Biology subjects

Moerland, P. D.

Publications and source records attributed to Moerland, P. D..

10 recordsLinked to original sources

bioRxiv2026

Single-cell characterization of 3D CLL spheroid model that recapitulates lymph node microenvironment-driven phenotypes and in vivo drug responses to ibrutinib

Lymph node (LN) microenvironmental signals promote chronic lymphocytic leukemia (CLL) cell survival, proliferation, and treatment resistance. We previously developed a 3D culture model that recapitulates key features of this protective LN niche. Using single-cell RNA sequencing of ex vivo paired peripheral blood and LN samples, together with matched patient-derived CLL spheroids, we investigated how microenvironmental cues shape CLL phenotypes and their response to ibrutinib. A total of 3 functional CLL states (resting, activated, proliferating) were identified, with expanded populations of activated and proliferating subsets in 3D cultures. After 5 days of in vitro ibrutinib treatment, CLL cells shifted toward a resting phenotype, reflecting published responses in patients at 6 months. Activated CLL cells showed relative resistance to ibrutinib, associated with TLR9-MALT1-MYC pathway activation. The 3D model recapitulated tissue-specific T cell phenotypes observed in patients, and computational analyses predicted several T-to-CLL cell-mediated interactions supporting the LN niche. Collectively, these data reveal distinct ibrutinib responses among CLL subsets and establish the 3D model as a physiologically relevant platform.

cancer biology↗

bioRxiv2025

Systematic evaluation of robustness to cell type mismatch of deconvolution methods for spatial transcriptomics data

Sequencing-based spatial transcriptomics (ST) approaches preserve spatial information but with limited cellular resolution, whereas single-cell RNA-sequencing (scRNA-seq) techniques provide single-cell resolution but lose spatial context during tissue dissociation. Given these complementary strengths, computational tools have been developed to combine scRNA-seq and ST data. These methods use deconvolution techniques to identify cell types and estimate their proportions at each spatial location in ST data, using scRNA-seq reference data. However, these methods are sensitive to missing cell types in the scRNA-seq reference, a problem known as cell type mismatch. Using two reference datasets, we performed extensive simulations to systematically evaluate the robustness to cell type mismatch of six deconvolution methods (CARD, cell2location, RCTD, Seurat, SPOTlight, Stereoscope) tailored for ST data, and two designed for bulk RNA-seq data (MuSiC, SCDC). At baseline, that is, with no cell types missing from the reference datasets, cell2location showed the strongest performance, while Seurat performed the worst. By simulating different cell type mismatch scenarios, we found that the performance of deconvolution methods decreases proportionally to the number of cell types missing from the reference. Moreover, compared to baseline, for most methods the relative decrease in performance is similar. Additionally, methods that perform well at baseline tend to assign the proportions of a missing cell type to the transcriptionally most similar cell types present in the reference data. Our results highlight the adverse effects of cell type mismatch on the performance of deconvolution methods for ST data and stress the need for more robust approaches to this issue.

bioinformatics↗

bioRxiv2024

Infiltrative classical monocyte-derived and SPP1 lipid-associated macrophages mediate inflammation and fibrosis in ANCA-associated glomerulonephritis

BackgroundKidney macrophage infiltration is a histological hallmark of vasculitic lesions and is strongly linked to disease activity in anti-neutrophil cytoplasmic antibodies (ANCA)-associated glomerulonephritis (AGN). The precise mechanisms by which kidney macrophages influence local inflammation and long-term damage remain largely unknown. MethodsHere, we investigate kidney macrophage diversity using single-cell transcriptome analysis of 25,485 freshly retrieved unfrozen, high-quality kidney CD45+ immune cells from five AGN patients, a lupus nephritis and nephrectomy control. Detailed subclustering of myeloid cells was performed to identify disease-specific macrophage subtypes. Next, transcriptome differences between macrophage subsets and disease serotypes were assessed. Findings were validated by immunostainings of an extended cohort of kidney biopsies and flow cytometric analysis of peripheral blood monocytes. ResultsFour main macrophage subsets were identified, including a classical monocyte-derived macrophage (MDM) subset expressing a chemotactic (CXCL2, CXCL3, CXCL8, CCL3) and pro-inflammatory (IL1{beta}, TNF) set of markers and a osteopontin/SPP1+ lipid-associated macrophage (SPP1 LAMs) subtype exhibiting distinctive upregulation of fibrotic genesets. AGN samples revealed a markedly increased proportion of CD163+ macrophages, predominantly composed of classical MDMs, accompanied by resident-like C1Q macrophages, and SPP1 LAMs. An analogous trend was observed in the expansion of peripheral blood classical monocytes during active disease. The proteinase 3 (PR3)-AGN subtype exhibited heightened classical MDM infiltration and markers of acute inflammation, while interferon signaling and markers of chronicity were reduced compared to myeloperoxidase (MPO)-AGN. ConclusionsOur findings highlight the expression of inflammatory and fibrotic genes by kidney macrophage subsets in AGN. Classical monocyte dysregulation might contribute to inflammation in the pathogenesis of AGN. Targeting these specific monocyte/macrophage subsets may potentially control the inflammatory cascade and attenuate resulting fibrosis in AGN and kidney disease in general. Key points- Classical monocyte-derived macrophages are predominant in ANCA-associated glomerulonephritis and exhibit chemotactic and pro-inflammatory markers - Osteopontin/SPP1+ lipid-associated macrophages (SPP1 LAMs) show distinctive upregulation of fibrotic genesets - Understanding of the macrophage immune response supports exploration of macrophage-directed therapies for the treatment of autoimmune kidney diseases

