Issue published November 9, 2026 Previous issue
- Volume 11, Issue 21
On the cover:
αTAT1 links microtubules to stiffness sensing, fibroblast activation, and fibrosis
Santos et al. report that stiffness sensing through microtubules drives fibroblast activation in mouse models of fibrotic disease. In the cover image, fluorescence microscopy of activated fibroblasts highlights the polarized microtubule network (green), revealing an unexpected role for microtubules as active drivers of cellular mechanosensing in fibrotic disease. By converting tissue stiffness into profibrotic signals, this microtubule network sustains fibroblast activation and reinforces fibrosis progression. These findings identify microtubule mechanotransduction as a potential therapeutic target across fibrotic diseases. Microtubules are shown in green, F-actin stress fibers in red, and nuclei in blue.
Santos et al. report that stiffness sensing through microtubules drives fibroblast activation in mouse models of fibrotic disease. In the cover image, fluorescence microscopy of activated fibroblasts highlights the polarized microtubule network (green), revealing an unexpected role for microtubules as active drivers of cellular mechanosensing in fibrotic disease. By converting tissue stiffness into profibrotic signals, this microtubule network sustains fibroblast activation and reinforces fibrosis progression. These findings identify microtubule mechanotransduction as a potential therapeutic target across fibrotic diseases. Microtubules are shown in green, F-actin stress fibers in red, and nuclei in blue.
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Research Letter
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Abstract
Authors
David Baidoe-Ansah, Annelie Mayer, Jasmin Mattern, Katrin Ritter, Niklas Klinghammer, Lídia Cantacorps, Robert A. Chesters, Joanne E. Falck, Selma Yagoub, Jiajie Zhu, Juri Felsberg, Rachel N. Lippert
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Research Articles
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Anti-Notch plus endocrine therapy in ER+ breast cancer inhibits cancer stem cells by increasing DAXX
Abstract
Resistance to endocrine therapy (ET) in estrogen receptor–positive (ER+) breast cancer is mediated by Notch signaling, but the clinical application of anti-Notch therapy has been limited by the lack of predictive biomarkers. To identify Notch-regulated biomarkers, we conducted a presurgical window study evaluating ET combined with the γ-secretase inhibitor (GSI) MK-0752. RNA expression in tumors was measured using an Affymetrix array and by real-time PCR (RT-PCR). ET plus GSI showed more genes were decreased than ET alone. Specifically, DAXX, NOXA, and LFNG RNAs were increased, while 15 additional transcripts were decreased. Mechanistically, GSI reduced Notch1 occupancy at CSL-binding elements within HES1, HEY2, HEYL, CCND1, MKI67, and DAXX genes, and inhibited cancer stem cells (CSCs) by 90%–100%. This anti-CSC effect required DAXX, while GSI treatment or Notch1/4 knockdown increased DAXX expression, suggesting transcriptional repression by Notch. Using mouse tumor xenograft studies, ET plus MK-0752 resulted in complete regression of MCF-7 tumors, with DAXX-high tumors showing greater treatment sensitivity. Clinically, high DAXX expression was associated with improved recurrence-free and overall survival. This study found that anti-Notch plus ET in ER+ breast cancer inhibits CSCs by increasing DAXX, a promising predictive biomarker. These findings support clinical evaluation of therapies that increase DAXX expression.
Authors
Kathy S. Albain, Debra Wyatt, Andrei Zlobin, Susan G. Hilsenbeck, Cheryl M. Czerlanis, Daniel S. Peiffer, Kyle R. Covington, Constantine Godellas, Shelly S. Lo, Patricia A. Robinson, Kathy Czaplicki, Barbara Busby, Davide Bova, Ping Tang, Patrick J. Stiff, Suzanne A.W. Fuqua, Lucio Miele, Clodia Osipo
×Abstract
Type 2 innate lymphoid cells (ILC2) are a population of lineage-negative cells in the lung, gastrointestinal tract, and skin that have emerged as a significant component of type 2 allergic inflammation. The regulation of cyclooxygenase-2 (COX-2) metabolites is critical to the pathophysiology of many inflammatory disorders, including allergic asthma. While COX-2 regulates Th9 and Th17 cell differentiation and function during allergic lung inflammation, it remains unknown whether COX-2 also regulates ILC2 cell differentiation and function under similar conditions. To address this question, we examined lung ILC2 cells from COX-2+/+ and COX-2–/– mice after ovalbumin-induced (OVA-induced) or Alternaria-induced allergic lung inflammation. ILC2 cells were significantly increased in COX-2–/– lungs compared with COX-2+/+ lungs after OVA exposure in vivo. The increase in ILC2 cells was accompanied by an increase in expression of the cytokines IL-5 and IL-13 and of the transcription factor GATA3. Both genetic disruption and selective inhibition of COX-2 significantly increased ILC2 cell differentiation from isolated common lymphoid progenitor cells (CLP) in vitro. Furthermore, COX-2–derived PGE2 acting via EP2 receptors significantly reduced IL-33 and TSLP expression, and it attenuated ILC2 cell differentiation in vitro and in vivo. Thus, during allergic lung inflammation, COX-2–derived PGE2 signals through the EP2 receptor to negatively regulate lung ILC2 cell differentiation and function.
Authors
Hong Li, Matthew L. Edin, Daniel Menendez, J. Alyce Bradbury, Joan P. Graves, Artiom Gruzdev, Gregory S. Whitehead, Maria I. Sifre, Laura M. DeGraff, Darryl C. Zeldin
×Abstract
Obesity induces pathological remodeling of white adipose tissue (WAT), including chronic inflammation and fibrosis, which drive metabolic dysfunction. Although weight loss (WL) is the cornerstone of obesity treatment and improves systemic metabolic health, its effect on WAT remodeling remains incompletely understood. Using a mouse model of diet-induced obesity followed by nutritional WL, we show that visceral WAT (vWAT) failed to fully recover despite normalization of body weight and marked metabolic improvement. Notably, vWAT fibrosis persisted and continued to progress after WL. This occurred while extracellular matrix (ECM) production by adipose progenitor cells was markedly reduced following WL, explaining why pharmacological inhibition of ECM synthesis fails to prevent fibrosis during WL. Instead, we observed impaired collagen degradation by both progenitor cells and macrophages, along with lysosomal dysfunction, during WL. This suggests that defective ECM degradation, rather than continued ECM production, underlies fibrosis persistence and aggravation during WL. Together, these findings identify impaired collagenolysis as a critical barrier to adipose tissue recovery after WL and suggest that enhancing ECM clearance, rather than inhibiting ECM synthesis, may represent a more effective therapeutic strategy to restore adipose tissue function.
