Patients with the renal phosphate–wasting disease X-linked hypophosphatemia (XLH) and Hyp mice, the murine homolog of XLH, are characterized by loss-of-function mutations in phosphate-regulating endopeptidase homolog X-linked (PHEX), leading to excessive secretion of the bone-derived phosphotropic hormone FGF23. The mineralization defect in patients with XLH and Hyp mice is caused by a combination of hypophosphatemia and local accumulation of mineralization-inhibiting molecules in bone. However, the mechanism by which PHEX deficiency regulates bone cell metabolism remains elusive. Here, we used spatial metabolomics by employing matrix-assisted laser desorption/ionization (MALDI) Fourier-transform ion cyclotron resonance mass spectrometry imaging (MSI) of undecalcified bone cryosections to characterize in situ metabolic changes in bones of Hyp mice in a holistic, unbiased manner. We found complex changes in Hyp bone metabolism, including perturbations in pentose phosphate, purine, pyrimidine, and phospholipid metabolism. Importantly, our study identified an upregulation of several biochemical pathways involved in intra- and extracellular production of the mineralization inhibitor pyrophosphate in the bone matrix of Hyp mice. Our data emphasize the utility of MSI–based spatial metabolomics in bone research and provide holistic in situ insights as to how Phex deficiency–induced changes in biochemical pathways in bone cells are linked to impaired bone mineralization.
Achim Buck, Verena M. Prade, Thomas Kunzke, Reinhold G. Erben, Axel Walch
Mass cytometry, or cytometry by TOF (CyTOF), provides a robust means of determining protein-level measurements of more than 40 markers simultaneously. While the functional states of immune cells occur along continuous phenotypic transitions, cytometric studies surveying cell phenotypes often rely on static metrics, such as discrete cell-type abundances, based on canonical markers and/or restrictive gating strategies. To overcome this limitation, we applied single-cell trajectory inference and nonnegative matrix factorization methods to CyTOF data to trace the dynamics of T cell states. In the setting of cancer immunotherapy, we showed that patient-specific summaries of continuous phenotypic shifts in T cells could be inferred from peripheral blood–derived CyTOF mass cytometry data. We further illustrated that transfer learning enabled these T cell continuous metrics to be used to estimate patient-specific cell states in new sample cohorts from a reference patient data set. Our work establishes the utility of continuous metrics for CyTOF analysis as tools for translational discovery.
Dimitrios N. Sidiropoulos, Genevieve L. Stein-O’Brien, Ludmila Danilova, Nicole E. Gross, Soren Charmsaz, Stephanie Xavier, James Leatherman, Hao Wang, Mark Yarchoan, Elizabeth M. Jaffee, Elana J. Fertig, Won Jin Ho
Bronchiolitis obliterans syndrome (BOS) is the main reason for poor outcomes after lung transplantation (LTx). We and others have recently identified B cells as major contributors to BOS after LTx. The extent of B cell heterogeneity and the relative contributions of B cell subpopulations to BOS, however, remain unclear. Here, we provide a comprehensive analysis of cell population changes and their gene expression patterns during chronic rejection after orthotopic LTx in mice. Of 11 major cell types, Mzb1-expressing plasma cells (PCs) were the most prominently increased population in BOS lungs. These findings were validated in 2 different cohorts of human BOS after LTx. A Bhlhe41, Cxcr3, and Itgb1 triple-positive B cell subset, also expressing classical markers of the innate-like B-1 B cell population, served as the progenitor pool for Mzb1+ PCs. This subset accounted for the increase in IgG2c production within BOS lung grafts. A genetic lack of Igs decreased BOS severity after LTx. In summary, we provide a detailed analysis of cell population changes during BOS. IgG+ PCs and their progenitors — an innate B cell subpopulation — are the major source of local Ab production and a significant contributor to BOS after LTx.
Natalia F. Smirnova, Kent Riemondy, Marta Bueno, Susan Collins, Pavan Suresh, Xingan Wang, Kapil N. Patel, Carlyne Cool, Melanie Königshoff, Nirmal S. Sharma, Oliver Eickelberg
Cancer cells release large quantities of cell-free DNA (cfDNA) into the surrounding tissue and circulation. As cfDNA is a common source of biomarkers for liquid biopsy and has been implicated as a functional mediator for intercellular communication, fundamental characterization of cfDNA topology has widespread biological and clinical ramifications. Whether the topology of cfDNA is such that it exists predominantly in membrane-bound extracellular vesicles (EVs) or in non-vesicular DNA-protein complexes remains poorly understood. Here, we employed a DNA-targeted approach to comprehensively assess total cfDNA topology in cancer. Using preclinical models and patient samples, we demonstrate that nuclear cfDNA is predominantly associated with nucleosomal particles and not EVs, while a substantial subset of mitochondrial cfDNA is membrane-protected and disproportionately derived from non-tumour cells. In addition, discrimination between membrane-protected and accessible mitochondrial cfDNA added diagnostic and prognostic value in a cohort of head and neck cancer patients. Our results support a revised model for cfDNA topology in cancer. Due to its abundance, nuclear cfDNA within nucleosomal particles is the most compelling liquid biopsy substrate, while EV-bound and accessible mitochondrial cfDNA represent distinct reservoirs of potential cancer biomarkers whose structural conformations may also influence their extracellular stability and propensity for uptake by recipient cells.
