Palmitate impairs autophagic degradation via oxidative stress/perilysosomal Ca2+ overload/mTORC1 activation pathway in pancreatic ß cells
Nguyen et al. report that palmitate triggers ROS-induced Ca2+ overload and mTORC1 activation at the lysosomal membrane, resulting in autophagy defects. Restoring perilysosomal Ca2+ homeostasis offers protection against β cell lipotoxicity. The cover shows an electron micrograph of mouse pancreatic β cells.
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Research Letter
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Abstract
Authors
Carolina M. Larrain, Jack H. Victory, Priyanka P. Desai, Lindsay R. Friedman, Hannah Stepp, Rachel Ashe, Kirsten Remmert, Surajit Sinha, Emily C. Smith, Nicole Russell, Tracey Pu, Alyssa V. Eade, Justine F. Burke, Jason Ho, Michael B. Yaffe, David E. Kleiner, Keith Schmidt, William D. Figg, Jonathan M. Hernandez
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Research Articles
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Abstract
We recently showed that cell surface translocation of the endoplasmic reticulum–resident protein GRP78, when bound by activated α 2-macroglobulin (α2M*), induces pro-fibrotic responses in glomerular mesangial cells in response to high glucose and regulates activation of the pro-fibrotic cytokine transforming growth factor-β1 (TGF-β1), implicating a pathogenic role in glomerulosclerosis. Interstitial fibrosis, largely mediated by proximal tubular epithelial cells (PTEC) and renal fibroblasts, develops later in kidney disease and correlates with functional decline. Here we investigated whether interstitial fibrosis was mediated by cell surface GRP78 (csGRP78)/α2M*. High glucose and TGF-β1 increased csGRP78 and α2M* in PTEC and renal fibroblasts, and their inhibition prevented fibrotic protein production. Interestingly, for TGF-β1, this depended on inhibition of noncanonical signaling through YAP/TAZ, with Smad3 activation unaffected. In vivo, type 1 diabetic Akita mice overexpressing TGF-β1 were treated with either a neutralizing antibody for csGRP78 (C38) or α2M* (Fα2M) or an inhibitory peptide blocking csGRP78/α2M* interaction, and mice with unilateral ureteral obstruction were treated with Fα2M or inhibitory peptide. Consistently, inhibition by antibody or peptide attenuated fibrosis and pro-fibrotic signaling. These findings show an important role for csGRP78/α2M* in mediating tubulointerstitial fibrosis in both diabetic and nondiabetic kidney disease and support their inhibition as a potential antifibrotic therapeutic intervention.
Authors
Jackie Trink, Ifeanyi Kennedy Nmecha, Katrine Pilely, Renzhong Li, Zi Yang, Sydney Kwiecien, Melissa MacDonald, Bo Gao, Mariam A. Mamai, Chao Lu, Urooj F. Bajwa, Nikhil Uppal, James C. Fredenburgh, Masao Kakoki, Salvatore V. Pizzo, Anthony F. Rullo, Matthew B. Lanktree, Jeffrey I. Weitz, Yaseelan Palarasah, Joan C. Krepinsky
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The present study aimed to explore the role and possible underlying mechanisms of histone lactylation (Kla) modifications in diabetes-associated cognitive impairment (DACD). In this study, behavioral tests, H&E staining, and immunohistochemistry were used to evaluate cognitive function and the extent of cerebral tissue injury. We quantified the levels of lactic acid and pan-lysine Kla (Pan-Kla) in the brains of type 2 diabetes mellitus (T2DM) mice and in high glucose–treated microglia. We also identified all Kla sites in isolated microglia. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were subsequently conducted to identify the functions and pathways that were enriched at the differentially expressed modification sites. Cleavage under targets and tagmentation (CUT&Tag) technology was used to identify candidate genes that are regulated by histone H3 lactylated at Lys-18 (H3K18la). siRNA and H3K18R mutant sequences were used to knock down crucial components in key signaling pathways to assess the effects of histone Kla on microglial polarization. We found that lactic acid levels were significantly greater in the brains of T2DM mice and high glucose–treated microglia than in those of their corresponding controls, which increased the level of Pan-Kla. We discovered that lactate can directly stimulate an increase in H3K18la. The global landscape of the lactylome reveals information about modification sites, indicating a correlation between the upregulation of H3K18la and protein Kla and Toll-like receptor (TLR) signaling. CUT&Tag demonstrated that enhanced H3K18la directly stimulates the NF-κB signaling pathway by increasing binding to the promoter of TLR4, thereby promoting M1 microglial polarization. The present study demonstrated that enhanced H3K18la directly stimulates TLR4 signaling to promote M1 microglial polarization, thereby facilitating DACD phenotypes. Targeting such loop may be a potential therapeutic approach for the treatment of DACD.
Authors
Ying Yang, Fei Chen, Lulu Song, Liping Yu, Jinping Zhang, Bo Zhang
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CD4+ T cells predominate lymphocytic foci found in the salivary glands (SGs) of Sjögren’s disease (SjD) cases. Yet little is known about T cell receptor (TCR) repertoire features that distinguish cases from healthy controls (HCs), the relationship between SG and peripheral blood (PB) repertoires of cases, and antigens recognized by pathogenic T cell clones. We performed deep sequencing of bulk-sorted CD4+CD45RA– PB T cells from SjD cases and matched HCs, and single-cell TCR sequencing of the same T cell population from labial SG biopsies of these cases. We found that clonally expanded SG CD4+ T cells expressed complementarity-determining region 3 (CDR3) sequences that were also detected in multiple copies in the blood of the same individuals with SjD. SjD cases displayed a “private” and restricted PB TCR repertoire with reduced clonotype diversity. We identified SjD-associated TCR motifs with the same putative antigen specificity shared between SGs and PB of cases. Their abundances in PB correlated with reduced salivary flow, linking these T cells with pathogenic disease features. Finally, we discovered 2 Ro60 epitopes eliciting an HLA-restricted immune response from expanded SG T cell clones. The comprehensive characterization of SjD TCR repertoires enables the discovery of target antigens and therapeutic strategies.
