Biology subjects

Knights, A. J.

Publications and source records attributed to Knights, A. J..

14 recordsLinked to original sources

bioRxiv2026

PIEZO1 mediates mechanosensing and myofibroblastic activation of synovial fibroblasts in post-traumatic osteoarthritis

Fibrosis and inflammation of the synovium are common pathological features of joint diseases. In osteoarthritis (OA), extensive evidence supports that synovial pathology drives symptoms and disease progression, yet the mechanisms underpinning pathological synovial remodeling, including fibrosis, remain unclear. Here, we identify the mechanosensitive ion channel PIEZO1 as a critical regulator of myofibroblast-like activation of synovial fibroblasts (SFs) in post-traumatic osteoarthritis (PTOA). Using multi-omic assessments in human and murine synovium, a murine model of joint injury, histopathology, and functional in vitro phenotyping, we demonstrate that joint injury triggers expansion of myofibroblast-like SFs concurrent with fibrotic remodeling and mechanical stiffening of the synovium. Myofibroblastic commitment of synovial progenitors was associated with activation of the YAP1 regulon, and PIEZO1 agonism drove nuclear accumulation of YAP1 in SFs. PIEZO1 was enriched in synovia of injured joints, and in cultured SFs pharmacologic PIEZO1 activation promoted SMA enrichment and increased matrix contraction, which were both ablated by YAP1 inhibition. PIEZO1 silencing abrogated TGF-{beta}-induced contractility and markedly altered the transcriptomic response to mechanical loading in vitro, including ECM production and neuroinflammatory programs. In vivo, acute intra-articular pharmacologic PIEZO1 activation induced a pro-fibrotic, pro-inflammatory, and anti-adipogenic transcriptomic program in synovium. Collectively, these findings establish PIEZO1 as a key mediator of the pathogenic activation of SFs and identify aberrant mechanosensing as a mechanism underlying synovial fibrosis in PTOA.

cell biology↗

bioRxiv2026

Hepatic cholinergic T cell signaling mediates an adaptive response to alcohol-induced liver stress

Alcohol-associated liver disease (ALD) is a major cause of liver-related morbidity and mortality, yet no targeted, disease-modifying therapy exists and the immune mechanisms driving disease progression remain poorly defined. Acetylcholine-producing cholinergic immune cells have emerged as regulators of homeostasis across tissues and in disease, but their role in alcohol-induced hepatic stress is unknown. Here, we characterized the hepatic cholinergic immune landscape during alcohol-induced stress using flow cytometry and single-cell RNA-sequencing of ChAT-eGFP reporter mice. Alcohol feeding expanded hepatic cholinergic immune cells, with T cells constituting a substantial and dynamic fraction of this population that showed a broad increase in acetylcholine signaling and shifted towards a repressed, anergy-like transcriptional state. CD4 T cells underwent coordinated suppression of cholesterol biosynthesis, while CD8 T cells instead adopted a cytotoxic activation program. Together, these findings identify cholinergic T cell signaling, and cholesterol metabolism within CD4 T cells specifically, as a previously unrecognized adaptive response to alcohol-induced liver stress, nominating candidate therapeutic targets for a disease that is marred by critically limited treatment options.

immunology↗

bioRxiv2026

Thrombospondin-2 deficiency primes the synovial joint for aberrant tissue remodeling and injury response

