Biology subjects

MacParland, S.

Publications and source records attributed to MacParland, S..

10 recordsLinked to original sources

bioRxiv2026

Identification and characterization of inflammatory LILR and fibrotic SPP1 macrophages in chronic lung allograft dysfunction

Chronic lung allograft dysfunction (CLAD) is a major cause of death after lung transplantation and manifests principally as bronchiolitis obliterans syndrome (BOS) or restrictive allograft syndrome (RAS). Although pulmonary macrophages are key regulators of lung injury and repair, their contributions to CLAD pathogenesis remain under-examined. We performed single-cell RNA sequencing-based transcriptomic analysis of CLAD (7 BOS, 7 RAS) and control lung tissue (n = 6), complemented by comparative and functional analyses. Two distinct macrophage populations were identified in CLAD: inflammatory macrophages expressing leukocyte immunoglobulin-like receptors (LILR), and fibrotic macrophages characterized by expression of osteopontin (SPP1). LILR macrophages were present in both BOS and RAS, whereas SPP1 macrophages were selectively enriched in RAS. Both populations were identified in idiopathic pulmonary fibrosis and cross-tissue comparisons. Surface marker-based sorting strategies were developed to isolate both populations. Functional studies demonstrated that LILR macrophages exhibited enhanced phagocytic activity, promoted T cell chemotaxis and secreted inflammatory mediators, whereas SPP1 macrophages produced soluble factors that drove fibroblast activation and contraction. These findings identify distinct inflammatory and fibrotic macrophage programs associated with CLAD, link RAS to conserved fibrotic macrophage states observed in pulmonary fibrosis and highlight macrophage populations as potential therapeutic targets for inflammatory and fibrotic allograft injury.

immunology↗

bioRxiv2026

Cholangiocyte RUNX1 Orchestrates Fibrogenic and Inflammatory Signaling to Drive Biliary Fibrosis

IntroductionBiliary fibrosis and inflammation are central to the pathogenesis of cholangiopathies such as primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC). Inflammatory and fibrogenic stimuli, such as transforming growth factor-{beta} (TGF{beta}) and lipopolysaccharide (LPS) signaling, drive these processes, but their underlying transcriptional mechanisms in cholangiocytes remain incompletely defined. We investigated the role of Runt-related transcription factor 1 (RUNX1) as a transcriptional co-regulator of fibroinflammatory signaling in cholangiocytes. MethodsHuman PSC-derived cholangiocytes (PSC-Cs) and mouse large biliary epithelial cells (MLEs) were subjected to RUNX1 knockdown or pharmacologic inhibition (Ro5-3335 or AI-10-104). Cytokine secretion was profiled by Luminex multiplexing; RUNX1 genomic binding and protein interactome were assessed by ChIP-qPCR, ChIP-seq, and LC-MS/MS. In vivo, Mdr2-/- mice received Ro5-3335, and cholangiocyte-selective Runx1 knockout mice (Krt19-CreERT) were challenged with a DDC diet, followed by evaluation of fibrosis and inflammation. ResultsRUNX1 expression was significantly increased in cholangiocytes from PSC and PBC patients, and Mdr2-/- mice. RUNX1 knockdown or inhibition reduced IL6, TNF, and other proinflammatory cytokines in PSC-Cs and attenuated TGF{beta}-, LPS-, and TNF-induced Il6 and Ccl2 expression in MLEs. ChIP-qPCR and ChIP-seq revealed TGF{beta}-induced RUNX1 binding to the Il6 promoter and 727 additional genomic sites enriched for fibrosis and inflammatory pathways; predicted upstream regulators included TGF{beta}, TNF, and NF{kappa}B signaling. Proteomic analysis identified TGF{beta}-induced RUNX1 interactions with SMAD2 and NF{kappa}B2. In vivo, Ro5-3335 treatment in Mdr2-/- mice reduced hepatic collagen, ECM gene expression, immune cell infiltration, and serum liver injury markers and bile acids. Similarly, cholangiocyte-specific Runx1 deletion mitigated fibrosis, inflammation, and liver injury in DDC-fed mice. ConclusionRUNX1 is a central transcriptional hub integrating TGF{beta} and inflammatory signals in cholangiocytes. Its inhibition attenuates biliary fibrosis and inflammation in cholestatic models, supporting RUNX1 as a potential therapeutic target in fibroinflammatory cholangiopathies.

