Acquisition of epithelial plasticity in human chronic liver disease.

For many adult human organs, tissue regeneration during chronic disease remains a controversial subject. Regenerative processes are easily observed in animal models, and their underlying mechanisms are becoming well characterized1-4, but technical challenges and ethical aspects are limiting the validation of these results in humans. We decided to address this difficulty with respect to the liver. This organ displays the remarkable ability to regenerate after acute injury, although liver regeneration in the context of recurring injury remains to be fully demonstrated. Here we performed single-nucleus RNA sequencing (snRNA-seq) on 47 liver biopsies from patients with different stages of metabolic dysfunction-associated steatotic liver disease to establish a cellular map of the liver during disease progression. We then combined these single-cell-level data with advanced 3D imaging to reveal profound changes in the liver architecture. Hepatocytes lose their zonation and considerable reorganization of the biliary tree takes place. More importantly, our study uncovers transdifferentiation events that occur between hepatocytes and cholangiocytes without the presence of adult stem cells or developmental progenitor activation. Detailed analyses and functional validations using cholangiocyte organoids confirm the importance of the PI3K-AKT-mTOR pathway in this process, thereby connecting this acquisition of plasticity to insulin signalling. Together, our data indicate that chronic injury creates an environment that induces cellular plasticity in human organs, and understanding the underlying mechanisms of this process could open new therapeutic avenues in the management of chronic diseases.

Human surface ectoderm and amniotic ectoderm are sequentially specified according to cellular density.

Mechanisms specifying amniotic ectoderm and surface ectoderm are unresolved in humans due to their close similarities in expression patterns and signal requirements. This lack of knowledge hinders the development of protocols to accurately model human embryogenesis. Here, we developed a human pluripotent stem cell model to investigate the divergence between amniotic and surface ectoderms. In the established culture system, cells differentiated into functional amnioblast-like cells. Single-cell RNA sequencing analyses of amnioblast differentiation revealed an intermediate cell state with enhanced surface ectoderm gene expression. Furthermore, when the differentiation started at the confluent condition, cells retained the expression profile of surface ectoderm. Collectively, we propose that human amniotic ectoderm and surface ectoderm are specified along a common nonneural ectoderm trajectory based on cell density. Our culture system also generated extraembryonic mesoderm-like cells from the primed pluripotent state. Together, this study provides an integrative understanding of the human nonneural ectoderm development and a model for embryonic and extraembryonic human development around gastrulation.

Proinflammatory cytokines driving cardiotoxicity in COVID-19.

AIMS: Cardiac involvement is common in patients hospitalized with COVID-19 and correlates with an adverse disease trajectory. While cardiac injury has been attributed to direct viral cytotoxicity, serum-induced cardiotoxicity secondary to serological hyperinflammation constitutes a potentially amenable mechanism that remains largely unexplored. METHODS AND RESULTS: To investigate serological drivers of cardiotoxicity in COVID-19 we have established a robust bioassay that assessed the effects of serum from COVID-19 confirmed patients on human embryonic stem cell (hESC)-derived cardiomyocytes. We demonstrate that serum from COVID-19 positive patients significantly reduced cardiomyocyte viability independent of viral transduction, an effect that was also seen in non-COVID-19 acute respiratory distress syndrome (ARDS). Serum from patients with greater disease severity led to worse cardiomyocyte viability and this significantly correlated with levels of key inflammatory cytokines, including IL-6, TNF-α, IL1-ß, IL-10, CRP, and neutrophil to lymphocyte ratio with a specific reduction of CD4+ and CD8+ cells. Combinatorial blockade of IL-6 and TNF-α partly rescued the phenotype and preserved cardiomyocyte viability and function. Bulk RNA sequencing of serum-treated cardiomyocytes elucidated specific pathways involved in the COVID-19 response impacting cardiomyocyte viability, structure, and function. The observed effects of serum-induced cytotoxicity were cell-type selective as serum exposure did not adversely affect microvascular endothelial cell viability but resulted in endothelial activation and a procoagulant state. CONCLUSION: These results provide direct evidence that inflammatory cytokines are at least in part responsible for the cardiovascular damage seen in COVID-19 and characterise the downstream activated pathways in human cardiomyocytes. The serum signature of patients with severe disease indicates possible targets for therapeutic intervention.

