Biology subjects

Phillips, T.

Publications and source records attributed to Phillips, T..

14 recordsLinked to original sources

bioRxiv2026

Activation of α7 Nicotinic Acetylcholine Receptor Inhibits Neutrophil ex vivo Swarming and Pulmonary Recruitment in Inflamed Mice through an Integrin αMβ2-dependent Mechanism

Background. The cholinergic anti-inflammatory pathway regulates inflammation through vagus signaling that activates the 7 nicotinic acetylcholine receptor (7nAChR) on leukocytes. Suppression of macrophage inflammatory cytokine production through 7nAChR-mediated inhibition of NF-{kappa}B activation is considered a central mechanism of this pathway. However, the effects of cholinergic activation of 7nAChR on neutrophils remain poorly understood. Here, we investigated whether and how activation of 7nAChR in neutrophils regulates neutrophil migration, defined the underlying molecular mechanism, and evaluated its contribution to anti-inflammatory protection during endotoxemia. Results. The ex vivo swarming assays revealed that activation of 7nAChR with the selective agonists PNU-282987 and AR-R17779 significantly inhibited human and mouse neutrophil migration. The inhibitory effect of PNU-282987 was abolished in 7nAChR-deficient neutrophils, confirming receptor specificity. Mechanistically, 7nAChR activation reduced integrin M{beta}2 activation, decreased F-actin polymerization, and impaired neutrophil polarization. These effects required M{beta}2, as PNU-282987 failed to inhibit actin remodeling in M-deficient neutrophils. Modulation of M{beta}2 activation with an activating antibody (MEM48) demonstrated that 7nAChR regulates neutrophil migration by limiting excessive M{beta}2 activation and maintaining an optimal adhesive state. In vivo, adoptively transferred fluorescently labeled PNU-282987-treated neutrophils exhibited reduced recruitment to the lungs compared with vehicle-treated neutrophils during LPS-induced endotoxemia. Furthermore, neutrophil depletion markedly diminished the protective effect of PNU-282987 on survival, demonstrating that neutrophils contribute to 7nAChR-mediated protection during systemic inflammation. Conclusions. Our findings identify neutrophils as an important cellular target of the cholinergic anti-inflammatory pathway and demonstrate that 7nAChR activation limits neutrophil migration by suppressing integrin M{beta}2 activation and actin cytoskeletal remodeling. Together, these findings establish M{beta}2 as a critical downstream effector of 7nAChR signaling in neutrophils and identify regulation of integrin activation as a previously unrecognized mechanism of the cholinergic anti-inflammatory pathway.

immunology↗

bioRxiv2026

Intact learning and memory in mice incapable of de novo myelination

Motor skill learning stimulates and requires generation of oligodendrocytes (OLs) from their precursors (OLPs) in the adult mouse brain, but the functional role(s) of the newly formed OLs is not known. We asked whether new compact myelin sheaths are required, by genetic block of myelin basic protein (MBP) synthesis in adult OLPs and their newly-differentiating OL progeny, using tamoxifen-inducible Cre-lox recombination. Newly-differentiating OLs in these Mbp-cKO mice are unable to assemble compact myelin or normal nodes of Ranvier. Despite this, Mbp-cKOs learned a motor skill just as well as their wild type littermates. They also demonstrated normal contextual fear conditioning. Therefore, neither motor nor fear learning depends on rapid saltatory conduction in newly-myelinated circuits. Mbp-cKOs also formed normal long-term motor and contextual fear memories. Moreover, OL lineage-specific knockout of Monocarboxylate transporter-1 (Mct1), believed to be responsible for transferring metabolic substrates from OLs into axons, did not affect learning or memory consolidation. Myelin regulatory factor (Myrf)-cKOs, in which newly-differentiating OLs die and are rapidly eliminated, confirmed that newly formed OLs are required for learning and memory. Together, the data suggest that learning and memory depends on a non-canonical property of pre-myelinating or myelinating OLs, distinct from myelins cardinal role in speeding action potentials.

