ABSTRACT
Pigs are the most suitable model to study various therapeutic strategies and drugs for human beings, although knowledge about cell type-specific transcriptomes and heterogeneity is poorly available. Through single-cell RNA sequencing and flow cytometry analysis of the types in the jejunum of pigs, we found that innate lymphoid cells (ILCs) existed in the lamina propria lymphocytes (LPLs) of the jejunum. Then, through flow sorting of live/dead-lineage (Lin)-CD45+ cells and single-cell RNA sequencing, we found that ILCs in the porcine jejunum were mainly ILC3s, with a small number of NK cells, ILC1s, and ILC2s. ILCs coexpressed IL-7Rα, ID2, and other genes and differentially expressed RORC, GATA3, and other genes but did not express the CD3 gene. ILC3s can be divided into four subgroups, and genes such as CXCL8, CXCL2, IL-22, IL-17, and NCR2 are differentially expressed. To further detect and identify ILC3s, we verified the classification of ILCs in the porcine jejunum subgroup and the expression of related hallmark genes at the protein level by flow cytometry. For systematically characterizing ILCs in the porcine intestines, we combined our pig ILC dataset with publicly available human and mice ILC data and identified that the human and pig ILCs shared more common features than did those mouse ILCs in gene signatures and cell states. Our results showed in detail for the first time (to our knowledge) the gene expression of porcine jejunal ILCs, the subtype classification of ILCs, and the markers of various ILCs, which provide a basis for an in-depth exploration of porcine intestinal mucosal immunity.
Subject(s)
Immunity, Innate , Lymphocytes , Humans , Animals , Mice , Swine , Jejunum , Killer Cells, Natural , Mucous MembraneABSTRACT
In maintaining organismal homeostasis, gut immunity plays a crucial role. The coordination between the microbiota and the immune system through bidirectional interactions regulates the impact of microorganisms on the host. Our research focused on understanding the relationships between substantial changes in jejunal intestinal flora and metabolites and intestinal immunity during porcine epidemic diarrhea virus (PEDV) infection in piglets. We discovered that Lactobacillus rhamnosus GG (LGG) could effectively prevent PEDV infection in piglets. Further investigation revealed that LGG metabolites interact with type 3 innate lymphoid cells (ILC3s) in the jejunum of piglets through the aryl hydrocarbon receptor (AhR). This interaction promotes the activation of ILC3s and the production of interleukin-22 (IL-22). Subsequently, IL-22 facilitates the proliferation of IPEC-J2 cells and activates the STAT3 signaling pathway, thereby preventing PEDV infection. Moreover, the AhR receptor influences various cell types within organoids, including intestinal stem cells (ISCs), Paneth cells, and enterocytes, to promote their growth and development, suggesting that AhR has a broad impact on intestinal health. In conclusion, our study demonstrated the ability of LGG to modulate intestinal immunity and effectively prevent PEDV infection in piglets. These findings highlight the potential application of LGG as a preventive measure against viral infections in livestock.IMPORTANCEWe observed high expression of the AhR receptor on pig and human ILC3s, although its expression was negligible in mouse ILC3s. ILC3s are closely related to the gut microbiota, particularly the secretion of IL-22 stimulated by microbial signals, which plays a crucial regulatory role in intestinal immunity. In our study, we found that metabolites produced by beneficial gut bacteria interact with ILC3s through AhR, thereby maintaining intestinal immune homeostasis in pigs. Moreover, LGG feeding can enhance the activation of ILC3s and promote IL-22 secretion in the intestines of piglets, ultimately preventing PEDV infection.