Multiparameter prediction of myeloid neoplasia risk.

The myeloid neoplasms encompass acute myeloid leukemia, myelodysplastic syndromes and myeloproliferative neoplasms. Most cases arise from the shared ancestor of clonal hematopoiesis (CH). Here we analyze data from 454,340 UK Biobank participants, of whom 1,808 developed a myeloid neoplasm 0-15 years after recruitment. We describe the differences in CH mutational landscapes and hematology/biochemistry test parameters among individuals that later develop myeloid neoplasms (pre-MN) versus controls, finding that disease-specific changes are detectable years before diagnosis. By analyzing differences between 'pre-MN' and controls, we develop and validate Cox regression models quantifying the risk of progression to each myeloid neoplasm subtype. We construct 'MN-predict', a web application that generates time-dependent predictions with the input of basic blood tests and genetic data. Our study demonstrates that many individuals that develop myeloid neoplasms can be identified years in advance and provides a framework for disease-specific prognostication that will be of substantial use to researchers and physicians.

Prevalence and significance of DDX41 gene variants in the general population.

Germ line variants in the DDX41 gene have been linked to myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) development. However, the risks associated with different variants remain unknown, as do the basis of their leukemogenic properties, impact on steady-state hematopoiesis, and links to other cancers. Here, we investigate the frequency and significance of DDX41 variants in 454 792 United Kingdom Biobank (UKB) participants and identify 452 unique nonsynonymous DNA variants in 3538 (1/129) individuals. Many were novel, and the prevalence of most varied markedly by ancestry. Among the 1059 individuals with germ line pathogenic variants (DDX41-GPV) 34 developed MDS/AML (odds ratio, 12.3 vs noncarriers). Of these, 7 of 218 had start-lost, 22 of 584 had truncating, and 5 of 257 had missense (odds ratios: 12.9, 15.1, and 7.5, respectively). Using multivariate logistic regression, we found significant associations of DDX41-GPV with MDS, AML, and family history of leukemia but not lymphoma, myeloproliferative neoplasms, or other cancers. We also report that DDX41-GPV carriers do not have an increased prevalence of clonal hematopoiesis (CH). In fact, CH was significantly more common before sporadic vs DDX41-mutant MDS/AML, revealing distinct evolutionary paths. Furthermore, somatic mutation rates did not differ between sporadic and DDX41-mutant AML genomes, ruling out genomic instability as a driver of the latter. Finally, we found that higher mean red cell volume (MCV) and somatic DDX41 mutations in blood DNA identify DDX41-GPV carriers at increased MDS/AML risk. Collectively, our findings give new insights into the prevalence and cognate risks associated with DDX41 variants, as well as the clonal evolution and early detection of DDX41-mutant MDS/AML.

Clearance of senescent macrophages ameliorates tumorigenesis in KRAS-driven lung cancer.

The accumulation of senescent cells in the tumor microenvironment can drive tumorigenesis in a paracrine manner through the senescence-associated secretory phenotype (SASP). Using a new p16-FDR mouse line, we show that macrophages and endothelial cells are the predominant senescent cell types in murine KRAS-driven lung tumors. Through single cell transcriptomics, we identify a population of tumor-associated macrophages that express a unique array of pro-tumorigenic SASP factors and surface proteins and are also present in normal aged lungs. Genetic or senolytic ablation of senescent cells, or macrophage depletion, result in a significant decrease in tumor burden and increased survival in KRAS-driven lung cancer models. Moreover, we reveal the presence of macrophages with senescent features in human lung pre-malignant lesions, but not in adenocarcinomas. Taken together, our results have uncovered the important role of senescent macrophages in the initiation and progression of lung cancer, highlighting potential therapeutic avenues and cancer preventative strategies.

Patterns of Oncogene Coexpression at Single-Cell Resolution Influence Survival in Lymphoma.

