Ming-An Sun

Enterotoxigenic Escherichia coli F18 (E. coli F18) ranks as the leading bacterial cause of diarrhea in piglets. Exosomes and other extracellular vesicles ferry diverse RNA species, among them mRNAs and non-coding RNAs, and act as central intermediaries in intercellular communication. Their contribution to the biology of E. coli F18-infected IPEC-J2 cells, however, has received no attention. In this study, we characterized the morphology, particle dimensions, and output of exosomes secreted by control and E. coli F18-infected cells, then profiled their circRNA cargo by RNA-seq. Nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Western blotting verified that the vesicle-like particles recovered from IPEC-J2 cultures were bona fide exosomes. We then catalogued the circRNAs within IPEC-J2-derived exosomes during E. coli F18 infection. Comparing the two groups yielded 280 differentially expressed circRNAs (DECs), of which circCHUK displayed the largest shift. circCHUK abundance rose sharply in E. coli F18-treated IPEC-J2 cells. Knocking down or overexpressing circCHUK markedly altered the adherence of E. coli F18 to IPEC-J2 cells. ceRNA network construction pinpointed the exo-circCHUK/ssc-miR-339-3p/FUT2 as candidate axis. Collectively, we speculated that exo-circCHUK is a new biomarker that regulates E. coli F18 susceptibility, offering a conceptual framework for tackling bacterial diarrhea in piglets.

Interferons (IFNs) are a family of cytokines which serve as the first line of defense against pathogen infections while also exerting critical immunomodulatory roles. IFNs can induce hundreds of IFN-stimulated genes (ISGs) with cell-specificity, yet a high-resolution comparison of time-serial ISG induction across cell types is lacking. By using RNA sequencing, we conducted a comparative transcriptomic profiling during time-serial IFN-γ stimulation for up to 24 h in HeLa and HEK293T cells, the two most widely used immortalized human cell lines. We uncovered remarkably stronger IFN responses in HeLa cells, regarding the global transcriptomic dynamics, the number of induced ISGs, and the level of ISG expression. Both cell lines share a core set of ISGs associated with canonical JAK-STAT signaling, yet HeLa uniquely activates additional inflammatory and adaptive immunity-related pathways. Despite the much weaker IFN response in HEK293T cells, we also identified a few HEK293T-specific ISGs, including several with crucial immune-related functions. Notably, transposable elements—including many adjacent to ISGs—are also highly up-regulated in HeLa cells, implying their potential links to ISG induction. Collectively, this study provides a high-resolution temporal atlas of IFN-γ-stimulated transcriptomic dynamics in HeLa and HEK293T cells, revealing the shared core module and cell-specific patterns of their interferon responses.

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic virus with pig as its major amplifying host. Despite that JEV can persistently infect the placentae of pregnant sows and cause severe reproductive failures, the molecular responses and potential regulators of JEV infection in pig placenta remain unclear. By using porcine trophectoderm (pTr) cells as model, we investigated the transcriptomic changes during JEV infection, with special focus on microRNA-centric regulation over immune genes and JEV infection. To avoid currently poor microRNA annotation for pig, we first performed small RNA-seq to achieve refined microRNA annotation, with hundreds of novel microRNAs annotated. After confirming that JEV can efficiently infect pTr cells and induce severe cytopathic effects, we conducted routine and small RNA-seq to determine the changes of mRNA and microRNA genes during JEV infection. We identified > 1000 differentially expressed genes and found JEV-induced genes are tightly associated with antiviral immune responses. Moreover, we identified 94 JEV-affected microRNAs, including many with their roles on viral infection still unclear. Focusing on JEV-affected microRNAs, we constructed a microRNA - mRNA negative regulatory network, with multiple hub microRNAs and their targets inspected. Among these microRNAs, we experimentally validated that ssc-miR-149 and ssc-miR-483 both can inhibit JEV replication in pTr cells, with the well-recognized immune suppressor SOCS1 validated as a direct target of ssc-miR-149. Overall, this study comprehensively characterized the microRNA-centric regulation of JEV infection in pTr cells, which provides mechanistical insights on JEV infection in pig placenta and will facilitate the development of novel therapies against JEV infection.