ObjectiveThis study investigates joint injury-induced angiogenesis and the effects of genetic deficiency of thrombospondin-2 (TSP2), an anti-angiogenic factor, in joint homeostasis and post-traumatic osteoarthritis (PTOA). MethodWe utilized a murine non-invasive anterior cruciate ligament rupture (ACLR) model of PTOA and mined published synovial transcriptomics datasets to investigate injury-induced synovial angiogenesis. Spatial transcriptomics and flow cytometry of TSP2-GFP reporter mice were used to assess injury-induced thrombospondin-2 and its cellular origins in synovium. Global TSP2 knockout mice (TSP2-KO) were used to assess the effect of TSP2 deficiency on early and late stages of PTOA development via molecular imaging of inflammation and angiogenesis, histopathology, micro-computed tomography, Raman spectroscopy, and synovium bulk RNA-sequencing. ResultsIntra-articular angiogenesis peaked at 7d post-ACLR and declined but remained elevated above baseline at 28d post-ACLR. We identified synovial crosstalk between endothelial cells and sublining fibroblasts as a key driver of angiogenesis and source of thrombospondin-2 signaling, with TSP2 primarily upregulated in sublining fibroblasts. TSP2-KO mice exhibited increased peri-articular inflammation at 7d post-ACLR and inferior bone quality. Histopathology revealed greater PTOA severity but paradoxically lower synovitis in TSP2-KOs. Additionally, aberrant structural remodeling of the entire knee joint was observed in uninjured and ACLR TSP2-KO limbs. The uninjured TSP2-KO synovial transcriptome demonstrated elevated immune, fibrotic, and angiogenic activation; however, TSP2-KO and WT synovial transcriptomes partially converged upon injury. ConclusionTSP2 is essential for joint homeostasis and trauma response. Global TSP2 deficiency causes premature OA and worsened PTOA, suggesting that therapeutic targeting with TSP2 mimetic could be used to prevent OA.

physiology↗

bioRxiv2026

Wnt/β-catenin signaling regulates fibrotic atrophy of intra-articular adipose tissue in post-traumatic osteoarthritis

Synovial joints like the knee are home to adipose tissue depots whose anatomy and functions are closely intertwined with that of other intra-articular soft tissues such as synovium, underscoring the growing understanding that joints are multi-tissue organs. Traumatic joint injury and the onset of osteoarthritis (OA) dramatically remodel the intra-articular adipose niche, marked by infiltration of fibrotic tissue postulated to underpin OA-associated joint stiffness and pain, yet we know very little about the disease-associated dynamics of joint adipose remodeling nor the mechanisms driving these phenomena. Here, we employed 2D histomorphometry and spatial transcriptomics, alongside 3D osmium tetroxide-enhanced micro-computed tomography to comprehensively define the spatiotemporal, structural, and transcriptional rewiring of joint adipose tissue in a non-invasive mouse model of post-traumatic osteoarthritis (PTOA). These revealed marked loss of intra-articular adiposity accompanied by expansion of fibroblast-rich, collagen-dense tissue with pro-fibrotic hallmarks and Wnt/{beta}-catenin-enriched gene programs. Joint adipose exhibited a distinct transcriptional signature compared to subcutaneous white adipose tissue, pointing to unique, depot-specific functions. Stromal cells isolated from PTOA joints had heightened baseline expression of fibrotic and Wnt pathway genes and exhibited impaired de novo adipogenesis, in contrast to cells derived from healthy joints. In accordance with the destabilized biomechanics of PTOA joints, in vitro modeling demonstrated that prolonged, injurious loading and perturbed Wnt/{beta}-catenin signaling were convergent anti-adipogenic cues that suppressed lipid droplet formation and adipogenic gene induction, while promoting markers of fibrosis in joint-derived stromal cells. Complementary gain-of-function studies using ex vivo joint adipose explants and in vivo joint injections demonstrated that chronic Wnt/{beta}-catenin activation, as seen in OA joints, is sufficient to diminish the intra-articular adipogenic program and shift adipose to a more fibrotic phenotype, independent of joint injury. Collectively, these findings establish a multi-modal framework for quantifying joint adipose atrophy and implicate aberrant Wnt/{beta}-catenin signaling and pathological mechanical loading as key factors impairing de novo adipogenesis and driving fibrotic remodeling of intra-articular adipose tissue in PTOA.

cell biology↗

bioRxiv2026

Mechano-activation of synovial fibroblasts and macrophages during OA progression in the dynamically stiffening synovial microenvironment

