The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused the current worldwide pandemic and the associated coronavirus disease 2019 with potentially lethal outcome. Although... Show moreThe severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused the current worldwide pandemic and the associated coronavirus disease 2019 with potentially lethal outcome. Although effective vaccines strongly contributed to reduce disease severity, establishing a toolbox to control current and newly emerging coronaviruses of epidemic concern requires the development of novel therapeutic compounds, to treat severely infected individuals and to prevent virus transmission. Here we present a therapeutic strategy targeting the SARS-CoV-2 RNA genome using antisense oligonucleotides (ASOs). We demonstrate that selected locked nucleic acid gapmers have the potency to reduce the in vitro intracellular viral load by up to 96%. Our promising results strongly support the case for further development of our preselected ASOs as therapeutic or prophylactic antiviral agents. Show less
In response to inflammatory cytokines and chemokines, monocytes differentiate into macrophages. Comprehensive analysis of gene expression regulation of neuronal guidance cue (NGC) ligands and... Show moreIn response to inflammatory cytokines and chemokines, monocytes differentiate into macrophages. Comprehensive analysis of gene expression regulation of neuronal guidance cue (NGC) ligands and receptors in the monocyte-to-macrophage differentiation process is not available yet. We performed transcriptome profiling in both human primary PBMCs/PBMC-derived macrophages and THP-1 cells/THP-1-macrophages using microarray or RNA sequencing methods. Pathway analysis showed that the axonal guidance pathway is significantly regulated upon monocyte differentiation. We confirmed NGC ligands and receptors which were consistently regulated, including SEMA4D, SEMA7A, NRP1, NRP2, PLXNA1 and PLXNA3. The involvement of RNA-binding protein quaking (QKI) in the regulation of NGC expression was investigated using monocytes and macrophages from a QKI haplo-insufficient patient and her healthy sibling. This revealed a positive correlation of SEMA7A expression with QKI expression. In silico analysis of 3 ' UTRs of NGCs proposed the competitive binding of QKI to proximal microRNA targeting sites as the mechanism of QKI-dependent regulation of SEMA7A. RNA immunoprecipitation confirmed an interaction of QKI with the 3 ' UTR of SEMA7A. Loss of SEMA7A resulted in monocyte differentiation towards a more anti-inflammatory macrophage. Taken together, the axonal guidance pathway is regulated during monocyte-to-macrophage differentiation, and the regulation is in line with the necessary functional adaption for the specialised role of macrophages. Show less
Atherosclerosis is the underlying pathology in a major part of cardiovascular disease, the leading cause of mortality in developed countries. The infiltration of monocytes into the vessel walls of... Show moreAtherosclerosis is the underlying pathology in a major part of cardiovascular disease, the leading cause of mortality in developed countries. The infiltration of monocytes into the vessel walls of large arteries is a key denominator of atherogenesis, making monocytes accountable for the development of atherosclerosis. With the development of high-throughput transcriptome profiling platforms and cytometric methods for circulating cells, it is now feasible to study in-depth the predicted functional change of circulating monocytes reflected by changes of gene expression in certain pathways and correlate the changes to disease outcome. Neuroimmune guidance cues comprise a group of circulating- and cell membrane-associated signaling proteins that are progressively involved in monocyte functions. Here, we employed the CIRCULATING CELLS study cohort to classify cardiovascular disease patients and healthy individuals in relation to their expression of neuroimmune guidance cues in circulating monocytes. To cope with the complexity of human datasets featured by noisy data, nonlinearity and multidimensionality, we assessed various machine-learning methods. Of these, the linear discriminant analysis, Naive Bayesian model and stochastic gradient boost model yielded perfect or near-perfect sensibility and specificity and revealed that expression levels of the neuroimmune guidance cues SEMA6B, SEMA6D and EPHA2 in circulating monocytes were of predictive values for cardiovascular disease outcome. Show less
Bruin, R.G. de; Vogel, G.; Prins, J.; Duijs, J.M.J.G.; Bijkerk, R.; Zande, H.J.P. van der; ... ; Richard, S. 2020
In the pathophysiologic setting of acute and chronic kidney injury, the excessive activation and recruitment of blood-borne monocytes prompts their differentiation into inflammatory macrophages, a... Show moreIn the pathophysiologic setting of acute and chronic kidney injury, the excessive activation and recruitment of blood-borne monocytes prompts their differentiation into inflammatory macrophages, a process that leads to progressive glomerulosclerosis and interstitial fibrosis. Importantly, this differentiation of monocytes into macrophages requires the meticulous coordination of gene expression at both the transcriptional and post-transcriptional level. The transcriptomes of these cells are ultimately determined by RNA-binding proteins such as QUAKING (QKI), that define their pre-mRNA splicing and mRNA transcript patterns. Using two mouse models, namely (1) quaking viable mice (qk(v)) and (2) the conditional deletion in the myeloid cell lineage using the lysozyme 2-Cre (QKI(FL/FL;LysM-Cre) mice), we demonstrate that the abrogation of QKI expression in the myeloid cell lineage reduces macrophage infiltration following kidney injury induced by unilateral urethral obstruction (UUO). The qk(v) and QKI(FL/FL;LysM-Cre) mice both showed significant diminished interstitial collagen deposition and fibrosis in the UUO-damaged kidney, as compared to wild-type littermates. We show that macrophages isolated from QKI(FL/FL;LysM-Cre) mice are associated with defects in pre-mRNA splicing. Our findings demonstrate that reduced expression of the alternative splice regulator QKI in the cells of myeloid lineage attenuates renal interstitial fibrosis, suggesting that inhibition of this splice regulator may be of therapeutic value for certain kidney diseases. Show less
