Biology subjects

Henderson, N. C.

Publications and source records attributed to Henderson, N. C..

At least 19 recordsLinked to original sources

bioRxiv2026

In utero barcoding of mouse mesoderm reveals the clonal architecture of organ mesenchyme

The mesoderm coordinates organogenesis and generates the cardiovascular, visceral and musculoskeletal systems, yet in mammals this germ layer is difficult to access, impeding its genetic manipulation and study. Here, we establish embryonic day (E) 7.5 exocoelomic cavity nano-injection as a scalable route for selective genetic targeting of mouse mesoderm. Combining in utero lentiviral barcoding with single-cell and spatial transcriptomics, we reconstructed mesodermal and neural crest clonal relationships across >590,000 cells and 31,000 multicellular clones from E9.5-E10.5 embryos and E16.5 livers and hearts. In liver, capsular mesothelium was closely related to hepatic stellate cells, whereas fibroblast and vascular smooth muscle lineages diverged earlier. In heart, epicardium was clonally coupled to three spatially restricted mesenchymal sub-lineages but not to cardiomyocytes, and lineage tracing separated epicardial- and neural crest-derived populations within transcriptionally convergent valve mesenchyme. This approach enables Cre-independent manipulation of mammalian mesoderm and resolves organ mesenchyme development at clonal resolution.

developmental biology↗

bioRxiv2025

A Human Single-cell Atlas Identifies OLR1+ Scar-associated Macrophages as a Therapeutic Target for Chronic Liver Disease

Chronic liver disease (CLD) is a major global healthcare problem. Irrespective of cause, chronic damage to the liver results in fibrosis, which is associated with adverse clinical outcomes. Immune cells, in particular monocyte-derived macrophages (MDMs), are key regulators of fibrosis and represent an attractive therapeutic target for CLD. However, it has remained unclear which specific subpopulation of MDMs drives pro-inflammatory and pro-fibrotic functions in human CLD and how they might be selectively modulated. Here, we generate an annotated human liver single cell atlas from 42 healthy and 35 CLD patients, identifying 125 transcriptionally distinct cellular states. Leveraging large patient and cell numbers, our atlas resolves rare liver cell states and distinguishes two types of disease-expanded scar-associated macrophages (SAMac), including a specific subpopulation with a pro-inflammatory pro-fibrotic phenotype. The scavenger receptor OLR1 was enriched in pro-inflammatory SAMacs and high hepatic OLR1 expression was associated with increased mortality in CLD patients. A corresponding monocyte-derived OLR1+ SAMac subpopulation expanded in a mouse model of CLD and exhibited a pro-inflammatory phenotype based on single-cell RNA-seq, single-cell ATAC-seq, and flow cytometry analyses. Primary human OLR1+ MDMs promoted fibrogenic signalling in multilineage liver spheroid cultures, whilst specific targeting of OLR1 reduced IL-1{beta} production by human macrophages and attenuated myofibroblast activation. Overall, our annotated human liver single-cell atlas provides a valuable reference to study disease-associated cell states in CLD. We utilise this resource to identify a distinct pro-inflammatory subpopulation of SAMacs and highlight OLR1 as a potential therapeutic target to specifically modulate SAMac function and attenuate liver fibrosis.

immunology↗

bioRxiv2023

Cellular heterogeneity of the developing worker honey bee (Apis mellifera) pupa: a single cell transcriptomics analysis