ObjectiveDuring osteoarthritis (OA) progression, the synovial membrane undergoes profound structural and compositional remodeling and fibrosis. We sought to elucidate how evolving synovial microenvironmental mechanics during fibrotic remodeling influence cell behavior and drive the progression of synovial pathology. MethodsSkeletally-mature male C57BL/6J mice were subjected to destabilization of the medial meniscus (DMM). To control for surgical confounders, both sham-operated and unoperated mice were included, with evaluation at 4- and 8-weeks. Synovial micromechanics were quantified via atomic force microscopy (AFM). Single-cell RNA sequencing (scRNA-seq), RNA fluorescence in situ hybridization (FISH), and flow cytometry were employed to investigate cellular heterogeneity, spatial organization, and crosstalk within fibrotic and non-fibrotic synovial niches. ResultsProgressive fibrotic remodeling and marked matrix stiffening were observed in DMM-operated synovium but absent in sham- and un-operated controls. While both sham and DMM joints mounted an acute stromal and immune response to surgery, these changes resolved over time in sham conditions but persisted in DMM synovium. During disease progression, distinct functional subsets of synovial fibroblasts and immune cells emerged, with mechanosignalling pathways and distinct immune cell-fibroblast crosstalk robustly activated within DMM-induced fibrotic microenvironments. ConclusionThis study demonstrates the complex cellular dynamics and crosstalk that differentiate the evolution of the pathological synovial response in the fibrotic DMM condition relative to surgical sham controls. Our findings highlight mechanotransduction as a central mechanism driving OA synovial pathogenesis and underscore the utility of the DMM model as a platform to dissect the molecular underpinnings of synovial fibrosis.

bioengineering↗

bioRxiv2025

Spatial transcriptomic profiling of decalcified murine musculoskeletal samples via Xenium Prime 5K

Successful generation of high-quality spatial transcriptomics data from murine musculoskeletal tissues has been impeded by the challenge of preserving RNA integrity through the harsh tissue processing steps required for histological sectioning. In particular, the need to thoroughly fix and decalcify mineralized tissues has proven problematic. We detail a comprehensive sample processing pipeline for three common murine musculoskeletal tissue samples, enabling high-quality transcript detection via imaging-based spatial transcriptomics using the Xenium Prime 5K platform from 10x Genomics. Our protocol outlines methodological details for transcardiac perfusion, fixation, decalcification, paraffin processing, and a sample co-embedding strategy facilitating anatomically consistent and simultaneous sectioning of multiple samples onto the spatial transcriptomics slide. Rigorous quality control demonstrates high-quality tissue-specific outcomes across intact knee joints, tibiae, and lumbar spines from adult mice. Our pipeline enabled 70-91% high-quality transcripts across synovium, meniscus, patellar tendon, articular cartilage, subchondral bone, cortical bone, bone marrow, muscle, fracture callus, and dorsal root ganglion tissues. The average number of detected transcripts varied markedly between tissue types - soft tissues such as synovium, patellar tendon, muscle, bone marrow, and callus exhibited ~200 - 400 transcript per cell; mineralized tissues such as subchondral bone, meniscus, and cortical bone exhibited ~ 13 - 150 transcripts per cell; highly active neuronal tissues such as dorsal root ganglion neurons yielded 750 - 1100 transcripts per cell. Canonical cell markers within each tissue confirmed successful identification and representation of key cell types. Through rigorous sample quality assessment at multiple stages of processing, this protocol yields high-quality RNA transcript detection while preserving critical anatomical context and will serve as a valuable tool enabling spatial transcriptomic profiling of intact musculoskeletal tissue samples. Lay summarySpatial transcriptomics is a powerful scientific tool that characterizes genetic coding material ("transcripts") of specific cell types in their native anatomical context. However, successfully applying this tool to musculoskeletal tissues has been challenging because preserving transcript integrity in these tissues requires additional care. The authors of this publication developed a specialized method for preparing tissue samples from mice that works with the spatial transcriptomics platform, 10x Genomics Xenium Prime 5K. This protocol improves sample quality while preserving high-quality genetic information in various mouse musculoskeletal tissues, making it easier to explore their functions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/693132v1_ufig1.gif" ALT="Figure 1"> View larger version (87K): org.highwire.dtl.DTLVardef@4c4389org.highwire.dtl.DTLVardef@e607bcorg.highwire.dtl.DTLVardef@5f957org.highwire.dtl.DTLVardef@34ce27_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