physiology↗

bioRxiv2026

Multiscale volume electron microscopy of the human liver maps vascular-cellular architecture, organelle dynamics and inter-organelle communication

The human liver depends on multiscale structural organization from vasculature to cells to organelles to perform its diverse metabolic functions. A unified three-dimensional view linking these hierarchical scales in intact human tissue would be useful for better understanding these levels and how they relate to each other. We present a high-resolution volume electron microscopy reconstruction of human periportal liver tissue (152 x 140 x 33 {micro}m3 at 8 nm pixel size) acquired by serial block-face scanning electron microscopy. Using a multiscale deep learning approach, we performed automated segmentation across the entire volume, enabling comprehensive annotation of vasculature, cells, and organelles. Quantitative analysis of bile duct architecture revealed coordinated scaling between lumen geometry and cholangiocyte number and size. Sinusoidal capillary branches exhibited distinct structural profiles with differential endothelial coverage. Analysis of 35,790 complete mitochondria identified substantial morphological heterogeneity, with elongated mitochondria displaying preferential endoplasmic reticulum (ER) contacts at narrowing sites, a pattern consistent with ER-mediated fission or fusion. This multiscale reconstruction establishes an ultrastructural reference for the healthy human liver and provides a quantitative framework for investigating hepatic physiology and disease.

cell biology↗

bioRxiv2026

NINJ1 mediates hepatic ischemia-reperfusion injury

Hepatic ischemia-reperfusion injury (IRI) results from interrupted perfusion to the liver and contributes to acute liver dysfunction such as following liver transplantation. Lytic cell death pathways are major drivers of IRI and the subsequent inflammatory response. The transmembrane protein ninjurin-1 (NINJ1) was identified as the key executor of terminal plasma membrane rupture across multiple lytic cell death pathways implicated in hepatic IRI. We hypothesized that NINJ1-mediated lytic cell death drives IRI and that its therapeutic inhibition would mitigate liver IRI. Using human liver specimens, we found that NINJ1 is highly expressed in human liver tissue and that its activation correlates with early allograft dysfunction in patients undergoing liver transplantation. Utilizing a segmental hepatic IRI model in mice and rats, Ninj1 genetic deletion or pharmacologic inhibition diminished acute liver injury. Mice with hepatocyte- or macrophage-specific Ninj1 knockout both had reduced hepatocellular injury following IRI, suggesting that NINJ1 within both populations contributes to the resulting liver injury. Mechanistically, we found that hepatocytes and Kupffer cells are highly susceptible to hypoxia-induced NINJ1-mediated plasma membrane rupture, which can be pharmacologically prevented. These data position NINJ1 as a potential new therapeutic target to limit hepatic IRI, with important implications for organ preservation during liver transplantation.

immunology↗

bioRxiv2025

Scalable expansion and hepatic zone maturation of hepatic progenitor cells from human pluripotent stem cells

Scalable generation of functionally mature hepatocytes from human pluripotent stem cells (hPSCs) is vital for regenerative medicine, disease modeling, and drug screening. We present a modular differentiation platform that enables serial expansion and cryopreservation of hepatic progenitors (hepatoblasts; HBs), supporting efficient downstream maturation. By modulating bile acid signaling with FGF19 and culturing under hypoxia, we preserved HB proliferation and bipotency. Subsequent thyroid hormone treatment induced alpha-fetoprotein-negative, albumin-positive hepatocytes, while WNT pathway modulation promoted zonal identity, mimicking in vivo hepatic metabolic heterogeneity. Zonation was confirmed by differential drug-metabolizing enzyme activity, mitochondrial function, and enhanced engraftment in a liver injury mouse model. To evaluate translational relevance, mature hepatocytes were incorporated into extrusion-based 3D bioprinted constructs, which maintained hepatic viability and function. This integrated strategy of combining progenitor cell banking, zonation control, and tissue engineering offers a scalable and clinically relevant approach for generating functional human liver tissue suitable for therapeutic development.