neuroscience↗

bioRxiv2025

The developing tendon and enthesis are hypoxic and rely on hypoxia-inducible factor 1a (Hif1a) during postnatal development

The tendon-bone enthesis is a specialized fibrocartilaginous tissue crucial for muscle-to-bone force transmission, yet its postnatal development is not fully understood. Emerging evidence suggests hypoxia plays a pivotal role in enthesis maturation akin to its function in skeletal growth plates, with Hypoxia Inducible Factor 1 alpha (HIF-1) acting as a key regulator of cellular adaptation (e.g., cell survival, extracellular matrix (ECM) deposition). Here, we investigated the spatial and temporal dynamics of hypoxia in the murine Achilles tendon enthesis and elucidated the role of Hif1a in enthesis cell survival and ECM formation using Scleraxis-lineage conditional knockout (ScxCre; Hif1a cKO) mice. We found that while neonatal tendons rapidly resolve hypoxia after birth, the enthesis maintains a hypoxic niche through postnatal day 5, mirroring a gradient observed in growth plates. Disruption of HIF-1 in enthesis-resident cells resulted in pronounced deficits in grip strength, abnormal tendon-bone attachment morphology, disrupted calcaneal architecture, impaired mineralization, and significant ECM disorganization. Histological analyses revealed persistent cell death and loss of the characteristic fibrocartilaginous gradient in cKO entheses, including dysregulated collagen alignment. In vitro, HIF-1-deficient tendon fibroblasts exhibited blunted transcriptional responses to hypoxia, altered metabolic gene expression, and changes in ECM deposition. Collectively, our findings illuminate hypoxia as a sustained niche in the postnatal enthesis, with HIF-1 critically required for cell survival, ECM organization, and enthesis structural integrity. This work advances our understanding of enthesis biology and provides insights relevant to tendon-bone attachment disorders and regenerative strategies.

developmental biology↗

bioRxiv2025

Profiling Lysosomal and Mitochondrial Dysfunction in Neurodegenerative Diseases Using Human Fibroblasts for Translational Therapeutic Screening

Lysosomal dysfunction and mitochondrial health are intricately connected, playing essential roles in cellular homeostasis. Lysosomes are acidic membrane-bound organelles responsible for degrading and recycling cellular waste, while mitochondria generate the energy required for cellular functions. Growing evidence implicates roles for lysosomal and mitochondrial dysfunction in neurodegenerative diseases, including Alzheimers and Parkinsons disease. With novel therapeutics targeting both the lysosomal and mitochondrial functions, robust assays for compound screening are becoming critical to evaluate modulation of both organelles in disease-relevant cellular models. Here, we investigated human fibroblasts derived from healthy donors, as well as patients with Alzheimers and Parkinsons disease, to assess their capacity to model key aspects of lysosomal and mitochondrial dysfunction. Lysosomal function was evaluated using various assays, including quantification of lysosomal proteins (TMEM175 and LAMP1), LysoTracker staining, measurement of lysosomal pH, and lysosomal enzymatic activity. Autophagic flux was assessed by measuring p62 levels as a marker of autophagy. Mitochondrial function was investigated by measuring mitochondrial calcium levels, membrane potential, oxidative stress, and mitochondrial content using MitoTracker. To explore the potential of using human fibroblasts for in vitro compound screening, we validated these assays in a 384-well high-throughput format using compounds such as chloroquine and ammonium chloride. Our findings demonstrate that human fibroblasts faithfully recapitulate lysosomal and mitochondrial dysfunctions characteristic of neurodegenerative diseases. Moreover, the use of robust assays positions these cells as a valuable platform for high-throughput screening to identify novel therapeutics targeting lysosomal and mitochondrial pathways.

neuroscience↗

bioRxiv2025

AMPK signaling is dysregulated in human tendinopathy and loss of AMPKα1 leads to cell, matrix and mechanical dysfunction in mouse Achilles tendon