CXCL16 mediates nociception and inflammation in murine post-traumatic osteoarthritis

This study investigates the role of the chemokine CXCL16 and its receptor, CXCR6, in post-traumatic osteoarthritis (PTOA) and joint nociception, highlighting the potential of targeting the CXCL16-CXCR6 axis for therapeutically managing joint inflammation and pain. Following joint injury in mice, the CXCL16-CXCR6 signaling axis is activated in synovium, driven by synovial fibroblasts and macrophages. Human OA synovium also exhibited increased CXCL16 and CXCR6 gene expression. CXCL16 stimulated a pro-inflammatory response in fibroblasts and macrophages, contrasting with an anti-inflammatory response observed in mesenchymal progenitor cells. In mice, repeated intra-articular CXCL16 injections induced histological synovitis and sex-dependent activation of inflammatory and fibrotic transcriptional programs in synovium. Repeated CXCL16 joint injections also induced knee hyperalgesia, which was mitigated by co-administration of the CXCR6 antagonist, ML339. A single intra-articular injection of CXCL16 induced acute knee hyperalgesia as early as 30 minutes post-injection, which was completely abrogated by ML339 co-treatment, suggesting direct CXCL16 binding to nociceptor-expressed CXCR6. In a murine PTOA model, systemic CXCR6 antagonism with ML339 alleviated knee hyperalgesia and altered circulating immune cell profiles. Direct stimulation of mouse dorsal root ganglion-derived nociceptive neurons with CXCL16 induced rapid calcium signaling, which was abolished by co-treatment with ML339. These findings establish CXCL16 as a regulator of joint inflammation and identifies the CXCL16-CXCR6 binding mechanism as key in mediating pain-related behaviors and nociceptor activation, offering a therapeutic target for PTOA-related inflammation and pain management. One Sentence SummaryCXCL16 regulates synovial inflammation and mediates joint nociception via CXCR6, highlighting its potential as a therapeutic target for post-traumatic osteoarthritis.

physiology↗

bioRxiv2025

A standardized, open-source, portable model for noninvasive joint injury in mice

Preclinical models of osteoarthritis (OA) are crucial for the study of disease mechanisms and for the development of critically-needed disease-modifying therapeutics. While surgical OA models, such as the destabilization of the medial meniscus (DMM), have been the gold standard in the field for decades, noninvasive joint loading-based models have increased in popularity and utility. To facilitate standardization of the noninvasive anterior cruciate ligament rupture (ACLR) model in mice, we present the Mobile Joint-Injury Operator (MoJO) - an open-source protocol with accompanying fixtures and data, designed for a low-cost, commercially-available, portable uniaxial testing system with a small footprint. We provide 3d-printable fixture designs and a rapid, highly-repeatable ACLR-mediated joint injury protocol that results in the expected post-traumatic osteoarthritis phenotype in male and female C56Bl/6 mice. We then describe the expected mechanical data from the injury procedure and offer various troubleshooting strategies. Finally, we summarize the resultant PTOA phenotype by knee hyperalgesia testing, {micro}CT imaging, flow cytometry, and histological assessment. Increased standardization of this model is a critical aspect of the overall refinement of animal models of OA.

physiology↗

bioRxiv2023

Synovial macrophage diversity and activation of M-CSF signaling in post-traumatic osteoarthritis