Authors
Ananth Aditya Jupudi, Michelle L. Joachims, Christina Lawrence, Charmaine Lopez-Davis, Bhuwan Khatri, Astrid Rasmussen, Kiely Grundahl, R. Hal Scofield, Judith A. James, Joel M. Guthridge, Christopher J. Lessard, Linda F. Thompson, A. Darise Farris
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Pulmonary fibrosis (PF) is a pathology associated with interstitial lung diseases (ILDs), including idiopathic pulmonary fibrosis (IPF). Fibrosis promotes continual secretion of extracellular matrix (ECM), producing nonfunctional scar tissue and causing organ failure. This study investigated the tyrosine kinase receptor Ephrin type-B receptor 4 (EphB4) as a mediator of PF. To this end, we generated mice with conditional Col1a2-driven deletion of Ephb4 and used a preclinical mouse model of PF, total and single nuclei RNA (snRNA) sequencing, NanoString, previously published single-cell data, computational analysis, and functional assays of mouse and human healthy control and IPF lung fibroblasts. Col1a2-CreERT–driven Ephb4 deletion, or EphB4 inhibition via NVP-BHG712, markedly protected against bleomycin-induced PF. Total RNA-Seq of fibroblasts isolated from Ephb4-deficient fibrotic mouse lungs exhibited reduced expression of ECM, ER Cargo, and protein trafficking–related genes. NVP-BHG712 reduced expression of these identified genes in mouse lung fibroblasts under fibrotic conditions in vitro. snRNA-Seq of mouse lungs treated with NVP-BHG712 identified transcriptomic changes of ECM genes in specific fibroblast subpopulations. RNA-Seq, computational, and functional assays using mouse and human IPF fibroblasts identified elastin as a key mediator involved in EphB4 signaling. Combined, our data show that EphB4 is a crucial mediator of PF.
Authors
Brian Wu, Starlee S. Lively, Shabana Vohra, Noah Fine, Chiara Pastrello, Anca Maglaviceanu, Osvaldo Espin-Garcia, Evan Pollock-Tahiri, Sayaka Nakamura, Paramvir Kaur, Keemo Delos Santos, Jason S. Rockel, Pratibha Potla, Himanshi Gupta, Poulami Datta, Laura Tang, Jacob Kwon, Akihiro Nakamura, Matthew B. Buechler, Rajiv Gandhi, Jiangping Wu, Boris Hinz, Igor Jurisica, Mohit Kapoor
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Insulin/insulin growth factor signaling is a conserved pathway that regulates lifespan. However, long-lived loss-of-function mutants often produce insulin resistance, slow growth, and impair reproduction. Recently, a gain-of-function mutation in the kinase insert domain (KID) of the Drosophila insulin/IGF receptor was seen to dominantly extend lifespan without impairing insulin sensitivity, growth, or reproduction. This substitution occurs within residues conserved in mammalian insulin receptor (IR) and insulin growth factor-1 receptor (IGF-1R). We produced 2 knock-in mouse strains that carry the homologous KID Arg/Cys substitution in murine IR or IGF-1R, and we replicated these genotypes in human cells. Cells with heterodimer receptors of IR or IGF-1R induce receptor phosphorylation and phospho-Akt when stimulated with insulin or IGF. Heterodimer receptors of IR fully induce pERK, but ERK was less phosphorylated in cells with IGF-1R heterodimers. Adults with a single KID allele (producing heterodimer receptors) have normal growth and glucose regulation. At 4 months, these mice variably display hormonal markers that associate with successful aging counteraction, including elevated adiponectin and FGF21, as well as reduced leptin and IGF-1. Livers of IGF-1R females show decreased transcriptome-based biological age, which may point toward delayed aging and warrants an actual lifespan experiment. These data suggest that KID mutants may slow mammalian aging while they avoid the complications of insulin resistance.
Authors
Ulalume Hernández-Arciga, Jun Kyoung Kim, Jacob L. Fisher, Alexander Tyshkovskiy, Alibek Moldakozhayev, Catherine Hall, Souvik Ghosh, Yashvandhini Govindaraj, Ian J. Sipula, Jake Kastroll, Diana Cooke, Jinping Luo, Jonathan K. Alder, Stacey J. Sukoff Rizzo, Gene P. Ables, Eunhee Choi, Vadim N. Gladyshev, Michael J. Jurczak, Marc Tatar, Andrey A. Parkhitko
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Reproductive disorders can result from a defective action of the neuropeptide gonadotropin-releasing hormone (GnRH), the master regulator of reproduction. We have previously shown that selenoprotein T (SELENOT), a newly described thioredoxin-like selenoprotein highly expressed in endocrine and neuroendocrine cells, plays a role in hormone secretion and neuroprotection. However, whether SELENOT is involved in neuroendocrine regulation in vivo is totally unknown. We found that SELENOT deficiency in the brain impaired sexual behavior, leading to a decline in fertility in both male and female mice. Biochemical and histological analyses of the gonadotrope axis of these mice revealed a higher expression of GnRH, which is associated with circulating luteinizing hormone (LH) excess, and elevated steroid hormones in males and a polycystic ovary syndrome–like phenotype in females. In addition, SELENOT deficiency impaired LH pulse secretion in both male and female mice. These changes were reverted after administration of a GnRH antagonist. Together, our data demonstrate for the first time to our knowledge the role of a selenoprotein in the central control of sexual behavior and reproduction, and identify a redox effector of GnRH neuron activity impacting both male and female reproductive function.
