Gene expression analysis of intestinal stem cells (ISCs) from polyI:C treated WT mice or Ifnar1-/- mice.
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ABSTRACT: To explore the influence of IFNR-mediated singnaling in ISCs, we performed microarray analysis of the ISCs from control Lgr5ki mice versus Ifnar1-/-Lgr5ki mice after treating for 1 weeks with low doses of poly(I:C), which potentially induce type I and -III IFNs. The Gene Ontology (GO) analysis indicated that the GO terms overrepresented among the genes upregulated in ISCs from poly(I:C)-treated WT mice compared with those from poly(I:C)-treated Ifnar1−/− mice included “defense response to virus”, “immune system process”, “response to virus”, and “innate immune response”, all from the GOTERM_Biological Processes (BP) category. We compared these results against the Interferome database, which contains genes regulated by type I, II, or III IFN, compiled by analyzing the expression data of IFN-treated cells (http://www.interferome.org), and found that most of the genes found for the GO term “defense response to virus” were IFN-inducible genes, indicating that IFNs act directly on ISCs. The GO analysis also showed an enrichment of defense response to bacteria. In the GOTERM_Cellular Component (CC) category, “extracellular space” and “extracellular region” ranked at the top, indicating a dramatic change in the expression pattern of secretory molecules. Notably, these GO terms included genes encoding antibacterial proteins and endocrine hormones such as angiogenin, defensin, lysozyme, chromogranin, and glucagon, all of which are produced by secretory IECs. Thus, we hypothesized that excess IFN signals force ISCs to lose stemness and differentiate into secretory progenitors.
Project description:To analyze global gene-expression changes caused by IRF2 loss in ISCs, ISCs were prepared from Irf2fl/fl: Lgr5-EGFP-Ires-CreERT2(Irf2fl/fl: Lgr5ki) mice or Irf2fl/fl: Ah-Cre: Lgr5-EGFP-Ires-CreERT2 (Irf2AhcKO: Lgr5ki) mice 1 month after 5 consecutive days of βNF treatment. The Gene Ontology (GO) analysis indicated that the GO terms overrepresented among the genes upregulated in ISCs of βNF treated Irf2AhcKO: Lgr5ki mice compared with those of Irf2fl/fl: Lgr5ki mice included “immune system process”, “immune response”, and “cellular response to Interferon-beta”, all from the GOTERM_Biological Processes (BP) category. Because these GO_term inculded many IFN-inducibl genes, IFN signaling augumented in Irf2 deleted ISCs.
Project description:Type I interferons (IFNs) are a family of cytokines that play an important role in regulating immune responses to pathogens and tumors and are used therapeutically. All IFNs are considered to signal via the heterodimeric IFNAR1-IFNAR2 complex, yet some subtypes such as IFN? can exhibit distinct functional properties, although the molecular basis of this is unclear. Here we demonstrate IFN uniquely and specifically ligates to IFNAR1 in an IFNAR2-independent manner and provide the structural basis of the IFNAR1-IFN interaction. We show that the IFNAR1-IFN complex transduces signals to modulate the expression of a set of genes independently of IFNAR2. Moreover, we show the in vivo importance of the IFNAR1-IFN signaling axis in a murine model of LPS-induced septic shock. Thus, we provide a molecular basis for understanding specific functions of IFN?. Interferon b induced gene expression in peritoneal exudate cells was measured 3hr post intra-peritoneal injection of 10,000IU/ml of interferon beta or saline into wildtype and Ifnar2-/- mice. Three independant experiments were performed for each treatment in both genotypes using different mice for each sample.
Project description:Type I interferons (IFNs) are a family of cytokines that play an important role in regulating immune responses to pathogens and tumors and are used therapeutically. All IFNs are considered to signal via the heterodimeric IFNAR1-IFNAR2 complex, yet some subtypes such as IFN? can exhibit distinct functional properties, although the molecular basis of this is unclear. Here we demonstrate IFN uniquely and specifically ligates to IFNAR1 in an IFNAR2-independent manner and provide the structural basis of the IFNAR1-IFN interaction. We show that the IFNAR1-IFN complex transduces signals to modulate the expression of a set of genes independently of IFNAR2. Moreover, we show the in vivo importance of the IFNAR1-IFN signaling axis in a murine model of LPS-induced septic shock. Thus, we provide a molecular basis for understanding specific functions of IFN?.
Project description:We provide the first comprehensive analysis of nasal cell responses to SARS-COV-2 using single cell transcriptomics and proteomics. The immune response to SARS-CoV-2 is dominated by a delayed type I and III IFN response, which is too slow to contain viral replication. Cells transitioning from secretory to ciliated states are highly permissive to SARS-CoV-2, whereas goblet cells are relatively resistant. Cell-type differences in the production and response to IFN-I/III correlate with permissiveness. Pre-treatment with recombinant IFNs potently restricts SARS-CoV-2. Nasal delivery of recombinant IFNs is a promising prophylactic strategy for SARS-CoV-2
Project description:Type I interferons (IFNs) are consequential cytokines in antibacterial defense. Whether and how bacterial pathogens inhibit innate immune receptor-driven type I IFN expression remains mostly unknown. By screening a library of enterohemorrhagic Escherichia coli (EHEC) mutants, we uncovered EhaF, an uncharacterized protein, as an inhibitor of innate immune responses including IFNs. Further analyses identified EhaF as a secreted autotransporter—a type of bacterial secretion system with no known innate immune-modulatory function—that translocates into host cell cytosol and inhibit IFN response to EHEC. Mechanistically, EhaF interacts with and inhibits the MiT/TFE family transcription factor TFE3 resulting in impaired TANK phosphorylation and consequently, reduced IRF3 activation and type I IFN expression. Notably, EhaF-mediated innate immune suppression promotes EHEC colonization and pathogenesis in vivo. Overall, this study has uncovered a previously unknown autotransporter-based bacterial strategy that targets a specific transcription factor to subvert innate host defense.
