Research
Abstract
Targeted injury to the biliary epithelium and impaired bile flow are central mechanisms underlying childhood cholangiopathies. In biliary atresia (BA), immune-mediated cholangiocyte injury drives progressive bile duct obstruction, yet epithelial-intrinsic programs regulating injury susceptibility remain incompletely defined. Here, we modeled virus-epithelium interactions using rhesus rotavirus (RRV) infection of human liver-derived cholangiocyte organoids from normal donors (NCO) and infants with BA at diagnosis (BACO-Dx) or transplantation (BACO-Tx). Compared with NCO, BA organoids displayed baseline defects in tight junction architecture and barrier function, with prominent epithelial disruption and apoptosis following RRV exposure. Transcriptomic profiling revealed that while NCO and BACO-Dx mounted predominantly interferon-associated antiviral responses, BACO-Tx exhibited an expanded inflammatory program characterized by enrichment of NF-κB (RELA/NFKB1) regulatory network at baseline and amplified by infection. Inhibition of NF-κB signaling with the hydrogen sulfide donor GYY4137 restored organoid growth, improved tight junction organization, reduced permeability, and attenuated RRV-induced apoptosis. In the murine BA model, GYY4137 improved disease severity, preserved bile duct architecture, and protected epithelial junctional integrity while limiting cholangiocyte apoptosis. These findings identify an epithelial NF-κB-predominant program as a key determinant of intrinsic susceptibility to viral injury in BA and support targeting cholangiocyte NF-κB signaling to suppress disease progression.
Authors
Wenqi Li, Ashley Fonseca-Mejia, Reena Mourya, Pavithra Sudhakara, Pranavkumar Shivakumar, Jorge A. Bezerra
Abstract
Advillin (AVIL) is an actin-binding protein expressed in peripheral sensory neurons and Merkel cells, but aberrantly upregulated in several malignancies, including low-grade glioma, glioblastoma, and rhabdomyosarcoma. AVIL’s oncogenic activity and restricted adult tissue expression make it an attractive therapeutic target; however, its physiological function and the safety of AVIL inhibition remain incompletely understood. We evaluated genetic and pharmacologic targeting of Avil using neurobehavioral and somatosensory assays in vivo. Constitutive and conditional Avil knockout mice, generated by exons 7 and 8 deletion, underwent behavioral testing assessing anxiety-related and exploratory behaviors, motor coordination, affective responses, and learning and memory. Nociceptive and tactile sensory function were evaluated alongside histologic analysis of peripheral mechanosensory architecture. In parallel, a first-in-class AVIL inhibitor was assessed in vivo. Avil-deficient mice exhibited normal exploratory and anxiety-related behavior, intact motor coordination, preserved affective responses, and unaltered learning and memory. Loss or pharmacologic inhibition of advillin did not impair nociceptive processing, tactile sensitivity, or peripheral mechanosensory architecture, and histologic abnormalities were not detected. Together, these findings show preserved baseline behavioral and sensory outcomes following lifelong Avil loss, with complementary evidence of tolerability following pharmacologic inhibition under the conditions tested, supporting AVIL as a potentially well-tolerated therapeutic target in cancer.
Authors
Sarah A. Lynch, Martyna Glowczyk-Gluc, Yiwei Wang, Summer Johnson, Xinrui Shi, Abdul-Salam Gidado, Alexis Johnson, Christopher Deppmann, John Lukens, Hui Li
Abstract
Matrix stiffness is a hallmark of aggressive solid tumors and strongly associated with therapeutic resistance and poor prognosis. This mechanical barrier represents a major unmet challenge in oncology, as current strategies rarely address how matrix mechanics themselves drive treatment resistance. Here, we identified ferroptosis, an iron-dependent form of cell death, as a potential long-sought vulnerability that can be exploited to eliminate mechanically resistant tumors. Using genetic and chemical approaches, we investigated how matrix stiffness sensitizes cancer cells to ferroptosis and whether this vulnerability can be therapeutically targeted. We found that cancer cells cultured on stiff matrices were more sensitive to ferroptosis than those on soft matrices, as evidenced by reduced cell viability and elevated lipid peroxidation. Furthermore, we found that matrix stiffness induced YAP activation, which upregulated its target gene SKP2, a key mediator required for the heightened ferroptosis sensitivity observed under stiff conditions. Finally, using an injectable hydrogel system, we demonstrated that stiffened tumors were resistant to chemotherapy but remained highly susceptible to ferroptosis induction, resulting in markedly reduced tumor volume and weight. Our findings established ferroptosis as a stiffness-dependent vulnerability mediated by a YAP-SKP2 signaling axis, revealing a mechanotransductive pathway that may be leveraged to target treatment-refractory tumors.