Pig is an important model for biomedical studies, yet the immune cell heterogeneity, the subtle relationship between immune cells and the inter-breed immun

Alveolar macrophages (AMs) play essential roles in maintaining homeostasis and immunity in the lung. The transcription factor PU.1, encoded by SPI1, is a core regulator in multiple immune cell lineages. However, its binding property and regulatory role in AMs remain unclear. The pig serves as an important livestock species and a valuable biomedical model. Using porcine AMs (PAMs) as a model, we combined gene knockdown experiments with multiomic profiling to elucidate the regulatory role of PU.1 in AMs. By integrating the RNA-seq data before and after SPI1 knockdown, we demonstrate that disruption of PU.1 impairs the expression of numerous immune-related genes, including many crucial for innate immune responses. We further employed CUT&Tag to characterize the genome-wide occupancy of PU.1 and the active histone modification H3K27ac, and found that PU.1 primarily binds active cis-regulatory elements (CREs), including a large proportion of enhancers derived from transposable elements. Moreover, integrative analysis identifies a set of CREs and their associated genes, which are putative direct targets of PU.1. Overall, this study provides novel insights into the regulatory role of PU.1 in AMs and extends our knowledge about this core regulator in the mammalian immune system.

Transposable elements (TEs) are mobile DNA elements abundant in eukaryote genomes. While recognized as key contributors to cis-regulatory elements (CREs),

Motor neurons (MNs) control muscle movement and are essential for breathing, walking and fine motor skills. MN and Pancreas Homeobox 1 (MNX1) has long been recognized as a key marker of the MN lineage, and its deficiency in mice results in early postnatal lethality—likely by causing abnormal MN development and respiratory malfunction. However, its genome-wide targets and regulatory mechanism remain unresolved. Using an in vitro model for efficient MN induction, we identified ∼6000 MNX1-bound loci, of which half are conserved enhancers frequently overlap peaks for core MN-inducing factors ISL1 and LHX3. Despite its widespread binding, Mnx1 knockout affects only a few dozen bound loci and causes mis-regulation of ∼100 genes, the majority are up-regulated neuronal genes which routinely have comparable or even higher expression in brain relative to MNs. Integrative analysis predicts Pbx3 and Pou6f2 as two putative direct targets—both are homeobox genes highly expressed in the central nervous system, and adjacent to MNX1-repressed enhancers according to luciferase reporter assay. Collectively, our results suggest MNX1 restrains the expression of non-MN-specific neuronal genes, likely mainly through an indirect fashion. Further, the rarity of direct targets in contrast to widespread binding reflects a distinctive mode of transcriptional regulation.

Deoxynivalenol (DON), a prevalent environmental toxin produced by Fusarium fungi, frequently contaminates feed and food products. However, the critical metabolites and regulatory factors involved in DON toxicity remain poorly understood. Building upon our established DON-induced porcine intestinal epithelial cells (IPEC-J2) injury model, this study employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to conduct metabolomic analysis, and integrated analysis with transcriptomic data from DON-exposed IPEC-J2. Results identified 1524 differentially expressed metabolites, and revealed significant enrichment in Glutathione metabolism and Mucin-type O-glycan biosyn-thesis pathways. Notably, γ-glutamylcysteine (γGC), the rate-limiting precursor for glutathione synthesis, showed significant reduction following DON exposure. To explore the biological function of γGC, this study found through exogenous supplementation experiments that γGC pretreatment could significantly alleviate the inhibition of IPEC-J2 cell viability, effectively reduce intracellular ROS accumulation and inhibit DON-induced apoptosis in IPEC-J2 cells. These results indicated that the severe oxidative stress induced by DON is closely related to the blockage of glutathione synthesis caused by the exhaustion of intracellular γGC, and revealed the application potential of γGC as an exogenous supplement in the prevention and treatment of DON exposure. In conclusion, this study offers valuable insights into the metabolic and transcriptional alterations, along with the key metabolites and regulators involved in the cellular response to DON pollution. It also lays a theoretical foundation for more effective prevention and treatment strategies against DON pollution.

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, has inflicted substantial economic losses on the global swine industry. While the severity of infectious disease depends on the dynamic interplay between inoculum dose and host response, the molecular mechanism by which PDCoV dose modulates host immunity remains unclear. Hence, we systematically compared the transcriptomic changes in intestinal epithelial cells infected with different doses of PDCoV, and investigated the relationships between inoculum dose, host immune responses, and disease progression. PDCoV replication peaked at 24 h post-infection, and host responses showed a distinct dose-dependent pattern, with high-dose infection inducing more extensive transcriptional remodeling than low-dose infection. Both doses significantly activated the transcription of STAT1 and its downstream interferon-stimulated genes, while high-dose infection additionally triggered a cytokine storm characterized by excessive IL-6 and TNF-α expression. Functional validation demonstrated that STAT1 overexpression markedly inhibited PDCoV infection by enhancing ISRE promoter activity, and overexpression of its downstream ISG15 and MX2 also exerted independent and significant antiviral effects. These findings reveal the biphasic nature of PDCoV dose-dependent regulation of immunopathological mechanisms and identify STAT1 and specific ISGs (ISG15, MX2) as potent antiviral effectors, providing crucial insights into PDCoV pathogenicity and offering promising targets for developing immunomodulatory therapeutics or vaccines to control PDCoV outbreaks in swine.