ObjectiveSynovium is home to immune and stromal cell types that orchestrate inflammation following a joint injury; in particular, macrophages are central protagonists in this process. We sought to define the cellular and temporal dynamics of the synovial immune niche in a mouse model of post-traumatic osteoarthritis (PTOA), and to identify stromal-immune crosstalk mechanisms that coordinate macrophage function and phenotype. DesignWe induced PTOA in mice using a non-invasive tibial compression model of anterior cruciate ligament rupture (ACLR). Single cell RNA-seq and flow cytometry were used to assess immune cell populations in healthy (Sham) and injured (7d and 28d post-ACLR) synovium. Characterization of synovial macrophage polarization states was performed, alongside computational modeling of macrophage differentiation, as well as implicated transcriptional regulators and stromal-immune communication axes. ResultsImmune cell types are broadly represented in healthy synovium, but experience drastic expansion and speciation in PTOA, most notably in the macrophage portion. We identified several polarization states of macrophages in synovium following joint injury, underpinned by distinct transcriptomic signatures, and regulated in part by stromal-derived macrophage colony-stimulating factor signaling. The transcription factors Pu.1, Cebp, Cebp{beta}, and Jun were predicted to control differentiation of systemically derived monocytes into pro-inflammatory synovial macrophages. ConclusionsWe defined different synovial macrophage subpopulations present in healthy and injured mouse synovium. Nuanced characterization of the distinct functions, origins, and disease kinetics of macrophage subtypes in PTOA will be critical for targeting these highly versatile cells for therapeutic purposes.

immunology↗

bioRxiv2022

Synovial fibroblasts assume distinct functional identities and secrete R-spondin 2 to drive osteoarthritis

ObjectivesSynovium is acutely affected following joint trauma and contributes to post-traumatic osteoarthritis (PTOA) progression. Little is known about discrete cell types and molecular mechanisms in PTOA synovium. We aimed to describe synovial cell populations and their dynamics in PTOA, with a focus on fibroblasts. We also sought to define mechanisms of synovial Wnt/{beta}-catenin signaling, given its emerging importance in arthritis. MethodsWe subjected mice to non-invasive anterior cruciate ligament rupture as a model of human joint injury. We performed single-cell RNA-sequencing to assess synovial cell populations, subjected Wnt-GFP reporter mice to joint injury to study Wnt-active cells, and performed intra-articular injections of the Wnt agonist R-spondin 2 (Rspo2) to assess whether gain-of-function induced pathologies characteristic of PTOA. Lastly, we used cultured fibroblasts, macrophages, and chondrocytes to study how Rspo2 orchestrates crosstalk between joint cell types. ResultsWe uncovered seven distinct functional subsets of synovial fibroblasts in healthy and injured synovium, and defined their temporal dynamics in early and established PTOA. Wnt/{beta}-catenin signaling was overactive in PTOA synovium, and Rspo2 was strongly induced after injury and secreted exclusively by Prg4hi lining fibroblasts. Trajectory analyses predicted that Prg4hi lining fibroblasts arise from a pool of Dpp4+ mesenchymal progenitors in synovium, with SOX5 identified as a potential regulator of this emergence. We also showed that Rspo2 orchestrated pathological crosstalk between synovial fibroblasts, macrophages, and chondrocytes. ConclusionsSynovial fibroblasts assume distinct functional identities during PTOA, and Prg4hi lining fibroblasts secrete the Wnt agonist Rspo2 to drive pathological crosstalk in the joint after injury.

cell biology↗

bioRxiv2021

Robust temporal map of human in vitro myelopoiesis using single-cell genomics

Myeloid cells have a central role in homeostasis and tissue defence. Characterising the current in vitro protocols of myelopoiesis is imperative for their use in research and immunotherapy as well as for understanding the early stages of myeloid differentiation in humans. Here, we profiled the transcriptome of more than 400k cells and generated a robust molecular map of the differentiation of human induced pluripotent stem cells (iPSC) into macrophages. By integrating our in vitro datasets with in vivo single-cell developmental atlases, we found that in vitro macrophage differentiation recapitulates features of in vivo yolk sac hematopoiesis, which happens prior to the appearance of definitive hematopoietic stem cells (HSC). During in vitro myelopoiesis, a wide range of myeloid cells are generated, including erythrocytes, mast cells and monocytes, suggesting that, during early human development, the HSC-independent immune wave gives rise to multiple myeloid cell lineages. We leveraged this model to characterize the transition of hemogenic endothelium into myeloid cells, uncovering poorly described myeloid progenitors and regulatory programs. Taking advantage of the variety of myeloid cells produced, we developed a new protocol to produce type 2 conventional dendritic cells (cDC2) in vitro. We found that the underlying regulatory networks coding for myeloid identity are conserved in vivo and in vitro. Using genetic engineering techniques, we validated the effects of key transcription factors important for cDC2 and macrophage identity and ontogeny. This roadmap of early myeloid differentiation will serve as an important resource for investigating the initial stages of hematopoiesis, which are largely unexplored in humans, and will open up new therapeutic opportunities.