Tendinopathy is a debilitating tendon disorder affecting millions of people, characterized by pain, swelling, and diminished biomechanical properties. While the precise mechanisms underlying tendon homeostasis remain unclear, metabolic regulation plays a critical role. In this study, we combine transcriptomic analysis of human tendinopathic samples with a conditional mouse model in which Prkaa1 (encoding AMPK1) is selectively deleted in tendon progenitors to elucidate the role of AMPK signaling in tendon homeostasis. RNA sequencing of diseased human tendons revealed downregulation of key metabolic genes, including several involved in the mitochondrial electron transport chain and AMPK signaling pathways, alongside an increase in markers associated with senescence and a secretory inflammatory profile. In parallel, mice with loss of Prkaa1 function exhibited normal postnatal development; however, by one month of age, tendons demonstrated widespread transcriptional alterations related to cell cycle regulation and ECM organization. By three months, AMPK1-deficient tendons showed significant reductions in mechanical strength and increased expression of senescence markers p21 and p16, progressing to prominent ectopic calcification with age. In vitro studies further confirmed that tendon fibroblasts lacking AMPK1 have altered ECM substrate adhesion profiles. Importantly, voluntary exercise partially rescued these deficits by enhancing ECM organization and reducing senescence marker expression. Collectively, our findings demonstrate that AMPK1 is critical for maintaining energy balance, regulating ECM remodeling, and preventing premature cellular senescence in tendon. These insights highlight AMPK signaling as a promising therapeutic target and underscore the beneficial role of exercise in mitigating tendinopathic changes.

pathology↗

bioRxiv2024

A hybrid computational model of cancer spheroid growth with ribose-induced collagen stiffening

Metastasis, the leading cause of death in cancer patients, arises when cancer cells disseminate from a primary solid tumour to distant organs. Growth and invasion of the solid tumour often involve collective cell migration, which is profoundly influenced by cell-cell interactions and the extracellular matrix (ECM). The ECMs biochemical composition and mechanical properties, such as stiffness, regulate cancer cell behaviour and migration dynamics. Mathematical modelling serves as a pivotal tool for studying and predicting these complex dynamics, with hybrid discrete-continuous models offering a powerful approach by combining agent-based representations of cells with continuum descriptions of the surrounding microenvironment. In this study, we investigate the impact of ECM stiffness, modulated via ribose-induced collagen cross-linking, on cancer spheroid growth and invasion. We employed a hybrid discrete-continuous model implemented in PhysiCell to simulate spheroid dynamics, successfully replicating three-dimensional in vitro experiments. The model incorporates detailed representations of cell-cell and cell-ECM interactions, ECM remodelling, and cell proliferation. Our simulations align with experimental observations of two breast cancer cell lines, non-invasive MCF7 and invasive HCC1954, under varying ECM stiffness conditions. The results demonstrate that increased ECM stiffness due to ribose-induced cross-linking inhibits spheroid invasion in invasive cells, whereas non-invasive cells remain largely unaffected. Furthermore, our simulations show that higher ECM degradation by the cells not only enables spheroid growth and invasion but also facilitates the formation of multicellular protrusions. Conversely, increasing the maximum speed that cells can reach due to cell-ECM interactions enhances spheroid growth while promoting single-cell invasion. This hybrid modelling approach enhances our understanding of the interplay between cancer cell migration, proliferation, and ECM mechanical properties, paving the way for future studies incorporating additional ECM characteristics and microenvironmental conditions.

systems biology↗

bioRxiv2024

Functional Changes to Achilles Tendon and Enthesis in a Mouse Model of an Adolescent Masculine Gender-Affirming Hormone Treatment