Authors
Emma Villares, Nico Taber, Shao-Chin Wu, Connor Amelung, Jianli Wu, Chao-Chieh Lin, Jen-Tsan Chi, Sharon Gerecht
Abstract
Castleman disease (CD) is an inflammatory disorder characterized by lymphadenopathy with distinctive histologic features, yet its pathophysiology remains poorly understood. This delays diagnoses and limits treatments, leading to additional morbidity and mortality. We used single- cell and spatial transcriptomics to characterize lymph nodes from unicentric CD (UCD) compared with non-specific inflammatory lymphadenopathies. While hematopoietic cells showed few compositional or transcriptional differences, lymph node stromal cells exhibited marked alterations in UCD, most notably an increased abundance of vascular smooth muscle cells (VSMCs) and broadly increased stromal signaling activity. A generalized linear model distinguished histologically indeterminate lymph nodes resembling UCD from non-specific inflammation most accurately using stromal rather than hematopoietic cell composition. Immunohistochemical staining for the VSMC marker CD146 (Melanoma Cell Adhesion Molecule, MCAM) further discriminated UCD from other causes of lymphadenopathy. Together, these findings identify increased VSMC abundance as a hallmark of UCD and suggest that this stromal signature can be leveraged to improve pathologic diagnosis and patient stratification.
Authors
Joshua D. Brandstadter, Samuel Meili, Joseph Zinski, Hung-Wei Cheng, Dorottya Laczko, Michael V. Gonzalez, Subin S. George, Megan S Lim, Adam Bagg, Yves Stanossek, Lucas Onder, Mechthild Lϋtge, Angelina De Martin, Bridget Austin, Melanie Mumau, Katherine S. Forsyth, Vinodh Pillai, Antonio Ferreira, Jon C. Aster, Sunita Nasta, Burkhard Ludewig, David C. Fajgenbaum, Ivan Maillard
Abstract
An immunosuppressive, macrophage-rich tumor microenvironment (TME) is a barrier to immunotherapy across many cancers including osteosarcoma, where immunotherapy has largely failed. cGAS-STING agonism has been explored to inflame such tumors, yet the mechanisms of STING-mediated TME remodeling remain poorly understood, especially in sarcomas. Using multi-modal single cell analysis, we show that systemic STING agonism in osteosarcoma triggers a myeloid-predominant inflammatory response and rapid tumor necrosis, followed by a T cell-mediated response, immunologic memory, and long-term cure in some models. Within two hours of treatment, STING agonism induces pro-inflammatory interferon programs in tumor-associated macrophages and monocytes, with additional contributions from endothelial cells. Efficacy persists in the absence of tumor-intrinsic STING or IFNAR, confirming that the therapeutic axis resides within the host TME rather than the tumor cell compartment. While cGAS-STING is frequently repressed in human osteosarcoma models of aggressive disease, a STING activation gene signature predicts survival in newly diagnosed patients, suggesting that this pathway is engaged during tumor progression. Together, these data identify an opportunity to exploit the myeloid compartment of osteosarcoma with STING pathway therapies, charting a path forward for engaging the TME to secure long term cure in an aggressive malignancy still treated with highly toxic chemotherapy.