Authors
Clémentine Rebière, Amanda Oliveira, Sébastien Dussaud, Iman Haddad, Agatha Assis-Ferreira, Adma M. Babetto, Christine Rouault, Fatiha Merabtene, Cheïma Benbida, Jimon Abatan, Simone Vargas da Silva, Laís Bhering Martins, Licia Torres, Ana Letícia M. Silveira, Cindy Rose, Laurent Genser, Fabrizio Andreelli, Isabelle Dugail, Marina Chaves de Oliveira, Joelle Vinh, Simon Lecoutre, Adaliene V.M. Ferreira, Karine Clément, Emmanuel L. Gautier, Genevieve Marcelin
×Abstract
Patients who survive sepsis are predisposed to new hospitalizations for respiratory failure, but the underlying mechanisms are unknown. Using a murine model in which prior sepsis predisposes to enhanced lung injury, we previously discovered that classical monocytes persist in the lungs after long-term recovery from sepsis and exhibit enhanced cytokine expression after secondary challenge with intranasal LPS. Here, we hypothesized that immune reprogramming of post-sepsis monocytes and altered ontogeny predispose to enhanced lung injury. Monocyte depletion and/or adoptive transfer was performed 3 weeks and 3 months after sepsis. Monocytes from post-sepsis mice were necessary and sufficient for enhanced LPS-induced lung injury and promoted neutrophil degranulation. Prior sepsis enhanced JAK/STAT signaling and AP-1 accessibility in monocytes and shifted monocytes toward the neutrophil-like monocyte lineage. Neutrophil-like monocytes demonstrated a proinflammatory phenotype with enhanced IL-1β expression and reduced phagocytic capacity. In human sepsis and/or pneumonia survivors, monocytes were predictive of 90-day mortality and exhibited transcriptional and proteomic neutrophil-like signatures. We conclude that sepsis reprograms monocytes into a proinflammatory phenotype and skews bone marrow progenitors and monocytes toward the neutrophil-like lineage, predisposing them to induce neutrophil degranulation and lung injury.
Authors
Scott J. Denstaedt, Breanna McBean, Alan P. Boyle, Brett C. Arenberg, Matthias Mack, Bethany B. Moore, Michael W. Newstead, Yamei Deng, Alexey I. Nesvizhskii, Benjamin H. Singer, Jennifer Cano, Hallie C. Prescott, Helen S. Goodridge, Rachel L. Zemans
×Abstract
Matrix stiffening sustains fibrosis by recruiting fibroblasts to stiff fibrotic areas through durotaxis. Once recruited, fibroblasts become mechanically activated and promote scar formation. Although actin-based mechanotransduction and focal adhesion signaling are well established, how microtubules contribute to stiffness sensing and fibroblast activation in fibrosis remains incompletely understood. Here, we identify αTAT1-mediated microtubule acetylation as a key regulator of fibroblast mechanosensing, durotaxis, and profibrotic activation. Through α-tubulin K40 acetylation, αTAT1 promotes microtubule flexibility, directional alignment, and persistent polarity along stiffness gradients, thereby enabling stiffness-directed migration and fibroblast activation. Mechanistically, αTAT1-dependent microtubule acetylation regulates focal adhesion dynamics and downstream mechanotransduction. αTAT1 loss abolishes K40 acetylation, impairs FAK-mediated YAP/TAZ signaling, and prevents stiffness- and TGF-β–induced myofibroblast differentiation. Accordingly, global or fibroblast-specific Atat1 depletion reduces fibroblast durotaxis and the accumulation of profibrotic fibroblasts, protecting mice from lung, dermal, and kidney fibrosis without affecting inflammation or vascular integrity. Our findings establish αTAT1-dependent microtubule acetylation as a conserved mechanosensing pathway that links fibroblast durotaxis and activation to fibrosis across organs in vivo, and identify αTAT1 catalytic activity as a potential mechano-therapeutic target in fibrotic disease.
Authors
Alba Santos, Tamanna Islam, Maria-Anna Chrysovergi, Paula E. Grasberger, Vera Auernheimer, Taslim A. Al-Hilal, Santiago Lamas, David Lagares
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Recent ACC/AHA guidelines recommend ≥ 5% weight loss over 6 months to reduce cardiovascular risk in obesity. However, some populations paradoxically exhibit increased cardiovascular disease (CVD) following weight loss. We investigated whether mild food restriction (MfR) accelerates atherogenesis through proatherogenic lipid remodeling. ApoE-deficient mice were fed Chow or a high-cholesterol/high-fat (HCHF) diet for 6 months, with MfR producing 5% lower body-weight gain. Atherosclerotic burden was quantified histologically, and hepatic lipidomes were analyzed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and compared with plasma lipidomic profiles from patients with CVD. MfR improved insulin sensitivity by lowering blood glucose and triglycerides but increased circulating cholesterol and accelerated atherosclerosis. HCHF-fed mice developed larger, more numerous plaques with increased necrotic core formation, thinner fibrous caps, higher intima/media ratios, and inflammatory cell infiltration. Similar proatherogenic changes occurred in Chow-fed mice subjected to MfR. Mechanistically, MfR increased hepatic free cholesterol and enriched proatherogenic phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, sphingomyelin, ceramide, and cholesterol ester species, yielding a CERT1 score of 8.1 indicative of cardiovascular risk. These lipid alterations mirrored plasma lipidomic signatures from patients with CVD with diabetes. Collectively, our findings demonstrate that mild caloric restriction is not universally atheroprotective and can accelerate atherosclerosis in hypercholesterolemic states by disrupting cholesterol homeostasis and promoting proatherogenic lipid remodeling.
