Biology subjects

O'Brien, C. A.

Publications and source records attributed to O'Brien, C. A..

7 recordsLinked to original sources

bioRxiv2026

A transcriptomic-driven segmentation and cell simulation framework for high-resolution spatial transcriptomics and cell-cell communication

The Visium HD spatial transcriptomics platform enables transcriptome-wide profiling at near-single-cell resolution. However, accurate segmentation of cells to define spatial boundaries relies heavily on histological images. Previous approaches struggle to define cells when the tissues have high cell density, are inflamed, or are mineralized, leading to transcriptomic bleed-through and inaccurate clustering. To address this, we developed TENGU (Transcript-signal Enrichment and Grouping Unit), a comprehensive end-to-end bioinformatic software package. Unlike existing tools, TENGU employs a transcript-first segmentation approach, prioritizing transcript-signal density as the primary modality and utilizing histological images only as a secondary supplement in unresolved regions. These initial boundaries are further optimized through a novel transcriptomic-driven cell simulation algorithm. Iterative refinement of boundaries based on localized gene expression probabilities effectively minimizes spatial scattering and preserves biologically distinct molecular signatures. The pipeline seamlessly integrates tissue segmentation, high-resolution cell-type annotation, and basic spatially aware cell-cell communication (CCC) analysis. We rigorously benchmarked TENGU against the 10X Genomics and Bin2cell pipelines for cell segmentation across diverse and technically challenging microenvironments. TENGU demonstrated superior transcriptomic distinctness in the murine brain, successfully captured matrix-embedded osteocytes, and localized critical osteoimmune CCC networks (Tgfb and Il1a) in a murine model of osteomyelitis. TENGU also resolved species-specific, pro-tumorigenic signaling hubs (MDK-SDC4) within a highly compacted human colorectal cancer xenograft. By mitigating the constraints of traditional image-dependent segmentation, TENGU provides a highly adaptable and robust computational framework that empowers researchers to accurately decode the complex functional micro-anatomy of both healthy and pathological tissues.

bioinformatics↗

bioRxiv2025

Deletion of the scavenger receptor Scarb1 in osteoblast progenitors and myeloid cells does not affect bone mass

The scavenger receptor class B member 1 (SCARB1), encoded by Scarb1, is a cell surface receptor for high density lipoproteins, low density lipoproteins (LDL), oxidized LDL (OxLDL), and phosphocholine-containing oxidized phospholipids (PC-OxPLs). Scarb1 is expressed in multiple cell types, including osteoblasts and macrophages. PC-OxPLs, present on OxLDL and apoptotic cells, adversely affect bone metabolism. Overexpression of E06 IgM - a natural antibody that recognizes PC-OxPLs- increases cancellous and cortical bone at 6 months of age in both sexes and protects against age- and high fat diet- induced bone loss, by increasing bone formation. We have reported that SCARB1 is the most abundant scavenger receptor for OxPLs in osteoblastic cells, and osteoblasts derived from Scarb1 knockout mice (Scarb1 KO) are protected from the pro-apoptotic and anti-differentiating effects of OxLDL. Skeletal analysis of Scarb1 KO mice produced contradictory results, with some studies reporting elevated bone mass and others reporting low bone mass. To clarify if Scarb1 mediates the negative effects of PC-OxPLs in bone, we deleted it in osteoblast lineage cells using Osx1-Cre transgenic mice. Bone mineral density (BMD) measurements and micro-CT analysis of cancellous and cortical bone at 6 months of age did not reveal any differences between Scarb1{Delta}OSX-l mice and their wild-type (WT), Osx1-Cre, or Scarb1fl/fl littermate controls. We then investigated whether PC-OxPLs could exert their anti-osteogenic effects via activation of SCARB1 in myeloid cells by deleting Scarb1 in LysM-Cre expressing cells. BMD measurements and micro-CT analysis at 6 months of age did not show any differences between Scarb1{Delta}LysM mice and their WT, LysM-Cre, or Scarb1fl/fl controls. Based on this evidence, we conclude that Based on this evidence, we conclude that the adverse skeletal effects of PC-OxPLs in adult mice are not mediated by Scarb1 expressed in osteoblast lineage cells or myeloid cells.

cell biology↗

bioRxiv2023

A framework for defining mesenchymal cell types associated with murine periosteal and endosteal bone