It is estimated that animals pollinate 87.5% of flowering plants worldwide and that managed honey bees (Apis mellifera) account for 30-50% of this ecosystem service to agriculture. In addition to their important role as pollinators, honey bees are well-established insect models for studying learning and memory, behaviour, caste differentiation, epigenetic mechanisms, olfactory biology, sex determination and eusociality. Despite their importance to agriculture, knowledge of honey bee biology lags behind many other livestock species. In this study we have used scRNA-Seq to map cell types to different developmental stages of the worker honey bee (prepupa at day 11 and pupa at day 15), and sought to determine their gene signatures and thereby provide potential functional annotations for as yet poorly characterized genes. To identify cell type populations we examined the cell-to-cell network based on the similarity of the single-cells transcriptomic profiles. Grouping similar cells together we identified 63 different cell clusters of which 15 clusters were identifiable at both stages. To determine genes associated with specific cell populations or with a particular biological process involved in honey bee development, we used gene co-expression analysis. We combined this analysis with literature mining, the honey bee protein atlas and Gene Ontology analysis to determine cell cluster identity. Of the cell clusters identified, 9 were related to the nervous system, 7 to the fat body, 14 to the cuticle, 5 to muscle, 4 to compound eye, 2 to midgut, 2 to hemocytes and 1 to malpighian tubule/pericardial nephrocyte. To our knowledge, this is the first whole single cell atlas of honey bees at any stage of development and demonstrates the potential for further work to investigate their biology of at the cellular level.

genomics↗

bioRxiv2023

Phenotypic and spatial heterogeneity of brain myeloid cells after stroke is associated with cell ontogeny, tissue damage, and brain connectivity

Acute stroke causes substantial mortality and morbidity and provokes extensive changes to myeloid immune cell populations in the brain that may be targets for limiting brain damage and enhancing brain repair. The most effective immunomodulatory approaches will require precise manipulation of discrete myeloid cell phenotypes in time and space to avoid harmful effects of indiscriminate neuroimmune perturbation. We sought to define how stroke alters the composition and phenotypes of mononuclear myeloid cells with particular attention to how cell ontogeny and spatial organisation combine to expand myeloid cell diversity across the brain after stroke. Multiple reactive microglial states and dual monocyte-derived populations contributed to an extensive repertoire of myeloid cells in post-stroke brain. We identified important overlap and distinctions among different cell types and states that involved ontogeny- and spatial-related properties. Notably, brain connectivity with infarcted tissue underpinned the pattern of local and remote altered cell accumulation and reactivity. Our discoveries suggest a global but anatomically-governed brain myeloid cell response to stroke that comprises diverse phenotypes arising through intrinsic cell ontogeny factors interacting with exposure to spatially-organised brain damage and neuroaxonal cues.

neuroscience↗

bioRxiv2023

Multimodal decoding of human liver regeneration

The liver has a unique ability to regenerate1,2, however in the setting of acute liver failure (ALF) this regenerative capacity is often overwhelmed and emergency liver transplantation is the only curative option3-5. To advance our understanding of human liver regeneration and to inform design of pro-regenerative therapies, we use paired single-nuclei RNA sequencing (snRNA-seq) combined with spatial profiling of healthy and ALF explant human livers to generate the first single-cell, pan-lineage atlas of human liver regeneration. We uncover a novel ANXA2+ migratory hepatocyte subpopulation which emerges during human liver regeneration, and a corollary migratory hepatocyte subpopulation in a mouse model of acetaminophen (APAP)-induced liver regeneration. Importantly, interrogation of necrotic wound closure and hepatocyte proliferation across multiple timepoints following APAP-induced liver injury in mice demonstrates that wound closure precedes hepatocyte proliferation. 4-D intravital imaging of APAP-induced mouse liver injury identifies motile hepatocytes at the edge of the necrotic area, enabling collective migration of the hepatocyte sheet to effect wound closure. Depletion of hepatocyte ANXA2 expression reduces HGF-induced human and mouse hepatocyte migration in vitro, and abrogates necrotic wound closure following APAP-induced mouse liver injury. Taken together, our work dissects unanticipated aspects of liver regeneration, demonstrating an uncoupling of wound closure and hepatocyte proliferation and uncovering a novel migratory hepatocyte subpopulation which mediates wound closure following liver injury. Therapies designed to promote rapid reconstitution of normal hepatic microarchitecture and reparation of the gut-liver barrier may open up new areas of therapeutic discovery in regenerative medicine.