Authors
Elizabeth P. Young, Christine A. Johnson, Alex G. Lee, Douglass Morris II, Georgia M. Edmondson, Alfonso G. Ramirez-Ristori, Jessica Tsui, Marcus R. Breese, Juan Antonio Camara Serrano, Courtney R. Schott, Clare L. Abram, Stanley G. Leung, Leanne C. Sayles, Aafrin M. Pettiwala, Vanessa S. Gutierrez Vera, Clifford A. Lowell, Alexis J. Combes, Troy A. McEachron, E. Alejandro Sweet-Cordero
Abstract
Differentiating keratinocytes break down their organelles and nuclei to become the compacted cornified layers of the epidermal barrier in a poorly understood catabolic process. Live confocal imaging of stratified human organotypic epidermis revealed endoplasmic reticulum (ER) fragmentation and lysosomal engulfment in the cornifying layers, where we found up-regulation of TEX264, a receptor that mediates selective autophagy of the ER (reticulophagy). TEX264 expression was increased by ER stress, which caused precocious cornification of organotypic epidermis. In undifferentiated keratinocytes, ectopic TEX264 was sufficient to induce ER fragments, while in highly differentiated keratinocytes, it accelerated ER elimination and nuclear shrinkage; these effects were abolished by mutating the LC3 interacting region required for its autophagic function. Knockout of TEX264 or inhibiting its activation disrupted the maturation of organotypic cultures, pointing to a critical role for reticulophagy in cornification. Finally, in patient biopsies and an organotypic model of Darier disease, a genetic cornification disorder linked to ER dysfunction, we found increased TEX264 in areas of premature cornification (dyskeratosis). Our results identified TEX264 as a key driver of late-stage epidermal differentiation and led us to propose a model of cornification in which keratinocytes activate selective autophagy receptors to orchestrate orderly organelle elimination during cutaneous barrier formation.
Authors
Christopher J. Johnson, Afua Tiwaa, Arti Parihar, Edwin Antonio, Brooke D. Lorenz, Reeteka Kudallur, Aaron Ramonett, Anthony Coon, Mrinal K. Sarkar, Johann Gudjonsson, Cory L. Simpson
Abstract
Obesity is a chronic progressive disease strongly linked to numerous comorbidities. This is underpinned by a chronic inflammatory environment and significant immune dysregulation. γδ T cells are unconventional T cells that display innate and adaptive characteristics, the predominant one being their independence of MHC restriction. In humans, γδ T cells represent up to 5% of the peripheral blood T cell compartment and are capable of rapidly producing a range of effector cytokines and driving cytotoxic responses. This is critical for protection from both bacterial and viral infections. We investigated circulating γδ T cell metabolic and functional responses in cohorts of people living with obesity (PWO) and people without obesity. We showed that PWO have reduced Vδ2+ T cell frequencies, and their effector responses are diminished following stimulation. We identified that the CD98-LAMTOR5-mTORC1 metabolic axis was critical for functional responses, and demonstrated significant defects in each component of this axis in PWO. Furthermore, STAT5 the initiator of amino acid metabolism was also decreased in PWO. Finally, we showed restoration of this metabolic axis after bariatric surgery, combined with robust improvements in functional responses. Collectively, our data highlighted that obesity drives a reversible dysregulation of Vδ2+ T cells by disrupting cellular metabolism.
Authors
Ciara L. Flynn, Eimear K. Ryan, Cian Davis, Czara A. Kennedy, Katherine G. Egan, Aimee Brennan, Philip Mc Entee, Kirstan Murphy, Niamh Lynam-Lennon, Linda V. Sinclair, Nicholas Jones, Donal O'Shea, Helen M. Heneghan, Andrew E. Hogan
Abstract
Exposure of humans to food insecurity (FI), involving sporadic access and low nutritional quality, is associated with later life development of metabolic disease phenotypes, but mechanisms are poorly understood. We exposed very young rats to a 20-day FI intervention or ad-lib (AL) feeding, using either standard chow (SC) or high fat/high sucrose (HF/HS) diets, followed by continued AL feeding on the respective diets for 10-13 weeks. We found that male, but not female rats subjected to FI on the HF/HS diet had increased body fat percentage, ratio of adipose to body weight, and fat cell size compared to AL rats, coupled with metabolomic and transcriptomic profiles consistent with reduced fatty acid oxidation in the liver and maladaptive remodeling of adipose tissue. These changes were specific to HF/HS feeding and did not occur with SC diet. Our study defines sex- and diet-specific metabolic remodeling responses to FI that may contribute to future disease phenotypes.
