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 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

Cas9-mediated excision of proximal DNaseI/H3K4me3 signatures confers robust silencing of microRNA and long non-coding RNA genes

<div><p>CRISPR/Cas9-based approaches have greatly facilitated targeted genomic deletions. Contrary to coding genes however, which can be functionally knocked out by frame-shift mutagenesis, non-coding RNA (ncRNA) gene knockouts have remained challenging. Here we present a universal ncRNA knockout approach guided by epigenetic hallmarks, which enables robust gene silencing even in provisionally annotated gene loci. We build on previous work reporting the presence of overlapping histone H3 lysine 4 tri-methylation (H3K4me3) and DNaseI hypersensitivity sites around the transcriptional start sites of most genes. We demonstrate that excision of this gene-proximal signature leads to loss of microRNA and lincRNA transcription and reveals ncRNA phenotypes. Exemplarily we demonstrate silencing of the constitutively transcribed MALAT1 lincRNA gene as well as of the inducible miR-146a and miR-155 genes in human monocytes. Our results validate a role of miR-146a and miR-155 in negative feedback control of the activity of inflammation master-regulator NFκB and suggest that cell-cycle control is a unique feature of miR-155. We suggest that our epigenetically guided CRISPR approach may improve existing ncRNA knockout strategies and contribute to the development of high-confidence ncRNA phenotyping applications.</p></div

Excision of H3K4me3/DNaseI signatures by dual guideRNA CRISPR vectors.

<p><b>A)</b> Primary human monocyte DNase- and ChIP-Seq (H3K4me3 and K3K27me3) coverages at the transcriptional start sites of the MALAT1, miR-146a and miR-155 ncRNA genes. Regions selected for targeted excision are marked (dashed lines). <b>B)</b> Genomic PCR with primers flanking the respective H3K4me3/DNaseI element to be excised. Besides the wild-type band (WT) a band of reduced size (ΔTSS) is detected after transfection of HEK293 cells with the respective ncRNA knockout CRISPR construct (KO) but not after transfection of a control CRISPR construct (Ctrl.). Position of the DNA ladder bands (2.5 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, 250 bp) is indicated to the left of each gel image. <b>C)</b> Sanger-sequencing of ΔTSS bands validates the deletion in the MALAT1, miR155 and miR146a promoter, respectively. Positions of guideRNAs (gRNA) and protospacer-adjacent motifs (PAM) are indicated.</p

Knockout of miR-155 affects monocyte cell cycle.

<p><b>A)</b> Gating scheme for determining cell fractions in G1/G0-, S- or G2/M-phase through propidium iodide staining. Representative data from LPS-treated wild-type (WT) or ncRNA knockout monocytes are shown. <b>B)</b> Quantification of percent-distribution of cell fractions in G1/G0-, S- or G2/M-phase in 4, 8 or 16 h mock or LPS (1 μg / ml) treated monocytes (upper and middle panels) and representation of individual mean- and replicate values for the 16 h time-point (lower panel). Data refer to wild-type (WT), MALAT1, miR-146a and miR-155 knockout U937 cells as indicated below the lower panel. Statistical significance was determined by one-way ANOVA with multiple comparisons (* p<0.05, ** p<0.01).</p

Model summarizing the observed ncRNA knockout phenotypes.

<p>NFκB p65 phosphorylation data indicate a negative feed-back function of both miR-146a and miR-155 in TLR signaling, while ERK1/2 signaling is not affected. Promoting entry into the G2/M cell cycle phase is a function unique to miR-155.</p

Silencing ncRNA genes based on proximal H3K4me3/DNaseI signatures.