Authors
Yun Zhu, Yanzhe Xu, Gerhard Liebisch, Juergen Borlak
×Abstract
In chronic infection, T cell clones recognizing the same epitope are variably expanded, with some clones evolving to become dominant, while others are lost. Clonal expansion and changes in the repertoire reflect both T cell receptor–intrinsic (TCR-intrinsic) and -extrinsic factors, yet the individuality of TCR repertoires makes it difficult to disentangle how TCR sequence shapes clonal behaviors. To address this, we exploited the observation that TCRs can be grouped into clusters based on sequence similarity. Using complementarity-determining region 3β (CDR3β) sequencing data derived from lymphocytic choriomeningitis virus–specific CD8+ T cells from mice infected early and late in the course of chronic infection, we identified CDR3β clusters ubiquitously shared by all mice. The pattern of CDR3β cluster abundance was highly similar between mice at the early time point in both self-limiting and persistent infection models. Over the course of chronic infection, the pattern of cluster abundance diverged between mice and dominant clones emerged that did not share sequence similarity between mice. This repertoire divergence was not observed in self-limiting infection, where the pattern of cluster abundance established early in infection was stable between mice beyond resolution of infection. In chronic infection, the repertoires of stem-like and exhausted antigen-specific T cells remained similar over time within individual mice, suggesting clonal selection acts on the stem-like pool. Our findings suggest that antigen-specific CD8+ T cell clones expanded early in infection have CDR3β motifs that are shared between mice, supporting a role for TCR structural properties in determining the repertoire structure. Divergent patterns of repertoire composition later in chronic infection suggest that these shared CDR3β motifs are less important in patterning the repertoire than during the initial response and there is greater complexity in factors that sculpt the repertoire over the course of persistent antigen exposure.
Authors
Daniel Y. Chang, Barbara Bravi, Sakshi Malik, Donald J. McGuire, Hasan Ahmed, Rustom Antia, Rafi Ahmed, Ehsan Ghorani
×Abstract
Congenital and early-life Zika virus (ZIKV) infection can result in neurologic deficits. Precise mechanisms of injury, especially in the more subtle presentation of postnatal infection, are not fully elucidated. Here, we defined the effects of ZIKV on the developing brain using single-cell transcriptomics, histopathology, design-based stereology, diffusion MRI, and neurobehavioral assessments in infant rhesus macaques. ZIKV upregulated interferon-stimulated genes in activated microglia and cell death pathways in neurons and downregulated metabolism and differentiation genes in mature oligodendrocytes. Abnormal micro-organization of the corpus callosum and limbic white matter tracts was seen on diffusion-weighted imaging. A curated gene set associated with autism spectrum disorder risk was negatively enriched in inhibitory and excitatory neurons from ZIKV-infected infants, with increased emotional reactivity already evident 2 weeks after infection. From single cells to organism-level behaviors, these results define the pathways and processes disrupted by early-life ZIKV infection.
Authors
Venkata-Viswanadh Edara, Sienna Freeman, Maureen Sampson, Kathryn M. Moore, Rebecca Richardson, Nils Schoof, Divine Burgess, Michelle J. Lee, Gregory K. Tharp, Roza Vlasova, Shane Taylor, Ariana Hodjatzadeh, Yihana P. Melendez-Alejandro, Samia M. Abdallah, Esther W. Ndungu, Ava K. Mascarenhas, Chao-Hsiung Hsu, Tsang-Wei Tu, Martin Styner, Mehul S. Suthar, Mar M. Sanchez, Steven E. Bosinger, Mark W. Burke, Steven A. Sloan, Jessica Raper, Ann Chahroudi
×Abstract
Cancer cachexia is a debilitating syndrome of involuntary body mass loss featuring profound skeletal muscle wasting and high mortality. Cardiac muscle wasting is prominent in cancer patients and cancer survivors. We developed a human induced pluripotent stem cell–derived cardiac myocyte (hiPSC-CM) platform modeling cancer cachexia using conditioned media and Transwell coculture with C26 or HCT116 tumor cells. Cachectic hiPSC-CMs exhibited atrophy, reduced contraction amplitude, prolonged relaxation time, and elevated oxygen consumption rate, reflecting wasting, contractile dysfunction, and metabolic dysregulation. Mechanistically, suppression of the AKT/mTORC1/S6 signaling axis notably reduced total and p-AKT, decreased the p-S6/S6 ratio, and downregulated RPTOR, which coincided with FOXO1/3 activation and FOXO1 nuclear translocation, promoting downstream atrophic remodeling. Concurrently, Atrogin-1 upregulation reduced calcineurin A, impairing NFAT nuclear translocation, thereby suppressing downstream growth signaling. Increased autophagic flux with an elevated LC3BII/LC3BI ratio accompanied these changes. Following withdrawal of cachexia-inducing stimuli, Atrogin-1 and autophagy markers normalized within 1 week; however, contractile and metabolic impairments persisted, indicating delayed functional recovery. These findings establish the FOXO/Atrogin-1/calcineurin A/NFAT axis, operating within the broader context of AKT/mTORC1 suppression, as a central regulatory mechanism of cachexia-induced cardiac wasting, unveiling a tractable therapeutic target.
Authors
Nora Hosny, Houda Cohen, Mohamed Waleed Elafany, Jeff Schreifels, John Bauer, Rishi Gulati, Brian R. Thompson, Julia C. Liu, Joseph M. Metzger
×Abstract
Multiple myeloma (MM) orchestrates immune evasion by subverting natural killer (NK) cell function. CD48, one of the most abundant NK ligands on MM cells, paradoxically enhances NK cell activation yet is associated with high-risk cytogenetics and poor patient survival. We integrated multiomics (bulk and single-cell RNA-seq, assay for transposase-accessible chromatin using sequencing [ATAC-seq], chromatin immunoprecipitation sequencing [ChIP-seq]), genome-wide CRISPR knockout/activation screens, and machine learning to dissect CD48 regulation and function. In human MM and Vκ*MYC mouse scRNA-seq datasets, NK cells exhibit stepwise increases in inflammatory and exhaustion signatures and loss of cytotoxic potential as disease progresses. In vitro coculture assays show that CD48 overexpression on MM enhances NK cell cytokine secretion, whereas chronic exposure leads to ex vivo NK dysfunction. In vivo, CD48-overexpressing Vκ*MYC tumors progress more slowly and extend host survival, while NK cell depletion accelerates disease. These findings support a context-dependent role for CD48, potentiating acute NK responses while coexisting with chronic NK exhaustion, and suggest strategies to modulate CD48 for therapeutic benefit.