Authors
Ben Yamine Mallouki, Loubna Boukhzar, Ludovic Dumont, Azénor Abgrall, Marjorie Gras, Agathe Prieur, David Alexandre, David Godefroy, Yves Tillet, Nathalie Rives, Luca Grumolato, Fatiha Chigr, Youssef Anouar
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Saturated fatty acids impose lipotoxic stress on pancreatic β cells, leading to β cell failure and diabetes. In this study, we investigate the critical role of organellar Ca2+ disturbance on defective autophagy and β cell lipotoxicity. Palmitate, a saturated fatty acid, induced perilysosomal Ca2+ elevation, sustained mTOR complex 1 (mTORC1) activation on the lysosomal membrane, suppression of the lysosomal transient receptor potential mucolipin 1 (TRPML1) channel, and accumulation of undigested autophagosomes in β cells. These Ca2+ aberrations with autophagy defects by palmitate were prevented by an mTORC1 inhibitor or a mitochondrial superoxide scavenger. To alleviate perilysosomal Ca2+ overload, strategies such as lowering extracellular Ca2+, employing voltage-gated Ca2+ channel blocker or ATP-sensitive K+ channel opener, effectively abrogated mTORC1 activation and preserved autophagy. Furthermore, redirecting perilysosomal Ca2+ into the endoplasmic reticulum (ER), with an ER Ca2+ ATPase activator, restored TRPML1 activity, promoted autophagic flux, and improved survival of β cells exposed to palmitate-induced lipotoxicity. Our findings suggest oxidative stress/Ca2+ overload/mTORC1 pathway involvement in TRPML1 suppression and defective autophagy during β cell lipotoxicity. Restoring perilysosomal Ca2+ homeostasis emerges as a promising therapeutic strategy for metabolic diseases.
Authors
Ha Thu Nguyen, Luong Dai Ly, Thuy Thi Thanh Ngo, Soo Kyung Lee, Carlos Noriega Polo, Subo Lee, Taesic Lee, Seung-Kuy Cha, Xaviera Riani Yasasilka, Kae Won Cho, Myung-Shik Lee, Andreas Wiederkehr, Claes B. Wollheim, Kyu-Sang Park
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Cardiac arrhythmias increase during acute SARS-CoV-2 infection and in long COVID syndrome, by unknown mechanisms. This study explored the acute and long-term effects of COVID-19 on cardiac electrophysiology and the cardiac conduction system (CCS) in a hamster model. Electrocardiograms and subpleural pressures were recorded by telemetry for 4 weeks after SARS-CoV-2 infection, and interferon-stimulated gene expression and macrophage infiltration of the CCS were assessed at 4 days and 4 weeks postinfection. COVID-19 induced pronounced tachypnea and cardiac arrhythmias, including bradycardia and persistent atrioventricular block, though no viral protein expression was detected in the heart. Arrhythmias developed rapidly, partially reversed, and then redeveloped, indicating persistent CCS injury. COVID-19 induced cardiac cytokine expression, connexin mislocalization, and CCS macrophage remodeling. Interestingly, sterile innate immune activation by direct cardiac injection of polyinosinic:polycytidylic acid (PIC) induced arrhythmias similar to those of COVID-19. PIC strongly induced cytokine secretion and interferon signaling in hearts, human induced pluripotent stem cell–derived cardiomyocytes, and engineered heart tissues, accompanied by alterations in excitation-contraction coupling. Importantly, the pulmonary and cardiac effects of COVID-19 were blunted by JAK/STAT inhibition or a mitochondrially targeted antioxidant, indicating that SARS-CoV-2 infection indirectly leads to arrhythmias by innate immune activation and redox stress, which could have implications for long COVID syndrome.
Authors
Deepthi Ashok, Ting Liu, Misato Nakanishi-Koakutsu, Joseph Criscione, Meghana Prakash, Alexis Tensfeldt, Byunggik Kim, Bryan Ho, Julian Chow, Morgan Craney, Mark J. Ranek, Brian L. Lin, Kyriakos Papanicolaou, Agnieszka Sidor, D. Brian Foster, Hee Cheol Cho, Andrew Pekosz, Jason Villano, Deok-Ho Kim, Brian O’Rourke
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BACKGROUND Icotrokinra is the first and only targeted oral peptide that selectively binds the IL-23 receptor with high affinity to precisely inhibit IL-23 signaling. Icotrokinra demonstrated high rates of complete skin clearance and durable disease control in the phase IIb trial, FRONTIER-1, and its long-term extension, FRONTIER-2, in participants with moderate-to-severe plaque psoriasis. This study evaluated systemic and skin pharmacodynamic response of icotrokinra and its relationship to clinical response in FRONTIER participants.METHODS FRONTIER-1 participants received icotrokinra or placebo for 16 weeks. FRONTIER-2 followed participants for up to 1 year of treatment; placebo participants transitioned to icotrokinra after week 16. Systemic pharmacodynamic changes were assessed in serum through week 52. Skin pharmacodynamic changes were assessed using transcriptomic analysis of skin biopsies and protein quantification in tape-strip samples through week 16.RESULTS Icotrokinra dose-dependently reduced serum levels of the IL-23/IL-17 axis and psoriasis disease biomarkers through week 52, with maximal reductions observed with the highest 100 mg twice-daily dose. Proteomic analyses showed icotrokinra selectively blocked IL-23–driven inflammation without broader impacts on circulating proteins, including serum IL-23 levels. Sixteen weeks of icotrokinra, but not placebo, reduced expression of psoriasis-associated genes in lesional skin. Icotrokinra treatment also reduced psoriasis-relevant proteins in week 16 lesional skin tape-strips to levels comparable to nonlesional samples.CONCLUSION Icotrokinra induced a dose-dependent pharmacodynamic response, with early (week 4) and sustained (week 52) reductions in biomarkers of IL-23 pathway activation and psoriasis disease severity, which correlated with clinical response.TRIAL REGISTRATION ClinicalTrials.gov: NCT05223868, NCT05364554.FUNDING Johnson & Johnson.