cell biology↗

bioRxiv2022

Mapping the glial transcriptome in Huntington's disease using snRNAseq: Selective disruption of glial signatures across brain regions

Huntingtons disease (HD) is a severely debilitating, autosomal dominant neurodegenerative disease with a fatal outcome. There is accumulating evidence of a prominent role of glia in the pathology of HD, and we investigated this by conducting single nuclear RNA sequencing (snRNAseq) of human post mortem brain in four differentially affected regions; caudate nucleus, frontal cortex, hippocampus and cerebellum. Across 127,205 nuclei from people with HD, and age/sex matched controls, we found heterogeneity of glia which is altered in HD. We describe prominent changes in the abundance of certain subtypes of astrocytes, microglia, oligodendrocyte precursor cells and oligodendrocytes between HD and control samples, and these differences are widespread across brain regions. Furthermore, we highlight two possible mechanisms that characterise the glial contribution to disease pathology. Firstly, we show that upregulation of molecular chaperones represents a cross-glial signature in HD, which likely reflects an adaptive response to the accumulation of mutant Huntingtin (mHTT). Secondly, we show an oligodendrocyte-specific upregulation of the calmodulin-dependent 3,5-cyclic nucleotide phosphodiesterase 1A (PDE1A) in HD brain compared to controls, which may cause dysfunction of key cellular functions due to the downregulation of the important second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Our results support the hypothesis that glia have an important role in the pathology of HD, and show that all types of glia are affected in the disease. As glia are more tractable to treat than neurons, our findings may be of therapeutic relevance.

neuroscience↗

bioRxiv2022

Single cell transcriptomics of Atlantic salmon (Salmo salar L.) liver reveals cellular heterogeneity and immunological responses to challenge by Aeromonas salmonicida

The liver is a multitasking organ with essential functions for vertebrate health spanning metabolism and immunity. In contrast to mammals, our understanding of liver cellular heterogeneity and its role in regulating immunological status remains poorly defined in fishes. Addressing this knowledge gap, we generated a transcriptomic atlas of 47,432 nuclei isolated from the liver of Atlantic salmon (Salmo salar L.) contrasting control fish with those challenged with a pathogenic strain of Aeromonas salmonicida, a problematic bacterial pathogen in global aquaculture. We identified the major liver cell types and their sub-populations, revealing poor conservation of many hepatic cell marker genes utilized in mammals, while identifying novel heterogeneity within the hepatocyte, lymphoid, and myeloid lineages. This included polyploid hepatocytes, multiple T cell populations including {gamma}{delta} T cells, and candidate populations of monocytes/macrophages/dendritic cells. A dominant hepatocyte population radically remodeled its transcriptome following infection to activate the acute phase response and other defense functions, while repressing routine functions such as metabolism. These defense-specialized hepatocytes showed strong activation of genes controlling protein synthesis and secretion, presumably to support the release of acute phase proteins into circulation. The infection response further involved up-regulation of numerous genes in an immune-cell specific manner, reflecting functions in pathogen recognition and killing, antigen presentation, phagocytosis, regulation of inflammation, B cell differentiation and T cell activation. Overall, this study greatly enhances our understanding of the multifaceted role played by liver cells in immune defense and metabolic remodeling following infection and provides many novel cell-specific marker genes to empower future studies of this organ in fishes.

immunology↗

bioRxiv2022

Identification of a Broadly Fibrogenic Macrophage Subset Induced by Type 3 Inflammation in Human and Murine Liver and Lung Fibrosis