developmental biology↗

bioRxiv2019

A map of transcriptional heterogeneity and regulatory variation in human microglia

Microglia, the tissue resident macrophages of the CNS, are implicated in a broad range of neurological pathologies, from acute brain injury to dementia. Here, we profiled gene expression variation in primary human microglia isolated from 141 patients undergoing neurosurgery. Using single cell and bulk RNA sequencing, we defined distinct cellular populations of acutely in vivo-activated microglia, and characterised a dramatic switch in microglial population composition in patients suffering from acute brain injury. We mapped expression quantitative trait loci (eQTLs) in human microglia and show that many disease-associated eQTLs in microglia replicate well in a human induced pluripotent stem cell (hIPSC) derived macrophage model system. Using ATAC-seq from 95 individuals in this hIPSC model we fine-map candidate causal variants at risk loci for Alzheimers disease, the most prevalent neurodegenerative condition in acute brain injury patients. Our study provides the first population-scale transcriptional map of a critically important cell for neurodegenerative disorders.

genomics↗

bioRxiv2017

Genetic effects on chromatin accessibility foreshadow gene expression changes in macrophage immune response

Noncoding regulatory variants play an important role in the genetics of complex traits. Although quantitative trait locus (QTL) mapping is a powerful approach to identify these variants, many genetic effects may remain unobserved when cells are sampled in only one of a large number of possible environments. Using a novel induced pluripotent stem cell-derived system, we mapped QTLs regulating chromatin accessibility and gene expression in macrophages in four conditions mimicking the interplay between interferon-gamma response and Salmonella infection. We found that approximately 50% of condition-specific effects on gene expression altered chromatin accessibility prior to stimulation. Furthermore, 6% of the chromatin accessibility QTLs regulated multiple neighbouring regions and these interactions were modulated by stimulation, occasionally producing condition-specific changes in gene expression. Profiling additional states also doubled the number of expression QTLs that could be confidently colocalised with disease associations. Thus, a substantial fraction of disease-associated variants may affect primed regulatory elements in naive cells.

genomics↗

bioRxiv2017

Molecular and functional variation in iPSC-derived sensory neurons

Induced pluripotent stem cells (iPSCs), and cells derived from them, have become key tools to model biological processes and disease mechanisms, particularly in cell types such as neurons that are difficult to access from living donors. Here, we present the first map of regulatory variants in an iPSC-derived cell type. To investigate genetic contributions to human sensory function, we performed 123 differentiations of iPSCs from 103 unique donors to a sensory neuronal fate, and measured gene expression, chromatin accessibility, and neuronal excitability. Compared with primary dorsal root ganglion, where sensory nerves collect near the spinal cord, gene expression was more variable across iPSC-derived neuronal cultures, particularly in genes related to differentiation and nervous system development. Single cell RNA-sequencing revealed that although the majority of cells are neuronal and express the expected marker genes, a substantial fraction have a fibroblast-like expression profile. By applying an allele-specific method we identify 3,778 quantitative trait loci influencing gene expression, 6,318 for chromatin accessibility, and 2,097 for RNA splicing at FDR 10%. A number of these overlap with common disease associations, and suggest candidate causal variants and target genes. These include known causal variants at SNCA for Parkinsons disease and TNFRSF1A for multiple sclerosis, as well as new candidates for migraine, Parkinsons disease, and schizophrenia.

genomics↗