Authors
David Strawn, James G. Krueger, Robert Bissonnette, Kilian Eyerich, Laura K. Ferris, Amy S. Paller, Andreas Pinter, Dylan Richards, Elizabeth Y. Chen, Kate Paget, Daniel Horowitz, Roohid Parast, Joshua J. Rusbuldt, Jocelyn Sendecki, Sunita Bhagat, Lynn P. Tomsho, Ching-Heng Chou, Marta E. Polak, Brice E. Keyes, Emily Bozenhardt, Yuan Xiong, Wangda Zhou, Cynthia DeKlotz, Paul Newbold, Dawn M. Waterworth, Megan Miller, Takayuki Ota, Ya-Wen Yang, Monica W.L. Leung, Lloyd S. Miller, Carolyn A. Cuff, Bradford McRae, Darren Ruane, Arun K. Kannan
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The immune mechanisms induced by the Bacillus Calmette-Guérin (BCG) vaccine, and the subset of which that mediate protection against tuberculosis (TB), remain poorly understood. This is further complicated by difficulties in verifying vaccine-induced protection in humans. Although research in animal models, namely mice and nonhuman primates (NHPs), has begun to close this knowledge gap, discrepancies in the relative importance of biological pathways across species limit the utility of animal model–derived biological insights in humans. To address these challenges, we applied a systems modeling framework, Translatable Components Regression (TransCompR), to identify human blood transcriptional variability that could predict Mycobacterium tuberculosis challenge outcomes in BCG-vaccinated NHPs. These protection-associated pathways included both innate and adaptive immune activation mechanisms, along with signaling via type I IFNs and antimycobacterial Th cytokines. We further partially validated the associations between these mechanisms and protection in humans using publicly available microarray data collected from BCG-vaccinated infants who either developed TB or remained healthy during 2 years of follow-up. Overall, our work demonstrates how species translation modeling can leverage animal studies to generate hypotheses about the mechanisms that underlie human infectious disease and vaccination outcomes, which may be difficult or impossible to ascertain using human data alone.
Authors
Kate Bridges, Denis Awany, Anele Gela, Temwa-Dango Mwambene, Sherry L. Kurtz, Richard E. Baker, Karen L. Elkins, Christopher M. Sassetti, Thomas J. Scriba, Douglas A. Lauffenburger
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X-linked myotubular myopathy (XLMTM) due to MTM1 mutations is a rare and often lethal congenital myopathy. Its downstream molecular and cellular mechanisms are currently incompletely understood. The most abundant protein in muscle, myosin, has been implicated in the pathophysiology of other congenital myopathies. Hence, in the present study, we aimed to define whether myosin is also dysfunctional in XLMTM and whether it, thus, may constitute a potential drug target. To this end, we used skeletal muscle tissue from patients and canine/mouse models; we performed Mant-ATP chase experiments coupled with x-ray diffraction analyses and LC/MS-based proteomics studies. In patients with XLMTM, we found that myosin molecules are structurally disordered and preferably adopt their ATP-consuming biochemical state. This phosphorylation-related (mal)adaptation was mirrored by a striking remodeling of the myofiber energetic proteome in XLMTM dogs. In line with these, we confirmed an accrued myosin ATP consumption in mice lacking MTM1. Hence, we treated these with a myosin ATPase inhibitor, mavacamten. After a 4-week treatment period, we observed a partial restoration of the myofiber proteome, especially proteins involved in cytoskeletal, sarcomeric, and energetic pathways. Altogether, our study highlights myosin inhibition as a potentially new drug mechanism for the complex XLMTM muscle phenotype.