BACKGROUND:Treatment of inflammatory kidney diseases with systemic high-dose glucocorticoids (GCs) has severe side effects. Liposomal encapsulation could facilitate local delivery of GCs to the... Show moreBACKGROUND:Treatment of inflammatory kidney diseases with systemic high-dose glucocorticoids (GCs) has severe side effects. Liposomal encapsulation could facilitate local delivery of GCs to the inflamed kidney, as liposomes encapsulate their payload until extravasation at sites of inflammation, potentially resulting in local bioactivity. Our aim was to evaluate the ability of liposomes to accumulate locally after renal ischaemia-reperfusion injury in the rat and to study its effect on macrophages.METHODS:In vitro, human macrophages were incubated with fluorescent liposomes, liposomal prednisolone, prednisolone, empty liposomes or saline. Uptake was studied microscopically and treatment effect was assessed by interkeukin 6 (IL-6) enzyme-linked immunosorbent assay. The mechanism of action was evaluated by analysing GC receptor activation by microscopy and quantitative polymerase chain reaction (qPCR). In vivo, rats were subjected to ischaemia-reperfusion injury and were injected intravenously with fluorescent liposomes, liposomal prednisolone, prednisolone, empty liposomes or saline. Uptake was measured by the FLARE camera and the treatment effect by immunohistochemistry for myeloid cells and qPCR for inflammatory markers.RESULTS:In vitro, macrophages internalized liposomes after 8 hours. Prednisolone or liposomal prednisolone treatment reduced IL-6 production and both compounds induced translocation of the GC receptor to the nucleus and upregulation of PER1 messenger RNA (mRNA), indicating a similar mechanism of action. In vivo, fluorescent liposomes accumulated in the inflamed kidney. Liposomal prednisolone treatment increased the presence of ED2-positive anti-inflammatory macrophages and both prednisolone and liposomal prednisolone reduced monocyte chemoattractant protein-1 (MCP-1) mRNA production, indicating a reduced pro-inflammatory profile in the kidney.CONCLUSIONS:Liposomal encapsulation is a promising strategy for local delivery of glucocorticoids to the inflamed kidney. Show less
Florijn, B.; Bijkerk, R.; Veer, E.P. van der; Zonneveld, A.J. van 2017
CVD is the primary cause of death among men and women worldwide. Nevertheless, our comprehension of how CVD progresses in women and elicits clinical outcomes is lacking, leading CVD to be under... Show moreCVD is the primary cause of death among men and women worldwide. Nevertheless, our comprehension of how CVD progresses in women and elicits clinical outcomes is lacking, leading CVD to be under-diagnosed and under-treated in women. A clear example of this differential presentation of CVD pathophysiologies in females is the strikingly higher prevalence of heart failure with preserved ejection fraction (HFpEF). Women with a history of preeclampsia, or those that present co-morbidities such as obesity, hypertension and diabetes mellitus are at increased risk to develop HFpEF. Long understood to be a critical CVD risk factor, our understanding of how gender differentially impacts the development of CVD has been greatly expanded by extensive genomic and transcriptomic studies. These studies uncovered a pivotal role for differential miRNA expression in response to systemic inflammation, where their coordinated expression forms a post-transcriptional regulatory network that instigates microcirculation defects. Importantly, the potential sex-biased expression of given miRNAs may explain sex-specific cardiovascular pathophysiologies in women, such as HFpEF. Sex-biased miRNAs are regulated by estrogen (E2) in their transcription and processing or are expressed from loci on the X-chromosome due to incomplete X-chromosome inactivation. Interestingly, while E2-induced miRNAs predominantly appear to serve protective functions, it could be argued that many X-linked miRNAs have been found to challenge microvascular and myocardial integrity. Therefore, menopausal E2 deficiency, resulting in protective miRNA loss, and the augmentation of X-linked miRNA expression, may well contribute to the molecular mechanisms that underlie the female-specific cardiovascular aetiology in HFpEF. Show less
A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to... Show moreA hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce transcriptional changes associated with macrophage phenotype, but posttranscriptional control of human macrophage differentiation is less well understood. Here we show that expression levels of the RNA-binding protein Quaking (QKI) are low in monocytes and early human atherosclerotic lesions, but are abundant in macrophages of advanced plaques. Depletion of QKI protein impairs monocyte adhesion, migration, differentiation into macrophages and foam cell formation in vitro and in vivo. RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal striking changes in QKI-dependent messenger RNA levels and splicing of RNA transcripts. The biological importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in posttranscriptionally guiding macrophage identity and function. Show less