Many transgender youth seek gender affirming care, such as puberty suppression, to prolong decision-making and to align their physical sex characteristics with their gender identity. During peripubertal growth, connective tissues such as tendon rapidly adapt to applied mechanical loads (e.g., exercise) yet if and how tendon adaptation is influenced by sex and gender affirming hormone therapy during growth remains unknown. The goal of this study was to understand the how pubertal suppression influences the structural and functional properties of the Achilles tendon using an established mouse model of transmasculine gender affirming hormone therapy. C57BL/6N female-born mice were assigned to experimental groups to mimic gender-affirming hormone therapy in human adolescents, and treatment was initiated prior to the onset of puberty (at postnatal day 26, P26). Experimental groups included controls and mice serially treated with gonadotropin release hormone analogue (GnRHa), delayed Testosterone (T), or GnRHa followed by T. We found that puberty suppression using GnRHa, with and without T, improved the overall tendon load capacity in female-born mice. Treatment with T resulted in an increase in the maximum load that tendon can withstand before failure. Additionally, we found that GnRHa, but not T, treatment resulted in a significant increase in cell density at the Achilles enthesis. NEW & NOTEWORTHYThese findings demonstrate that puberty suppression or testosterone does not negatively influence tendon structural or functional properties in a mouse model of transmasculine gender affirming care. In all treatment groups, the ability of the tendon to withstand load was significantly increased. Puberty suppression with GnRHa significantly increased enthesis cell density, suggesting an extended growth phase. These findings elucidate the effects of gender affirming care on the structural and functional properties of the tendon and enthesis.

physiology↗

bioRxiv2023

Uncoupling of synaptic loss from amyloid burden by an Alzheimer's disease protective variant of PLCγ2.

A rare coding missense variant (rs72824905; P522R) in PLCG2 decreases the risk of late-onset Alzheimers disease, but how this protective effect is mediated is unclear. Here we demonstrate a mechanism for this protection, the R522 variant of PLC{gamma}2 alters microglial activity leading to a marked preservation of synaptic integrity and reduced peri-plaque microglial engulfment of synapses independently of amyloid burden. Our data advocate for a direct central role of PLC{gamma}2 in mediating synaptic loss as part of the pathological process of Alzheimers disease (AD), prioritising it as a therapeutic target and modulator of disease.

neuroscience↗

bioRxiv2022

Astrocyte MCT1 expression does not contribute to the axonal degenerative phenotype observed with ubiquitous MCT1 depletion.

We recently reported that loss of oligodendrocyte metabolic support through the lactate and pyruvate transporter Monocarboxylate Transporter 1 (MCT1) is well tolerated into adulthood. Only with advanced aging did we observe axonal degeneration and hypomyelination due to loss of MCT1 from oligodendroglia lineage cells. MCT1 is also expressed by other glial subtypes, such as astrocytes and endothelial cells where it has been suggested to be essential for learning and memory tasks. However, the importance of MCT1 in these cell types for long-term axonal metabolic support is still unknown. We therefore addressed whether conditional loss of MCT1 from either of these cell types would lead to widespread axonal degeneration with aging. Using a conditional null approach, similar to what was used for oligodendrocyte MCT1 depletion, we observed that conditional knockout of MCT1 from either astrocytes or endothelial cells did not cause neuronal injury. On the other hand, inducible ubiquitous depletion of MCT1 causes late-onset axonal degeneration, comparable with what was observed in our previous study using the oligodendrocyte lineage MCT1 null mice. In summary, we conclude that unlike oligodendrocyte MCT1, astrocyte MCT1 is not an essential driver of astrocyte mediated axonal energy homeostasis with aging.

neuroscience↗

bioRxiv2020

The Alzheimer's disease protective P522R variant of PLCG2, consistently enhances stimulus-dependent PLCγ2 activation, depleting substrate and altering cell function.