Subject(s)
Coronavirus Infections , Immunity, Innate , Interleukin-22 , Interleukins , Lymphocytes , Porcine epidemic diarrhea virus , Receptors, Aryl Hydrocarbon , Animals , Receptors, Aryl Hydrocarbon/metabolism , Swine , Interleukins/metabolism , Porcine epidemic diarrhea virus/immunology , Lymphocytes/immunology , Lymphocytes/metabolism , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Coronavirus Infections/veterinary , Coronavirus Infections/virology , Coronavirus Infections/metabolism , Gastrointestinal Microbiome/immunology , Swine Diseases/immunology , Swine Diseases/virology , Swine Diseases/prevention & control , Swine Diseases/microbiology , Jejunum/immunology , Jejunum/metabolism , Signal Transduction , Ligands , Intestines/immunology , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolismABSTRACT
African swine fever (ASF) is a devastating infectious disease of pigs caused by the African swine fever virus (ASFV), which poses a great danger to the global pig industry. Many viral proteins can suppress with interferon signaling to evade the host's innate immune responses. Therefore, the development of an effective vaccine against ASFV has been dampened. Recent studies have suggested that the L83L gene may be integrated into the host genome, weakening the host immune system, but the underlying mechanism is unknown. Our study found that L83L negatively regulates the cGAS-STING-mediated type I interferon (IFN-I) signaling pathway. Overexpression of L83L inhibited IFN-ß promoter and ISRE activity, and knockdown of L83L induced higher transcriptional levels of interferon-stimulated genes (ISGs) and phosphorylation levels of IRF3 in primary porcine alveolar macrophages. Mechanistically, L83L interacted with cGAS and STING to promote autophagy-lysosomal degradation of STING by recruiting Tollip, thereby blocking the phosphorylation of the downstream signaling molecules TBK1, IRF3, and IκBα and reducing IFN-I production. Altogether, our study reveals a negative regulatory mechanism involving the L83L-cGAS-STING-IFN-I axis and provides insights into an evasion strategy involving autophagy and innate signaling pathways employed by ASFV. IMPORTANCE African swine fever virus (ASFV) is a large double-stranded DNA virus that primarily infects porcine macrophages. The ASFV genome encodes a large number of immunosuppressive proteins. Current options for the prevention and control of this pathogen remain pretty limited. Our study showed that overexpression of L83L inhibited the cGAS-STING-mediated type I interferon (IFN-I) signaling pathway. In contrast, the knockdown of L83L during ASFV infection enhanced IFN-I production in porcine alveolar macrophages. Additional analysis revealed that L83L protein downregulated IFN-I signaling by recruiting Tollip to promote STING autophagic degradation. Although L83L deletion has been reported to have little effect on viral replication, its immune evade mechanism has not been elucidated. The present study extends our understanding of the functions of ASFV-encoded pL83L and its immune evasion strategy, which may provide a new basis for developing a live attenuated vaccine for ASF.
Subject(s)
African Swine Fever Virus , Interferon Type I , Viral Proteins , Animals , African Swine Fever , African Swine Fever Virus/immunology , Immunity, Innate/immunology , Interferon Type I/immunology , Nucleotidyltransferases/metabolism , Swine , Viral Proteins/genetics , Viral Proteins/immunologyABSTRACT
E3 ubiquitin ligase (E3) plays a vital role in regulating inflammatory responses by mediating ubiquitination. Previous studies have shown that ankyrin repeat and SOCS box-containing protein 3 (ASB3) is involved in immunomodulatory functions associated with cancer. However, the impact of ASB3 on the dynamic interplay of microbiota and inflammatory responses in inflammatory bowel disease (IBD) is unclear. Here, we systematically identify the E3 ligase ASB3 as a facilitative regulator in the development and progression of IBD. We observed that ASB3 exhibited significant upregulation in the lesions of patients with IBD. ASB3-/- mice are resistant to dextran sodium sulfate-induced colitis. IκBα phosphorylation levels and production of proinflammatory factors IL-1ß, IL-6, and TNF-α were reduced in the colonic tissues of ASB3-/- mice compared to WT mice. This colitis-resistant phenotype was suppressed after coprophagic microbial transfer and reversed after combined antibiotics removed the gut commensal microbiome. Mechanistically, ASB3 specifically catalyzes K48-linked polyubiquitination of TRAF6 in intestinal epithelial cells. In contrast, in ASB3-deficient organoids, the integrity of the TRAF6 protein is shielded, consequently decelerating the onset of intestinal inflammation. ASB3 is associated with dysregulation of the colitis microbiota and promotes proinflammatory factors' production by disrupting TRAF6 stability. Strategies to limit the protein level of ASB3 in intestinal epithelial cells may help in the treatment of colitis. IMPORTANCE: Ubiquitination is a key process that controls protein stability. We determined the ubiquitination of TRAF6 by ASB3 in intestinal epithelial cells during colonic inflammation. Inflammatory bowel disease patients exhibit upregulated ASB3 expression at focal sites, supporting the involvement of degradation of TRAF6, which promotes TLR-Myd88/TRIF-independent NF-κB aberrant activation and intestinal microbiota imbalance. Sustained inflammatory signaling in intestinal epithelial cells and dysregulated protective probiotic immune responses mediated by ASB3 collectively contribute to the exacerbation of inflammatory bowel disease. These findings provide insights into the pathogenesis of inflammatory bowel disease and suggest a novel mechanism by which ASB3 increases the risk of colitis. Our results suggest that future inhibition of ASB3 in intestinal epithelial cells may be a novel clinical strategy.
