Automatic cell-type harmonization and integration across Human Cell Atlas datasets.

Harmonizing cell types across the single-cell community and assembling them into a common framework is central to building a standardized Human Cell Atlas. Here, we present CellHint, a predictive clustering tree-based tool to resolve cell-type differences in annotation resolution and technical biases across datasets. CellHint accurately quantifies cell-cell transcriptomic similarities and places cell types into a relationship graph that hierarchically defines shared and unique cell subtypes. Application to multiple immune datasets recapitulates expert-curated annotations. CellHint also reveals underexplored relationships between healthy and diseased lung cell states in eight diseases. Furthermore, we present a workflow for fast cross-dataset integration guided by harmonized cell types and cell hierarchy, which uncovers underappreciated cell types in adult human hippocampus. Finally, we apply CellHint to 12 tissues from 38 datasets, providing a deeply curated cross-tissue database with ∼3.7 million cells and various machine learning models for automatic cell annotation across human tissues.

Dynamic regulation of hypoxia-inducible factor-1α activity is essential for normal B cell development.

B lymphocyte development and selection are central to adaptive immunity and self-tolerance. These processes require B cell receptor (BCR) signaling and occur in bone marrow, an environment with variable hypoxia, but whether hypoxia-inducible factor (HIF) is involved is unknown. We show that HIF activity is high in human and murine bone marrow pro-B and pre-B cells and decreases at the immature B cell stage. This stage-specific HIF suppression is required for normal B cell development because genetic activation of HIF-1α in murine B cells led to reduced repertoire diversity, decreased BCR editing and developmental arrest of immature B cells, resulting in reduced peripheral B cell numbers. HIF-1α activation lowered surface BCR, CD19 and B cell-activating factor receptor and increased expression of proapoptotic BIM. BIM deletion rescued the developmental block. Administration of a HIF activator in clinical use markedly reduced bone marrow and transitional B cells, which has therapeutic implications. Together, our work demonstrates that dynamic regulation of HIF-1α is essential for normal B cell development.

Reticular Fibroblasts Expressing the Transcription Factor WT1 Define a Stromal Niche that Maintains and Replenishes Splenic Red Pulp Macrophages.

Located within red pulp cords, splenic red pulp macrophages (RPMs) are constantly exposed to the blood flow, clearing senescent red blood cells (RBCs) and recycling iron from hemoglobin. Here, we studied the mechanisms underlying RPM homeostasis, focusing on the involvement of stromal cells as these cells perform anchoring and nurturing macrophage niche functions in lymph nodes and liver. Microscopy revealed that RPMs are embedded in a reticular meshwork of red pulp fibroblasts characterized by the expression of the transcription factor Wilms' Tumor 1 (WT1) and colony stimulating factor 1 (CSF1). Conditional deletion of Csf1 in WT1+ red pulp fibroblasts, but not white pulp fibroblasts, drastically altered the RPM network without altering circulating CSF1 levels. Upon RPM depletion, red pulp fibroblasts transiently produced the monocyte chemoattractants CCL2 and CCL7, thereby contributing to the replenishment of the RPM network. Thus, red pulp fibroblasts anchor and nurture RPM, a function likely conserved in humans.

Anti-commensal IgG Drives Intestinal Inflammation and Type 17 Immunity in Ulcerative Colitis.

Inflammatory bowel disease is a chronic, relapsing condition with two subtypes, Crohn's disease (CD) and ulcerative colitis (UC). Genome-wide association studies (GWASs) in UC implicate a FCGR2A variant that alters the binding affinity of the antibody receptor it encodes, FcγRIIA, for immunoglobulin G (IgG). Here, we aimed to understand the mechanisms whereby changes in FcγRIIA affinity would affect inflammation in an IgA-dominated organ. We found a profound induction of anti-commensal IgG and a concomitant increase in activating FcγR signaling in the colonic mucosa of UC patients. Commensal-IgG immune complexes engaged gut-resident FcγR-expressing macrophages, inducing NLRP3- and reactive-oxygen-species-dependent production of interleukin-1ß (IL-1ß) and neutrophil-recruiting chemokines. These responses were modulated by the FCGR2A genotype. In vivo manipulation of macrophage FcγR signal strength in a mouse model of UC determined the magnitude of intestinal inflammation and IL-1ß-dependent type 17 immunity. The identification of an important contribution of IgG-FcγR-dependent inflammation to UC has therapeutic implications.

