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Darmanis, S.

Publications and source records attributed to Darmanis, S..

At least 19 recordsLinked to original sources

Combined antagonism of Oncostatin M (OSM) and Interleukin 6 (IL-6) provides both anti-fibrotic and anti-inflammatory benefit in pulmonary fibrosis

Interstitial lung diseases (ILDs), including idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated ILD (SSc-ILD), are irreversible fibrosing diseases with a mean survival time of less than 5 years for IPF. Poorly understood etiology and complex pathogenesis of these diseases have hampered the identification and development of effective therapeutics. Existing treatments can slow progressive fibrosis and lung function decline but do not stop it entirely, resulting in a minimal impact on patient survival. Thus, novel therapeutic interventions are needed. Tocilizumab, an anti-IL-6 receptor antibody, was recently approved by FDA for the treatment of SSc-ILD based on evidence demonstrating a reduction in the rate of lung function decline. In this study, we have characterized an IL-6-driven feed-forward myeloid axis contributing to lung inflammation providing a mechanistic hypothesis for tocilizumab. Concomitantly we have identified an oncostatin M (OSM) orchestrated lung injury response contributing to epithelial and endothelial cell disruption, myofibroblast activation and fibrosis. Despite dysregulated expression of IL11 in IPF, SSc-ILD, and murine models of fibrosis, we found no evidence for a pro-fibrotic role for IL-11 in vitro or in vivo. Instead, in pre-clinical models of IPF we demonstrate that antagonism of OSM alone, or to a greater degree in combination with IL-6, reduced lung fibrosis and inflammation. Translating these observations, we validated gp130:OSMR, rather than gp130:LIFR, as the dominant human receptor complex used by OSM, identifying OSMR as a potential therapeutic target to stall fibrosis.

immunology↗

IRF2 degradation tunes the innate immune response

The transcription factor IRF2 protects against skin inflammation in mice and humans but, paradoxically, promotes pyroptosis by inducing Gsdmd. How IRF2 activates some proinflammatory genes, but suppresses others is unclear. We show that skin inflammation in Irf2-deficient mice is driven by IRF1 activation of interferon-stimulated genes (ISGs). Chromatin profiling revealed that IRF1 and IRF2 occupy the same ISG regulatory sites, but as a weaker transcriptional activator, IRF2 limited ISG transcription by IRF1. Toll-like receptor signaling favored IRF1-driven transcription by inducing Irf1. In addition, IRF1 recruited the ubiquitin ligase SPOP to ISG sites, resulting in proteasomal degradation of IRF2. This shift from IRF2 to IRF1 occupancy enhanced ISG transcription. Collectively, these findings define a hierarchical transcriptional circuit in which IRF2 limits IRF1 activity under homeostatic conditions but is displaced during an immune response, allowing IRF1-dependent gene programs central to innate immunity and autoinflammation.

immunology↗

Maladaptive inflammatory signaling in old mice impairs colonic regeneration by promoting a sustained fetal-like epithelial state

Aging is associated with a decline in the regenerative capacity of many tissues. Central to this decline is a complex interplay between inflammation and stem cell function. How these two processes are linked and influence regenerative capacity remains unclear. Here, we undertake a comprehensive assessment of age-related changes in the mouse colon at single-cell resolution. A survey of immune and epithelial compartments revealed a hyperactivated inflammatory state in the colon of old mice characterized by the induction of an interferon {gamma} (IFN{gamma}) response signature in immune cells. This does not result in increased inflammation under homeostasis, but triggers a disproportionate inflammatory response, disrupting regeneration after challenge with the enteropathogen Citrobacter rodentium. Colons of old mice exhibit higher production of IFN{gamma} by T and innate lymphoid cells (ILCs) that are associated with reduced Lgr5+ stem cells and decreased epithelial proliferation. Interestingly, we find aged intestinal epithelial cells to be hypersensitive to IFN{gamma} signaling, inducing a regeneration-associated fetal-like gene expression signature that, in turn, renders these cells more sensitive to IFN{gamma}-induced apoptosis. Our findings reveal an age-related imbalance in the interaction between the immune and epithelial compartments in the colon, priming the system for excessive inflammatory responses and the emergence of a hypersensitive epithelial cell state thus derailing proper repair of the intestinal epithelium after injury.