Ethan Z. Malkin, Steven De Michino, Meghan Lambie, Rita G. Gill, Zhen Zhao, Ariana Rostami, Andrea Arruda, Mark D. Minden, Scott V. Bratman
The kidney contains a population of resident macrophages from birth that expands as it grows and forms a contiguous network throughout the tissue. Kidney resident macrophages (KRMs) are important in homeostasis and the response to acute kidney injury (AKI). While the kidney contains many microenvironments, it is unknown whether KRMs are a heterogeneous population differentiated by function and location. We combined single-cell RNA sequencing (scRNAseq), spatial transcriptomics, flow cytometry, and immunofluorescence imaging to localize, characterize, and validate KRM populations during quiescence and following 19 minutes of bilateral ischemic kidney injury. scRNAseq and spatial transcriptomics revealed seven distinct KRM subpopulations, which are organized into zones corresponding to regions of the nephron. Each subpopulation was identifiable by a unique transcriptomic signature suggesting distinct functions. Specific protein markers were identified for two clusters allowing analysis by flow cytometry or immunofluorescence imaging. Following injury, the original localization of each subpopulation is lost, either from changing locations or transcriptomic signatures. The original spatial distribution of KRMs is not fully restored for at least 28 days post-injury. The change in KRM localization confirms a long hypothesized dysregulation of the local immune system following acute injury and may explain the increased risk for chronic kidney disease.
Matthew D. Cheung, Elise N. Erman, Kyle H. Moore, Jeremie M.P. Lever, Zhang Li, Jennifer R. LaFontaine, Gelare Ghajar-Rahimi, Shanrun Liu, Zhengqin Yang, Rafay Karim, Bradley K. Yoder, Anupam Agarwal, James F. George
Histopathology, the standard method to assess bone marrow in hematologic malignancies such as myeloproliferative neoplasms (MPNs), suffers from notable limitations in both research and clinical settings. Bone marrow biopsies in patients fail to detect disease heterogeneity; may yield a non-diagnostic sample; and cannot be repeated frequently in clinical oncology. Endpoint histopathology precludes monitoring disease progression and response to therapy in the same mouse over time, missing likely variations among mice. To overcome these shortcomings, we used magnetic resonance imaging (MRI) to measure changes in cellularity, macromolecular constituents, and fat versus hematopoietic cells in bone marrow using diffusion-weighted imaging, magnetization transfer, and chemical shift encoded fat imaging. Combining metrics from these imaging parameters revealed dynamic alterations in bone marrow following myeloablative radiation and transplantation. In a mouse MPLW515L bone marrow transplant model of MPN, MRI detected effects of a JAK2 inhibitor, ruxolitinib, within five days of initiating treatment and identified differing kinetics of treatment responses in sub-regions of the tibia. Histopathology validated MRI results for bone marrow composition and heterogeneity. Anatomic MRI scans also showed reductions in spleen volume during treatment. These findings establish an innovative, clinically translatable MRI approach to quantify spatial and temporal changes in bone marrow in MPN.
Tanner H. Robison, Manisha Solipuram, Kevin Heist, Ghoncheh Amouzandeh, Winston Y. Lee, Brock A. Humphries, Johanna M. Buschhaus, Avinash Bevoor, Anne Zhang, Kathryn E. Luker, Kristen Pettit, Moshe Talpaz, Dariya Malyarenko, Thomas L. Chenevert, Brian D. Ross, Gary D. Luker
The epidermis is the outermost layer of skin. Here, we use targeted lipid profiling to characterize the biogeographic alterations of human epidermal lipids across 12 anatomically distinct body sites, and use single-cell RNA sequencing to compare keratinocyte gene expression at acral and non-acral surfaces. We demonstrate that acral skin has low expression of EOS acyl-ceramides and the genes involved in their synthesis, as well as low expression of genes involved in filaggrin and keratin citrullination (PADI1 and PADI3) and corneodesmosome degradation, changes consistent with increased corneocyte retention. Several overarching principles governing epidermal lipid expression were also noted. For example, there is a strong negative correlation between the expression of 18-carbon and 22-carbon sphingoid base ceramides. Disease-specific alterations in epidermal lipid gene expression and their corresponding alterations to the epidermal lipidome were characterized. Lipid biomarkers with diagnostic utility for inflammatory and precancerous conditions were identified, and a two-analyte diagnostic model of psoriasis was constructed using a step-forward algorithm. Finally, gene co-expression analysis revealed a strong connection between lipid and immune gene expression. This work highlights mechanisms by which the epidermis is uniquely adapted for the specific environmental insults encountered at different body surfaces, and how inflammation-associated alterations in gene expression affect the epidermal lipidome.