<p><b>A)</b> Schematic representation of the ncRNA knockout strategy. To abrogate transcription a signature consisting of H3K4me3 and DNaseI hyper-sensitivity (DNase HSS) signals overlapping at the gene proximal promoter is excised through two flanking guideRNAs. <b>B)</b> Cloning strategy to obtain a pX458 CRISPR vector construct simultaneously expressing two guideRNAs driven by repeated U6 promoters for knockout of a given ncRNA gene TSS signature. The depicted insert is generated by gene synthesis.</p

Elevated NFκB p65 but not ERK1/2 activity on miR-146a and miR-155 knockout.

<p><b>A)</b> Representative FACS scatter plots showing a right-shift of 30 min LPS-stimulated (1 μg / ml) compared to mock-treated monocytes stained with phospho-p65 antibody (PE-channel). <b>B)</b> Representative histogram plots showing an increased right-shift of miR-146a and miR-155 deficient compared to control or MALAT1 deficient monocytes after 30 min LPS-stimulation (1 μg / ml) and staining with a phospho-p65 antibody (PE-channel). <b>C)</b> Fold change in phospho-p65 signal in monocytes stimulated with LPS (1 μg / ml) for 15, 30 or 100 min compared to mock-treatment (ctrl) in wild-type (WT) or the indicated ncRNA knockout (KO) cells. All fold-changes are relative to the respective WT mock control. <b>D-F)</b> Same as A-C) but with phospho-ERK1/2 staining (APC-channel). Statistical significance was determined by a one-way ANOVA test with multiple comparisons (* p<0.05).</p

Excision of H3K4me3/DNaseI signatures abolishes ncRNA expression.

<p><b>A)</b> Experimental overview. U937 monocytes are spin-lipofected with dual guideRNA CRISPR constructs and clonally expanded after single cell sorting of cells positive for GFP (co-expressed from the CRISPR vector). <b>B)</b> Genomic PCR showing homozygous deletion of the proximal promoter elements identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193066#pone.0193066.g002" target="_blank">Fig 2</a> of the MALAT1, miR155, miR146a genes after clonal expansion of monocytes transfected with the respective dual guideRNA CRISPR constructs (KO). Transfection of control CRISPR construct does not result in the deletion, respectively (WT). <b>C)</b> Real-time PCR analysis of IL1β mRNA expression in wild type (WT) and MALAT1, miR-146a or miR-155 TSS knockout monocytes after control-stimulation (mock) or activation with 1 μg / ml of LPS for 4h, 8h or 16h. Fold-changes compared to mock-stimulation of control cells are shown. <b>D)</b> Real-time PCR analysis of MALAT-1 expression in wild-type (WT) or in MALAT1 H3K4me3/DNaseI element deficient cell clones stimulated as in C). <b>E)</b> Northern blot with miR-146a, and miR-17 detection probes showing loss of miR-146a induction on stimulation with LPS (1 μg / ml) in monocytes deficient in the gene-proximal H3K4me3/DNaseI element (KO) but not in wild-type (WT) cell clones. <b>F)</b> Same as E) but with miR-155 gene proximal H3K4me3/DNaseI element excised and miR-155 instead of miR-146a detection.</p

Biofilm formation on human immune cells is a multicellular predation strategy of Vibrio cholerae

Biofilm formation is generally recognized as a bacterial defense mechanism against environmental threats, including antibiotics, bacteriophages, and leukocytes of the human immune system. Here, we show that for the human pathogen Vibrio cholerae, biofilm formation is not only a protective trait but also an aggressive trait to collectively predate different immune cells. We find that V. cholerae forms biofilms on the eukaryotic cell surface using an extracellular matrix comprising primarily mannose-sensitive hemagglutinin pili, toxin-coregulated pili, and the secreted colonization factor TcpF, which differs from the matrix composition of biofilms on other surfaces. These biofilms encase immune cells and establish a high local concentration of a secreted hemolysin to kill the immune cells before the biofilms disperse in a c-di-GMP-dependent manner. Together, these results uncover how bacteria employ biofilm formation as a multicellular strategy to invert the typical relationship between human immune cells as the hunters and bacteria as the hunted