immunology↗

A single MHCII neoepitope mRNA vaccine elicits CD4 T- and B- cell responses promoting endogenous CD8 anti-tumor immunity

Recent progress in therapeutic cancer vaccines has shown promising clinical activity, especially when targeting MHC class I (MHCI) neoantigen-specific CD8+ T cell responses in post-surgical patients. To explore the role of CD4+ T cells in vaccine-dependent tumor rejection, we constructed an mRNA lipoplex vaccine encoding a single MHCII-restricted neoantigen. The vaccine elicited Tfh and Th1 cell responses while decreasing Tregs, leading to rejection of established tumors in mice. IL-21 and IFN-{gamma}, crucial for Tfh and Th1 function respectively, contributed to anti-tumor activity. B cells and neoantigen-specific antibodies were also shown to participate in vaccine efficacy. Additionally, conventional type 1 dendritic cells (cDC1s) were essential for eliciting vaccine-induced CD4+ T cells, and both cDC1s and CD4+ T cells were required to enhance endogenous CD8+ responses, which were crucial for tumor control. Our results suggest that immunizing against MHCII neoantigens alone is sufficient to orchestrate a potent and cooperative immune response against cancer.

immunology↗

G-CSF and IL-6 drive myeloid dysregulation during severe viral infection

Dysregulated myeloid states are associated with disease severity in both sepsis and COVID-19. However, their relevance in non-COVID-19 viral infection, the factors driving their induction, and their role in tissue injury remain poorly understood. We performed a meta-analysis of 1,622,180 myeloid cells from 890 COVID-19 or sepsis patients and controls across 19 published blood scRNA-seq datasets, which revealed severity-associated gene programs in both neutrophils and monocytes pointing to emergency myelopoiesis (EM). Using published bulk transcriptional data from 562 individuals with non-COVID-19 viral disease, we show that these signatures are similarly upregulated during severe influenza and RSV infection. Analysis of transcriptional and proteomic responses in tocilizumab-treated COVID-19 patients show that IL-6 signaling blockade results in a partial reduction of EM signatures and a compensatory increase in the growth factor G-CSF. Using a cellular model of human myelopoiesis, we show that both IL-6 and G-CSF stimulate the production of myeloid cells that express EM signatures in vitro. Using a mouse model of severe influenza infection, we demonstrate the effect of IL-6 and G-CSF signaling blockade on EM-associated myeloid cells, and highlight the opposing effects of EM-induced neutrophils and monocytes on tissue injury. Our study demonstrates the link between systemic cytokines and myeloid dysregulation during severe infection in humans, and highlights the cooperative role of IL-6 and G-CSF signaling in driving infection-induced myelopoiesis.

immunology↗

Comparative Analysis of Commercial Single-Cell RNA Sequencing Technologies

This study evaluates ten commercially available single-cell RNA sequencing (scRNA-seq) approaches across four technology groups: Emulsion-based kits from 10x Genomics and Fluent Biosciences; Microwell-based kits from Becton Dickinson, Honeycomb Technologies and Singerlon Technologies; Combinatorial-indexing kits from Parse Biosciences and Scale Biosciences; and a Matrigel-based kit from Scipio Biosciences. Peripheral blood mononuclear cells (PBMCs) from a single donor were used to assess analytical performance. Key features such as sample compatibility, cost, and experimental duration were also compared. Notably, superior analytical performance was demonstrated by the Chromium Fixed RNA Profiling kit from 10x Genomics, which uniquely features probe hybridization for transcript detection. Additionally, the Rhapsody WTA kit from Becton Dickinson provided a cost-effective balance of performance and expense per cell. With a rich dataset of 218,154 cells, this work provides a basis for differentiating commercial scRNA-seq technologies, which is intended to facilitate the effective application and further methodological development of single cell transcriptomics.