Authors
Elise Gerlach Melhedegaard, Fanny Rostedt, Charlotte Gineste, Robert A.E. Seaborne, Hannah F. Dugdale, Vladimir Belhac, Edmar Zanoteli, Michael W. Lawlor, David L. Mack, Carina Wallgren-Pettersson, Anthony L. Hessel, Heinz Jungbluth, Jocelyn Laporte, Yoshihiko Saito, Ichizo Nishino, Julien Ochala, Jenni Laitila
×Abstract
Chronic liver injury results in activation of quiescent hepatic stellate cells (HSCs) into collagen type I–producing activated HSCs that make the liver fibrotic. We identified ETS1 and ETS2 (ETS1/2) as lineage-specific transcription factors regulating HSC phenotypes. Here, we investigated the role of ETS1/2 in HSCs in liver fibrosis using toxic liver injury models and 3D human liver spheroids. Liver fibrosis was induced in WT and HSC-specific Ets1-KO (Ets1ΔHSC) and Ets2-KO (Ets2ΔHSC) mice by administration of CCl4 for 6 weeks, followed by cessation of liver injury for 2 weeks. Liver fibrosis was more severe in Ets1ΔHSC and to a lesser extent Ets2ΔHSC mice compared with WT mice. Regression of liver fibrosis was suppressed only in Ets1ΔHSC mice, indicating Ets1 is the predominant isoform maintaining a quiescent-like phenotype in HSCs. Similar results were obtained in a metabolic dysfunction–associated steatohepatitis (MASH) model using 3D human liver spheroids. Knockdown of ETS1 in human HSCs caused upregulation of fibrogenic genes in MASH human liver spheroids and prevented fibrosis regression. ETS1 regulated the quiescent HSC phenotype via the CREB-regulated transcription coactivator 2 (CRTC2)/PGC1α/PPARγ pathway. Knockdown of CRTC2 abrogated PPARγ responses and facilitated HSC activation. These findings suggest that ETS1 may represent a therapeutic target for antifibrotic therapy.
Authors
Wonseok Lee, Xiao Liu, Sara Brin Rosenthal, Charlene Miciano, Sadatsugu Sakane, Kanani Hokutan, Debanjan Dhar, Hyun Young Kim, David A. Brenner, Tatiana Kisseleva
×Abstract
The tumor microenvironment plays a key role in cancer progression and therapy resistance, with cancer-associated fibroblasts (CAFs) contributing to desmoplasia, extracellular matrix (ECM) remodeling, and elevated interstitial fluid pressure, all of which hinder drug delivery. We investigated fibroblast activation protein–targeted (FAP-targeted) near-infrared photoimmunotherapy (NIR-PIT) as a strategy to improve drug penetration in CAF-rich tumors. In clinical esophageal cancer samples, FAP expression strongly correlated with increased collagen I, hyaluronic acid, and microvascular collapse. CAF-rich 3D spheroids demonstrated elevated ECM deposition and significantly impaired drug uptake compared with CAF-poor models. FAP-targeted NIR-PIT selectively reduced CAFs, reduced ECM components, and restored drug permeability. In vivo, FAP-targeted NIR-PIT enhanced the accumulation of panitumumab and Abraxane in CAF-rich tumors and improved antitumor efficacy when combined with chemotherapy. These findings highlight FAP-targeted NIR-PIT as a promising therapeutic approach to remodel the tumor stroma and overcome drug resistance in desmoplastic solid tumors.
Authors
Seitaro Nishimura, Kazuhiro Noma, Tasuku Matsumoto, Yasushige Takeda, Tatsuya Takahashi, Hijiri Matsumoto, Kento Kawasaki, Hotaka Kawai, Tomoyoshi Kunitomo, Masaaki Akai, Teruki Kobayashi, Noriyuki Nishiwaki, Hajime Kashima, Takuya Kato, Satoru Kikuchi, Shunsuke Tanabe, Toshiaki Ohara, Hiroshi Tazawa, Yasuhiro Shirakawa, Peter L. Choyke, Hisataka Kobayashi, Toshiyoshi Fujiwara
×Abstract
BACKGROUND Platelets are increasingly recognized as active participants in immune signaling and systemic inflammation. Upon activation, platelets form monocyte platelet aggregates (MPA) representing the crossroads of thrombosis and inflammation. We hypothesized that platelet transcriptomics could capture this thromboinflammatory axis and identify individuals at elevated cardiovascular risk.METHODS: MPA levels, defined as CD14+CD61+ cells, were measured using flow cytometry at 2 time points, 4 weeks apart, in healthy individuals Platelets were isolated and sequenced. Individuals were categorized as MPAhi or MPAlo based on consistently high or low MPA levels across time points.RESULTS Among 149 participants (median age 52 years, 57% female, 50% non-White), MPAhi individuals exhibited increased expression of platelet activation markers P-selectin (P < 0.001), PAC-1 (P = 0.021), and CD40L (P < 0.001) and enriched immune signaling pathways. Informed by MPA levels and derived from the platelet transcriptome, we developed a 42-gene thromboinflammation platelet signature (TIPS), which correlated with MPA levels in multiple cohorts and was reproducible over time. TIPS was elevated in patients with COVID-19 (P = 0.0002) and myocardial infarction (Padj = 0.008), and as in predicted future cardiovascular events in patients who underwent lower extremity revascularization after a median follow-up of 18 months (adjusted for age, sex, race, and ethnicity [adjHR] 1.55, P = 0.006). Notably, TIPS was modifiable by ticagrelor (P = 0.002) but not aspirin.CONCLUSION These findings establish MPA as a biomarker of thromboinflammation and introduce TIPS, a platelet RNA signature, that captures thromboinflammation and provides a promising tool for cardiovascular risk stratification and a potential therapeutic target.TRIAL REGISTRATION NCT04369664FUNDING NIH R35HL144993, NIH R01HL139909, and AHA 16SFRN2873002 to JSB, DFG Walter-Benjamin-Programme 537070747 to AB.