Project description:The innate defense mechanisms that control infections with intracellular bacteria are still incompletely understood. Here we show that type I and II IFNs are key regulators of the early gene expression and the host-protective immune response during Legionella pneumophila-induced pneumonia. Using mixed bone marrow-chimeric mice and isolated cells we indicate that both IFNs protect against L. pneumophila by activating an alveolar macrophage-intrinsic antibacterial defense pathway. Quantitative mass spectrometry analysis reveals that both IFNs markedly alter the protein composition of purified Legionella-containing vacuoles, and integrated network analysis defines distinct subsets of IFN-regulated proteins. Subsequent experiments uncover Immunoresponsive gene 1 (Irg1) as a central effector that restricts the bacteria through production of itaconic acid. Collectively, we provide a comprehensive analysis of IFN-mediated effects on gene expression and the bacterial vacuole proteome, and show that L. pneumophila is restricted by an Irg1-dependent production of a bactericidal metabolite. Microarray experiments were performed as dual-color hybridizations on Agilent mouse whole genome catalog 44K arrays. To compensate for dye-specific effects, a dye-reversal color-swap was applied.
Project description:Candida albicans is the most common human fungal pathogen causing mucosal and systemic infections. However, human antifungal immunity remains poorly defined. By integrating transcriptional analysis and functional genomics we identified Candida-specific host defense mechanisms in humans. Candida induced significant (p<10-35) expression of genes from the type I interferon (IFN) pathway in human peripheral blood mononuclear cells. This unexpectedly prominent role of type I IFN pathway in anti-Candida host defense was supported by additional evidence. Polymorphisms in type I IFN genes modulated Candida-induced cytokine production and were correlated with susceptibility to systemic candidiasis. In in-vitro experiments, type I IFNs skewed Candida-induced inflammation from a Th17-response toward a Th1-response. Patients with chronic mucocutaneaous candidiasis displayed defective expression of genes in the type I IFN pathway. These findings indicate that the type I IFN pathway is a main signature of Candida-induced inflammation and plays a crucial role in anti-Candida host defense in humans. 3 healthy controls and 2 CMC patients
Project description:Deciphering the intricate dynamic events governing type I interferon (IFN) signaling is critical to unravel key regulatory mechanisms in host antiviral defense. Here, we leveraged TurboID-based proximity labeling coupled with affinity purification-mass spectrometry to comprehensively map temporal changes to the proximal human proteomes of all seven canonical type I IFN signaling cascade members following IFN stimulation. This established a network of 108 proteins in close proximity to the core members IFNAR1, IFNAR2, JAK1, TYK2, STAT1, STAT2, and IRF9, and validated several known protein assemblies, while also revealing novel, transient associations between key signaling molecules.
Project description:Type I Interferons (IFNs) are potent inhibitors of viral replication. Here, we reformatted the natural murine and human type I Interferon-α/β receptors IFNAR1 and IFNAR2 into fully synthetic biological switches. The transmembrane and intracellular domains of natural IFNAR1 and IFNAR2 were conserved, whereas the extracellular domains were exchanged by nanobodies directed against the fluorescent proteins Green fluorescent protein (GFP) and mCherry. Using this approach, multimeric single-binding GFP-mCherry ligands induced synthetic IFNAR1/IFNAR2 receptor complexes and initiated STAT1/2 mediated signal transduction via Jak1 and Tyk2. Homodimeric GFP and mCherry ligands showed that IFNAR2 but not IFNAR1 homodimers were sufficient to induce sustained STAT1/2 signaling. Transcriptome analysis revealed that synthetic murine type I IFN signaling was highly comparable to IFNα4 signaling and resulted in efficient elimination of vesicular stomatitis virus (VSV) in a cell culture based viral infection model using MC57 cells stimulated with synthetic type IFN ligands. Using intracellular deletion variants and point mutations, Y510 and Y335 in murine IFNAR2 were verified as unique phosphorylation sites for STAT1/2 activation, whereas the other tyrosine residues in IFNAR1 and IFNAR2 were not involved in STAT1/2 phosphorylation. Comparative analysis of synthetic human IFNARs supporting this finding. In summary, our data showed that synthetic type I IFN signal transduction is originating from IFNAR2 rather than IFNAR1.
Project description:The innate defense mechanisms that control infections with intracellular bacteria are still incompletely understood. Here we show that type I and II IFNs are key regulators of the early gene expression and the host-protective immune response during Legionella pneumophila-induced pneumonia. Using mixed bone marrow-chimeric mice and isolated cells we indicate that both IFNs protect against L. pneumophila by activating an alveolar macrophage-intrinsic antibacterial defense pathway. Quantitative mass spectrometry analysis reveals that both IFNs markedly alter the protein composition of purified Legionella-containing vacuoles, and integrated network analysis defines distinct subsets of IFN-regulated proteins. Subsequent experiments uncover Immunoresponsive gene 1 (Irg1) as a central effector that restricts the bacteria through production of itaconic acid. Collectively, we provide a comprehensive analysis of IFN-mediated effects on gene expression and the bacterial vacuole proteome, and show that L. pneumophila is restricted by an Irg1-dependent production of a bactericidal metabolite.