molecular biology↗

Overloading And unpacKing (OAK) - droplet-based combinatorial indexing for ultra-high throughput single-cell multiomic profiling

Multiomic profiling of single cells by sequencing is a powerful technique for investigating cellular diversity in complex biological systems. Although the existing droplet-based microfluidic methods have advanced single-cell sequencing, they produce a plethora of cell-free droplets and underutilize barcoding capacities due to their low cell concentration prerequisites. Meanwhile, combinatorial indexing on microplates can index cells in a more effective way; however, it requires time-consuming and laborious protocols involving multiple splitting and pooling steps. Addressing these constraints, we have developed "Overloading And unpacKing" (OAK). With reduced labor intensity, OAK can provide cost-effective multiomic profiling for hundreds of thousands of cells, offering detection sensitivity on par with commercial droplet-based methods. To demonstrate OAKs versatility, we conducted single-cell RNA sequencing (scRNA-Seq) as well as joint single-nucleus RNA sequencing (snRNA-Seq) and single-nucleus Assay for Transposase Accessible Chromatin with sequencing (snATAC-Seq) using cell lines. We further showcased OAKs performance on more complex samples, including in vitro differentiated bronchial epithelial cells and primary retinal tissues. Finally, we examined transcriptomic responses of 408,000 melanoma cells across around 1,000 starting lineages over a 90-day treatment with a RAF inhibitor, belvarafenib. We discovered a rare cell population (0.12%) that underwent a sequence of transcriptomic changes, resulting in belvarafenib resistance. Ultra-high throughput, broad compatibility with diverse molecular modalities, high detection sensitivity, and simplified experimental procedures distinguish OAK from previous methods, and render OAK a powerful tool for large-scale analysis of molecular signatures, even for rare cells.

molecular biology↗

Glomerular immune injury promotes anti-tumor activity

Recent evidence suggests that the interaction between the tumor microenvironment (TME) and systemic host environment can alter the host immune system to promote anti-tumor activity. Here, we investigated whether glomerular immune injury affects cancer progression. We used nephrotoxic serum nephritis (NTN), a model for glomerular immune injury, and followed it by cancer cell implantation. NTS-injected mice developed smaller primary tumors compared with controls. Tumors of NTS-injected mice had more activated CD8 T cells, suggesting a role for the immune system in the anti-tumor phenotype. Using RNA-seq data, we identified transcriptomic alterations in the bone marrow following NTN. Moreover, using scRNA-seq of white blood cells following NTN we found these transcriptomic alterations were reflected in {gamma}{delta} T cells and neutrophils. This is the first study to show that glomerular immune injury changes the transcription of cells in the bone marrow to advance anti-tumor activity. Our study highlights the pivotal role of BM-mediated transcriptional alterations underlying the enhanced host immunity to tumor growth.

cancer biology↗

Aging and senescence-associated analysis of the aged kidney glomerulus highlights the role of mesangial cells in renal aging

Most causes of chronic kidney disease begin with injury to the glomerulus and involve progressive loss of kidney function. The glomerulus is a capillary bed where blood filtration to produce urine in the kidney occurs. During aging, there is progressive loss of glomeruli and filtration capacity of the kidney because podocytes, the glomerular epithelial cell, are lost with aging and after injury. Podocytes cannot divide and therefore cannot be replaced. Our histological analysis confirmed the presence of glomerulosclerosis, generalized interstitial fibrosis and glomerular hypertrophy in the aged mouse kidney. One barrier to studies of glomeruli is their low frequency in the kidney, less than 1.5% of the cells, and as such, they are often underrepresented in whole kidney analyses. To address this challenge, we used both bulk and single cell RNA sequencing (scRNA-Seq) to characterize purified glomeruli from young and aged mice. Aged glomeruli displayed increased inflammation and expressed a variety of injury and senescence-associated markers, most notably in mesangial cells and macrophages. This increased expression of senescence markers in mesangial cells of aged kidneys suggests a potential cellular target to address age-related renal dysfunction and chronic kidney disease (CKD), which represent a tremendous unmet medical need.