ABSTRACT
Zbtb1 (zinc finger and BTB domain containing 1) is a member of mammalian zbtb gene family. A series of bioinformatics analysis was carried out for the EL4 cell and the Zbtb1-deficient EL4 cell by Hi-C, ATAC-seq and RNA-seq techniques. Finally, Hi-C results showed that the intensity of chromatin interaction in the deletion group decreased with distance, the degree of chromosome interaction decreased significantly, the AB division region changed significantly, and the compactness of TAD structure decreased; The results of ATAC-seq showed that the open area and degree of chromatin in the deletion group decreased; 7778 differentially expressed mRNAs were found by RNA-seq. Our experimental results for the first time expounded the significance of Zbtb1 gene for T cell development, lymphocyte production and apoptosis from the aspects of chromosome spatial structure and chromatin opening degree, and provided relevant theoretical basis and data support for the in-depth study of related Zbtb1 genes in the future.
ABSTRACT
In previous experiments, we identified the effect of deletion of the Zbtb1 gene on circRNAs and microRNAs. In this study, we examined the expression profiles of lncRNAs and mRNAs using the RNA-seq method for Zbtb1-deficient EL4 cells and performed a clustering analysis of differentially expressed lncRNAs and mRNAs. GO term histograms and KEGG scatter plots were drawn. For the experimental results, a joint analysis was performed, which predicted the regulatory relationships among lncRNAs, mRNAs, microRNAs and circRNAs. For the regulatory relationship between lncRNAs and target genes, the chromatin structure and the degree of openness were verified for the possible target gene locations regulated by lncRNA using experimental methods such as Hi-C and ATAC-seq. Ultimately, the possible differential regulation of the Brcal and Dennd5d genes by lncRNAs and the differential changes in transcription factor binding sites in the promoter region were identified. For neRNA-regulated target genes with significantly differentially expressed mRNAs, a combined screen was performed, and the final obtained candidate target genes were subjected to GO and KEGG term enrichment analyses. Our results illustrate that the Zbtb1 gene can not only function as a regulatory factor but also regulate EL4 cells from multiple perspectives based on ceRNA theory.
Subject(s)
MicroRNAs , RNA, Long Noncoding , Cell Line , Gene Regulatory Networks , MicroRNAs/genetics , RNA, Circular , RNA, Long Noncoding/genetics , RNA, Messenger/geneticsABSTRACT
Zinc finger and BTB domain containing 1(Zbtb1) is a transcriptional suppressor protein, and a member of the mammalian Zbtb gene family. Previous studies have shown that Zbtb1 is essential for T-cell development. However, the role of Zbtb1 in T-cell lymphoma is undetermined. In this study, an EL4 cell line with Zbtb1 deletion was constructed using the CRISPR-Cas9 technique. The expression profiles of microRNA and circRNA produced by the control and gene deletion groups were determined by RNA-seq. In general, 24 differentially expressed microRNA and 16 differentially expressed circRNA were found between normal group and gene deletion group. Through further analysis of differentially expressed genes, GO term histogram and KEGG scatter plot were drawn, and three pairs of miRNA and circRNA regulatory relationships were found. This study describes the differentially expressed microRNA and circRNA in normal and Zbtb1-deficient EL4 cell lines, thus providing potential targets for drug development and clinical treatment of T-cell lymphoma.
