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Long non-coding RNAs involved in myeloid cell differentiation and macrophage activation
The human genome encodes for ~ 20.000 long non-coding RNAs (lncRNAs), yet their molecular functions, especially in the immune system, remain largely unknown. In this study, two main aspects were addressed: the participation of lncRNAs in the differentiation of myeloid immune cells and their involve...
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| Format: | Dissertation |
| Sprache: | Englisch |
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Philipps-Universität Marburg
2020
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| Zusammenfassung: | The human genome encodes for ~ 20.000 long non-coding RNAs (lncRNAs), yet their molecular functions, especially in the immune system, remain largely unknown. In this study, two main aspects were addressed: the participation of lncRNAs in the differentiation of myeloid immune cells and their involvement in pro-inflammatory activation of human macrophages. In order to address the first aspect, RNA sequencing results from distinct immune cell subsets were analysed. These data showed that lncRNAs define immune-cell identity equally well as protein-coding genes, such as surface receptors considered as precise markers of leukocyte subsets. In the present work, non-coding RNA LINC00211 was identified as a specific myeloid cell lineage marker. Functional characterisation demonstrated that this lncRNA regulates the expression of several genes, including CHI3L1 and S100A9, which participate in myeloid cell differentiation. Furthermore, LINC00211 was regulated by PU.1, a transcription factor with fundamental roles in immune cell lineage commitment. Additionally, LINC00211 could be characterised as a biomarker of pulmonary inflammation, since high expression was observed in bronchoalveolar lavage fluid from infected individuals and in lung extracts from IPF patients, correlating with the degree of neutrophil infiltration.
In order to investigate the involvement of lncRNAs in pro-inflammatory activation of human macrophages, RNA sequencing experiments were performed and unveiled several differentially expressed lncRNAs in resting and immune-activated human macrophages. Furthermore, a multidimensional approach was established to categorize human lncRNAs according to their subcellular localization and co-sedimentation with cellular protein complexes in macrophages. The resulting data revealed that lncRNAs constitute a highly heterogeneous class of RNA co-sedimenting with various cellular machineries, including ribosomes. Using these data, lncRNA MaIL1 was identified as a highly immune-responsive, cytosolic and non-ribosome associated intergenic lncRNA (lincRNA). Functional analysis associated MaIL1 with type I interferon production after Toll-like Receptor (TLR) activation. RNA antisense purification and mass spectrometry (RAP-MS) showed that MaIL1 interacts with Optineurin, a protein known to be required for signal transduction within the TBK1-IRF3 axis, thus facilitating type I interferon production. More specifically, MaIL1 regulates Optineurin ubiquitination, a modification essential for Optineurin function. When MaIL1 was knocked down, IRF3 phosphorylation and subsequently type I interferon production was impaired. Moreover, MaIL1 was found to be essential for defence against Legionella pneumophila, a Gram-negative bacterium that predominantly replicates inside alveolar macrophages and causes pneumonia. In addition, MaIL1 levels were increased during pulmonary infections and correlated linearly with IFNβ mRNA levels in human bronchoalveolar lavage fluid. Thus, the present work identifies MaIL1 as a critical regulator of TLR-induced IFN responses to infection. In summary, both studies revealed detailed information about the function of lncRNAs in myeloid immune cells and provide a rich resource and blueprint for future investigations of lncRNA functions in the immune system. |
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| Umfang: | 191 Seiten |
| DOI: | 10.17192/z2020.0162 |