cell biology↗

Coordinated wound responses in a regenerative animal-algal photosymbiotic metaorganism

Animal regeneration requires coordinated responses of many cell types throughout the animal body. In animals carrying endosymbionts, cells from the other species may also participate in regeneration, but how cellular responses are integrated across species is yet to be unraveled. Here, we study the acoel Convolutriloba longifissura, which hosts symbiotic Tetraselmis green algae and can regenerate entire bodies from small tissue fragments. We show that animal injury leads to a decline in the photosynthetic efficiency of the symbiotic algae and concurrently induces upregulation of a cohort of photosynthesis-related genes. A deeply conserved animal transcription factor, runt, is induced after injury and required for the acoel regeneration. Knockdown of runt also dampens algal transcriptional responses to the host injury, particularly in photosynthesis related pathways, and results in further reduction of photosynthetic efficiency post-injury. Our results suggest that the runt-dependent animal regeneration program coordinates wound responses across the symbiotic partners and regulates photosynthetic carbon assimilation in this metaorganism.

developmental biology↗

The renal capsule: a vibrant and adaptive cell environment of the kidney in homeostasis, disease and aging

The kidney is a complex organ that governs many physiological parameters. It is roughly divided into three parts, the renal pelvis, medulla, and cortex. Covering the cortex is the renal capsule, a serosal tissue that provides protection and forms a barrier for the kidney. Serosal tissues of many organs have been recently shown to play a vital role in homeostasis and disease. Analyses of the cells that reside in these tissues have identified distinct cell types with unique phenotypes. Here, we characterized this niche and found that it is mainly comprised of fibroblasts and macrophages, but also includes other diverse cell types. Characterizing renal capsule-associated macrophages, we found that they consist of a distinct subset (i.e., TLF+ macrophages) that is nearly absent in the kidney parenchyma. Injury, disease, and other changes that involve the kidney, affected the cell composition of the renal capsule, indicating its dynamic response to changes within the organ parenchyma. Lastly, we studied age-related changes in the renal capsule and found that aging affected the cell composition and inflammatory phenotype of macrophages, increased CD8 T cells and other lymphocyte counts, and promoted a senescence-associated phenotype in fibroblasts. Taken together, our data illustrate the complexity and heterogeneity of the renal capsule and its underlying changes during aging and disease, improving our understanding of the kidney serosa that may be valuable for novel renal therapies.

immunology↗

Generation of Schwann cell derived melanocytes from hPSCs identifies pro-metastatic factors in melanoma

Summary/AbstractThe neural crest (NC) is highly multipotent and generates diverse lineages in the developing embryo. However, spatiotemporally distinct NC populations display differences in fate potential, such as increased gliogenic and parasympathetic potential from later migrating, nerve-associated Schwann cell precursors (SCPs). Interestingly, while melanogenic potential is shared by both early migrating NC and SCPs, differences in melanocyte identity resulting from differentiation through these temporally distinct progenitors have not been determined. Here, we leverage a human pluripotent stem cell (hPSC) model of NC temporal patterning to comprehensively characterize human NC heterogeneity, fate bias, and lineage development. We captured the transition of NC differentiation between temporally and transcriptionally distinct melanogenic progenitors and identified modules of candidate transcription factor and signaling activity associated with this transition. For the first time, we established a protocol for the directed differentiation of melanocytes from hPSCs through a SCP intermediate, termed trajectory 2 (T2) melanocytes. Leveraging an existing protocol for differentiating early NC-derived melanocytes, termed trajectory 1 (T1), we performed the first comprehensive comparison of transcriptional and functional differences between these distinct melanocyte populations, revealing differences in pigmentation and unique expression of transcription factors, ligands, receptors and surface markers. We found a significant link between the T2 melanocyte transcriptional signature and decreased survival in melanoma patients in the cancer genome atlas (TCGA). We performed an in vivo CRISPRi screen of T1 and T2 melanocyte signature genes in a human melanoma cell line and discovered several T2-specific markers that promote lung metastasis in mice. We further demonstrated that one of these factors, SNRPB, regulates the splicing of transcripts involved in metastasis relevant functions such as migration, cell adhesion and proliferation. Overall, this study identifies distinct developmental trajectories as a source of diversity in melanocytes and implicates the unique molecular signature of SCP-derived melanocytes in metastatic melanoma.