Authors
Bonell Patiño-Escobar, Torsten Steinbrunn, Lucy Perez-Lugo, Sham Rampersaud, Daniel D. Waller, Daniela Ordoñez, Huimin Geng, Fernando Salangsang, Paul Phojanakong, Juan Antonio Camara Serrano, Yan Zeng, Veronica Steri, Oscar A. Aguilar, Constantine S. Mitsiades, Arun P. Wiita
×Abstract
Both elevated plasma cell-free hemoglobin (CFH) and heparanase-driven endothelial glycocalyx shedding are contributors to microvascular dysfunction and organ injury in sepsis. However, the mechanisms governing heparanase activation, and the potential role of CFH in this process, are not understood. Utilizing patient samples, mice with cecal slurry–induced (CS-induced) peritonitis and elevated CFH, and human lung microvascular endothelial cells (HLMVECs), we tested the hypothesis that CFH upregulates heparanase production to drive endothelial glycocalyx degradation. In human sepsis, elevated circulating CFH was associated with higher heparanase and heparan sulfate levels, which in turn correlated with adverse clinical outcomes. CS+CFH-treated mice had increased plasma heparanase, glycocalyx degradation, and pulmonary and systemic inflammation; endothelial heparanase deletion abrogated these effects. Additionally, in both pulmonary endothelial cells isolated from CS+CFH-treated mice and HLMVECs exposed to CFH and TNF-α, heparanase transcription and active enzyme production were increased. The deleterious effects of CFH were attenuated by acetaminophen, a hemoprotein reductant. In summary, we demonstrate that CFH oxidation stimulates endothelial heparanase expression and activation during sepsis, leading to endothelial glycocalyx degradation, which may disrupt the endothelial barrier and result in organ injury. Our findings highlight the CFH-heparanase axis as a potential therapeutic target for endothelial glycocalyx preservation in sepsis.
Authors
Avery M. Bogart, Anisa S. Haffizulla, Jason Lin, Nathan D. Putz, Han Noo Ri Lee, David M. Aslaner, Samantha K. Gonski, Nancy Wickersham, Kyle Riedmann, Jamie E. Meegan, Kaori Oshima, Ciara M. Shaver, Julie A. Bastarache, Eric P. Schmidt, Lorraine B. Ware
×Abstract
ADAMTS9 mutations cause the ciliopathies nephronophthisis and Joubert syndrome. Here we demonstrated that deletion of ADAMTS9 in the proximal nephron led to polycystic kidney development in mice. In males, Adamts9 deletion caused kidneys to become highly cystic while remaining small without undergoing enlargement. In contrast, female mice developed cystic kidneys at a slower rate. ADAMTS9 deletion disrupted ciliogenesis through the loss of cleavage of the ciliary transition zone (TZ) protein TMEM67, which led to loss of the MKS/B9 module — a key component of the ciliary gate. Functional analysis of all 8 ciliopathy patient variants of ADAMTS9 identified to date showed that the TMEM67 C-terminus failed to localize to the TZ, thus disrupting a key regulatory mechanism in patient renal ciliogenesis. Modeling ADAMTS9-mediated TMEM67 cleavage using TMEM67 cleavage–deficient mice revealed loss of TZ formation, but not elevated canonical Wnt signaling, as the underlying mechanism driving cystogenesis. Adamts9 deletion led to comparatively intense interstitial collagen deposition, which likely restricted kidney enlargement and resulted in the characteristically small kidney phenotype seen in nephronophthisis. By comparative analysis of 4 interconnected polycystic kidney models, in addition to Pkd1- and Pkd2-deleted kidneys, we identified differential collagen homeostasis as a principal factor determining cystic kidney size and type.
Authors
Sydney Fischer, Karyn L. Robert, Manu Ahmed, Griffin I. Kane, Matthew A. Kavanaugh, Wei Wang, Pamela V. Tran, Prabhani U. Atukorale, Sumeda Nandadasa
×Abstract
Sterile tissue injury triggers a rapid neutrophil response that can be either pathogenic or protective, reflecting substantial functional heterogeneity of neutrophils; however, the neutrophil subsets underlying these divergent functions remain poorly defined. Here, using a well-established sterile corneal injury model, we delineate the time-dependent functional and transcriptional heterogeneity of neutrophils following sterile injury. Temporal neutrophil depletion revealed that neutrophils recruited at day 1, but not day 7, are essential for suppressing inflammation, promoting epithelial healing, and preserving nerve density. Single-cell RNA-seq uncovered substantial transcriptional heterogeneity among circulating neutrophils and their rapid reprogramming within 24 hours after injury. Specifically, Il1r2 was highly enriched in these early injury-responsive neutrophils. Functional validation demonstrated that adoptive transfer of IL-1R2+ neutrophils markedly attenuated inflammation and accelerated epithelial and nerve repair, restoring tissue integrity, whereas IL-1R2– neutrophils exacerbated inflammatory responses. Together, these findings identify IL-1R2+ neutrophils as an early protective neutrophil subset expanded by sterile injury that restrains excessive inflammation and preserves tissue homeostasis, providing mechanistic insight into injury-induced neutrophil reprogramming and highlighting a potential therapeutic target for enhancing tissue repair.
Authors
Hyun Ju Lee, Jung Hwa Ko, Joo Youn Oh
×Abstract
Acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) limits their therapeutic benefits in patients with high-grade serous ovarian cancer (HGSC). Identifying therapeutic vulnerabilities that function across distinct forms of acquired PARPi resistance is an urgent need. In BRCA2-deficient PARPi-naive cells, targeting RAD52 can be synthetic lethal with a PARPi, making RAD52 an attractive therapeutic target. However, this approach has not been tested in HGSC models of acquired PARPi resistance. Here, we assessed the effects of RAD52 loss in 2 distinct Brca2-deficient mouse models of HGSC with acquired PARPi resistance. RAD52 loss suppressed homologous recombination and single-strand annealing DNA repair activity in both models. Genetic depletion and pharmacologic inhibition of RAD52 restored PARPi sensitivity both in vitro and in vivo. Furthermore, RAD52 expression was elevated in a newly derived human model of BRCA2-deficient HGSC with acquired PARPi resistance. In this cell line, RAD52 inhibition synergized with a PARPi. Finally, elevated RAD52 expression was associated with poor survival in patients with HGSC. Collectively, these findings establish RAD52 as a clinically relevant therapeutic vulnerability in multiple models of HGSC with acquired PARPi resistance.