Macrophages are central orchestrators of the tissue response to injury, with distinct macrophage activation states playing key roles in the progression and resolution of fibrosis. Identifying the unique fibrogenic macrophages that are found in human fibrotic tissues could lead to new and more effective treatments for fibrosis. Here we used human liver and lung single cell RNA sequencing datasets to identify a unique subset of CD9+ TREM2+ macrophages expressing SPP1, GPNMB, FABP5, and CD63 with strong pro-fibrotic activity. This population was validated across orthogonal techniques, species and tissues. These macrophages were enriched at the outside edges of scarring adjacent to activated mesenchymal cells, and in the fibrotic niche across species and organs. Neutrophils producing the type 3 cytokines GM-CSF and IL-17A, and expressing MMP9, which participates in the activation of TGF-{beta}1, clustered with these scar-associated macrophages. Using in vitro primary human cell assays, we determined that GM-CSF, IL-17A and TGF-{beta}1 drive the differentiation of these scar-associated macrophages, and that co-culture of monocyte-derived macrophages with hepatic stellate cells and TGF-{beta}1 augmented type 1 collagen deposition. In vivo blockade of GM-CSF, IL-17A or TGF-{beta}1 with small or large molecules reduced scar-associated macrophage expansion and fibrosis in multiple models of hepatic and pulmonary fibrosis. Our work demonstrates that a specific scar-associated macrophage population is linked with fibrosis across species and tissues. It further provides a strategy for unbiased discovery, triage and preclinical validation of therapeutic targets within this fibrogenic macrophage population.

immunology↗

bioRxiv2022

Marked regional glial heterogeneity in the human white matter of the central nervous system

The myelinated white matter tracts of the central nervous system (CNS) are essential for fast transmission of electrical impulses and are commonly affected in neurodegenerative diseases. However, these often uniquely human diseases differentially affect white matter regions, at various ages and between males and females, and we hypothesised that this is secondary to physiological variation in white matter glia with region, age and sex. Using single nucleus RNA sequencing of healthy human post-mortem samples, we find marked glial heterogeneity with tissue region (primary motor cortex, cerebellum, cervical spinal cord), with tissue-specific cell populations of oligodendrocyte precursor cells and astrocytes, and a spinal cord-enriched oligodendrocyte type that appears human-specific. Spinal cord microglia but not astrocytes show a more activated phenotype compared to brain. These regional effects, with additional differentially expressed genes with age and sex in all glial lineages, help explain pathological patterns of disease - essential knowledge for therapeutic strategies.

neuroscience↗

bioRxiv2021

Single cell RNA sequencing and lineage tracing confirm mesenchyme to epithelial transformation (MET) contributes to repair of the endometrium at menstruation

The human endometrium experiences repetitive cycles of tissue wounding characterised by piecemeal shedding of the surface epithelium and rapid restoration of tissue homeostasis. In this study we used a mouse model of endometrial repair and three transgenic lines of mice to investigate whether epithelial cells that become incorporated into the newly formed luminal epithelium have their origins in one or more of the mesenchymal cell types present in the stromal compartment of the endometrium. Using scRNAseq we identified a novel population of PDGFRb+ mesenchymal stromal cells that developed a unique transcriptomic signature in response to endometrial breakdown/repair. These cells expressed genes usually considered specific to epithelial cells and in silico trajectory analysis suggested they were stromal fibroblasts in transition to becoming epithelial cells. To confirm our hypothesis we used a lineage tracing strategy to compare the fate of stromal fibroblasts (PDGFRa+) and stromal perivascular cells (NG2/CSPG4+). We demonstrated that stromal fibroblasts can undergo a mesenchyme to epithelial transformation and become incorporated into the re-epithelialised luminal surface of the repaired tissue. This study is the first to discover a novel population of wound-responsive, plastic endometrial stromal fibroblasts that contribute to the rapid restoration of an intact luminal epithelium during endometrial repair. These findings form a platform for comparisons both to endometrial pathologies which involve a fibrotic response (Ashermans syndrome, endometriosis) as well as other mucosal tissues which have a variable response to wounding. HighlightsO_LISingle cell RNA sequencing identified a population of PDGFR{beta}+ mesenchymal cells with a unique transcriptomic signature that arises in response to induction of a menses-like wound C_LIO_LIThe repair-specific mesenchymal cells express genes considered specific to both mesenchymal and epithelial lineages indicative of an intermediate phenotype and a mesenchymal to epithelial transition (MET) C_LIO_LIin silico trajectory analysis revealed that repair-specific cells originate from the fibroblast cell clusters, were distinct from perivascular cells, and had a predicted trans-differentiation trajectory towards definitive epithelial cells C_LIO_LIin vivo lineage tracing in adult mice provides definitive evidence that PDGFR+ endometrial fibroblasts, and not NG2+ perivascular cells, undergo MET and can become incorporated into the luminal epithelium of the post repair tissue C_LI