immunology↗

bioRxiv2024

Cardiovascular risk in ANCA-associated vasculitis: monocyte phenotyping reveals distinctive signatures between serological subsets

ObjectivesAnti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitides (AAV) is associated with an increased cardiovascular risk, particularly the myeloperoxidase AAV serotype (MPO-AAV). Distinct alterations in monocyte phenotypes may cause accelerated atherosclerotic disease in AAV. MethodsA cohort including 43 AAV patients and 19 healthy controls were included for downstream analyses. Extensive phenotyping of monocytes and monocyte-derived macrophages was performed using bulk RNA-sequencing and flow cytometry. An in vitro transendothelial migration assay reflecting intrinsic adhesive and migratory capacities of monocytes was employed. Subsequent sub-analyses were performed to investigate differences between serological subtypes. ResultsMonocyte subset analysis showed increased classical monocytes during active disease, whereas non-classical monocytes were decreased. RNA-sequencing revealed upregulation of distinct inflammatory pathways and lipid metabolism-related markers in monocytes of active AAV patients. No differences were detected in the intrinsic monocyte adhesion and migration capacity. Monocytes of MPO-AAV patients in remission expressed genes related to inflammation, coagulation, platelet-binding and interferon signalling, whereas the expression of chemokine receptors indicative of acute inflammation and monocyte extravasation (i.e., CCR2 and CCR5) was increased in monocytes of proteinase-3(PR3)-AAV patients. During active disease, PR3-AAV was linked with elevated serum CRP and increased platelet counts compared to MPO-AAV. ConclusionThese findings highlight changes in monocyte subset composition and activation, but not in the intrinsic migration capacity of AAV monocytes. MPO-AAV monocytes are associated with sustained upregulation of inflammatory genes, whereas PR3-AAV monocytes exhibit chemokine receptor upregulation. These molecular changes may play a role in elevating cardiovascular risk as well as in the underlying pathophysiology of AAV. Key messages- Monocytes are activated during active ANCA-associated vasculitis (AAV) and upregulate lipid metabolism-related markers - AAV monocytes have a normal intrinsic adhesion and migration capacity, although overall monocyte migration likely rises by other mechanisms - The two serological subsets MPO-AAV and PR3-AAV exhibit differences in monocyte activation and chemokine receptor expression

immunology↗

bioRxiv2023

Identification and mapping of human lymph node stromal cell subsets by combining single-cell RNA sequencing with spatial transcriptomics.

Lymph node stromal cells (LNSCs) have a crucial immunomodulatory function, but their heterogeneity in human is incompletely understood. Here, we report the single cell RNA sequencing (scRNA-seq) of 12000 LNSCs isolated from a human lymph node (LN). This study comprehensively defines the gene signatures of 10 fibroblast subtypes: CCL21+SC, CCL19+SC, CD34+CXCL14+SC, pericytes, DES+SC, LAMP5+SC, NR4A1+BCAM+ SC, HLA-DR+SC, SEPT4+SC and GLDN+SC. To explore the heterogeneous stromal compartment within the complex LN tissue architecture, we integrated the scRNA-seq profiles of the identified LNSC subsets with a publicly available human spatial transcriptomic LN dataset and predicted their location within the complex LN tissue architecture. Each LNSC subtype was spatially restricted to specific LN regions, indicating different LNSC-lymphocyte interactions which was further investigated using NicheNet. The positioning of distinct LNSC subtypes in different LN regions sets the stage for future research on the relationship between LNSC-specific niches and immunomodulatory function during health and disease.

cell biology↗

bioRxiv2021

A public antibody class recognizes a novel S2 epitope exposed on open conformations of SARS-CoV-2 spike