Authors
Yukihide Ota, Maia L. Elizagaray, Vijayalaxmi Gupta, Bisiayo E. Fashemi, Moreniola Akande, Preedia Babu, Prasanth Kumar Thuthika, Elena Lomonosova, Joshua Brill, Lulu Sun, Brooke Sanders, Lindsay M. Kuroki, Carolyn K. McCourt, Andrea R. Hagemann, Ian S. Hagemann, Premal H. Thaker, David G. Mutch, Matthew A. Powell, Krzysztof Hyrc, Priyanka Verma, John Krais, Benjamin G. Bitler, Mary M. Mullen, Dineo Khabele
×Abstract
The clinical success of chimeric antigen receptor T cells in treating hematological malignancies has highlighted the immense therapeutic potential of engineering T cells with synthetic receptors. However, the limited availability of tumor-specific antigens and the poor ability of T cells to infiltrate solid tumor cores remain major obstacles for applying this approach to solid tumors. In this study, we identify a subset of CD8+ T cells at inflammatory sites that acquire FcγRI through trogocytosis from infiltrating monocytes, enabling them to perform transient TCR-independent antibody-dependent cellular cytotoxicity (ADCC). Analysis of FcγRI signaling in these T cells revealed that phosphorylation occurs only when antigen density on the membrane exceeds 30 molecules/μm². Exploiting this threshold, we engineered peripheral blood CD8+ T cells to express a modified FcγRI receptor, allowing them to selectively kill tumor cells while largely sparing healthy tissues expressing normal physiological levels of the same antigen. Moreover, expression of FcγRI receptor in T cells was associated with reduced basement membrane interactions, enhancing their capacity to infiltrate tumors and establish long-term immune memory. Collectively, this work establishes T cell–mediated ADCC as a potent therapeutic strategy that leverages antigen density, rather than antigen identity, to achieve selective cancer targeting.
Authors
Alon Richter, Shahar Dotan, Petr Shendrik, Diana Rasoulouniriana, Amichai Zarfin, Nadine Santana-Magal, Aseel Khateeb, Adi Frisch, Gal Verbin, Ariel Galor, Eran Sadot, Oran Zlotnik, Avishai Maliah, Ori Moskowitz, Tom Ben-Dov, Maya Halpern, Chen Luxenburg, Alexander Leithner, Yu Zhang, Natalia T. Freund, Merav Cohen, Carmit Levy, Matthew H. Spitzer, Raya Sorkin, Michael L. Dustin, Peleg Rider, Yaron Carmi
×Abstract
Defective feedback inhibition of serine palmitoyltransferase (SPT) caused by pathogenic SPTSSA variants underlies childhood-onset complex hereditary spastic paraplegia, yet the developmental timing and therapeutic reversibility of sphingolipid dysregulation remain unclear. We generated a knock-in mouse carrying the disease-associated SptssaT51I variant and show that heterozygous animals exhibit preserved intrinsic SPT activity but impaired ORMDL-mediated regulation, leading to sustained elevation of bioactive sphingolipid intermediates that peak during postnatal myelination. Although gross myelin formation was initially maintained, excess sphingolipid flux rendered oligodendrocytes and neurons selectively vulnerable. Dietary L-serine, which augments SPT substrate availability, amplified sphingolipid accumulation in mutant but not WT mice, unmasking progressive spasticity, axonal injury, myelin ultrastructural defects, and — when administered during early postnatal development — severe pulmonary pathology likely responsible for lethality. Pharmacologic SPT inhibition with myriocin normalized sphingolipid synthesis, prevented serine-induced lethality and reversed neurological and metabolic abnormalities. Translating these findings, treatment of a child with SPTSSA-T51I–associated complex hereditary spastic paraplegia using the FDA-approved SPT inhibitor D-cycloserine resulted in sustained improvement in spasticity, reduced baclofen requirement, and decreased plasma levels of neurofilament light chain (NFL). These data define dysregulated sphingolipid biosynthesis as a developmentally and metabolically sensitive driver of neurodegeneration and suggest SPT inhibition as a mechanistically grounded therapeutic strategy.
Authors
Yi Gong, Robert Thompson, Ashley M. Glover, Kenneth Gable, Sita D. Gupta, Natalie Golovanov, Julie Tassinari, Nathan Casey, Brian D. Wishart, Elise L. Townsend, April Qian, Martin Selig, Armen Yerevanian, Teresa M. Dunn, Florian Eichler
×Abstract
Cellular senescence, a hallmark of aging, is accompanied by a striking expansion of the cell’s lysosomal compartment, yet the pathway leading to increased lysosomal content remains unresolved. Here, we demonstrate that the master lysosomal transcriptional regulator TFEB is activated during senescence through a pathway that involves conjugation of ATG8 onto single membranes (CASM). Using various senescence inducers, we show that senescence induction triggers robust TFEB nuclear translocation and upregulation of TFEB target genes. In contrast, inhibition of the core CASM machinery by CRISPR-mediated deletion of either ATG16L1, ATG12, or ATG5 markedly reduced senescence-induced TFEB nuclear translocation. In ATG16L1-KO cells, reconstitution with WT ATG16L1 but not the CASM-defective ATG16L1F467A mutant restored both ATG8 lipidation on the lysosomal surface and TFEB activation. Moreover, expression of the Salmonella effector protein SopF, a previously described specific CASM inhibitor, similarly blocked senescence-induced TFEB translocation. Functionally, upon stress, CASM-deficient cells exhibited a pronounced shift away from senescence and toward a cell death fate, a phenotype reversed by expressing a constitutively active form of TFEB. Together, these findings identify CASM-dependent TFEB activation as an essential mechanism enabling senescence-associated lysosomal expansion and suggest that lysosomal capacity modulates cell fate decisions in the aftermath of acute stress.