physiology↗

bioRxiv2020

Milo: differential abundance testing on single-cell data using k-NN graphs

Single-cell omic protocols applied to disease, development or mechanistic studies can reveal the emergence of aberrant cell states or changes in differentiation. These perturbations can manifest as a shift in the abundance of cells associated with a biological condition. Current computational workflows for comparative analyses typically use discrete clusters as input when testing for differential abundance between experimental conditions. However, clusters are not always an optimal representation of the biological manifold on which cells lie, especially in the context of continuous differentiation trajectories. To overcome these barriers to discovery, we present Milo, a flexible and scalable statistical framework that performs differential abundance testing by assigning cells to partially overlapping neighbourhoods on a k-nearest neighbour graph. Our method samples and refines neighbourhoods across the graph and leverages the flexibility of generalized linear models, making it applicable to a wide range of experimental settings. Using simulations, we show that Milo is both robust and sensitive, and can reveal subtle but important cell state perturbations that are obscured by discretizing cells into clusters. We illustrate the power of Milo by identifying the perturbed differentiation during ageing of a lineage-biased thymic epithelial precursor state and by uncovering extensive perturbation to multiple lineages in human cirrhotic liver. Milo is provided as an open-source R software package with documentation and tutorials at https://github.com/MarioniLab/miloR.

bioinformatics↗

bioRxiv2020

The proportion of periportal mesenchyme to ductal epithelial cells acts as a proliferative rheostat in liver regeneration

In the homeostatic liver, ductal cells intermingle with a microenvironment of endothelial and mesenchymal cells to form the functional unit of the portal tract. Ductal cells proliferate rarely in homeostasis but do so transiently after tissue injury to replenish any lost epithelium. We have shown that liver ductal cells can be expanded as liver organoids that recapitulate several of the cell-autonomous mechanisms of regeneration, but lack the stromal cell milieu of the biliary tract in vivo. Here, we describe a subpopulation of SCA1+ periportal mesenchymal cells that closely surrounds ductal cells in vivo and exerts a dual control on their proliferative capacity. Mesenchymal-secreted mitogens support liver organoid formation and expansion from differentiated ductal cells. However, direct mesenchymal-to-ductal cell-cell contact, established following a microfluidic co-encapsulation that enables the cells to self-organize into chimeric organoid structures, abolishes ductal cell proliferation in a mesenchyme-dose dependent manner. We found that it is the ratio between mesenchymal and epithelial cell contacts that determines the net outcome of ductal cell proliferation both in vitro, and in vivo, during damage-regeneration. SCA1+ mesenchymal cells control ductal cell proliferation dynamics by a mechanism involving, at least in part, Notch signalling activation. Our findings underscore how the relative abundance of cell-cell contacts between the epithelium and its mesenchymal microenvironment are key regulatory cues involved in the control of tissue regeneration. SummaryIn the homeostatic liver, the ductal epithelium intermingles with a microenvironment of stromal cells to form the functional unit of the portal tract. Ductal cells proliferate rarely in homeostasis but do so transiently after tissue injury. We have shown that these cells can be expanded as liver organoids that recapitulate several of the cell-autonomous mechanisms of regeneration, but lack the stromal cell milieu of the portal tract in vivo. Here, we describe a subpopulation of SCA1+ periportal mesenchymal niche cells that closely surrounds ductal cells in vivo and exerts a dual control on their proliferative capacity. Mesenchymal-secreted mitogens support liver organoid formation and expansion from differentiated ductal cells. However, direct mesenchymal-to-ductal cell-cell contact, established through a microfluidic co-encapsulation method that enables the cells to self-organize into chimeric organoid structures, abolishes ductal cell proliferation in a mesenchyme-dose dependent manner. We found that it is the ratio between mesenchymal and epithelial cell contacts that determines the net outcome of ductal cell proliferation both in vitro, and in vivo, during damage-regeneration. SCA1+ mesenchymal cells control ductal cell proliferation dynamics by a mechanism involving, at least in part, Notch signalling activation. Our findings re-evaluate the concept of the cellular niche, whereby the proportions of cell-cell contacts between the epithelium and its mesenchymal niche, and not the absolute cell numbers, are the key regulatory cues involved in the control of tissue regeneration.