Cancers often overexpress multiple clinically relevant oncogenes, but it is not known if combinations of oncogenes in cellular subpopulations within a cancer influence clinical outcomes. Using quantitative multispectral imaging of the prognostically relevant oncogenes MYC, BCL2, and BCL6 in diffuse large B-cell lymphoma (DLBCL), we show that the percentage of cells with a unique combination MYC+BCL2+BCL6- (M+2+6-) consistently predicts survival across four independent cohorts (n = 449), an effect not observed with other combinations including M+2+6+. We show that the M+2+6- percentage can be mathematically derived from quantitative measurements of the individual oncogenes and correlates with survival in IHC (n = 316) and gene expression (n = 2,521) datasets. Comparative bulk/single-cell transcriptomic analyses of DLBCL samples and MYC/BCL2/BCL6-transformed primary B cells identify molecular features, including cyclin D2 and PI3K/AKT as candidate regulators of M+2+6- unfavorable biology. Similar analyses evaluating oncogenic combinations at single-cell resolution in other cancers may facilitate an understanding of cancer evolution and therapy resistance. SIGNIFICANCE: Using single-cell-resolved multiplexed imaging, we show that selected subpopulations of cells expressing specific combinations of oncogenes influence clinical outcomes in lymphoma. We describe a probabilistic metric for the estimation of cellular oncogenic coexpression from IHC or bulk transcriptomes, with possible implications for prognostication and therapeutic target discovery in cancer. This article is highlighted in the In This Issue feature, p. 1027.

bulkAnalyseR: an accessible, interactive pipeline for analysing and sharing bulk multi-modal sequencing data.

Bulk sequencing experiments (single- and multi-omics) are essential for exploring wide-ranging biological questions. To facilitate interactive, exploratory tasks, coupled with the sharing of easily accessible information, we present bulkAnalyseR, a package integrating state-of-the-art approaches using an expression matrix as the starting point (pre-processing functions are available as part of the package). Static summary images are replaced with interactive panels illustrating quality-checking, differential expression analysis (with noise detection) and biological interpretation (enrichment analyses, identification of expression patterns, followed by inference and comparison of regulatory interactions). bulkAnalyseR can handle different modalities, facilitating robust integration and comparison of cis-, trans- and customised regulatory networks.

Microfluidic antibody profiling after repeated SARS-CoV-2 vaccination links antibody affinity and concentration to impaired immunity and variant escape in patients on anti-CD20 therapy.

Background: Patients with autoimmune/inflammatory conditions on anti-CD20 therapies, such as rituximab, have suboptimal humoral responses to vaccination and are vulnerable to poorer clinical outcomes following SARS-CoV-2 infection. We aimed to examine how the fundamental parameters of antibody responses, namely, affinity and concentration, shape the quality of humoral immunity after vaccination in these patients. Methods: We performed in-depth antibody characterisation in sera collected 4 to 6 weeks after each of three vaccine doses to wild-type (WT) SARS-CoV-2 in rituximab-treated primary vasculitis patients (n = 14) using Luminex and pseudovirus neutralisation assays, whereas we used a novel microfluidic-based immunoassay to quantify polyclonal antibody affinity and concentration against both WT and Omicron (B.1.1.529) variants. We performed comparative antibody profiling at equivalent timepoints in healthy individuals after three antigenic exposures to WT SARS-CoV-2 (one infection and two vaccinations; n = 15) and in convalescent patients after WT SARS-CoV-2 infection (n = 30). Results: Rituximab-treated patients had lower antibody levels and neutralisation titres against both WT and Omicron SARS-CoV-2 variants compared to healthy individuals. Neutralisation capacity was weaker against Omicron versus WT both in rituximab-treated patients and in healthy individuals. In the rituximab cohort, this was driven by lower antibody affinity against Omicron versus WT [median (range) KD: 21.6 (9.7-38.8) nM vs. 4.6 (2.3-44.8) nM, p = 0.0004]. By contrast, healthy individuals with hybrid immunity produced a broader antibody response, a subset of which recognised Omicron with higher affinity than antibodies in rituximab-treated patients [median (range) KD: 1.05 (0.45-1.84) nM vs. 20.25 (13.2-38.8) nM, p = 0.0002], underpinning the stronger serum neutralisation capacity against Omicron in the former group. Rituximab-treated patients had similar anti-WT antibody levels and neutralisation titres to unvaccinated convalescent individuals, despite two more exposures to SARS-CoV-2 antigen. Temporal profiling of the antibody response showed evidence of affinity maturation in healthy convalescent patients after a single SARS-CoV-2 infection, which was not observed in rituximab-treated patients, despite repeated vaccination. Discussion: Our results enrich previous observations of impaired humoral immune responses to SARS-CoV-2 in rituximab-treated patients and highlight the significance of quantitative assessment of serum antibody affinity and concentration in monitoring anti-viral immunity, viral escape, and the evolution of the humoral response.