Recent genome-wide association studies of Alzheimers disease (AD) have identified variants implicating immune pathways in disease development. A rare coding variant of PLCG2, which encodes PLC{gamma}2, shows a significant protective effect for AD (rs72824905, P522R, P=5.38x10-10, Odds Ratio = 0.68). Molecular dynamic modelling of the PLC{gamma}2-R522 variant, situated within the auto-inhibitory domain of PLC{gamma}2, suggests a structural change to the protein. Through CRISPR-engineering we have generated novel PLCG2-R522 harbouring human induced pluripotent cell lines (hiPSC) and a mouse knockin model, neither of which exhibits alterations in endogenous PLCG2 expression. Mouse microglia and macrophages and hiPSC-derived microglia-like cells with the R522 mutation, all demonstrate a consistent non-redundant hyperfunctionality in the context of normal expression of other PLC isoforms. This signalling alteration manifests as enhanced cellular Ca2+ store release ([~]20-40% increase) in response to physiologically-relevant stimuli (e.g. Fc receptor ligation and A{beta} oligomers). This hyperfunctionality resulted in increased PIP2 depletion in the cells with the PLC{gamma}2-R522 variant after exposure to stimuli and reduced basal detection of PIP2 levels in vivo. These PLC{gamma}2-R522 associated abnormalities resulted in impairments to phagocytosis (fungal and bacterial particles) and enhanced endocytosis (A{beta} oligomers and dextran). PLC{gamma}2 sits downstream of disease relevant pathways, such as TREM2 and CSF1R and alterations in its activity, direct impacts cell function, which in the context of the inherent drugability of enzymes such as PLC{gamma}2, raise the prospect of manipulation of PLC{gamma}2 as a therapeutic target in Alzheimers Disease.

neuroscience↗

bioRxiv2020

Quantitative single cell heterogeneity profiling of patient derived tumor initiating gliomaspheres reveals unique signatures of malignancy and response

BackgroundGlioblastoma is a deadly brain tumor with median patient survival of 14.6 months. At the core of this malignancy are rare, highly heterogenous malignant stem-like tumor initiating cells. Aberrant signaling across the EGFR-PTEN-AKT-mTOR signal transduction pathways are common oncogenic drivers in these cells. Though gene-level clustering has determined the importance of the EGFR signaling pathway as a treatment indicator, multiparameter protein-level analyses are necessary to discern functional attributes of signal propagation. Multiparameter single cell analyses is emerging as particularly useful in identifying such attributes. MethodsSingle cell targeted proteomic analysis of EGFR-PTEN-AKT-mTOR proteins profiled heterogeneity in a panel of fifteen patient derived gliomaspheres. A microfluidic cell array chip tool served as a low cost methodology to derive high quality quantitative single cell analytical outputs. Chip design specifications produced extremely high signal-to-noise ratios and brought experimental efficiencies of cell control and minimal cell use to accommodate experimentation with these rare and often slow-growing cell populations. Quantitative imaging software generated datasets to observe similarities and differences within and between cells and patients. Bioinformatic self-organizing maps (SOMs) and hierarchical clustering stratified patients into malignancy and responder groups which were validated by phenotypic and statistical analyses. ResultsFifteen patient dissociated gliomaspheres produced 59,464 data points from 14,866 cells. Forty-nine molecularly defined signaling phenotypes were identified across samples. Bioinformatics resolved two clusters diverging on EGFR expression (p = 0.0003) and AKT/TORC1 activation (p = 0.08 and p = 0.09 respectively). TCGA status of a subset showed genetic heterogeneity with proneural, classical and mesenchymal subtypes represented in both clusters. Phenotypic validation measures indicated drug responsive phenotypes to EGFR blocking were found in the EGFR expressing cluster. EGFR expression in the subset of drug-treated lines was statistically significant (p<.05). The EGFR expressing cluster was of lower tumor initiating potential in comparison to the AKT/TORC1 activated cluster. Though not statistically significant, EGFR expression trended with improved patient prognosis while AKT/TORC1 activated samples trended with poorer outcomes. ConclusionsQuantitative single cell heterogeneity profiling resolves signaling diversity into meaningful non-obvious phenotypic groups suggesting EGFR is decoupled from AKT/TORC1 signalling while identifying potentially valuable targets for personalized therapeutic approaches for deadly tumor-initiating cell populations.