Authors
Jie An, Jessica Cannavino, Ashley Williams, Akshay Bareja, Wenxin Tong, Olga Ilkayeva, Michael Muehlbauer, Danhong Lu, Robert A. Hollander, Sarah Armstrong, Rana K. Gupta, Christopher B. Newgard
Abstract
Bone morphogenetic protein-2 (BMP2) is used clinically to promote bone regeneration, but dose-limiting side effects restrict its clinical utility. Canonical Wnt signaling is essential for bone formation, and R-spondin-2 (RSPO2) is a secreted Wnt agonist highly expressed by osteoprogenitors. We found that BMP2 strongly induced RSPO2 during early human mesenchymal stem cell (hMSC) osteoblast differentiation. Recombinant RSPO2 synergized with BMP2 in both hMSCs and MC3T3-E1 preosteoblasts, whereas RSPO2 silencing impaired BMP2-driven osteogenic responses. In a murine critical-size calvarial defect model, co-delivery of RSPO2 and a suboptimal dose of BMP2 yielded significantly more new bone than BMP2 alone, while RSPO2 alone was ineffective. Enhanced canonical Wnt activation with BMP2-RSPO2 co-treatment was shown with an in vivo Wnt reporter mouse. Bulk RNA sequencing of MC3T3-E1 cells revealed that BMP2-RSPO2 co-treatment differentially activated osteogenesis-related genes and pathways compared to BMP2 alone. The guanine exchange factor (GEF), Prex1, was selectively upregulated by BMP2-RSPO2 co-treatment, and was required for RSPO2-mediated enhancement of BMP2-induced osteoblastogenesis. These findings identify RSPO2 as an enhancer of BMP2-driven osteogenesis, amplifying Wnt activation and involving PREX1-dependent signaling. RSPO2 or PREX1 activation may represent a co-therapy that could enhance BMP2 efficacy while mitigating the adverse effects of high-dose BMP2.
Authors
Buchan Jiang, Erin B. Meymarian, Shije Shahin, Jack T. Wilkening, Alexander J. Knights, Archana Sanjay, Tristan Maerz, Yadav Wagley, Kurt D. Hankenson
Abstract
The inflammasome is an innate immune complex responsible for aberrant interleukin-1β (IL-1β) signaling in numerous chronic inflammatory diseases, including gout and Alzheimer’s disease. The inflammasome is composed of several proteins, including the apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC). We developed vaccines targeting various regions of ASC using a Qβ virus-like particle (VLP) platform to assess the efficacy of anti-ASC antibodies for inhibiting inflammasome activity. Using a mouse model of gout, we assessed seven vaccines targeting mouse ASC for their ability to reduce inflammasome activation and neutrophil responses. Three vaccines targeting different regions of the ASC protein (pyrin domain, linker region, and the caspase activation and recruitment domain) successfully targeted ASC, reduced IL-1β, and reduced neutrophil activation. Combining these three vaccines into one cocktail vaccine yielded similar efficacy in the gout model with a favorable safety profile. The combined ASC vaccine also reduced inflammasome levels and pathologically phosphorylated tau in a transgenic mouse model of tauopathy and did not significantly impair host immune responses to bacterial or viral infections. Thus, Qβ-VLP vaccines against ASC can neutralize pathological inflammasome activity across multiple inflammatory disease models.
Authors
Jonathan Hulse, Zachary Villaseñor, Samuel Kress, Kathryn Sánchez, Julianne Peabody, Kimberly A. Morrissey, Alan McNolty, Benjamin Lantz, Laura Gonzalez Bosc, Judy L. Cannon, Seth Daly, Bryce Chackerian, Kiran Bhaskar
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