Authors
Yu-Wei Cheng, Michael Z. Gao, Jay Xiaojun Tan, Jie Liu, Toren Finkel
×Abstract
Early antiretroviral therapy (ART) limits viral reservoir establishment in infants but also constrains virus-specific immunity required to clear reactivated cells. In the early ART context, we evaluated whether combining adeno-associated virus serotype 9–vectored (AAV9-vectored) eCD4-IgG1 with pharmacological latency reversal using the second mitochondria-derived activator of caspases (SMAC) mimetic AZD5582 could promote reservoir reduction and control in SIV-infected infant macaques. AAV9 delivery achieved sustained eCD4-IgG1 expression, and AZD5582 induced on-ART viremia, but the combination did not reduce intact SIV proviral DNA relative to controls. Partial postintervention control of viremia during analytical treatment interruption (ATI) occurred in 2 of 6 infants receiving AAV9-eCD4-IgG1 plus AZD5582 with restricted reservoir expansion during recrudescence at week 13. Intact reservoir size during ATI correlated inversely with on-ART viremia during AZD5582 treatment. Animals were classified as controllers or non-controllers. Controllers did not show increased eCD4-IgG1 expression nor increased on-ART viremia during AZD5582 treatment but harbored less pre-ART intact SIV DNA in PBMCs than non-controllers. Controllers also exhibited higher frequencies and greater polyfunctionality of virus-specific CD8+ T cells prior to ATI. These findings suggest immune control of reservoir size and post-ART viral dynamics in early-treated infants and that AAV9-eCD4-IgG1 plus AZD5582 increases the likelihood of post-ART viral control.
Authors
Jairo A. Fonseca, Alexis C. King, Kaleaha S. Davis, Daniel O’Hagan, Adrian Khoei, Lucas Alves Britto Da Costa, Camryn Cockerham, Mackenzie Cottrell, Stephanie Ehnert, Jennifer S. Wood, Michael D. Alpert, Matthew R. Gardner, Jeffrey D. Lifson, Maud Mavigner, Mauricio A. Martins, Ann Chahroudi
×Abstract
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is the leading infectious cause of death globally. Despite wide use of antiretroviral therapy (ART) by people living with HIV, the risk of TB remains increased. To understand immune interactions within lung granulomas, we compared spatial transcriptomics of Mtb and simian immunodeficiency virus (SIV) in co-infected macaques with or without ART as a model for HIV/Mtb co-infection. Spatially differentiated transcriptional profiles were observed in Mtb-only granulomas, with myeloid cells enriched in metabolic/antimicrobial pathways within the inner ring and T cell costimulatory/activation pathways enriched in the outer ring. These spatially distinct patterns were lost in SIV/Mtb granulomas, with higher enrichment in type I IFN pathways compared with Mtb-only granulomas. SIV/ART/Mtb granulomas had an intermediate transcriptional pattern without restoration to Mtb-only granulomas, despite a lack of viral replication. Cell-cell communication was reduced among SIV/Mtb co-infected groups. These data suggested that HIV disrupted spatially organized immune functions of granulomas, which were not fully restored by ART.
Authors
Jessica M. Medrano, Persis Sunny, Collin R. Diedrich, Pauline Maiello, Christopher Kline, Tara Rutledge, Edwin Klein, Joshua T. Mattila, Jishnu Das, Zandrea Ambrose, Philana Ling Lin
×Abstract
Roberts syndrome is caused by biallelic ESCO2 pathogenic variants, but the mechanisms linking ESCO2 loss to embryonic endothelial failure remain poorly defined. Endothelial cells must maintain identity and resist endothelial-mesenchymal transition (EndMT), yet conventional transcriptomics measures gene abundance rather than isoform composition. Here, using endothelial-specific Esco2 deletion in a mouse model relevant to Roberts syndrome, we show that Esco2 loss causes yolk sac vascular remodeling failure, pathological EndMT, diversion from hemogenic endothelial fate, hemogenic endothelial depletion, impaired hematopoietic output, and embryonic lethality. Short-read single-cell RNA-seq revealed broad endothelial reprogramming, whereas single-cell long-read isoform sequencing uncovered a hidden layer of isoform dysregulation that conventional gene-level analysis failed to resolve. Esco2-deficient endothelium exhibited transcriptome-wide isoform switching, disruption of a multi-tier splicing-factor network, and predicted broad loss or rewiring of protein-coding output. Chromatin occupancy profiling localized Esco2 to exonic regions near alternatively spliced loci, supporting a cotranscriptional route to spliceopathy. Whole-genome sequencing showed that local DNA variation was insufficient to explain these changes, supporting a predominantly posttranscriptional mechanism. These findings identify isoform control as a disease-relevant layer of endothelial identity maintenance in Roberts syndrome.
Authors
Roy Jung, Sherin Saheera, Chinmay M. Trivedi
×Abstract
Sex-specific mechanisms in chronic obstructive pulmonary disease (COPD) remain poorly defined. Sphingolipids are bioactive mediators that regulate airway epithelial integrity, inflammation, and bronchial smooth muscle tone, and their dysregulation contributes to airway hyperresponsiveness. We tested whether sphingolipid metabolism is regulated by sex in COPD. Lung tissue and paired plasma from female and male never-smoking controls, ever-smoking controls, and individuals with COPD were analyzed for sphingolipid enzyme expression and metabolite levels. Bronchial rings from mice and human bronchial smooth muscle cells (HBSMCs) were exposed to estradiol or cigarette smoke extract (CSE) with and without ceramidase or sphingosine kinase inhibitors. Expression of sphingolipid regulators ASAH1 and ORMDL3 was increased in COPD airways, significantly higher in women, and correlated with airway smooth muscle remodeling. Plasma from women with COPD showed increased sphingosine-1-phosphate (S1P), whereas men exhibited higher ceramide levels. Female mice displayed greater CSE-induced bronchial reactivity, attenuated by sphingosine kinase inhibition. In male donor HBSMCs, estradiol increased ASAH1, ORMDL3, S1P, and smooth muscle protein expression and enhanced contractility. CSE further augmented molecular responses in estradiol-treated cells but reduced collagen gel contraction, whereas ceramidase inhibition attenuated estradiol-dependent effects. These findings suggest that estrogen-dependent alterations in sphingolipid metabolism contribute to airway hyperreactivity and remodeling in women with COPD.