Epigenetics and tissue immunity-Translating environmental cues into functional adaptations.

There is an increasing appreciation that many innate and adaptive immune cell subsets permanently reside within non-lymphoid organs, playing a critical role in tissue homeostasis and defense. The best characterized are macrophages and tissue-resident T lymphocytes that work in concert with organ structural cells to generate appropriate immune responses and are functionally shaped by organ-specific environmental cues. The interaction of tissue epithelial, endothelial and stromal cells is also required to attract, differentiate, polarize and maintain organ immune cells in their tissue niche. All of these processes require dynamic regulation of cellular transcriptional programmes, with epigenetic mechanisms playing a critical role, including DNA methylation and post-translational histone modifications. A failure to appropriately regulate immune cell transcription inevitably results in inadequate or inappropriate immune responses and organ pathology. Here, with a focus on the mammalian kidney, an organ which generates differing regional environmental cues (including hypersalinity and hypoxia) due to its physiological functions, we will review the basic concepts of tissue immunity, discuss the technologies available to profile epigenetic modifications in tissue immune cells, including those that enable single-cell profiling, and consider how these mechanisms influence the development, phenotype, activation and function of different tissue immune cell subsets, as well as the immunological function of structural cells.

Spatial and molecular profiling of the mononuclear phagocyte network in classic Hodgkin lymphoma.

Classic Hodgkin lymphoma (cHL) has a rich immune infiltrate, which is an intrinsic component of the neoplastic process. Malignant Hodgkin Reed-Sternberg cells (HRSCs) create an immunosuppressive microenvironment by the expression of regulatory molecules, preventing T-cell activation. It has also been demonstrated that mononuclear phagocytes (MNPs) in the vicinity of HRSCs express similar regulatory mechanisms in parallel, and their presence in tissue is associated with inferior patient outcomes. MNPs in cHL have hitherto been identified by a small number of canonical markers and are usually described as tumor-associated macrophages. The organization of MNP networks and interactions with HRSCs remains unexplored at high resolution. Here, we defined the global immune-cell composition of cHL and nonlymphoma lymph nodes, integrating data across single-cell RNA sequencing, spatial transcriptomics, and multiplexed immunofluorescence. We observed that MNPs comprise multiple subsets of monocytes, macrophages, and dendritic cells (DCs). Classical monocytes, macrophages and conventional DC2s were enriched in the vicinity of HRSCs, but plasmacytoid DCs and activated DCs were excluded. Unexpectedly, cDCs and monocytes expressed immunoregulatory checkpoints PD-L1, TIM-3, and the tryptophan-catabolizing protein IDO, at the same level as macrophages. Expression of these molecules increased with age. We also found that classical monocytes are important signaling hubs, potentially controlling the retention of cDC2 and ThExh via CCR1-, CCR4-, CCR5-, and CXCR3-dependent signaling. Enrichment of the cDC2-monocyte-macrophage network in diagnostic biopsies is associated with early treatment failure. These results reveal unanticipated complexity and spatial polarization within the MNP compartment, further demonstrating their potential roles in immune evasion by cHL.

Assessment of biological organ age using molecular pathology in pre-transplant kidney biopsies.