Alexander A. Merleev, Stephanie T. Le, Claire Alexanian, Atrin Toussi, Yixuan Xie, Alina I. Marusina, Steven M. Watkins, Forum Patel, Allison C. Billi, Julie Wiedemann, Yoshihiro Izumiya, Ashish Kumar, Ranjitha Uppala, J. Michelle Kahlenberg, Fu-Tong Liu, Iannis E. Adamopoulos, Elizabeth A. Wang, Chelsea Ma, Michelle Y. Cheng, Halani Xiong, Amanda Kirane, Guillaume Luxardi, Bogi Andersen, Lam C. Tsoi, Carlito B. Lebrilla, Johann E. Gudjonsson, Emanual Maverakis
Despite being a leading cause of advanced liver disease, alcohol-associated liver disease (ALD) has no effective medical therapies. The circulating proteome, which comprises proteins secreted by different cells and tissues in the context of normal physiological function or in the setting of disease and illness, represents an attractive target for uncovering novel biology related to the pathogenesis of ALD. In this work, we used the aptamer-based SomaScan proteomics platform to quantify the relative concentration of over 1300 proteins in a well-characterized cohort of patients with the spectrum of ALD. We found a distinct circulating proteomic signature that correlated with ALD severity, including over 600 proteins that differed significantly between ALD stages, many of which have not previously been associated with ALD to our knowledge. Notably, certain proteins that were markedly dysregulated in patients with alcohol-associated hepatitis were also altered, to a lesser degree, in patients with subclinical ALD and may represent early biomarkers for disease progression. Taken together, our work highlights the vast and distinct changes in the circulating proteome across the wide spectrum of ALD, identifies potentially novel biomarkers and therapeutic targets, and provides a proteomic resource atlas for ALD researchers and clinicians.
Jay Luther, Augustin G.L. Vannier, Esperance A. Schaefer, Russell P. Goodman
The androgen receptor (AR) is a master transcription factor that regulates prostate cancer (PC) development and progression. Inhibition of AR signaling by androgen deprivation is the first-line therapy with initial efficacy for advanced and recurrent PC. Paradoxically, supraphysiological levels of testosterone (SPT) also inhibit PC progression. However, as with any therapy, not all patients show a therapeutic benefit, and responses differ widely in magnitude and duration. In this study, we evaluated whether differences in the AR cistrome before treatment can distinguish between SPT-responding (R) and -nonresponding (NR) tumors. We provide the first preclinical evidence to our knowledge that SPT-R tumors exhibit a distinct AR cistrome when compared with SPT-NR tumors, indicating a differential biological role of the AR. We applied an integrated analysis of ChIP-Seq and RNA-Seq to the pretreatment tumors and identified an SPT-R signature that distinguishes R and NR tumors. Because transcriptomes of SPT-treated clinical specimens are not available, we interrogated available castration-resistant PC (CRPC) transcriptomes and showed that the SPT-R signature is associated with improved survival and has the potential to identify patients who would respond to SPT. These findings provide an opportunity to identify the subset of patients with CRPC who would benefit from SPT therapy.
Xintao Qiu, Lisha G. Brown, Jennifer L. Conner, Holly M. Nguyen, Nadia Boufaied, Sarah Abou Alaiwi, Ji-Heui Seo, Talal El Zarif, Connor Bell, Edward O’Connor, Brian Hanratty, Mark Pomerantz, Matthew L. Freedman, Myles Brown, Michael C. Haffner, Peter S. Nelson, Felix Y. Feng, David P. Labbé, Henry W. Long, Eva Corey
Atrial fibrillation (AF), the most common sustained cardiac arrhythmia and a major risk factor for stroke, often arises through ectopic electrical impulses derived from the pulmonary veins (PV). Sequence variants in enhancers controlling expression of the transcription factor PITX2, which is expressed in the cardiomyocytes (CMs) of the PV and left atrium (LA), have been implicated in AF predisposition. Single nuclei multiomic profiling of RNA and analysis of chromatin accessibility combined with spectral clustering uncovered distinct PV- and LA-enriched CM cell states. Pitx2 mutant PV and LA CMs exhibited gene expression changes consistent with cardiac dysfunction through cell-type-distinct, PITX2-directed, cis-regulatory grammars controlling target gene expression. The perturbed network targets in each CM were enriched in distinct human AF-predisposition genes, suggesting combinatorial risk for AF-genesis. Our data further reveals that PV and LA Pitx2 mutant CMs signal to endothelial and endocardial cells through BMP10 signaling with pathogenic potential. This work provides a multiomic framework for interrogating the basis of AF-predisposition in the PV of humans.
Jeffrey D. Steimle, Francisco J. Grisanti Canozo, Minjun Park, Zachary A. Kadow, Md. Abul Hassan Samee, James F. Martin
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