Authors
Elisabetta Granato, Xiaoyun Wang, Yun Zhang, Joselyn Rojas-Quintero, Ida Cerqua, Scott A. Ochsner, Jeff Thomas Kue, Luca Cecchetto, Maor Sauler, Farrah Kheradmand, Joshua Malo, Fiorentina Roviezzo, Irina Petrache, Francesca Polverino
×Abstract
BACKGROUND This prospective, single-arm, multicenter phase I/II trial evaluated vorinostat added to standard graft-versus-host disease (GVHD) prophylaxis in pediatric, adolescent, and young adult (AYA) patients undergoing allogeneic hematopoietic cell transplantation (HCT) from HLA-matched related, HLA-matched unrelated, and haploidentical donors.METHODS Patients aged 3–39 years received twice-daily vorinostat with tacrolimus/methotrexate after HLA-matched HCT from day –10 to +30, or with posttransplant cyclophosphamide/tacrolimus/mycophenolate mofetil after haploidentical HCT from day +5 to +30. The primary endpoint was cumulative incidence of grade 2–4 acute GVHD by day +100. All outcomes were based on intention-to-treat analysis.RESULTS Forty-three patients were enrolled; median age was 19 years, with 74% receiving HLA-matched and 26% haploidentical HCT. The recommended phase II dose was 60 mg/m2 twice daily. No dose-limiting toxicities, unexpected vorinostat-related serious adverse events, or primary graft failures occurred. Median neutrophil and platelet recovery occurred at 14 and 18 days, respectively. Day +100 grade 2–4 and 3–4 acute GVHD were 14% (95% CI, 5.6%–26%) and 4.7% (95% CI, 0.83%–14%), respectively. One-year overall survival was 88.4% (95% CI, 79.3%–98.5%), relapse 14% (95% CI, 5.6%–26%), nonrelapse mortality 4.7% (95% CI, 0.8%–14%), and GVHD-free/relapse-free survival 55.8% (95% CI, 42.8%–72.8%). Correlative studies demonstrated on-target HDAC activity, with increased histone.RESULTS Forty-three patients were enrolled; median age was 19 years, with 74% receiving HLA-matched and 26% haploidentical HCT. The recommended phase II dose was 60 mg/m2 twice daily. No dose-limiting toxicities, unexpected vorinostat-related serious adverse events, or primary graft failures occurred. Median neutrophil and platelet recovery occurred at 14 and 18 acetylation and lower proinflammatory cytokines.CONCLUSION These findings support randomized evaluation of vorinostat-based GVHD prophylaxis in pediatric and AYA HCT.TRIAL REGISTRY ClinicalTrials.gov NCT03842696.FUNDING NIH grants 1R01CA249211, UG1HL069330, 1K24HL156896, and NHLBI Training Grant T32HL007622-36.
Authors
Nicolas Parnell, Xiao Cao, Jan H. Beumer, Thomas M. Braun, Yilei Cui, Gary J. Fisher, Guoqing Hou, Julianne Holleran, Tracey Churay, Michelle Rozwadowski, Luna Heider, Mark T. Vander Lugt, Carrie L. Kitko, April L. Rahrig, Ed Peres, Kirsten M. Williams, Julie-An Talano, Vanessa A. Fabrizio, Ghada Abusin, Gregory A. Yanik, John Magenau, Mary Riwes, Marcus J. Geer, Sarah Anand, Monalisa Ghosh, Attaphol Pawarode, Kristen Votruba, Pavan Reddy, Sung Won Choi
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Abstract
TGF-β-activating integrins promote solid-organ fibrosis, suggesting their potential as disease markers. Chronic lung allograft dysfunction (CLAD), a progressive fibrotic complication that limits lung transplant survival, is driven by intragraft TGF-β activation. However, TGF-β-activating integrin expression patterns remain undefined in lung transplants. Single-cell RNA sequencing and proteomic analysis of tissue specimens from a mouse CLAD model and human explanted lungs with CLAD revealed high levels of the TGF-β-activating integrin αvβ6, primarily localized to fibrosis-associated Keratin 8+ alveolar transitional epithelial cells, which were greatly reduced in tolerant lungs. Molecular imaging with a newly optimized positron emission tomography radiotracer specific for αvβ6, [64Cu]Cu-DOTA-A20-K16R, showed significantly higher uptake in CLAD than in tolerant transplants. [64Cu]Cu-DOTA-A20-K16R activity was also reduced by experimental treatments that lowered αvβ6 expression and inhibited CLAD severity. Finally, [64Cu]Cu-DOTA-A20-K16R autoradiographic analysis of human explanted lungs with CLAD showed elevated activity that correlated with increased αvβ6 expression. Collectively, these findings illustrate the potential utility of αvβ6 molecular imaging for detecting CLAD pathogenesis.
Authors
Marlene Cano, Chandra S. Bathula, Fuyi Liao, Cristian Wieczorek Villas Boas, Yan Tao, Zhiyi Liu, Ricky Ebenezer, Victoria Davis, Allison Cannady, Dequan Zhou, Shunning Liang, Hanqiu Zhao, Derek E. Byers, Alexander S. Krupnick, Daniel Kreisel, Susan M. Lin, Allen Duong, Tereza Martinu, Zhiyu Dai, Buck E. Rogers, Andrew E. Gelman
Abstract
Inflammatory bowel diseases (IBD) including Crohn’s disease (CD) are characterized by impaired intestinal epithelial barrier (IEB) function. Alterations in the desmosomal cadherin desmoglein 2 (Dsg2) have been linked to loss of IEB and intestinal inflammation in IBD. We investigated the pathogenic impact of a cytoplasmatic-truncating mutation in the desmosomal cadherin desmoglein 2 gene (DSG2) in an IBD patient. Genetic analysis of a CD patient revealed a likely pathogenic DSG2 mutation leading to a truncated protein lacking part of the intracellular domain. Generation of an enterocyte-specific mouse model to recapitulate the human mutation resulted in a lethal intestinal barrier defect which was evident in homozygous but not in heterozygous mice. In vitro, barrier defects correlated with changes in Dsg2 mobility and upregulation of pore-forming tight junction protein claudin-2. Organoids derived from heterozygous Dsg2 truncated mice exhibited increased epithelial susceptibility to proinflammatory cytokines. Bulk RNAseq followed by gene ontology analysis in homozygous mice revealed a skewed IL17/IL23 response overlapping with transcriptional changes in patients with CD. Furthermore, 16S rRNA sequencing revealed alterations in microbiome composition in animals that carried the Dsg2 truncation. In summary, a patient-derived mutation in the DSG2 gene causing cytoplasmic truncation in homozygous state induced a lethal intestinal barrier breakdown in mice paralleled by altered gut microbiota and intestinal immune response.