Organ shortage is a major challenge in kidney transplantation but the use of older donors, often with co-morbidities, is hampered by inconsistent outcomes. Methods of accurately stratifying marginal donor organs by clinical and histological assessment are lacking. To better understand organ variability, we profiled the transcriptomes of 271 kidneys from deceased donors at retrieval. Following correction for biopsy composition, we assessed molecular pathways that associated with delayed, and sub-optimal one-year graft function. Analysis of cortical biopsies identified an adaptive immune gene-rich module that significantly associated with increasing age and worse outcomes. Cellular deconvolution using human kidney reference single cell transcriptomes confirmed an increase in kidney-specific B and T cell signatures, as well as kidney macrophage, myofibroblast and fibroblast gene sets in this module. Surprisingly, innate immune pathway and neutrophil gene signature enrichment was associated with better outcomes. Thus, our work uncovers cellular molecular features of pathological organ ageing, identifiable at kidney retrieval, with translational potential.

Lysis of cold-storage-induced microvascular obstructions for ex vivo revitalization of marginal human kidneys.

Thousands of kidneys from higher-risk donors are discarded annually because of the increased likelihood of complications posttransplant. Given the severe organ shortage, there is a critical need to improve utilization of these organs. To this end, normothermic machine perfusion (NMP) has emerged as a platform for ex vivo assessment and potential repair of marginal organs. In a recent study of 8 transplant-declined human kidneys on NMP, we discovered microvascular obstructions that impaired microvascular blood flow. However, the nature and physiologic impact of these lesions were unknown. Here, in a study of 39 human kidneys, we have identified that prolonged cold storage of human kidneys induces accumulation of fibrinogen within tubular epithelium. Restoration of normoxic conditions-either ex vivo during NMP or in vivo following transplant-triggered intravascular release of fibrinogen correlating with red blood cell aggregation and microvascular plugging. Combined delivery of plasminogen and tissue plasminogen activator during NMP lysed the plugs leading to a significant reduction in markers of renal injury, improvement in indicators of renal function, and improved delivery of vascular-targeted nanoparticles. Our study suggests a new mechanism of cold storage injury in marginal organs and provides a simple treatment with immediate translational potential.

Applying single-cell technologies to clinical pathology: progress in nephropathology.

Cells represent the basic building blocks of living organisms. Accurate characterisation of cellular phenotype, intercellular signalling networks, and the spatial organisation of cells within organs is crucial to deliver a better understanding of the processes underpinning physiology, and the perturbations that lead to disease. Single-cell methodologies have increased rapidly in scale and scope in recent years and are set to generate important insights into human disease. Here, we review current practices in nephropathology, which are dominated by relatively simple morphological descriptions of tissue biopsies based on their appearance using light microscopy. Bulk transcriptomics have more recently been used to explore glomerular and tubulointerstitial kidney disease, renal cancer, and the responses to injury and alloimmunity in kidney transplantation, generating novel disease insights and prognostic biomarkers. These studies set the stage for single-cell transcriptomic approaches that reveal cell-type-specific gene expression patterns in health and disease. These technologies allow genome-wide disease susceptibility genes to be interpreted with the knowledge of the specific cell populations within organs that express them, identifying candidate cell types for further study. Single-cell technologies are also moving beyond assaying individual cellular transcriptomes, to measuring the epigenetic landscape of single cells. Single-cell antigen-receptor gene sequencing also enables specific T- and B-cell clones to be tracked in different tissues and disease states. In the coming years these rich 'multi-omic' descriptions of kidney disease will enable histopathological descriptions to be comprehensively integrated with molecular phenotypes, enabling better disease classification and prognostication and the application of personalised treatment strategies. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Arginine reprogramming in ADPKD results in arginine-dependent cystogenesis.