cell biology↗

bioRxiv2022

Single cell profiling reveals strain-specific differences in myeloid inflammatory potential in the rat liver

Liver transplantation is currently the only treatment for end-stage liver disease and acute liver failure. Liver transplant rejection is among the most lethal complications of transplantation, and therapeutic development is limited by our lack of a comprehensive understanding of the cellular landscape of the liver. The laboratory rat (Rattus norvegicus), ideal in size as a model for surgical procedures, is a strong platform to study liver biology in the context of liver transplantation. Liver allograft rejection is known to be strain-specific in the rat model, although the transplantation is accepted without rejection in some strains, it leads to acute rejection in others. To shed light on the cellular landscape of the rat liver and build a foundation for strain comparison, we present a comprehensive single-cell transcriptomics map of the healthy rat liver of Lewis and Dark Agouti strains. Using a novel computational pipeline we developed to guide the detailed annotation of our rat liver atlas, we discovered that hepatic myeloid cells have strong Lewis and Dark Agouti strain-specific differences focused on inflammatory signaling pathways. We experimentally validated these strain-specific differences in myeloid inflammatory potential in vitro using intracellular cytokine staining. Our work provides the first examination of the multi-strain healthy rat liver by single cell transcriptomics and uncovers key insights into strain-specific differences in this valuable model animal. SummaryThe laboratory rat (Rattus norvegicus) is a standard model animal for orthotopic liver transplantation. Transplanting a liver from a Dark agouti (DA) to a Lewis (LEW) strain rat leads to transplant rejection and the reverse procedure leads to tolerance. Understanding this strain difference may help explain the cellular drivers of liver allograft rejection post-transplant. This study uses single-cell transcriptomics to better understand the complex cellular composition of the rat liver and unravels cellular and molecular sources of inter-strain hepatic variation. We generated single-cell transcriptomic maps of the livers of healthy DA and LEW rat strains and developed a novel, factor analysis-based bioinformatics pipeline to study data covariates, such as strain and batch. Using this approach, we discovered variations within hepatocyte and myeloid populations that explain how the states of these cells differ between strains in the healthy rat, which may explain why these strains respond differently to liver transplants.

genomics↗

bioRxiv2022

Selective Control of Parasitic Nematodes Using Bioactivated Nematicides

Parasitic nematodes are a major threat to global food security, particularly as the world amasses 10 billion people amidst limited arable land. Most traditional nematicides have been banned due to poor nematode-selectivity, leaving farmers with inadequate controls. Here, we use the model nematode Caenorhabditis elegans to identify a family of selective imidazothiazole nematicides, called selectivins, that undergo cytochrome p450-dependent bioactivation exclusively in nematodes. At low parts-per-million concentrations, selectivins perform comparably well with commercial nematicides to control root infection by Meloidogyne incognita - the worlds most destructive plant-parasitic nematode. Tests against a wide range of phylogenetically diverse non-target systems demonstrate that selectivins are more nematode-selective than nearly all marketed nematicides. Thus, selectivins are first-in-class bioactivated nematode controls that provide efficacy as well as much-needed nematode selectivity.

pharmacology and toxicology↗

bioRxiv2022

Single-cell atlas of human liver development reveals pathways directing hepatic cell fates

The liver has been studied extensively due to the broad number of diseases affecting its vital functions. However, therapeutic advances, especially in regenerative medicine, are currently hampered by the lack of knowledge concerning human hepatic cell development. Here, we addressed this limitation by describing the developmental trajectories of different cell types comprising the human fetal liver at single-cell resolution. These transcriptomic analyses revealed that sequential cell-to-cell interactions direct functional maturation of hepatocytes, with non-parenchymal cells playing critical, supportive roles during organogenesis. We utilised this information to derive bipotential hepatoblast organoids and then exploited this novel model system to validate the importance of key signalling pathways and developmental cues. Furthermore, these insights into hepatic maturation enabled the identification of stage-specific transcription factors to improve the functionality of hepatocyte-like cells generated from human pluripotent stem cells. Thus, our study establishes a new platform to investigate the basic mechanisms of human liver development and to produce cell types for clinical applications.