Authors
Daniela Kugelmann, Alexander García-Ponce, Nadja Thielemann, Mariya Y. Radeva, Maresa Anders, Da Eun Kang, Christian Siadjeu, Natalie Burkard, Anja K.E. Horn, Jessica Neubauer, Matthias Hiermaier, Desalegn T. Egu, Christoph Schmitz, Oliver Kurzai, Katri S. Vuopala, Outi Kuismin, Nicolas Schlegel, Jens Waschke
Abstract
Essential thrombocythemia (ET) and myelofibrosis (MF) are myeloproliferative neoplasms (MPN) that can result from heterozygous or homozygous mutations in the calreticulin (CRT) gene (CALR). Mutant CRT binds to the thrombopoietin receptor to induce signaling that drives ET and MF. CRT is also a chaperone for the assembly of the highly polymorphic human leukocyte antigen (HLA) class I molecules. The effects of heterozygous versus homozygous MPN CALR mutations on the expression of HLA class I variants are unknown. To assess mutant CRT-induced changes to HLA class I expression, we generated CRISPR-based mutant CALR knock-in cell lines and CALR-knockout cell lines reconstituted with wild-type or MPN mutant CALR and also undertook measurements in MPN patient platelets and monocytes. Collectively, our findings indicate that heterozygous CALR mutations preserve HLA class I surface expression, whereas full loss of wild-type CRT results in marked, allele-dependent reductions in HLA class I expression, which are rescued by wild-type CRT but not MPN mutants. Reduced expression of CRT-dependent HLA class I in cells lacking a functional CRT induces natural killer (NK) cell activation. These studies identify specific HLA class I alleles and homozygous CALR mutations as prerequisites for low HLA class I expression in MPN.
Authors
Amanpreet Kaur, Grace Pagnucco, Fawziyah S. Khatri, Harini Desikan, Malathi Kandarpa, Matthew Cusick, Moshe Talpaz, Malini Raghavan
Abstract
Tuberous sclerosis complex (TSC) is an inherited tumor predisposition syndrome caused by pathogenic variants in TSC1 or TSC2, yet no animal model faithfully recapitulates the cardiac rhabdomyomas (CR) that are among its earliest manifestations. We generated a heterozygous TSC2-mutant Yucatan minipig carrying the recurrent pathogenic TSC2 p.Arg505* variant and characterized its cardiac phenotype using histopathology, immunohistochemistry, and RNA sequencing. TSC2-mutant minipigs consistently developed multiple CR that closely resembled human lesions, including enlarged glycogen-rich spider cells and activation of mechanistic target of rapamycin complex 1 (mTORC1) signaling. Comparative transcriptomic analyses demonstrated enrichment of a conserved TSC-associated gene expression program and activation of microphthalmia/transcription factor E (MITF/TFE)-regulated lysosomal pathways in both human and minipig tumors. Although other manifestations of TSC were not observed during the first year of life, this large-animal model faithfully reproduced the principal histopathologic and molecular features of human CR. These findings have established the TSC2-mutant Yucatan minipig as what we believe to be the first large-animal model of TSC-associated CR and provide a physiologically relevant platform for investigating tumor pathogenesis, mechanisms of spontaneous regression, and therapeutic strategies targeting mTORC1- and MITF/TFE-dependent signaling.
Authors
Elias Bou Farhat, Heng Du, Chrystalle Katte Carreon, Jad Halawi, Sophie Kudler, Magdalena Losko, Zachary T. Herbert, Adrienne L. Watson, Daniel Carlson, Dennis A. Webster, Derek M. Korpela, David M. Meredith, Elizabeth P. Henske, David J. Kwiatkowski
Abstract
Osteoarthritis (OA) is a heterogeneous disease in which synovial inflammation drives structural progression and pain, yet the molecular diversity of synovitis remains poorly defined. Here, we performed a multimodal characterization of synovial tissues from 48 dogs with naturally occurring OA, integrating histopathology, immunohistochemistry, transcriptomics, MRI, and soluble mediator profiling. This analysis identified three discrete synovial pathotypes, pauci-immune, diffuse myeloid, and lympho-myeloid, defined by distinct immune cell composition and transcriptional programs reflecting biologically distinct endotypes. The lympho-myeloid pathotype, characterized by ectopic lymphoid structures and adaptive immune enrichment, was independently associated with greater structural joint damage on MRI despite comparable clinical scores, demonstrating that tissue stratification captures heterogeneity invisible to clinical assessment alone. Composite soluble mediator signatures correlated with disease severity across pathotypes. Cross-species analyses integrating a large human synovial cohort revealed conserved pathotype architecture and shared gene networks, including tyrosine kinase signaling and B cell receptor activation, validating the translational relevance of the canine model. Together, these findings provide a framework for patient stratification and therapeutic target discovery in OA, with broad implications for precision medicine within a One Medicine perspective.
Authors
Anaëlle Drieu-Vincent, Julien De Lima, Floriane M. Etienne, Romain Guiho, Noémie Podevin, Richard Danger, Florence Lezin, Nicolas Gaigeard, Sophie Allain, Jérôme Abadie, Thomas Rousseau, Pierre Maitre, Olivier Gauthier, Jérôme Guicheux, Marion S. Fusellier, Marie-Astrid Boutet