Research into metabolic reprogramming in cancer has become commonplace, yet this area of research has only recently come of age in nephrology. In light of the parallels between cancer and autosomal dominant polycystic kidney disease (ADPKD), the latter is currently being studied as a metabolic disease. In clear cell renal cell carcinoma (RCC), which is now considered a metabolic disease, we and others have shown derangements in the enzyme arginosuccinate synthase 1 (ASS1), resulting in RCC cells becoming auxotrophic for arginine and leading to a new therapeutic paradigm involving reducing extracellular arginine. Based on our earlier finding that glutamine pathways are reprogrammed in ARPKD, and given the connection between arginine and glutamine synthetic pathways via citrulline, we investigated the possibility of arginine reprogramming in ADPKD. We now show that, in a remarkable parallel to RCC, ASS1 expression is reduced in murine and human ADPKD, and arginine depletion results in a dose-dependent compensatory increase in ASS1 levels as well as decreased cystogenesis in vitro and ex vivo with minimal toxicity to normal cells. Nontargeted metabolomics analysis of mouse kidney cell lines grown in arginine-deficient versus arginine-replete media suggests arginine-dependent alterations in the glutamine and proline pathways. Thus, depletion of this conditionally essential amino acid by dietary or pharmacological means, such as with arginine-degrading enzymes, may be a novel treatment for this disease.

High Initial Sputter Rate Found for Vaccinia Virions Using Isotopic Labeling, NanoSIMS, and AFM.

High-lateral-resolution secondary ion mass spectrometry (SIMS) has the potential to provide functional and depth resolved information from small biological structures, such as viral particles (virions) and phage, but sputter rate and sensitivity are not characterized at shallow depths relevant to these structures. Here we combine stable isotope labeling of the DNA of vaccinia virions with correlated SIMS imaging depth profiling and atomic force microscopy (AFM) to develop a nonlinear, nonequilibrium sputter rate model for the virions and validate the model on the basis of reconstructing the location of the DNA within individual virions. Our experiments with a Cs+ beam show an unexpectedly high initial sputter rate (∼100 um2·nm·pA-1·s-1) with a rapid decline to an asymptotic rate of 0.7 um2·nm·pA-1·s-1 at an approximate depth of 70 nm. Correlated experiments were also conducted with glutaraldehyde-fixed virions, as well as O- and Ga+ beams, yielding similar results. Based on our Cs+ sputter rate model, the labeled DNA in the virion was between 50 and 90 nm depth in the virion core, consistent with expectations, supporting our conclusions. Virion densification was found to be a secondary effect. Accurate isotopic ratios were obtained from the initiation of sputtering, suggesting that isotopic tracers could be successfully used for smaller virions and phage.

An Aggravated Trajectory of Depression and Anxiety Co-morbid with Hepatitis C: A Within-groups Study of 61 Australian Outpatients.

BACKGROUND: This study aimed to explore the course of depression and anxiety in chronic hepatitis C patients. METHODS:   Data were combined from two studies: (1) Hospital Anxiety and Depression Scale (HADS) scores in 395 consecutive Australian outpatients from 2006 to 2010 formed the baseline measurement; and (2) Depression Anxiety Stress Scales (DASS) scores in a survey of a sub-sample of these patients in 2011 formed the follow-up measurement. After converting DASS to HADS scores, changes in symptom scores and rates of case-ness (≥8), and predictors of follow-up symptoms were assessed. RESULTS:   Follow-up data were available for 61 patients (70.5% male) whose age ranged from 24.5 to 74.6 years (M=45.6). The time to follow-up ranged from 20.7 to 61.9 months (M=43.8). Baseline rates of depression (32.8%) and anxiety (44.3%) increased to 62.3% and 67.2%, respectively. These findings were confirmed, independent of the conversion, by comparing baseline HADS and follow-up DASS scores with British community norms. Baseline anxiety and younger age predicted depression, while baseline anxiety, high school non-completion, and single relationship status predicted anxiety. CONCLUSION:  This study demonstrated a worsening trajectory of depression and anxiety. Further controlled and prospective research in a larger sample is required to confirm these findings.

Identification of a core transcriptional program driving the human renal mesenchymal-to-epithelial transition.