Authors
Antonia Beitzen-Heineke, Matthew A. Muller, Yuhe Xia, Elliot Luttrell-Williams, Florencia Schlamp, Deepak Voora, Kelly V. Ruggles, Michael S. Garshick, Tessa J. Barrett, Jeffrey S. Berger
×Abstract
Autoimmunity arises when self-reactive B and T cells target the body’s own tissues, with B cells contributing through antigen presentation as well as production of autoantibodies and proinflammatory cytokines. Genome wide association studies (GWAS) and recent identification of loss-of-function gene variants in individuals with young-onset autoimmunity have highlighted a role for protein tyrosine phosphatase nonreceptor type 2 (PTPN2) in development of autoimmunity. While prior studies have focused on the mechanism of Ptpn2 in T cells and other cell types, its function in B cells has not been explored. To test the B cell–intrinsic roles of Ptpn2, we generated a B cell–specific deletion of Ptpn2 in mice (Mb1-Cre;Ptpn2fl/fl). We found that loss of Ptpn2 in B cells promoted organ inflammation, increased the frequency of age/autoimmune-associated B cells (ABCs) and plasmablasts in the periphery, and increased circulating autoantibodies. Moreover, we found that Ptpn2 acted as a negative regulator of the JAK/STAT and TLR7 pathways in B cells. In line with this, treatment of B cells from Mb1-Cre;Ptpn2fl/fl mice with IFN-γ and TLR7 agonist lead to enhanced differentiation into ABCs. These findings highlight the critical roles of Ptpn2 in B cell function and its potential as a key regulator in preventing B cell associated autoimmunity.
Authors
Bridget N. Alexander, Soojin Kim, Kristen L. Wells, Maya J. Hunter, Kevin P. Toole, Scott M. Wemlinger, Daniel P. Regan, Andrew Getahun, Mia J. Smith
×Abstract
BACKGROUND Predictive biomarkers to guide chemotherapy decisions for metastatic castration–resistant prostate cancer (mCRPC) are lacking. Preclinical studies indicate that circulating tumor cell (CTC) studies of chromosomal instability (CTC-CIN) can predict taxane resistance.METHODS The CARD trial randomized individuals with mCRPC progressing within a year of treatment with an androgen receptor pathway inhibitor (ARPI; enzalutamide or abiraterone acetate plus prednisolone/prednisone) to cabazitaxel or the alternative ARPI. As a preplanned biomarker analysis, CTCs were isolated from blood samples obtained at baseline, cycle 2, and the end of treatment. Associations between baseline CTC and CTC-CIN counts with imaging-based progression-free survival (ibPFS), overall survival (OS), time to prostate-specific antigen (PSA) progression, RECIST 1.1 objective response rate (ORR), and PSA50 response rate were assessed. RESULTS High baseline CTC-CIN counts significantly associated with worse OS after adjustment for confounding variables (median OS, 15.3 vs. 8.9 months; univariate HR, 2.16; 95% CI, 1.52–3.06; P < 0.001; multivariate HR, 1.56; 95% CI, 1.01–2.43; P = 0.047). Detectable CTC-CIN counts at baseline may predict a lack of ibPFS and OS benefit when comparing cabazitaxel with ARPI. CONCLUSION This preplanned analysis of biomarker data from the CARD trial confirms that CTC-CIN counts are a clinically useful prognostic and predictive biomarker of taxane resistance in mCRPC. Detectable CTC-CIN at baseline defines a patient subpopulation with unmet clinical needs in which alternative therapeutics should be tested.TRIAL REGISTRATION ClinicalTrials.gov number NCT02485691.FUNDING Funded by Sanofi and Epic Sciences.
Authors
Ossian Longoria, Jan Rekowski, Santosh Gupta, Nick Beije, Klaus Pantel, Eleni Efstathiou, Cora Sternberg, Daniel Castellano, Karim Fizazi, Bertrand Tombal, Adam Sharp, Oliver Sartor, Sandrine Macé, Christine Geffriaud-Ricouard, Richard Wenstrup, Ronald de Wit, Johann de Bono
×Abstract
TLR7 agonists are promising immunostimulatory agents for the treatment of chronic infections and cancer. However, their systemic toxicity remains a challenge. In this study, SA-5, a potentially novel liver-targeted, orally available TLR7 agonist, was evaluated for pharmacokinetics, safety, and efficacy in young and aged macaques across 1–10 mg/kg repeated doses. Safety was evaluated through hematologic, biochemical, and flow cytometric profiling, while efficacy was assessed via IFN-α production, gene expression of IFN-stimulated genes, and plasmacytoid dendritic cell activation. A principal component analysis–based (PCA-based) composite scoring system was used to integrate multimodal parameters. SA-5 induced dose-dependent type I IFN with limited systemic inflammation, with 3 mg/kg showing optimal balance. SA-5 had comparable immunostimulatory activity to GS-9620 but with reduced adverse biomarker shifts. In aged macaques, efficacy was maintained with modestly increased safety responses. These findings support SA-5 as a safer next-generation TLR7 agonist effective across age groups, highlighting integrated biomarker profiling in preclinical immunomodulatory drug development.