developmental biology↗

bioRxiv2021

Interferon-stimulated and metallothionein-expressing macrophages are associated with acute and chronic allograft dysfunction after lung transplantation

Lung transplant (LT) recipients experience episodes of immune-mediated acute lung allograft dysfunction (ALAD). ALAD episodes are a risk factor for chronic lung allograft dysfunction (CLAD), the major cause of death after LT. We have applied single-cell RNA sequencing (scRNAseq) to bronchoalveolar lavage (BAL) cells from stable and ALAD patients and to cells from explanted CLAD lung tissue to determine key cellular elements in dysfunctional lung allografts, with a focus on macrophages. We identified two alveolar macrophage (AM) subsets uniquely represented in ALAD. Using pathway analysis and differentially expressed genes, we annotated these as pro-inflammatory interferon-stimulated gene (ISG) and metallothionein-mediated inflammatory (MT) AMs. Functional analysis of an independent set of AMs in vitro revealed that ALAD AMs exhibited a higher expression of CXCL10, a marker of ISG AMs, and increased secretion of pro-inflammatory cytokines compared to AMs from stable patients. Using publicly available BAL scRNAseq datasets, we found that ISG and MT AMs are associated with more severe inflammation in COVID-19 patients. Analysis of cells from four explanted CLAD lungs revealed similar macrophage populations. Using a single nucleotide variation calling algorithm, we also demonstrated contributions of donor and recipient cells to all AM subsets early post-transplant, with loss of donor-derived cells over time. Our data reveals extensive heterogeneity among lung macrophages after LT and indicates that specific sub-populations may be associated with allograft dysfunction, raising the possibility that these cells may represent important therapeutic targets.

immunology↗

bioRxiv2021

Pro-inflammatory alveolar macrophages associated with allograft dysfunction after lung transplantation

PurposeLung transplant (LT) recipients experience episodes of immune-mediated acute lung allograft dysfunction (ALAD). We have applied single-cell RNA sequencing (scRNAseq) to bronchoalveolar lavage (BAL) cells of stable and ALAD patients to determine key cellular elements in dysfunctional lung allografts. Our particular focus here is on studying alveolar macrophages (AMs) as scRNAseq enables us to elucidate their heterogeneity and possible association with ALAD where our knowledge from cytometry-based assays is very limited. MethodsFresh bronchoalveolar lavage (BAL) cells from 6 LT patients, 3 with stable lung function (3044 {+/-} 1519 cells) and 3 undergoing an episode of ALAD (2593 {+/-} 904 cells) were used for scRNAseq. R Bioconductor and Seurat were used to perform QC, dimensionality reduction, annotation, pathway analysis, and trajectory. Donor and recipient deconvolution was performed using single nucleotide variations. ResultsOur data revealed that AMs are highly heterogeneous (12 transcriptionally distinct subsets in stable). We identified two AM subsets uniquely represented in ALAD. Based on pathway analysis and the top differentially expressed genes in BAL we annotated them as pro-inflammatory interferon-stimulated genes (ISG) and metallothioneins-mediated inflammatory (MT). Pseudotime analysis suggested that ISG AMs represent an earlier stage of differentiation which may suggest them as monocyte drive macrophages. Our functional analysis on an independent set of BAL samples shows that ALAD samples have significantly higher expression of CXCL10, a marker of ISG AM, as we as higher secretion of pro-inflammatory cytokines. Single nucleotide variation calling algorithm has allowed us to identify macrophages of donor origin and demonstrated that donor AMs are lost with time post-transplant. ConclusionUsing scRNAseq, we observed AMs heterogeneity and identified specific subsets that may be associated with allograft dysfunction. Further exploration with scRNAseq will shed light on LT immunobiology and the role of AMs in allograft injury and dysfunction.

immunology↗