Project description:microRNA-126 is a microRNA predominately expressed by endothelial cells and controls angiogenesis. Unexpectedly, we found that mice deficient in miR-126 have a major impairment in their innate response to pathogen-associated nucleic acids, as well as HIV, which results in more widespread cell infection. Further examination revealed that this was due to miR-126 control of plasmacytoid DC (pDC) homeostasis and function, and that miR-126 regulates expression of TLR7, TLR9, NFkB1 and other innate response genes, as well as VEGF-receptor 2 (VEGFR2). Deletion of VEGFR2 on DCs resulted in reduced interferon production, supporting a role for VEGFR2 in miR-126 regulation of pDCs. These studies identify the miR-126/VEGFR2 axis as an important regulator of the innate response that operates through multiscale control of pDCs.
Project description:microRNA-126 is a microRNA predominately expressed by endothelial cells and controls angiogenesis. Unexpectedly, we found that mice deficient in miR-126 have a major impairment in their innate response to pathogen-associated nucleic acids, as well as HIV, which results in more widespread cell infection. Further examination revealed that this was due to miR-126 control of plasmacytoid DC (pDC) homeostasis and function, and that miR-126 regulates expression of TLR7, TLR9, NFkB1 and other innate response genes, as well as VEGF-receptor 2 (VEGFR2). Deletion of VEGFR2 on DCs resulted in reduced interferon production, supporting a role for VEGFR2 in miR-126 regulation of pDCs. These studies identify the miR-126/VEGFR2 axis as an important regulator of the innate response that operates through multiscale control of pDCs. Plasmactyoid dendritic cells were FACS-sorted from spleens from wildtype and miR-126 KO mice and their RNA extracted. RNA was amplified, labeled and hybridized to Mouse Gene 1.0 ST arrays with the data generation and quality control pipeline of 19 the Immunological Genome Project (www.immgen.org). Raw data were background-corrected and normalized using the RMA algorithm.
Project description:Dermal fibroblasts from megabat and microbat, stimulated with dsRNA (poly(I:C)) and controls. Bats can harbor some of the most deadliest viruses to humans while rarely displaying pathogenicity themselves. To study their innate immune response - the expression program that is initiated once a pathogen is senseds, we stimulated dermal fibroblast cells from two species (Rousettus aegyptiacus and Pipistrellus kuhlii) for four hours with dsRNA - a viral RNA mimic that triggers a rapid innate immune response. Subsequently, we profiled the response using bulk RNA-seq.
Project description:Dermal fibroblasts from bat and human, stimulated with dsRNA (poly(I:C)) and controls. Bats can harbor some of the most deadliest viruses to humans while rarely displaying pathogenicity themselves. To study the transcriptional divergence and cell-to-cell variability of their innate immune response - the expression program that is initiated once a pathogen is sensed, we stimulated dermal fibroblast cells from Rousettus aegyptiacus and from human for four hours with dsRNA - a viral RNA mimic that triggers a rapid innate immune response. Subsequently, we profiled the response using scRNA-seq.
Project description:Cutaneous lupus erythematosus (CLE) is a photosensitive autoimmune disease characterized by a strong type-I-interferon (IFN) associated inflammation. Keratinocytes are known to determine the interface-dermatitis-pattern in CLE by production of proinflammatory cytokines in the lower epidermis. These cytokines drive a cytotoxic anti-epithelial immune response resulting in keratinocytic cell death and release of endogenous nucleic acids (eNA). We hypothesized that these eNA (RNA- and DNA-motifs) have the capacity to activate innate immune pathways in keratinocytes via pathogen-recognition-receptors (PRR). Gene expression analyses revealed an excessive activation of innate immune response pathways with strong expression of IFN-regulated cytokines in CLE skin lesions. Cultured keratinocytes produce large amounts of these cytokines in response to stimulation of PRR with eNA. UV-stimulation enhances the immunogenicity of eNA and induces CLE-like skin lesions in knockout mice lacking the cytosolic DNase TREX1. Our results provide evidence for a pathogenetic role of endogenous nucleic acids in CLE. They are released within the cytotoxic inflammation along the dermo-epidermal junction and have the capacity to drive the LE-typical inflammation. UV-irradiation supports this inflammation by generation of highly immunostimulatory DNA motifs (8-OHG). These findings explain the photosensitivity of lupus patients and identify pathways of the innate immune system as targets for future therapies.