During kidney development, nephron epithelia arise de novo from fate-committed mesenchymal progenitors through a mesenchymal-to-epithelial transition (MET). Downstream of fate specification, transcriptional mechanisms that drive establishment of epithelial morphology are poorly understood. We used human iPSC-derived renal organoids, which recapitulate nephrogenesis, to investigate mechanisms controlling renal MET. Multi-ome profiling via snRNA-seq and ATAC-seq of organoids identified dynamic changes in gene expression and chromatin accessibility driven by activators and repressors throughout MET. CRISPR interference identified that paired box 8 (PAX8) is essential for initiation of MET in human renal organoids, contrary to in vivo mouse studies, likely by activating a cell-adhesion program. While Wnt/ß-catenin signaling specifies nephron fate, we find that it must be attenuated to allow hepatocyte nuclear factor 1-beta (HNF1B) and TEA-domain (TEAD) transcription factors to drive completion of MET. These results identify the interplay between fate commitment and morphogenesis in the developing human kidney, with implications for understanding both developmental kidney diseases and aberrant epithelial plasticity following adult renal tubular injury.

Directly coupled high-performance liquid chromatography-accelerator mass spectrometry measurement of chemically modified protein and peptides.

Quantitation of low-abundance protein modifications involves significant analytical challenges, especially in biologically important applications, such as studying the role of post-translational modifications in biology and measurement of the effects of reactive drug metabolites. (14)C labeling combined with accelerator mass spectrometry (AMS) provides exquisite sensitivity for such experiments. Here, we demonstrate real-time (14)C quantitation of high-performance liquid chromatography (HPLC) separations by liquid sample accelerator mass spectrometry (LS-AMS). By enabling direct HPLC-AMS coupling, LS-AMS overcomes several major limitations of conventional HPLC-AMS, where individual HPLC fractions must be collected and converted to graphite before measurement. To demonstrate LS-AMS and compare the new technology to traditional solid sample AMS (SS-AMS), reduced and native bovine serum albumin (BSA) was modified by (14)C-iodoacetamide, with and without glutathione present, producing adducts on the order of 1 modification in every 10(6) to 10(8) proteins. (14)C incorporated into modified BSA was measured by solid carbon AMS and LS-AMS. BSA peptides were generated by tryptic digestion. Analysis of HPLC-separated peptides was performed in parallel by LS-AMS, fraction collection combined with SS-AMS, and (for peptide identification) electrospray ionization and tandem mass spectrometry (ESI-MS/MS). LS-AMS enabled (14)C quantitation from ng sample sizes and was 100 times more sensitive to (14)C incorporated in HPLC-separated peptides than SS-AMS, resulting in a lower limit of quantitation of 50 zmol (14)C/peak. Additionally, LS-AMS turnaround times were minutes instead of days, and HPLC trace analyses required 1/6th the AMS instrument time required for analysis of graphite fractions by SS-AMS.

D-Lactate production as a function of glucose metabolism in Saccharomyces cerevisiae.

Methylglyoxal, a reactive, toxic dicarbonyl, is generated by the spontaneous degradation of glycolytic intermediates. Methylglyoxal can form covalent adducts with cellular macromolecules, potentially disrupting cellular function. We performed experiments using the model organism Saccharomyces cerevisiae, grown in media containing low, moderate and high glucose concentrations, to determine the relationship between glucose consumption and methylglyoxal metabolism. Normal growth experiments and glutathione depletion experiments showed that metabolism of methylglyoxal by log-phase yeast cultured aerobically occurred primarily through the glyoxalase pathway. Growth in high-glucose media resulted in increased generation of the methylglyoxal metabolite D-lactate and overall lower efficiency of glucose utilization as measured by growth rates. Cells grown in high-glucose media maintained higher glucose uptake flux than cells grown in moderate-glucose or low-glucose media. Computational modelling showed that increased glucose consumption may impair catabolism of triose phosphates as a result of an altered NAD⁺:NADH ratio.

Acceptability of psychotherapy, pharmacotherapy, and self-directed therapies in Australians living with chronic hepatitis C.