Subject(s)
Lymphoma, T-Cell/genetics , MicroRNAs , RNA, Circular , Repressor Proteins/genetics , Animals , Cell Differentiation , Cell Line , Gene Expression Profiling , Gene Knockout Techniques , Mice , MicroRNAs/genetics , RNA, Circular/geneticsABSTRACT
Lactic acid bacteria (LAB) are the primary genera of the intestinal flora and have many probiotic functions. In the present study, Lactobacillus rhamnosus GG (LGG) ATCC 53103 was used to treat BALB/c mice. After LGG intervention, both low and high LGG doses were shown to improve the observed OTU, Chao1, ACE, and Shannon indices, while the Simpson index decreased, demonstrating that LGG can promote intestinal microbiota abundance and diversity. Furthermore, LGG treatment increased the abundances of intestinal Firmicutes, Bacteroides and Actinomycetes while reducing that of Proteobacteria. In addition to its effect on gut the microbiota, LGG could also regulate the host immune system. In the present study, we showed that LGG could affect the percentage of CD3+ T lymphocytes in the spleens (SPLs), mesenteric lymph nodes (MLNs), Peyer's patches (PPs) and lamina propria lymphocytes (LPLs) of mice, including total CD3+ T, CD3+CD4+ T, and CD3+CD8+ T lymphocytes. Furthermore, LGG could effectively increase the expression of Th1-type cytokines (IFN-γ) and Th2 cytokines (IL-4) in CD4+ T cells, indicating that the proportion of Th1 and Th2 cells in mice with LGG treatment was in a high equilibrium state compared to the control group. In addition, the IFN-γ/IL-4 ratio was greater than 1 in mice with LGG intervention, suggesting that LGG tends to mediate the Th1 immune response. The results of the present study also showed that LGG upregulated the expression of IL-17 in CD4+ T cells and regulated the percentage of CD4+CD25+Foxp3+ Treg cells in various secondary immunological organs, indicating that LGG may promote the balance of Th-17 and Treg cells.
ABSTRACT
Supported Ni catalysts on three mesoporous SiO2 supports (i.e., SBA-15, MCM-41, and HMS) were prepared using a solid-state reaction between Ni(NO3)2 and organic template-occluded mesoporous SiO2. For comparison, supported Ni catalysts on mesoporous SiO2 synthesized by the conventional impregnation method were also included. The catalysts were characterized by scanning electron microscopy, X-ray diffraction, UV-vis diffuse reflectance spectroscopy, N2 adsorption, X-ray photoelectron spectroscopy, H2 temperature-programmed reduction, transmission electron microscopy, and transmission electron microscopy-energy-dispersive X-ray. The catalytic properties of the catalysts were evaluated using gas-phase catalytic hydrodechlorination of 1,2-dichloroethane. The results showed that upon grinding Ni(NO3)2 with template-occluded mesoporous SiO2, strong coordination between Ni2+ and dodecylamine was identified in the Ni(NO3)2-HMS system. Additionally, the results of H2 temperature-programmed reduction revealed that NiO in calcined NiO/HMS was reduced at higher temperature than those in calcined NiO/SBA-15 and NiO/MCM-41, reflecting the presence of a strong interaction between NiO and mesoporous SiO2 in NiO/HMS. Consistently, the average particle sizes of metallic Ni were found to be 2.7, 3.4, and 9.6 nm in H2-reduced Ni/HMS, Ni/SBA-15, and Ni/MCM-41, respectively, indicative of a much higher Ni dispersion in Ni/HMS. For the catalytic hydrodechlorination of 1,2-dichloroethane, Ni/MCM-41 synthesized by the solid-state reaction method exhibited a catalytic activity similar to that prepared by the impregnation method, while higher catalytic activities were observed on Ni/HMS and Ni/SBA-15 than on their counterparts prepared by the impregnation method. Furthermore, a higher conversion was identified on Ni/HMS than on Ni/SBA-15 and Ni/MCM-41, highlighting the importance of template type for the preparation of highly dispersed metal catalysts on mesoporous SiO2.