developmental biology↗

Dissecting the contributions of tumor heterogeneity on metastasis at single-cell resolution

Metastasis is the leading cause of cancer-related deaths, but metastasis research is challenged by limited access to patient material and a lack of experimental models that appropriately recapitulate tumor heterogeneity. Here, we analyzed single-cell transcriptomes of matched primary tumor and metastasis from patient-derived xenograft models of breast cancer, demonstrating that primary tumor and metastatic cells show profound transcriptional differences across heterogeneous tumors. While primary tumor cells upregulated several metabolic genes, metastatic cells displayed a motility phenotype in micrometastatic lesions and increased stress response signaling during metastatic progression. Additionally, we identified gene signatures that are associated with the metastatic potential and correlated with patient outcomes. Poorly metastatic primary tumors showed increased immune-regulatory control that may prevent metastasis, whereas highly metastatic primary tumors upregulated markers of epithelial-mesenchymal transition (EMT). We found that intra-tumor heterogeneity is dominated by epithelial-mesenchymal plasticity (EMP) which presented as a dynamic continuum with intermediate cell states that were characterized by novel, specific markers. These intermediate EMP markers correlated with worse patient outcomes and could serve as potential new therapeutic targets to block metastatic development.

cancer biology↗

Neuroendocrinology of the lung revealed by single cell RNA sequencing

Pulmonary neuroendocrine cells (PNECs) are sensory epithelial cells that transmit airway status to the brain via sensory neurons and locally via CGRP and GABA. Several other neuropeptides and neurotransmitters have been detected in various species, but the number, targets, functions, and conservation of PNEC signals are largely unknown. We used scRNAseq to profile hundreds of the rare mouse and human PNECs. This revealed >40 PNEC neuropeptide and peptide hormone genes, most cells expressing unique combinations of 5-18 genes. Peptides are packaged in separate vesicles, release presumably regulated by the distinct, multimodal combinations of sensors expressed by each PNEC. Expression of cognate receptors predicts an array of local targets, and we show the new PNEC signal angiotensin directly activates innervating sensory neurons. Many signals lack lung targets so may have endocrine activity like those of PNEC- derived carcinoid tumors. PNECs are an extraordinarily rich and diverse signaling hub rivaling the enteroendocrine system.

bioinformatics↗

Molecular hallmarks of heterochronic parabiosis at single cell resolution

Slowing or reversing biological ageing would have major implications for mitigating disease risk and maintaining vitality. While an increasing number of interventions show promise for rejuvenation, the effectiveness on disparate cell types across the body and the molecular pathways susceptible to rejuvenation remain largely unexplored. We performed single-cell RNA-sequencing on 13 organs to reveal cell type specific responses to young or aged blood in heterochronic parabiosis. Adipose mesenchymal stromal cells, hematopoietic stem cells, hepatocytes, and endothelial cells from multiple tissues appear especially responsive. On the pathway level, young blood invokes novel gene sets in addition to reversing established ageing patterns, with the global rescue of genes encoding electron transport chain subunits pinpointing a prominent role of mitochondrial function in parabiosis-mediated rejuvenation. Intriguingly, we observed an almost universal loss of gene expression with age that is largely mimicked by parabiosis: aged blood reduces global gene expression, and young blood restores it. Altogether, these data lay the groundwork for a systemic understanding of the interplay between blood-borne factors and cellular integrity.

genomics↗

Tuning TPO-R signaling to influence hematopoietic stem cell differentiation and inhibit essential thrombocythemia