Single-cell RNA sequencing has led to numerous novel designations for mesenchymal cell types associated with bone. Consequently, there are now multiple designations for what appear to be the same cell type. In addition, existing datasets contain relatively small numbers of mature osteoblasts and osteocytes and there has been no comparison of periosteal bone cells to those at the endosteum and trabecular bone. The main goals of this study were to increase the amount of single cell RNA sequence data for osteoblasts and osteocytes, to compare cells from the periosteum to those inside bone, and to clarify the major categories of cell types associated with murine bone. To do this, we created an atlas of murine bone-associated cells by harmonizing published datasets with in-house data from cells targeted by Osx1-Cre and Dmp1-Cre driver strains. Cells from periosteal bone were analyzed separately from those isolated from the endosteum and trabecular bone. Over 100,000 mesenchymal cells were mapped to reveal 11 major clusters designated fibro-1, fibro-2, chondrocytes, articular chondrocytes, tenocytes, adipo-CAR, osteo-CAR, pre-osteoblasts, osteoblasts, osteocytes, and osteo-X, the latter defined in part by Postn expression. Osteo-X, osteo-CAR, and pre-osteoblasts were closely associated with osteoblasts at the trabecular bone surface. Wnt16 was expressed in multiple cell types from the periosteum but not in any cells from endocortical or cancellous bone. Fibro-2 cells, which express markers of skeletal stem cells, localized to the periosteum but not trabecular bone in adult mice. Suppressing bone remodeling eliminated osteoblasts and altered gene expression in pre-osteoblasts but did not change the abundance or location of osteo-X or osteo-CAR cells. These results provide a framework for identifying bone cell types in murine single cell RNA sequencing datasets and suggest that osteoblast progenitors reside near the surface of remodeling bone. Author SummaryThe skeleton of vertebrate animals is produced by bone forming cells known as osteoblasts. New osteoblasts are continually produced throughout life to maintain the skeleton. However, the identity of the cells from which new osteoblasts are derived is not clear. Single cell RNA sequencing provides a gene expression profile of individual cells and allows them to be grouped into clusters of cells with similar profiles. In many cases, distinct clusters represent distinct cell types. Recent single cell RNA sequencing studies of mouse bones by several different laboratories have identified many novel cell types, some of which may be osteoblast progenitors. In many cases, different laboratories have created different names for what appear to be the same cell type. To help clarify this situation, we performed single cell RNA sequencing studies and combined our results with those from several published studies to create a harmonized map of the cell types associated with mouse bones. We also identified gene signatures for each cell type that can be used to improve the consistency of cell type designation in future single cell RNA sequencing studies of mouse bone.

genomics↗

bioRxiv2022

Microglial STAT1-sufficiency is required for resistance to toxoplasmic encephalitis

Toxoplasma gondii is a ubiquitous intracellular protozoan parasite that establishes a life-long chronic infection largely restricted to the central nervous system (CNS). Constant immune pressure, notably IFN-{gamma}-STAT1 signaling, is required for preventing fatal pathology during T. gondii infection. Here, we report that abrogation of STAT1 signaling in microglia, the resident immune cells of the CNS, is sufficient to induce a loss of parasite control in the CNS and susceptibility to toxoplasmic encephalitis during the early stages of chronic infection. Using a microglia-specific genetic labeling and targeting system that discriminates microglia from blood-derived myeloid cells that infiltrate the brain during infection, we find that, contrary to previous in vitro reports, microglia do not express inducible nitric-oxide synthase (iNOS) during T. gondii infection in vivo. Instead, transcriptomic analyses of microglia reveal that STAT1 regulates both (i) a transcriptional shift from homeostatic to "disease-associated microglia" (DAM) phenotype conserved across several neuroinflammatory models, including T. gondii infection, and (ii) the expression of anti-parasitic cytosolic molecules that are required for eliminating T. gondii in a cell-intrinsic manner. Further, genetic deletion of Stat1 from microglia during T. gondii challenge leads to fatal pathology despite largely equivalent or enhanced immune effector functions displayed by brain-infiltrating immune populations. Finally, we show that microglial STAT1-deficiency results in the overrepresentation of the highly replicative, lytic tachyzoite form of T. gondii, relative to its quiescent, semi-dormant bradyzoite form typical of chronic CNS infection. Our data suggest an overall protective role of CNS-resident microglia against T. gondii infection, illuminating (i) general mechanisms of CNS-specific immunity to infection (ii) and a clear role for IFN-STAT1 signaling in regulating a microglial activation phenotype observed across diverse neuroinflammatory disease states.

immunology↗

bioRxiv2020

Gasdermin-D-dependent IL-1α release from microglia promotes protective immunity during chronic Toxoplasma gondii infection