Delineating the origins and properties of antibodies elicited by SARS-CoV-2 infection and vaccination is critical for understanding their benefits and potential shortcomings. Therefore, we investigated the SARS-CoV-2 spike (S)-reactive B cell repertoire in unexposed individuals by flow cytometry and single-cell sequencing. We found that [~]82% of SARS-CoV-2 S-reactive B cells show a naive phenotype, which represents an unusually high fraction of total human naive B cells ([~]0.1%). Approximately 10% of these naive S-reactive B cells shared an IGHV1-69/IGKV3-11 B cell receptor pairing, an enrichment of 18-fold compared to the complete naive repertoire. A proportion of memory B cells, comprising switched ([~]0.05%) and unswitched B cells ([~]0.04%), was also reactive with S and some of these cells were reactive with ADAMTS13, which is associated with thrombotic thrombocytopenia. Following SARS-CoV-2 infection, we report an average 37-fold enrichment of IGHV1-69/IGKV3-11 B cell receptor pairing in the S-reactive memory B cells compared to the unselected memory repertoire. This class of B cells targets a previously undefined non-neutralizing epitope on the S2 subunit that becomes exposed on S proteins used in approved vaccines when they transition away from the native pre-fusion state because of instability. These findings can help guide the improvement of SARS-CoV-2 vaccines.

immunology↗

bioRxiv2020

4sUDRB-sequencing for genome-wide transcription bursting quantification in breast cancer cells

Epigenetics maintains cell-identity specific gene-expression patterns. However, within a population of isogenic cells of the same identity, a substantial variability in gene expression and responsiveness is still observed. Transcription bursting is a substantial source of this gene-expression variability or noise, contributing to phenotypic heterogeneity and potentially driving both physiological and pathological processes such as differentiation or tumorigenesis and drug resistance. Identification of transcription-bursting dynamics at a genome-wide scale has been restricted to inferring bursts in mRNA production computationally from the heterogeneity of mRNA levels in single cell transcriptomic data. Systematic characterisation of the genomic and epigenetic chromatin context of genes with defined transcription bursting behaviour has been incomplete. Here, we measured the bursting of transcription itself by genome-wide nascent RNA sequencing of breast cancer MCF-7 cells upon synchronisation of transcription with a transcription elongation inhibitor and by calibration using live cell imaging of nascent PP7-tagged GREB1 transcription. Comparing across the entire genome, we find transcription bursting to be ubiquitous, with burst sizes of up to 160 transcripts. Transcription bursting is strongly correlated with steady state gene expression between genes, whereas both burst frequency and nascent transcript degradation only correlate weakly. Individual genes deviate strongly from this trend and engage both in anomalous burst size and frequency. We find that the presence of the TATA box or Inr sequence within gene promoters are significantly associated with a larger burst size, as are promoter-associated YY1 and E2F1 transcription-factor binding motifs. Enrichment of the transcription start site with epigenetic marks such as H3K79me2 and H3K27ac is also strongly associated with the transcription burst size. Finally, we show that in these MCF-7 breast-cancer cells, genes with a larger transcription burst size exhibit a larger immediate transcriptional response following endocrine drug treatment. Our genome-wide transcription-bursting analysis method paves the way to elucidate the dynamic role of epigenetic regulation on dynamic transcription in pathophysiology.

molecular biology↗

bioRxiv2020

Core Circadian Clock Genes Per1 and Per2 regulate the Rhythm in Photoreceptor Outer Segment Phagocytosis

Retinal photoreceptors undergo daily renewal of their distal outer segments, a process indispensable for maintaining retinal health. Photoreceptor Outer Segment (POS) phagocytosis occurs as a daily peak, roughly about one hour after light onset. However, the underlying cellular and molecular mechanisms which initiate this process are still unknown. Here we show that, under constant darkness, mice deficient for core circadian clock genes (Per1 and Per2), lack a daily peak in POS phagocytosis. By qPCR analysis we found that core clock genes were rhythmic over 24h in both WT and Per1, Per2 double mutant whole retinas. More precise transcriptomics analysis of laser capture microdissected WT photoreceptors revealed no differentially, expressed genes between time-points preceding and during the peak of POS phagocytosis. By contrast, we found that microdissected WT retinal pigment epithelium (RPE) had a number of genes that were differentially expressed at the peak phagocytic time-point compared to adjacent ones. We also found a number of differentially expressed genes in Per1, Per2 double mutant RPE compared to WT ones at the peak phagocytic time-point. Finally, based on STRING analysis we found a group of interacting genes which potentially drive POS phagocytosis in the RPE. This potential pathway consists of genes such as: Pacsin1, Syp, Camk2b and Camk2d among others. Our findings indicate that Per1 and Per2 are necessary clock components for driving POS phagocytosis and suggest that this process is transcriptionally driven by the RPE. DeclarationsO_ST_ABSFundingC_ST_ABSThis project has been funded with support from the NeuroTime Erasmus+ grant (European Union), Rotterdamse Stichting Blindenbelangen (Netherlands), Nelly Reef fund (Netherlands), Stichting voor Ooglijders (Netherlands), Stichting tot Verbetering van het Lot der Blinden (Netherlands) and Retina France (France). Conflicts of interest/Competing interestsThe authors declare no competing interests. Availability of data and materialData supporting the conclusions of this article are included within the article and are available from the corresponding authors on reasonable request. Code availabilityThe R code for analysis is available from the corresponding authors on reasonable request. Ethics approvalAll experimental procedures were performed in accordance with the Association for Research in Vision and Ophthalmology Statement on Use of Animals in Ophthalmic and Vision Research, as well as with the European Union Directive (2010/63/EU). Consent to participateNot applicable Consent for publicationAll authors read and approve of the contents of this manuscript. Author contributionsN.M. performed experiments, analysis, prepared figures, wrote the manuscript and obtained funding. O.A.-H.H. performed experiments, data analysis, prepared figures and obtained funding. P.D.M. and A.J. performed bioinformatics analysis and edited the manuscript. U.B., J.B.t.B. and C.S. provided technical assistance, performed experiments, prepared figures and edited the manuscript. D.H., A.A.B. and M.-P.F.-S. conceptualized and directed the project, obtained funding, provided resources, performed analysis and edited the manuscript.

