Research Article
Abstract
Although refrigerated storage slows the metabolism of volunteer donor RBCs, which is essential in transfusion medicine, cellular aging still occurs throughout this in vitro process. Storage-induced microerythrocytes (SMEs) are morphologically altered senescent RBCs that accumulate during storage and are cleared from circulation following transfusion. However, the molecular and cellular alterations that trigger clearance of this RBC subset remain to be identified. Using a staining protocol that sorts long-stored SMEs (i.e., CFSEhi) and morphologically normal RBCs (CFSElo), these in vitro aged cells were characterized. Metabolomics analysis identified depletion of energy, lipid-repair, and antioxidant metabolites in CFSEhi RBCs. By redox proteomics, irreversible protein oxidation primarily affected CFSEhi RBCs. By proteomics, 96 proteins, mostly in the proteostasis family, had relocated to CFSEhi RBC membranes. CFSEhi RBCs exhibited decreased proteasome activity and deformability; increased phosphatidylserine exposure, osmotic fragility, and endothelial cell adherence; and were cleared from the circulation during human spleen perfusion ex vivo. Conversely, molecular, cellular, and circulatory properties of long-stored CFSElo RBCs resembled those of short-stored RBCs. CFSEhi RBCs are morphologically and metabolically altered, have irreversibly oxidized and membrane-relocated proteins, and exhibit decreased proteasome activity. In vitro aging during storage selectively alters metabolism and proteostasis in these storage-induced senescent RBCs targeted for clearance.
Authors
Sandy Peltier, Mickaël Marin, Monika Dzieciatkowska, Michaël Dussiot, Micaela Kalani Roy, Johanna Bruce, Louise Leblanc, Youcef Hadjou, Sonia Georgeault, Aurélie Fricot, Camille Roussel, Daniel Stephenson, Madeleine Casimir, Abdoulaye Sissoko, François Paye, Safi Dokmak, Papa Alioune Ndour, Philippe Roingeard, Emilie-Fleur Gautier, Steven L. Spitalnik, Olivier Hermine, Pierre A. Buffet, Angelo D’Alessandro, Pascal Amireault
Abstract
CD4+FOXP3+ Treg cells maintain self tolerance, suppress the immune response to cancer, and protect against tissue injury during acute inflammation. Treg cells require mitochondrial metabolism to function, but how Treg cells adapt their metabolic programs to optimize their function during an immune response occurring in a metabolically stressed microenvironment remains unclear. Here, we tested whether Treg cells require the energy homeostasis–maintaining enzyme AMPK to adapt to metabolically aberrant microenvironments caused by malignancy or lung injury, finding that AMPK is dispensable for Treg cell immune-homeostatic function but is necessary for full Treg cell function in B16 melanoma tumors and during influenza virus pneumonia. AMPK-deficient Treg cells had lower mitochondrial mass and exhibited an impaired ability to maximize aerobic respiration. Mechanistically, we found that AMPK regulates DNA methyltransferase 1 to promote transcriptional programs associated with mitochondrial function in the tumor microenvironment. During viral pneumonia, we found that AMPK sustains metabolic homeostasis and mitochondrial activity. Induction of DNA hypomethylation was sufficient to rescue mitochondrial mass in AMPK-deficient Treg cells, linking AMPK function to mitochondrial metabolism via DNA methylation. These results define AMPK as a determinant of Treg cell adaptation to metabolic stress and offer potential therapeutic targets in cancer and tissue injury.
Authors
Manuel A. Torres Acosta, Jonathan K. Gurkan, Qianli Liu, Nurbek Mambetsariev, Carla Reyes Flores, Kathryn A. Helmin, Anthony M. Joudi, Luisa Morales-Nebreda, Kathleen Cheng, Hiam Abdala-Valencia, Samuel E. Weinberg, Benjamin D. Singer
Abstract
Biological targeting is crucial for effective cancer treatment with reduced toxicity but is limited by the availability of tumor surface markers. To overcome this, we developed a nanoparticle-based (NP-based), tumor-specific surface marker–independent (TRACER) targeting approach. Utilizing the unique biodistribution properties of NPs, we encapsulated Ac4ManNAz (Maz) to selectively label tumors with azide-reactive groups. Surprisingly, while NP-delivered Maz was cleared by the liver, it did not label macrophages, potentially reducing off-target effects. To exploit this tumor-specific labeling, we functionalized anti–4-1BB Abs with dibenzocyclooctyne to target azide-labeled tumor cells and activate the immune response. In syngeneic B16F10 melanoma and orthotopic 4T1 breast cancer models, TRACER enhanced the therapeutic efficacy of anti–4-1BB, increasing the median survival time. Immunofluorescence analyses revealed increased tumor infiltration of CD8+ T and NK cells with TRACER. Importantly, TRACER reduced the hepatotoxicity associated with anti–4-1BB, resulting in normal serum ALT and AST levels and decreased CD8+ T cell infiltration into the liver. Quantitative analysis confirmed a 4.5-fold higher tumor-to-liver ratio of anti–4-1BB accumulation with TRACER compared with conventional anti–4-1BB Abs. Our work provides a promising approach for developing targeted cancer therapies that circumvent limitations imposed by the paucity of tumor-specific markers, potentially improving efficacy and reducing off-target effects to overcome the liver toxicity associated with anti–4-1BB.
