oxidation-reduction imbalance, inflammatory responses, and associated core modulators of the Nrf2 and MAPK pathways in DON-induced hepatotoxicity. In conclusion, LF performs hepatic antioxidative and anti-inflammatory functions by regulating the Nrf2/MAPK signaling pathways, thus reducing DON-induced hepatotoxicity.

Protective effects of melatonin on deoxynivalenol-induced oxidative stress and autophagy in IPEC-J2 cells.

This study explored protective effects of melatonin (MEL) on deoxynivalenol (DON)-induced toxicity in porcine jejunum epithelial cells (IPEC-J2). Cells were preexposed to MEL and then exposed to DON to detect cell viability, apoptosis, and oxidative stress indicators. Compared to DON treatment, pretreatment with MEL significantly increased cell proliferation. (P < 0.01), intracellular catalase (CAT) and superoxide dismutase (SOD) levels (P < 0.05), decreased apoptosis and oxidative stress, and significantly attenuated the inflammatory response. RNA-seq analysis revealed that MEL protects IPEC-J2 from the adverse effects of DON by affecting the expression of tight junction and autophagy pathway-related genes. Similarly, further experiments revealed that MEL partly prevented DON-induced disruption of intestinal barrier function and decreased autophagy induced by DON via activation of the AKT/mTOR pathway. In conclusion, these results demonstrated the preventive properties of MEL against DON-induced cell damage by activating the antioxidant system and Inhibition of autophagy.

Integrative ATAC-seq and RNA-seq analyses of IPEC-J2 cells reveals porcine transcription and chromatin accessibility changes associated with Escherichia coli F18ac inhibited by Lactobacillus reuteri.

Escherichia coli is the main cause of postweaning diarrhea in pigs, leading to economic loss. As a probiotic, Lactobacillus reuteri has been used to inhibit E. coli in clinical applications; however, its integrative interactions with hosts remain unclear, especially in pigs. Here, we found that L. reuteri effectively inhibited E. coli F18ac adhering to porcine IPEC-J2 cells, and explored the genome-wide transcription and chromatin accessibility landscapes of IPEC-J2 cells by RNA-seq and ATAC-seq. The results showed that some key signal transduction pathways, such as PI3K-AKT and MAPK signaling pathways, were enriched in the differentially expressed genes (DEGs) between E. coli F18ac treatment with and without L. reuteri groups. However, we found less overlap between RNA-seq and ATAC-seq datasets; we speculated that this might be caused by histones modification through ChIP-qPCR detection. Furthermore, we identified the regulation of the actin cytoskeleton pathway and a number of candidate genes (ARHGEF12, EGFR, and DIAPH3) that might be associated with the inhibition of E. coli F18ac adherence to IPEC-J2 cells by L. reuteri. In conclusion, we provide a valuable dataset that can be used to seek potential porcine molecular markers of E. coli F18ac pathogenesis and L. reuteri antibacterial activity, and to guide the antibacterial application of L. reuteri.

Metabolomic and Proteomic Profiling of Porcine Intestinal Epithelial Cells Infected with Porcine Epidemic Diarrhea Virus.

Porcine epidemic diarrhea virus (PEDV) infection results in severe epidemic diarrhea and the death of suckling pigs. Although new knowledge about the pathogenesis of PEDV has been improved, alterations in metabolic processes and the functional regulators involved in PEDV infection with host cells remain largely unknow. To identify cellular metabolites and proteins related to PEDV pathogenesis, we synergistically investigated the metabolome and proteome profiles of PEDV-infected porcine intestinal epithelial cells by liquid chromatography tandem mass spectrometry and isobaric tags for relative and absolute quantification techniques. We identified 522 differential metabolites in positive and negative ion modes and 295 differentially expressed proteins after PEDV infection. Pathways of cysteine and methionine metabolism, glycine, serine and threonine metabolism, and mineral absorption were significantly enriched by differential metabolites and differentially expressed proteins. The betaine-homocysteine S-methyltransferase (BHMT) was indicated as a potential regulator involved in these metabolic processes. We then knocked down the BHMT gene and observed that down-expression of BHMT obviously decreased copy numbers of PEDV and virus titers (p < 0.01). Our findings provide new insights into the metabolic and proteomic profiles in PEDV-infected host cells and contribute to our further understanding of PEDV pathogenesis.

Chromatin Accessibility and Transcriptional Landscape during Inhibition of Salmonella enterica by Lactobacillus reuteri in IPEC-J2 Cells.

Lactobacillus reuteri is a probiotic with bacteriostatic effects, which can effectively inhibit the activity of pathogens. However, the molecular mechanism underlying the inhibition of pathogens by L. reuteri in intestinal cells remains unclear. Using the porcine intestinal cell line IPEC-J2 as a model, we combined RNA-seq and ATAC-seq methods to delineate the porcine genome-wide changes in biological processes and chromatin accessibility in IPEC-J2 cells stimulated by Salmonella enterica BNCC186354, as well as L. reuteri ATCC 53608. Overall, we found that many porcine transcripts were altered after S. enterica BNCC186354 treatment, while L. reuteri ATCC 53608 treatment partially restored this alteration, such as salmonella infection and PI3K/AKT and MAPK pathways. Combined analysis of these two datasets revealed that 26 genes with similar trends overlapped between gene expression and chromatin accessibility. In addition, we identified potential host functional transcription factors (TFs), such as GATA1, TAL1, TBP, RUNX1, Gmeb1, Gfi1b, RARA, and RXRG, in IPEC-J2 cells that might play a critical role and are targeted by L. reuteri ATCC 53608. Moreover, we verified that PI3K/AKT, MAPK, and apoptosis pathways are potentially regulated by S. enterica BNCC186354 but restored by L. reuteri ATCC 53608. The PI3K/AKT pathway was activated by L. reuteri ATCC 53608, thereby potentially inhibiting S. enterica BNCC186354 infection. In conclusion, our data provide new insights into the expression pattern of functional genes and the epigenetic alterations in IPEC-J2 cells underlying the bacteriostatic action of L. reuteri ATCC 53608.