Despite the prevalence of psychiatric co-morbidity in chronic hepatitis C (CHC), treatment is under-researched. Patient preferences are likely to affect treatment uptake, adherence, and success. Thus, the acceptability of psychological supports was explored. A postal survey of Australian CHC outpatients of the Royal Adelaide Hospital and online survey of Australians living with CHC was conducted, assessing demographic and disease-related variables, psychosocial characteristics, past experience with psychological support, and psychological support acceptability. The final sample of 156 patients (58 % male) had significantly worse depression, anxiety, stress, and social support than norms. The most acceptable support type was individual psychotherapy (83 %), followed by bibliotherapy (61 %), pharmacotherapy (56 %), online therapy (45 %), and group psychotherapy (37 %). The most prominent predictor of support acceptability was satisfaction with past use. While individual psychotherapy acceptability was encouragingly high, potentially less costly modalities including group psychotherapy or online therapy may be hampered by low acceptability, the reasons for which need to be further explored.

HPLC-Parallel accelerator and molecular mass spectrometry analysis of 14C-labeled amino acids.

Accelerator mass spectrometry (AMS) is the method of choice for quantitation of low amounts of 14C-labeled biomolecules. Despite exquisite sensitivity, an important limitation of AMS is its inability to provide structural information about the analyte. This limitation is not critical when the labeled compounds are well-characterized prior to AMS analysis. However, analyte identity is important in other experiments where, for example, a compound is metabolized and the structures of its metabolites are not known. We previously described a moving wire interface that enables direct AMS measurement of liquid sample in the form of discrete drops or HPLC eluent without the need for individual fraction collection, termed liquid sample-AMS (LS-AMS). We now report the coupling of LS-AMS with a molecular mass spectrometer, providing parallel accelerator and molecular mass spectrometry (PAMMS) detection of analytes separated by liquid chromatography. The repeatability of the method was examined by performing repeated injections of 14C-labeled tryptophan, and relative standard deviations of the 14C peak areas were ≤10.57% after applying a normalization factor based on a standard. Five 14C-labeled amino acids were separated and detected to provide simultaneous quantitative AMS and structural MS data, and AMS results were compared with solid sample-AMS (SS-AMS) data using Bland-Altman plots. To demonstrate the utility of the workflow, yeast cells were grown in a medium with 14C-labeled tryptophan. The cell extracts were analyzed by PAMMS, and 14C was detected in tryptophan and its metabolite kynurenine.

Predicting the impact of sequence motifs on gene regulation using single-cell data.

The binding of transcription factors at proximal promoters and distal enhancers is central to gene regulation. Identifying regulatory motifs and quantifying their impact on expression remains challenging. Using a convolutional neural network trained on single-cell data, we infer putative regulatory motifs and cell type-specific importance. Our model, scover, explains 29% of the variance in gene expression in multiple mouse tissues. Applying scover to distal enhancers identified using scATAC-seq from the developing human brain, we identify cell type-specific motif activities in distal enhancers. Scover can identify regulatory motifs and their importance from single-cell data where all parameters and outputs are easily interpretable.

Atypical B cells and impaired SARS-CoV-2 neutralization following heterologous vaccination in the elderly.

Suboptimal responses to a primary vaccination course have been reported in the elderly, but there is little information regarding the impact of age on responses to booster third doses. Here, we show that individuals 70 years or older (median age 73, range 70-75) who received a primary two-dose schedule with AZD1222 and booster third dose with mRNA vaccine achieve significantly lower neutralizing antibody responses against SARS-CoV-2 spike pseudotyped virus compared with those younger than 70 (median age 66, range 54-69) at 1 month post booster. Impaired neutralization potency and breadth post third dose in the elderly is associated with circulating "atypical" spike-specific B cells expressing CD11c and FCRL5. However, when considering individuals who received three doses of mRNA vaccine, we did not observe differences in neutralization or enrichment in atypical B cells. This work highlights the finding that AdV and mRNA COVID-19 vaccine formats differentially instruct the memory B cell response.