Authors
Hyesun Hyun, Bo Sun, Mostafa Yazdimamaghani, Albert Wielgus, Yue Wang, Stephanie Ann Montgomery, Tian Zhang, Jianjun Cheng, Jonathan S. Serody, Andrew Z. Wang
Abstract
Osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) has been recognized as the principal mechanism underlying vascular calcification (VC). Runt-related transcription factor 2 (RUNX2) in VSMCs plays a pivotal role because it constitutes an osteogenic transcription factor essential for bone formation. As a key DNA demethylation enzyme, ten-eleven translocation 2 (TET2) is crucial in maintaining the VSMC phenotype. However, whether TET2 involves in VC progression remains elusive. Here we identified a substantial downregulation of TET2 in calcified human and mouse arteries, as well as human primary VSMCs. In vitro gain- and loss-of-function experiments demonstrated that TET2 regulated VC. Subsequently, in vivo knockdown of TET2 significantly exacerbated VC in both vitamin D3– and adenine diet–induced chronic kidney disease (CKD) mouse models. Mechanistically, TET2 bound to and suppressed activity of the P2 promoter within the RUNX2 gene; however, an enzymatic loss-of-function mutation of TET2 did not change its binding and suppressive effects. Furthermore, TET2 formed a complex with histone deacetylases 1/2 (HDAC1/2) to deacetylate H3K27ac on the P2 promoter, thereby inhibiting its transcription. Moreover, SNIP1 was indispensable for TET2 to interact with HDAC1/2 to exert an inhibitory effect on VC, and knockdown of SNIP1 accelerated VC in mice. Collectively, our findings imply that TET2 might serve as a potential therapeutic target for VC.
Authors
Dayu He, Jianshuai Ma, Ziting Zhou, Yanli Qi, Yaxin Lian, Feng Wang, Huiyong Yin, Huanji Zhang, Tingting Zhang, Hui Huang
Abstract
Postoperative atrial fibrillation (poAF) is AF occurring days after surgery, with a prevalence of 33% among patients undergoing open-heart surgery. The degree of postoperative inflammation correlates with poAF risk, but less is known about the cellular and molecular mechanisms driving postoperative atrial arrhythmogenesis. We performed single-cell RNA-seq comparing atrial nonmyocytes from mice with and without poAF, which revealed infiltrating CCR2+ macrophages to be the most altered cell type. Pseudotime trajectory analyses identified Il-6 as a gene of interest driving in macrophages, which we confirmed in pericardial fluid collected from human patients after cardiac surgery. Indeed, macrophage depletion and macrophage-specific Il6ra conditional knockout (cKO) prevented poAF in mice. Downstream STAT3 inhibition with TTI-101 and cardiomyocyte-specific Stat3 cKO rescued poAF, indicating a proarrhythmogenic role of STAT3 in poAF development. Confocal imaging in isolated atrial cardiomyocytes (ACMs) uncovered what we believe to be a novel link between STAT3 and CaMKII-mediated ryanodine receptor–2 (RyR2)-Ser(S)2814 phosphorylation. Indeed, nonphosphorylatable RyR2S2814A mice were protected from poAF, and CaMKII inhibition prevented arrhythmogenic Ca2+ mishandling in ACMs from mice with poAF. Altogether, we provide multiomic, biochemical, and functional evidence from mice and humans that IL-6-STAT3-CaMKII signaling driven by infiltrating atrial macrophages is a pivotal driver of poAF, which portends therapeutic utility for poAF prevention.