cancer biology↗

bioRxiv2019

A Transcriptome Fingerprinting Assay for Clinical Immune Monitoring

BackgroundWhile our understanding of the role that the immune system plays in health and disease is growing at a rapid pace, available clinical tools to capture this complexity are lagging. We previously described the construction of a third-generation modular transcriptional repertoire derived from genome-wide transcriptional profiling of blood of 985 subjects across 16 diverse immunologic conditions, which comprises 382 distinct modules.\n\nResultsHere we describe the use of this modular repertoire framework for the development of a targeted transcriptome fingerprinting assay (TFA). The first step consisted in down-selection of the number of modules to 32, on the basis of similarities in changes in transcript abundance and functional interpretation. Next down-selection took place at the level of each of the 32 modules, with each one of them being represented by four transcripts in the final 128 gene panel. The assay was implemented on both the Fluidigm high throughput microfluidics PCR platform and the Nanostring platform, with the list of assays target probes being provided for both. Finally, we provide evidence of the versatility of this assay to assess numerous immune functions in vivo by demonstrating applications in the context of disease activity assessment in systemic lupus erythematosus and longitudinal immune monitoring during pregnancy.\n\nConclusionsThis work demonstrates the utility of data-driven network analysis applied to large-scale transcriptional profiling to identify key markers of immune responses, which can be downscaled to a rapid, inexpensive, and highly versatile assay of global immune function applicable to diverse investigations of immunopathogenesis and biomarker discovery.

immunology↗

bioRxiv2019

A Novel Repertoire of Blood Transcriptome Modules Based on Co-expression Patterns Across Sixteen Disease and Physiological States

As the capacity for generating large scale data continues to grow the ability to extract meaningful biological knowledge from it remains a limitation. Here we describe the development of a new fixed repertoire of transcriptional modules. It is meant to serve as a stable reusable framework for the analysis and interpretation of blood transcriptome profiling data. It is supported by customized resources, which include analysis workflows, fingerprint grid plots data visualizations, interactive web applications providing access to a vast number of module-specific functional profiling reports, reference transcriptional profiles and give users the ability to visualize of changes in transcript abundance across the modular repertoire at different granularity levels. A use case focusing on a set of six modules comprising interferon-inducible genes is also provided. Altogether we hope that this resource will also serve as a framework for improving over time our collective understanding of the immunobiology underlying blood transcriptome profiling data.

immunology↗

bioRxiv2018

Detection of early stage pancreatic cancer using 5-hydroxymethylcytosine signatures in circulating cell free DNA

Pancreatic cancers are typically diagnosed at late stage where disease prognosis is poor as exemplified by a 5-year survival rate of 8.2%. Earlier diagnosis would be beneficial by enabling surgical resection or earlier application of therapeutic regimens. We investigated the detection of pancreatic ductal adenocarcinoma (PDAC) in a non-invasive manner by interrogating changes in 5-hydroxymethylation cytosine status (5hmC) of circulating cell free DNA in the plasma of a PDAC cohort (n=51) in comparison with a non-cancer cohort (n=41). We found that 5hmC sites are enriched in a disease and stage specific manner in exons, 3UTRs and transcription termination sites. Our data show that 5hmC density is reduced in promoters and histone H3K4me3-associated sites with progressive disease suggesting increased transcriptional activity. 5hmC density is differentially represented in thousands of genes, and a stringently filtered set of the most significant genes points to biology related to pancreas (GATA4, GATA6, PROX1, ONECUT1) and/or cancer development (YAP1, TEAD1, PROX1, ONECUT1, ONECUT2, IGF1 and IGF2). Regularized regression models were built using 5hmC densities in statistically filtered genes or a comprehensive set of highly variable 5hmC counts in genes and performed with an AUC = 0.94-0.96 on training data. We were able to test the ability to classify PDAC and non-cancer samples with the Elastic net and Lasso models on two external pancreatic cancer 5hmC data sets and found validation performance to be AUC = 0.74-0.97. The findings suggest that 5hmC changes enable classification of PDAC patients with high fidelity and are worthy of further investigation on larger cohorts of patient samples.

cancer biology↗