developmental biology↗

bioRxiv2020

Stretch Regulates Alveologenesis Via Mesenchymal Gαq/11-Mediated TGFβ2 Activation

Alveolar development and repair require tight spatiotemporal regulation of numerous signalling pathways that are influenced by chemical and mechanical stimuli. Mesenchymal cells play key roles in numerous developmental processes. Transforming growth factor-{beta} (TGF{beta}) is essential for alveologenesis and lung repair, and the G protein subunits Gq and G11 (Gq/11) transmit mechanical and chemical signals to activate TGF{beta} in epithelial cells. To understand the role of mesenchymal Gq/11 in lung development, we generated constitutive (Pdgfrb-Cre+/-;Gnaqfl/fl;Gna11-/-) and inducible (Pdgfrb-Cre/ERT2+/-;Gnaqfl/fl;Gna11-/-) mesenchymal Gq/11 deleted mice. Mice with constitutive Gq/11 gene deletion exhibited abnormal alveolar development, with suppressed myofibroblast differentiation, altered mesenchymal cell synthetic function, and reduced lung TGF{beta}2 deposition, as well as kidney abnormalities. Tamoxifen-induced mesenchymal Gq/11 gene deletion in adult mice resulted in emphysema associated with reduced TGF{beta}2 and elastin deposition. Cyclical mechanical stretch-induced TGF{beta} activation required Gq/11 signalling and serine protease activity, but was independent of integrins, suggesting an isoform-specific role for TGF{beta}2. These data highlight a previously undescribed mechanism of cyclical stretch-induced Gq/11-dependent TGF{beta}2 signalling in mesenchymal cells, which is imperative for normal alveologenesis and maintenance of lung homeostasis. Summary statementMesenchymal cell Gq/11 signalling regulates myofibroblast function and stretch-mediated TGF{beta}2 signalling, which are important for alveologenesis and organ homeostasis. These mechanisms are relevant to both developmental and adult lung disease.

developmental biology↗

bioRxiv2020

Single cell RNA sequencing redefines the mesenchymal cell landscape of mouse endometrium