neuroscience↗

bioRxiv2020

Time-resolved profiling reveals ATF3 as a novel mediator of endocrine resistance in breast cancer

Breast cancer is one of the leading causes of death for women worldwide. Patients whose tumors express Estrogen Receptor (ER) account for around 70% of cases and are mostly treated with targeted endocrine therapy. However, 40% of these tumors eventually relapse due to resistance development and further treatment of these patients is highly ineffective. In this study we profiled the early phases of the resistance development process to uncover drivers of this phenomenon. Time-resolved analysis revealed that ATF3, a member of the ATF/CREB family of transcription factors, acts as a novel regulator of the response to therapy via rewiring of central signaling processes towards the adaptation to endocrine treatment. ATF3 was found to be essential in controlling crucial processes such as proliferation, cell cycle and apoptosis during the early response to treatment through the regulation of MAPK/AKT signaling pathways. Its essential role was confirmed in vivo in a mouse model and elevated expression of ATF3 was verified in patient datasets, adding clinical relevance to our findings. This study proposes ATF3 as a novel mediator of endocrine resistance development in breast cancer and elucidates its role in the regulation of downstream pathways activities.

cancer biology↗

bioRxiv2019

Candidate methylation sites associated with endocrine therapy resistance in the TCGA ER+/HER2- breast cancer cohort

BackgroundEstrogen receptor (ER) positive breast cancer is often effectively treated with drugs that inhibit ER signaling, i.e., tamoxifen (TAM) and aromatase inhibitors (AIs). However, about 30% of ER+ breast cancer patients develop resistance to therapy leading to tumour recurrence. Changes in the methylation profile have been implicated as one of the mechanisms through which therapy resistance develops. Therefore, we aimed to identify methylation loci associated with endocrine therapy resistance. MethodsWe used genome-wide DNA methylation profiles of primary ER+ tumors from The Cancer Genome Atlas in combination with curated data on survival and treatment to predict development of endocrine resistance. Association of individual DNA methylation markers with survival was assessed using Cox proportional hazards models in a cohort of ER+/HER2- tumours (N=552) and two sub-cohorts corresponding to the endocrine treatment (AIs or TAM) that patients received (N=210 and N=172, respectively). Models were adjusted for clinical variables tumour stage, age, AI treatment and luminal subtype. We also identified signatures of multiple methylation loci associated with survival using Cox proportional hazards models with elastic net regularization. Individual markers and multivariable signatures were compared with DNA methylation profiles generated in a time course experiment using the T47D ER+ breast cancer cell line treated with tamoxifen or deprived from estrogen. ResultsWe identified 132, 9 and 1 CpGs for which DNA methylation is significantly associated with survival in the ER+/HER2-, TAM and AI cohorts respectively. Corresponding multi-locus signatures consisted of 171, 50 and 160 CpGs and showed a large overlap with the corresponding single-locus signatures. Single-locus signatures for the ER+/HER2- and TAM cohorts were conserved among the loci that were differentially methylated in endocrine-resistant T47D cells. Similarly, multi-locus signatures for the ER+/HER2- and AI cohorts were conserved in endocrine-resistant T47D cells. ConclusionsWe identified individual and multivariable DNA methylation markers associated with therapy resistance independently of luminal status. Our results suggest that these markers identified from primary tumours and prior to any endocrine treatment are associated with development of endocrine resistance.

bioinformatics↗