Authors
Shokichi Takahama, Takahiro Tomiyama, Sachiyo Yoshio, Yuta Nagatsuka, Hirotomo Murakami, Takuto Nogimori, Mami Kochi, Shoko Ochiai, Hidenori Kimura, Akihisa Fukushima, Tatsuya Kanto, Takuya Yamamoto
×Abstract
The transcription factor IKAROS, encoded by IKZF1, is crucial for lymphocyte development and differentiation. Germline heterozygous IKZF1 mutations cause B cell immunodeficiency, but also affect T cells. Patients with IKZF1 haploinsufficiency (HI) or dimerization-defective (DD) variants show reduced naive and increased memory T cells, while dominant-negative (DN) mutations result in the opposite phenotype. Gain-of-function patients display variable patterns. To investigate IKAROS’s role in shaping the human naive/memory T cell phenotype, we performed IKAROS immunomodulation and knockdown experiments and analyzed early T cell development in an artificial thymic organoid (ATO) system using CD34+ cells from patients with representative IKZF1 variants. IKAROS inhibition by lenalidomide or silencing by small hairpin RNA directly altered expression of HNRNPLL, the master regulator of CD45 isoform splicing that defines CD45RA+/naive and CD45RO+/memory phenotypes. In the ATO system, IKAROS-DN precursor cells were blocked at the CD4–CD8–/double-negative stage and retained a CD45RA+ phenotype, whereas IKAROS-HI cells inefficiently reached the CD4+CD8+/double-positive stage and partially transitioned from CD45RA to CD45RO. Analysis of public gene expression data showed high HNRNPLL expression in double-positive thymic cells, beyond the stages affected by IKZF1 DN and HI mutations. Collectively, these findings indicate that IKAROS regulates early and late T cell development by mechanisms, including HNRNPLL modulation.
Authors
Jennifer Stoddard, Hye Sun Kuehn, Ravichandra Tagirasa, Marita Bosticardo, Francesca Pala, Julie E. Niemela, Agustin A. Gil Silva, Kayla Amini, Eduardo Anaya, Mario Framil Seoane, Carolina Bouso, Dimana Dimitrova, Jennifer A. Kanakry, Laia Alsina, Matias Oleastro, Steven M. Holland, Thomas A. Fleisher, Richard L. Wasserman, Luigi D. Notarangelo, Sergio D. Rosenzweig
×Abstract
Host factors influencing susceptibility to rhinovirus-induced asthma exacerbations remain poorly characterized. Using organotypic bronchial epithelial cultures from well-characterized children with asthma and healthy children, this study investigated viral load kinetics and resultant host responses by bulk and single-cell transcriptomics and targeted protein analyses. Bronchial epithelium from exacerbation-prone children exhibited greater rhinovirus replication and a cascade of exaggerated downstream interferon (IFN), inflammatory, epithelial stress, and remodeling responses. These transcriptional patterns were confirmed and further refined using single-cell transcriptomics, revealing cell type–specific contributions — particularly from non-ciliated cell populations including secretory immune response, tuft, and basal cells. We observed that these post-infection differences were associated with lower pre-infection IFN-stimulated gene (ISG) expression and protein levels of the ISG CXCL10. Prophylactic IFN-β treatment reduced viral replication and normalized downstream responses, supporting low baseline (pre-infection) IFN tone as a modifiable causal determinant of host susceptibility to adverse rhinovirus-induced responses in exacerbation-prone children with asthma.
Authors
Naresh Doni Jayavelu, Basilin Benson, Patricia C. dela Cruz, Weston T. Powell, Lucille M. Rich, Elizabeth R. Vanderwall, Camile R. Gates, Andrew J. Nagel, Maria P. White, Nyssa B. Samanas, Kourtnie Whitfield, Teal S. Hallstrand, Steven F. Ziegler, Matthew C. Altman, Jason S. Debley
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Abstract
This investigation leverages single-cell RNA sequencing (scRNA-Seq) to delineate the contributions of muscle-resident Schwann cells to neuromuscular junction (NMJ) remodeling by comparing a model of stable innervation with models of reinnervation following partial or complete denervation. The study discovered multiple distinct Schwann cell subtypes, including a novel terminal Schwann cell (tSC) subtype integral to the denervation-reinnervation cycle, identified by a transcriptomic signature indicative of cell migration and polarization. The data also characterizes three myelin Schwann cell subtypes, which are distinguished based on enrichment of genes associated with myelin production, mesenchymal differentiation or collagen synthesis. Importantly, SPP1 signaling emerges as a pivotal regulator of NMJ dynamics, promoting Schwann cell proliferation and muscle reinnervation across nerve injury models. These findings advance our understanding of NMJ maintenance and regeneration and underscore the therapeutic potential of targeting specific molecular pathways to treat neuromuscular and neurodegenerative disorders.
Authors
Steve D. Guzman, Ahmad Abu-Mahfouz, Carol S. Davis, Lloyd P. Ruiz, Peter C.D. Macpherson, Susan V. Brooks
Abstract
C-type natriuretic peptide (CNP) is known to promote chondrocyte proliferation and bone formation; however, CNP’s extremely short half-life necessitates continuous intravascular administration to achieve bone-lengthening effects. Vosoritide, a CNP analog designed for resistance to neutral endopeptidase, allows for once daily administration. Nonetheless, it distributes systemically rather than localizing to target tissues, which may result in adverse effects such as hypotension. To enhance local drug delivery and therapeutic efficacy, we developed a novel synthetic protein by fusing a collagen-binding domain (CBD) to CNP, termed CBD-CNP. This fusion protein exhibited stability under heat conditions and retained the collagen-binding ability and bioactivity as CNP. CBD-CNP localized to articular cartilage in fetal murine tibiae and promoted bone elongation. Spatial transcriptomic analysis revealed that the upregulation of chondromodulin expression may contribute to its therapeutic effects. Treatment of CBD-CNP mixed with collagen powder to a fracture site of a mouse model increased bone mineral content and bone volume rather than CNP-22. Intra-articular injection of CBD-CNP to a mouse model of knee osteoarthritis suppressed subchondral bone thickening. By addressing the limitations of CNP’s rapid degeneration, CBD-CNP leverages its collagen-binding capacity to achieve targeted, sustained delivery in collagen-rich tissues, offering a promising strategy for enhancing chondrogenesis and osteogenesis.