The endometrium is a dynamic tissue that exhibits remarkable resilience to repeated episodes of differentiation, breakdown, regeneration and remodelling. Endometrial physiology relies on a complex interplay between the stromal and epithelial compartments with the former containing a mixture of fibroblasts, vascular and immune cells. There is evidence for rare populations of putative mesenchymal progenitor cells located in the perivascular niche of human endometrium, but the existence of an equivalent cell population in mouse is unclear. In the current study we used the Pdgfrb-BAC-eGFP transgenic reporter mouse in combination with bulk and single cell RNA sequencing (scRNAseq) to redefine the endometrial mesenchyme. Contrary to previous reports we show that CD146 is expressed in both PDGFR{beta}+ perivascular cells as well as CD31+ endothelial cells. Bulk RNAseq revealed cells in the perivascular niche which express high levels of Pdgfrb as well as genes previously identified in pericytes and/or vascular smooth muscle cells (Acta2, Myh11, Olfr78, Cspg4, Rgs4, Rgs5, Kcnj8, Abcc9). scRNAseq identified five subpopulations of cells including closely related pericytes/vascular smooth muscle cells and three subpopulations of fibroblasts. All three fibroblast populations were PDGFR+/CD34+ but were distinct in their expression of Spon2/Angptl7 (fibroblast 1), Smoc2/Rgs2 (fibroblast 2) and Clec3b/Col14a1/Mmp3 (fibroblast 3), with potential functions in regulation of immune responses, response to wounding and organisation of extracellular matrix respectively. In conclusion, these data are the first to provide a single cell atlas of the mesenchymal cell landscape in mouse endometrium. By identifying novel markers for subpopulations of mesenchymal cells we can use mouse models investigate their contribution to endometrial function, compare with other tissues and apply these findings to further our understanding of human endometrium. HighlightsO_LIGFP expression in the mouse endometrium, under the control of the Pdgfrb promoter, is restricted to two cell populations based on the intensity of GFP with GFPbright cells close to the vasculature C_LIO_LISingle cell RNAseq identified five subpopulations of GFP+ mesenchymal cells: pericytes, vascular smooth muscle cells (vSMC) and three closely related but distinct populations of fibroblasts C_LIO_LIBioinformatics revealed that pericytes and vSMC share functions associated with the circulatory system, actin-filament process and cell adhesion, and an apparent role for pericytes in smooth muscle cell migration and response to interferons C_LIO_LIComparisons between the fibroblast subpopulations suggest distinct roles in regulation of immune response, response to wound healing and collagen organisation. C_LI Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

physiology↗

bioRxiv2020

Kidney single-cell atlas reveals myeloid heterogeneity in progression and regression of kidney disease.

The kidney has a limited capacity to repair following injury, however, the endogenous reparative pathways are not well understood. Here we employ integrated droplet- and plate-based scRNA-seq in the murine reversible unilateral ureteric obstruction model to dissect the transcriptomic landscape at the single cell level during renal injury and resolution of fibrosis. We generate a comprehensive catalogue of the changes induced during injury and repair, revealing significant myeloid cell heterogeneity, which would not have been identifiable by conventional flow cytometry. We identify new markers for the myeloid populations within the kidney as well as identification of novel subsets including an Arg1+ monocyte population specific to early injury and a Mmp12+ macrophage subset exclusive to repair. Finally, using paired blood exchange to track circulating immune cells, we confirm that monocytes are recruited to the kidney early after injury and are the source of Ccr2+ macrophages that accumulate in late injury. Our data demonstrate the utility of complementary technologies to identify novel myeloid subtypes that may represent therapeutic targets to inhibit progression or promote regression of kidney disease.

immunology↗

bioRxiv2020

Transfer of hepatocellular microRNA regulates cytochrome P450 2E1 in renal tubular cells

Extracellular microRNAs have been demonstrated to have the ability to enter kidney tubular cells and modify gene expression. We have used a Dicer-hepatocyte-specific microRNA conditional knock-out (Dicer-CKO) mouse to investigate functional microRNA transfer from liver to kidney under physiological conditions and in the context of drug toxicity. Dicer-CKO mice demonstrated a time-dependent decrease in the hepatocyte-derived microRNA, miR-122, in the kidney in the absence of other microRNA changes. During hepatotoxicity, miR-122 increased in kidney tubular cells; this was abolished in Dicer-CKO mice. Depletion of hepatocyte microRNAs increased expression and activity of the miR-122 target - cytochrome (CYP) P450 2E1 - in the kidney. Serum extracellular vesicles (ECVs) from mice with hepatotoxicity increased proximal tubular cell miR-122 and prevented cisplatin proximal tubular cell toxicity. miR-122 also increased in urinary ECVs during hepatotoxicity in humans. Transfer of microRNA was not restricted to liver injury - we detected miR-499 release with murine cardiac injury, and this correlated with an increase in the kidney. In summary, a physiological transfer of microRNA to the kidney exists, which is increased by liver injury. Regulation of renal drug response due to signalling by microRNA of hepatic origin represents a new paradigm for understanding and preventing nephrotoxicity.