Microglia, the resident immune cells of the brain parenchyma, are thought to be first-line defenders against CNS infections. We sought to identify specific roles of microglia in the control of the eukaryotic parasite Toxoplasma gondii, an opportunistic infection that can cause severe neurological disease. In order to identify the specific function of microglia in the brain during infection, we sorted microglia and infiltrating myeloid cells from infected microglia reporter mice. Using RNA-sequencing, we find strong NF-{kappa}B and inflammatory cytokine signatures overrepresented in blood-derived macrophages versus microglia. Interestingly, we also find that IL-1 is enriched in microglia and IL-1{beta} in macrophages, which was also evident at the protein level. We find that mice lacking IL-1R1 or IL-1, but not IL-1{beta}, have impaired parasite control and immune cell infiltration specifically within the brain. Further, by sorting purified populations from infected brains, we show that microglia, not peripheral myeloid cells, release IL-1 ex vivo. Finally, using knockout mice as well as chemical inhibition, we show that ex vivo IL-1 release is gasdermin-D dependent, and that gasdermin-D and caspase-1/11 deficient mice show deficits in immune infiltration into the brain and parasite control. These results demonstrate that microglia and macrophages are differently equipped to propagate inflammation, and that in chronic T. gondii infection, microglia specifically can release the alarmin IL-1, a cytokine that promotes neuroinflammation and parasite control.

immunology↗

bioRxiv2018

IL-10 and ICOS differentially regulate T cell responses in the brain during chronic Toxoplasma gondii infection

Control of chronic CNS infection with the parasite Toxoplasma gondii requires an ongoing T cell response in the brain. Immunosuppressive cytokines are also important for preventing lethal immunopathology during chronic infection. To explore the loss of suppressive cytokine exclusively during the chronic phase of infection we blocked IL-10 receptor (IL-10R). Blockade was associated with widespread changes in the inflammatory response, including increased antigen presenting cell (APC) activation, expansion of CD4+ T cells, and increased neutrophil recruitment to the brain, consistent with previous reports. We then sought to identify regulatory mechanisms contributing to IL-10 production, focusing on ICOS (inducible T cell costimulator), a molecule that promotes IL-10 production in many systems. Unexpectedly, ICOS-ligand (ICOSL) blockade led to a local expansion of effector T cells in the inflamed brain without affecting IL-10 production or APC activation. Instead, we found that ICOSL blockade led to changes in T cells associated with their proliferation and survival. Specifically, we observed increased expression of IL-2 associated signaling molecules, including CD25, STAT5 phosphorylation, Ki67, and Bcl-2 in T cells in the brain. Interestingly, increases in CD25 and Bcl-2 were not observed following IL-10R blockade. Also unlike IL-10R blockade, ICOSL blockade led to an expansion of both CD8+ and CD4+ T cells in the brain, with no expansion of peripheral T cell populations or neutrophil recruitment to the brain Overall, these results suggest that IL-10 and ICOS differentially regulate T cell responses in the brain during chronic T. gondii infection.

immunology↗

bioRxiv2018

The damage signal IL-33 promotes a focal protective myeloid cell response to Toxoplasma gondii in the brain

Understanding how invading pathogens are sensed within the brain is necessary to uncover how effective immune response are mounted in immunoprivileged sites. The eukaryotic parasite Toxoplasma gondii colonizes the brain of its hosts and initiates robust immune cell recruitment, but little is known about innate recognition of T. gondii within brain tissue. The host damage signal IL-33 is one protein that has been implicated in control of chronic T. gondii infection, but the specific impact of IL-33 signaling within the brain is unclear. Here, we show that IL-33 is expressed by oligodendrocytes and astrocytes during T. gondii infection, is released into the cerebrospinal fluid of T. gondii-infected animals, and is required for control of infection. IL-33 signaling promotes chemokine expression within brain tissue and is required for the recruitment of peripheral anti-parasitic immune cells, including IFN-{gamma}-expressing T cells and iNOS-expressing monocytes. Importantly, we find that the beneficial effects of IL-33 during chronic infection are not a result of signaling on infiltrating immune cells, but rather on radio-resistant responders, and specifically, astrocytes. Mice with IL-33R-deficient astrocytes fail to promote an adaptive immune response in the CNS and control parasite burden, demonstrating that astrocytes can directly respond to IL-33 in vivo. Together, these results indicate a brain-specific mechanism by which IL-33 is released and sensed locally, to engage the peripheral immune system in controlling a neurotropic pathogen.

immunology↗