The Sum of Two Halves May Be Different from the Whole-Effects of Splitting Sequencing Samples Across Lanes.

The advances in high-throughput sequencing (HTS) have enabled the characterisation of biological processes at an unprecedented level of detail; most hypotheses in molecular biology rely on analyses of HTS data. However, achieving increased robustness and reproducibility of results remains a main challenge. Although variability in results may be introduced at various stages, e.g., alignment, summarisation or detection of differential expression, one source of variability was systematically omitted: the sequencing design, which propagates through analyses and may introduce an additional layer of technical variation. We illustrate qualitative and quantitative differences arising from splitting samples across lanes on bulk and single-cell sequencing. For bulk mRNAseq data, we focus on differential expression and enrichment analyses; for bulk ChIPseq data, we investigate the effect on peak calling and the peaks' properties. At the single-cell level, we concentrate on identifying cell subpopulations. We rely on markers used for assigning cell identities; both smartSeq and 10× data are presented. The observed reduction in the number of unique sequenced fragments limits the level of detail on which the different prediction approaches depend. Furthermore, the sequencing stochasticity adds in a weighting bias corroborated with variable sequencing depths and (yet unexplained) sequencing bias. Subsequently, we observe an overall reduction in sequencing complexity and a distortion in the biological signal across technologies, experimental contexts, organisms and tissues.

Viral transduction of primary human lymphoma B cells reveals mechanisms of NOTCH-mediated immune escape.

Hotspot mutations in the PEST-domain of NOTCH1 and NOTCH2 are recurrently identified in B cell malignancies. To address how NOTCH-mutations contribute to a dismal prognosis, we have generated isogenic primary human tumor cells from patients with Chronic Lymphocytic Leukemia (CLL) and Mantle Cell Lymphoma (MCL), differing only in their expression of the intracellular domain (ICD) of NOTCH1 or NOTCH2. Our data demonstrate that both NOTCH-paralogs facilitate immune-escape of malignant B cells by up-regulating PD-L1, partly dependent on autocrine interferon-γ signaling. In addition, NOTCH-activation causes silencing of the entire HLA-class II locus via epigenetic regulation of the transcriptional co-activator CIITA. Notably, while NOTCH1 and NOTCH2 govern similar transcriptional programs, disease-specific differences in their expression levels can favor paralog-specific selection. Importantly, NOTCH-ICD also strongly down-regulates the expression of CD19, possibly limiting the effectiveness of immune-therapies. These NOTCH-mediated immune escape mechanisms are associated with the expansion of exhausted CD8+ T cells in vivo.

Myeloid cell interferon secretion restricts Zika flavivirus infection of developing and malignant human neural progenitor cells.

Zika virus (ZIKV) can infect human developing brain (HDB) progenitors resulting in epidemic microcephaly, whereas analogous cellular tropism offers treatment potential for the adult brain cancer, glioblastoma (GBM). We compared productive ZIKV infection in HDB and GBM primary tissue explants that both contain SOX2+ neural progenitors. Strikingly, although the HDB proved uniformly vulnerable to ZIKV infection, GBM was more refractory, and this correlated with an innate immune expression signature. Indeed, GBM-derived CD11b+ microglia/macrophages were necessary and sufficient to protect progenitors against ZIKV infection in a non-cell autonomous manner. Using SOX2+ GBM cell lines, we found that CD11b+-conditioned medium containing type 1 interferon beta (IFNß) promoted progenitor resistance to ZIKV, whereas inhibition of JAK1/2 signaling restored productive infection. Additionally, CD11b+ conditioned medium, and IFNß treatment rendered HDB progenitor lines and explants refractory to ZIKV. These findings provide insight into neuroprotection for HDB progenitors as well as enhanced GBM oncolytic therapies.