Authors
Kenta Hirai, Kenta Sawamura, Ryusaku Esaki, Ryusuke Sawada, Yuka Okusha, Eriko Aoyama, Hiroki Saito, Kentaro Uchida, Takehiko Mima, Satoshi Kubota, Hirokazu Tsukahara, Shiro Imagama, Masaki Matsushita, Osamu Matsushita, Yasuyuki Hosono
Abstract
Clonal haematopoiesis of indeterminate potential (CHIP) is the expansion of blood stem cells and progeny after somatic mutation. CHIP associates with increased cardiovascular disease (CVD) with inflammation from macrophages a proposed common effector. However, mouse CHIP studies are discordant for clonal expansion and inflammation. Similarly, directionality of association between CHIP and CVD remains debated. We investigated effects of three CHIP mutations on macrophage cytokines, clonal expansion and atherosclerosis in parallel. We find that Tet2 and Dnmt3a mutations increase cytokines and inflammasome activation in Tet2 but decrease in Dnmt3a. However, Jak2 mutant macrophages produced equivalent cytokine as wild-type. In mice, Tet2 mutants clonally expanded, but Dnmt3a and Jak2 mutants didn’t. Expansion was unaffected by systemic inflammation, while hyperlipidemia expanded Tet2-/- cells, but not mono-allelic mutants. Similarly, human Mendelian randomisation showed no effect of serum cytokines or CVD on CHIP risk. Experimental atherosclerosis was increased in females with Tet2 and males with Jak2, but unchanged with Dnmt3a mutations. Together, common CHIP mutations have disparate effects on macrophage cytokines and clonal expansion, and sex-dependent effects on atherogenesis, suggesting a common mechanism across CHIP is unlikely. Thus, CHIP mutations differ in pathophysiology and clinical sequalae across sexes and should be treated as different entities.
Authors
Paul R. Carter, Lauren Kitt, Amanda Rodgers, Nichola Figg, Ang Zhou, Chengrui Zhu, Ziyang Wang, Peter Libby, Stephen Burgess, George S. Vassiliou, Murray CH. Clarke
Abstract
Background: Sleep is increasingly recognized as essential to human health, yet the adverse health consequences of acute sleep deprivation are unknown. We hypothesized that acute sleep deprivation is associated with health outcomes and modulated by sleep-associated genotypes. Methods: LOESS smoothing was performed on sleep estimates from Fitbit users (N = 14,681) between June 1, 2016 and July 1, 2022. Dates when population minutes slept were less than the 90% confidence interval of the LOESS regression were named acute sleep deprivation events (ASDEs). Phenome-wide disease incidence among the AoU population (N = 287,012) in the 10 days post-ASDE was compared to a preceding reference period by McNemar test. Circadian rhythm and sleep duration-associated SNPs were screened to identify genotypes associated with shorter ASDE sleep duration. Influences of sleep and circadian genotype on post-ASDE influenza risk were modeled using binomial family generalized estimating equations. Results: We identified 32 ASDEs spanning major national events. A phenome-wide screen found increased risk of influenza (OR = 1.54 [1.40, 1.70], P-value = 1.00 x 10-18) following ASDEs. 56 SNPs were associated with decreased sleep duration on ASDEs. Higher quantiles of ASDE-related SNP genotype burden were associated with less ASDE sleep duration and a greater risk of influenza-associated healthcare visits. Conclusion: Major national events are associated with acute sleep deprivation and greater influenza risk which is amplified by sleep genotypes. These findings should inform public health vigilance surrounding major national events.
Authors
Neil J. Kelly, Rahul Chaudhary, Wadih El Khoury, Nishita Kalepalli, Jesse Wang, Priya Patel, Irene Chan, Haris Rahman, Aisha Saiyed, Anisha N. Shah, Colleen A. McClung, Satoshi Okawa, Mehdi Nouraie, Stephen Y. Chan
Abstract
Hofbauer cells (HBC) are fetal-derived macrophages located in the placenta that contribute to antimicrobial defense, angiogenesis, tissue remodeling, and metabolic processes within the chorionic villi. Although their roles in placental biology are increasingly recognized, the mechanisms that regulate HBC identity and function are not yet fully defined. This study aimed to define the core transcriptomic and epigenomic features of HBCs in term placentas and to examine their capacity for transcriptional responsiveness and phenotypic variation. Using chromatin accessibility profiling and bulk RNA sequencing, we found that HBCs exhibit a unique gene expression and chromatin accessibility profile compared to other fetal and adult macrophages. We identified a coordinated transcriptional network involving nuclear receptors NR4A1–3, the glucocorticoid receptor (GR), and RFX family members (RFX1, RFX2, RFX5) that appears to shape HBC identity, particularly through pathways linked to lipid metabolism and angiogenesis. Although exploratory in nature, in vitro stimulation studies showed that HBCs exhibited increased transcriptional activity in response to combined IL-4 and RSG treatment, including induction of the lipid transporter CD36. Mass cytometry analysis revealed surface markers indicative of both immature and mature macrophage states. Together, these results indicated that HBCs represent a distinct and diverse macrophage population with specialized and adaptable regulatory program in the human placenta.
Authors
Benjámin R. Baráth, Dóra Bojcsuk, Krisztian Bene, Noemí Caballero-Sánchez, Tímea Cseh, João CR. de Freitas, Petros Tzerpos, Marta Toth, Zhonghua Tang, Seth Guller, Zoárd Tibor Krasznai, Patrícia Neuperger, Gabor J. Szebeni, Gergely Nagy, Tamás Deli, Laszlo Nagy