Thrombopoietin (TPO) and the TPO-receptor (TPO-R, or c-MPL)) are essential for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Agents that can modulate TPO-R signaling are highly desirable, both experimentally and clinically. We have developed a series of surrogate protein-ligands for TPO-R, in the form of diabodies, that homodimerize the TPO-R on the cell surface in different geometries, in effect tuning downstream signaling responses. These surrogate ligands exhibit diverse pharmacological properties, inducing graded signaling outputs, from full to partial TPO agonism and antagonism, thus decoupling the dual functions of TPO/TPO-R. Using scRNA sequencing and HSC self-renewal assays we find that partial agonistic diabodies preserved the stem-like properties of cultured HSCs, but also blocked oncogenic colony formation in Essential Thrombocythemia (ET) through inverse agonism. Our data suggest that dampening downstream TPO signaling is a powerful approach not only for HSC preservation in culture, but also for inhibiting oncogenic signaling through the TPO-R. Significance StatementThe TPO cytokine, which signals through its receptor c-MPL (or TPO-R), is essential for megakaryocyte differentiation and maintenance of hematopoietic stem cells (HSCs). Its signaling is deregulated in Essential Thrombocythemia (ET). Here, we engineered diabodies (DBs) against the TPO-R as surrogate TPO ligands to manipulate TPO-R signaling, from full to partial to antagonism, thus decoupling the dual functions of TPO/TPO-R (i.e, HSC maintenance versus megakaryopoiesis). We subsequently discovered that partial agonistic DBs, by reducing the strength of the TPO-R signal, not only preserved HSCs in culture, but also blocked oncogenic signaling in ET. This finding has the potential to improve HSC cultures for transplants, as well as serve as a unique therapeutic approach for ET.

cell biology↗

Single cell analysis of human site-specific melanocyte differentiation and the decoding of developmental programs in melanoma

In humans, epidermal melanocytes are responsible for skin pigmentation, defense against ultraviolet radiation, and the deadliest common skin cancer, melanoma. While there is substantial overlap in melanocyte development pathways between different model organisms, species dependent differences are frequent and the conservation of these processes in human skin remains unresolved1-3. Thus, the biology of developing and adult human melanocytes remains largely uncharacterized. Here, we used a single-cell enrichment and RNA-sequencing pipeline to study human epidermal melanocytes derived directly from skin, capturing transcriptomes across different anatomic sites, developmental age, sexes, and multiple skin tones. Using donor-matched skin from distinct volar and non-volar anatomic locations, we uncovered subpopulations of melanocytes exhibiting site-specific enrichment that occurs during gestation and persists through adulthood. In addition, we identified human melanocyte differentiation transcriptional programs that are distinct from gene signatures generated from model systems. Finally, we use these programs to define patterns of dedifferentiation that are predictive of melanoma prognosis. Overall, the characterization of human melanocytes fresh from skin revealed new subpopulations, human-specific transcriptional programs, and valuable insights into melanoma dedifferentiation.

cancer biology↗

Differential encoding in prefrontal cortex projection neuron classes across cognitive tasks

Single-cell transcriptomics has been widely applied to classify neurons in the mammalian brain, while systems neuroscience has historically analyzed the encoding properties of cortical neurons without considering cell types. Here we examine how specific transcriptomic types of mouse prefrontal cortex (PFC) projection neurons relate to axonal projections and encoding properties across multiple cognitive tasks. We found that most types projected to multiple targets, and most targets received projections from multiple types, except PFC[->]PAG (periaqueductal gray). By comparing Ca2+-activity of the molecularly homogeneous PFC[->]PAG type against two heterogeneous classes in several two-alternative choice tasks in freely-moving mice, we found that all task-related signals assayed were qualitatively present in all examined classes. However, PAG-projecting neurons most potently encoded choice in cued tasks, whereas contralateral PFC-projecting neurons most potently encoded reward context in an uncued task. Thus, task signals are organized redundantly, but with clear quantitative biases across cells of specific molecular-anatomical characteristics.

neuroscience↗