Host Respiratory Transcriptome Signature Associated with Poor Outcome in Children with Influenza-Staphylococcus aureus Pneumonia.

Respiratory coinfection of influenza with Staphylococcus aureus often causes severe disease; methicillin-resistant S. aureus (MRSA) coinfection is frequently fatal. Understanding disease pathogenesis may inform therapies. We aimed to identify host and pathogen transcriptomic (messenger RNA) signatures from the respiratory compartment of pediatric patients critically ill with influenza-S. aureus coinfection (ISAC), signatures that predict worse outcomes. Messenger RNA extracted from endotracheal aspirate samples was evaluated for S. aureus and host transcriptomic biosignatures. Influenza-MRSA outcomes were worse, but of 190 S. aureus virulence-associated genes, 6 were differentially expressed between MRSA-coinfected versus methicillin-susceptible S. aureus-coinfected patients, and none discriminated outcome. Host gene expression in patients with ISAC was compared with that in patients with influenza infection alone. Patients with poor clinical outcomes (death or prolonged multiorgan dysfunction) had relatively reduced expression of interferons and down-regulation of interferon γ-induced immune cell chemoattractants CXCL10 and CXCL11. In ISAC, airway host but not pathogen gene expression profiles predicted worse clinical outcomes.

Mapping the biogenesis of forward programmed megakaryocytes from induced pluripotent stem cells.

Platelet deficiency, known as thrombocytopenia, can cause hemorrhage and is treated with platelet transfusions. We developed a system for the production of platelet precursor cells, megakaryocytes, from pluripotent stem cells. These cultures can be maintained for >100 days, implying culture renewal by megakaryocyte progenitors (MKPs). However, it is unclear whether the MKP state in vitro mirrors the state in vivo, and MKPs cannot be purified using conventional surface markers. We performed single-cell RNA sequencing throughout in vitro differentiation and mapped each state to its equivalent in vivo. This enabled the identification of five surface markers that reproducibly purify MKPs, allowing us insight into their transcriptional and epigenetic profiles. Last, we performed culture optimization, increasing MKP production. Together, this study has mapped parallels between the MKP states in vivo and in vitro and allowed the purification of MKPs, accelerating the progress of in vitro-derived transfusion products toward the clinic.

noisyR: enhancing biological signal in sequencing datasets by characterizing random technical noise.

High-throughput sequencing enables an unprecedented resolution in transcript quantification, at the cost of magnifying the impact of technical noise. The consistent reduction of random background noise to capture functionally meaningful biological signals is still challenging. Intrinsic sequencing variability introducing low-level expression variations can obscure patterns in downstream analyses. We introduce noisyR, a comprehensive noise filter to assess the variation in signal distribution and achieve an optimal information-consistency across replicates and samples; this selection also facilitates meaningful pattern recognition outside the background-noise range. noisyR is applicable to count matrices and sequencing data; it outputs sample-specific signal/noise thresholds and filtered expression matrices. We exemplify the effects of minimizing technical noise on several datasets, across various sequencing assays: coding, non-coding RNAs and interactions, at bulk and single-cell level. An immediate consequence of filtering out noise is the convergence of predictions (differential-expression calls, enrichment analyses and inference of gene regulatory networks) across different approaches.

ZAP-70 constitutively regulates gene expression and protein synthesis in chronic lymphocytic leukemia.

The expression of ZAP-70 in a subset of chronic lymphocytic leukemia (CLL) patients strongly correlates with a more aggressive clinical course, although the exact underlying mechanisms remain elusive. The ability of ZAP-70 to enhance B-cell receptor (BCR) signaling, independently of its kinase function, is considered to contribute. We used RNA-sequencing and proteomic analyses of primary cells differing only in their expression of ZAP-70 to further define how ZAP-70 increases the aggressiveness of CLL. We identified that ZAP-70 is directly required for cell survival in the absence of an overt BCR signal, which can compensate for ZAP-70 deficiency as an antiapoptotic signal. In addition, the expression of ZAP-70 regulates the transcription of factors regulating the recruitment and activation of T cells, such as CCL3, CCL4, and IL4I1. Quantitative mass spectrometry of double-cross-linked ZAP-70 complexes further demonstrated constitutive and direct protein-protein interactions between ZAP-70 and BCR-signaling components. Unexpectedly, ZAP-70 also binds to ribosomal proteins, which is not dependent on, but is further increased by, BCR stimulation. Importantly, decreased expression of ZAP-70 significantly reduced MYC expression and global protein synthesis, providing evidence that ZAP-70 contributes to translational dysregulation in CLL. In conclusion, ZAP-70 constitutively promotes cell survival, microenvironment interactions, and protein synthesis in CLL cells, likely to improve cellular fitness and to further drive disease progression.

Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Human Developmental Hematopoiesis.

Regulation of hematopoiesis during human development remains poorly defined. Here we applied single-cell RNA sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) to over 8,000 human immunophenotypic blood cells from fetal liver and bone marrow. We inferred their differentiation trajectory and identified three highly proliferative oligopotent progenitor populations downstream of hematopoietic stem cells (HSCs)/multipotent progenitors (MPPs). Along this trajectory, we observed opposing patterns of chromatin accessibility and differentiation that coincided with dynamic changes in the activity of distinct lineage-specific transcription factors. Integrative analysis of chromatin accessibility and gene expression revealed extensive epigenetic but not transcriptional priming of HSCs/MPPs prior to their lineage commitment. Finally, we refined and functionally validated the sorting strategy for the HSCs/MPPs and achieved around 90% enrichment. Our study provides a useful framework for future investigation of human developmental hematopoiesis in the context of blood pathologies and regenerative medicine.

Gene expression during larval caste determination and differentiation in intermediately eusocial bumblebees, and a comparative analysis with advanced eusocial honeybees.

The queen-worker caste system of eusocial insects represents a prime example of developmental polyphenism (environmentally-induced phenotypic polymorphism) and is intrinsic to the evolution of advanced eusociality. However, the comparative molecular basis of larval caste determination and subsequent differentiation in the eusocial Hymenoptera remains poorly known. To address this issue within bees, we profiled caste-associated gene expression in female larvae of the intermediately eusocial bumblebee Bombus terrestris. In B. terrestris, female larvae experience a queen-dependent period during which their caste fate as adults is determined followed by a nutrition-sensitive period also potentially affecting caste fate but for which the evidence is weaker. We used mRNA-seq and qRT-PCR validation to isolate genes differentially expressed between each caste pathway in larvae at developmental stages before and after each of these periods. We show that differences in gene expression between caste pathways are small in totipotent larvae, then peak after the queen-dependent period. Relatively few novel (i.e., taxonomically-restricted) genes were differentially expressed between castes, though novel genes were significantly enriched in late-instar larvae in the worker pathway. We compared sets of caste-associated genes in B. terrestris with those reported from the advanced eusocial honeybee, Apis mellifera, and found significant but relatively low levels of overlap of gene lists between the two species. These results suggest both the existence of low numbers of shared toolkit genes and substantial divergence in caste-associated genes between Bombus and the advanced eusocial Apis since their last common eusocial ancestor.

PAREameters: a tool for computational inference of plant miRNA-mRNA targeting rules using small RNA and degradome sequencing data.

MicroRNAs (miRNAs) are short, non-coding RNAs that modulate the translation-rate of messenger RNAs (mRNAs) by directing the RNA-induced silencing complex to sequence-specific targets. In plants, this typically results in cleavage and subsequent degradation of the mRNA. Degradome sequencing is a high-throughput technique developed to capture cleaved mRNA fragments and thus can be used to support miRNA target prediction. The current criteria used for miRNA target prediction were inferred on a limited number of experimentally validated A. thaliana interactions and were adapted to fit these specific interactions; thus, these fixed criteria may not be optimal across all datasets (organisms, tissues or treatments). We present a new tool, PAREameters, for inferring targeting criteria from small RNA and degradome sequencing datasets. We evaluate its performance using a more extensive set of experimentally validated interactions in multiple A. thaliana datasets. We also perform comprehensive analyses to highlight and quantify the differences between subsets of miRNA-mRNA interactions in model and non-model organisms. Our results show increased sensitivity in A. thaliana when using the PAREameters inferred criteria and that using data-driven criteria enables the identification of additional interactions that further our understanding of the RNA silencing pathway in both model and non-model organisms.