Project description:The innate immune system is the organism’s first line of defense against pathogens. Pattern recognition receptors (PRRs) are responsible for sensing the presence of pathogen-associated molecules. The prototypic PRRs, the membrane-bound receptors of the Toll-like receptor (TLR) family, recognize pathogen-associated molecular patterns (PAMPs) and initiate an innate immune response through signaling pathways that depend on the adaptor molecules MyD88 and TRIF. Deciphering the differences in the complex signaling events that lead to pathogen recognition and initiation of the correct response remains challenging. Here we report the discovery of temporal changes in the protein signaling components involved in innate immunity. Using an integrated strategy combining unbiased proteomics, transcriptomics and macrophage stimulations with three different PAMPs, we identified differences in signaling between individual TLRs and revealed specifics of pathway regulation at the protein level.
Project description:The interactions between proteins and nucleic acids have a fundamental function in many biological processes well beyond nuclear gene transcription and include RNA homeostasis, protein translation and pathogen sensing for innate immunity. While our knowledge of the ensemble of proteins binding individual mRNAs in mammalian cells has greatly been augmented by recent surveys, no systematic study on the native proteins of human cells differentially engaging various types of nucleic acids in a non sequence-specific manner has been reported. We designed an experimental approach to cover the non sequence-specific RNA and DNA binding space broadly, including methylation, and test for its ability to interact with the human proteome. We used 25 rationally designed nucleic acid probes in an affinity purification mass spectrometry and bioinformatics workflow to identify proteins from whole cell extracts of three different human cell lines. The proteins were profiled for their binding preferences to the different general types of nucleic acids. The study identified 746 high confidence direct binders, 249 of which were devoid of previous experimental evidence for binding nucleic acids. We could assign 513 specific affinities for sub-types of nucleic acid probes to 219 distinct proteins and to individual domains. The evolutionary conserved protein YB-1, previously associated with cancer and gene regulation, is shown to bind methylated cytosine preferentially conferring YB-1 a potential epigenetic function. Collectively, the dataset represents a rich resource of experimentally determined nucleic acid-specific binding proteins in humans and, indirectly, for other species. Identification of genomic YB-1 binding sites in HEK293 cells
Project description:MicroRNA-122 (miR-122) is the most abundant microRNA in hepatocytes and a central player in liver biology and disease. Herein, we report a previously unknown role for miR-122 in hepatocyte intrinsic innate immunity. Restoring miR-122 levels in hepatoma cells markedly enhanced the activation of interferons (IFNs) in response to a variety of viral nucleic acids or simulations, especially of hepatitis C virus RNA and poly (I:C). Mechanistically, miR-122 down-regulated the phosphorylation (Tyr705) of STAT3 and thereby removed the negative regulation of STAT3 on IFN-signaling. While STAT3 represses IFN expression by inhibiting interferon regulatory factor 1 (IRF1); miR-122 targets MERTK, FGFR1 and IGF1R, three receptor tyrosine kinases (RTKs) that directly promote STAT3 phosphorylation. This work identifies a miR-122–RTKs/STAT3–IRF1–IFNs regulatory circuitry, which may play a pivotal role in regulating hepatocyte innate immunity. These findings renewed our knowledge about miR-122’s function and have important implications for treating hepatitis viruses. We used microarrays to analyze which genes were down-regulated by miR-122.
Project description:Toll-like receptors (TLRs) are mediators in the innate immune response by recognizing nucleic acids derived from microbial components as for TLR9 binding ability for CpG rich bacterial DNA. We studied the differential expression by microarrays of all known T-UCRs and near genes in a series of 6 primary B-CLL cases cells comparing cells treated in vitro with CpG ODN versus mock controls.