pharmacology and toxicology↗

bioRxiv2019

Single-cell RNA-seq reveals CD16- monocytes as key regulators of human monocyte transcriptional response to Toxoplasma

Monocytes are among the major myeloid cells that respond to Toxoplasma, a ubiquitous foodborne that infects [≥]1 billion people worldwide, in human peripheral blood. As such, a molecular understanding of human monocyte-Toxoplasma interactions can expedite the development of novel human toxoplasmosis control strategies. Current molecular studies on monocyte-Toxoplasma interactions are based on average cell or parasite responses across bulk cell populations. Although informative, population-level averages of monocyte responses to Toxoplasma have sometimes produced contradictory results, such as whether CCL2 or IL12 define effective monocyte response to the parasite. Here, we used single-cell dual RNA sequencing (scDual-Seq) to comprehensively define, for the first time, the monocyte and parasite transcriptional responses that underpin human monocyte-Toxoplasma encounters at the single cell level. We report extreme transcriptional variability between individual monocytes. Furthermore, we report that Toxoplasma-exposed and unexposed monocytes are transcriptionally distinguished by a reactive subset of CD14++CD16- monocytes. Functional cytokine assays on sorted monocyte populations show that the infection-distinguishing monocytes secrete high levels of chemokines, such as CCL2 and CXCL5. These findings uncover the Toxoplasma-induced monocyte transcriptional heterogeneity and shed new light on the cell populations that largely define cytokine and chemokine secretion in human monocytes exposed to Toxoplasma.

genomics↗

bioRxiv2019

The circadian clock protein REVERBα inhibits pulmonary fibrosis development

Pulmonary inflammatory responses lie under circadian control; however the importance of circadian mechanisms in fibrosis is not understood. Here, we identify a striking change to these mechanisms resulting in a gain of amplitude and lack of synchrony within pulmonary fibrotic tissue. These changes result from an infiltration of mesenchymal cells, an important cell type in the pathogenesis of pulmonary fibrosis. Mutation of the core clock protein REVERB in these cells exacerbated the development of bleomycin-induced fibrosis, whereas mutation of REVERB in club or myeloid cells had no effect on the bleomycin phenotype. Knockdown of REVERB revealed regulation of the poorly described transcription factor TBPL1. Both REVERB and TBPL1 altered integrin{beta}1 focal adhesion formation, resulting in increased myofibroblast activation. The translational importance of our findings was established through analysis of two human cohorts. In the UK Biobank circadian strain markers (sleep length, chronotype and shift work) are associated with pulmonary fibrosis making them novel risk factors. In a separate cohort REVERB expression was increased in human idiopathic pulmonary fibrosis (IPF) lung tissue. Pharmacological targeting of REVERB inhibited myofibroblast activation in IPF fibroblasts and collagen secretion in organotypic cultures from IPF patients, suggesting targeting REVERB could be a viable therapeutic approach.\n\nSignificanceThe circadian clock plays an essential role in energy metabolism, and inflammation. In contrast the importance of the clock in the pathogenesis of fibrosis remains poorly explored. This study describes a striking alteration in circadian biology during pulmonary fibrosis where the relatively arrhythmic alveolar structures gain circadian but desynchronous rhythmicity due to infiltration by fibroblasts. Disruption of the clock in these cells, which are not widely implicated in circadian pathophysiology, results in a pro-fibrotic phenotype. Translation of these findings in humans revealed previously unrecognised important circadian risk factors for pulmonary fibrosis (sleep length, chronotype and shift work). In addition, targeting REVERB repressed collagen secretion from human fibrotic lung tissue making this protein a promising therapeutic target.

cell biology↗