Authors
Joshua A. Keefe, Yuriana Aguilar-Sanchez, J. Alberto Navarro-Garcia, Isabelle Ong, Luge Li, Amelie Paasche, Issam Abu-Taha, Marcel A. Tekook, Florian Bruns, Shuai Zhao, Markus Kamler, Ying H. Shen, Mihail G. Chelu, Na Li, Dobromir Dobrev, Xander H.T. Wehrens
Abstract
Aortic aneurysms are potentially fatal focal enlargements of the aortic lumen; the disease burden is increasing as the human population ages. Pathological oxidative stress is implicated in the development of aortic aneurysms. We pursued a chemogenetic approach to create an animal model of aortic aneurysm formation using a transgenic mouse line, DAAO-TGTie2, that expresses yeast d-amino acid oxidase (DAAO) under control of the endothelial Tie2 promoter. In DAAO-TGTie2 mice, DAAO generated the ROS hydrogen peroxide (H2O2) in endothelial cells only when provided with d-amino acids. When DAAO-TGTie2 mice were chronically fed d-alanine, the animals became hypertensive and developed abdominal, but not thoracic, aortic aneurysms. Generation of H2O2 in the endothelium led to oxidative stress throughout the vascular wall. Proteomics analyses indicated that the oxidant-modulated protein kinase JNK1 was dephosphorylated by the phosphoprotein phosphatase DUSP3 (dual specificity phosphatase 3) in abdominal, but not thoracic, aorta, causing activation of Kruppel-like Factor 4 (KLF4)-dependent transcriptional pathways that triggered phenotypic switching and aneurysm formation. Pharmacological DUSP3 inhibition completely blocked the aneurysm formation caused by chemogenetic oxidative stress. These studies establish that regional differences in oxidant-modulated signaling pathways lead to differential disease progression in discrete vascular beds and identify DUSP3 as a potential pharmacological target for the treatment of aortic aneurysms.
Authors
Apabrita Ayan Das, Markus Waldeck-Weiermair, Shambhu Yadav, Fotios Spyropoulos, Arvind Pandey, Tanoy Dutta, Taylor A. Covington, Thomas Michel
Abstract
Clostridioides difficile infection (CDI) recurs in 1 of 5 patients. Monoclonal antibodies targeting the virulence factor TcdB reduce disease recurrence, suggesting that an inadequate anti-TcdB response to CDI leads to recurrence. In patients with CDI, we discovered that IL-33 measured at diagnosis predicts future recurrence, leading us to test the role of IL-33 signaling in the induction of humoral immunity during CDI. Using a mouse recurrence model, IL-33 was demonstrated to be integral for anti-TcdB antibody production. IL-33 acted via ST2+ ILC2 cells, facilitating germinal center T follicular helper (GC-Tfh) cell generation of antibodies. IL-33 protection from reinfection was antibody-dependent, as μMT KO mice and mice treated with anti-CD20 mAb were not protected. These findings demonstrate the critical role of IL-33 in generating humoral immunity to prevent recurrent CDI.
Authors
Farha Naz, Md Jashim Uddin, Nicholas Hagspiel, Mary K. Young, David Tyus, Rachel Boone, Audrey C. Brown, Girija Ramakrishnan, Isaura Rigo, Claire Fleming, Gregory R. Madden, William A. Petri Jr.
Abstract
Phosphorylation of Smad3 is a critical mediator of TGF-β signaling, which plays an important role in regulating innate immune responses. However, whether Smad3 activation can be regulated in innate immune cells in TGF-β–independent contexts remains poorly understood. Here, we show that Smad3 is activated through the phosphorylation of its C-terminal residues (pSmad3C) in murine and human macrophages in response to bacterial and viral ligands, and this activation is mediated by activin A in a TGF-β–independent manner. Specifically, infectious ligands, such as LPS, induced secretion of activin A through the transcription factor STAT5 in macrophages, and activin A signaling in turn activated pSmad3C. This activin A/Smad3 axis controlled mitochondrial ATP production and ATP conversion into adenosine by CD73 in macrophages, enforcing an antiinflammatory mechanism. Consequently, mice with a deletion of activin A receptor 1b specifically in macrophages (Acvr1bfl/fl-Lyz2cre) succumbed more to sepsis as a result of uncontrolled inflammation and exhibited exacerbated skin disease in a mouse model of imiquimod-induced psoriasis. Thus, we have revealed a previously unrecognized natural brake to inflammation in macrophages that occurs through the activation of Smad3 in an activin A–dependent manner.
Authors
Thierry Gauthier, Yun-Ji Lim, Wenwen Jin, Na Liu, Liliana C. Patiño, Weiwei Chen, James Warren, Daniel Martin, Robert J. Morell, Gabriela Dveksler, Gloria H. Su, WanJun Chen
Abstract
Fibroblastic reticular cells (FRCs) are the master regulators of the lymph node (LN) microenvironment. However, the role of specific FRC subsets in controlling alloimmune responses remains to be studied. Single-cell RNA sequencing (scRNA-Seq) of naive and draining LNs (DLNs) of heart-transplanted mice and human LNs revealed a specific subset of CXCL12hi FRCs that expressed high levels of lymphotoxin-β receptor (LTβR) and are enriched in the expression of immunoregulatory genes. CXCL12hi FRCs had high expression of CCL19, CCL21, indoleamine 2,3-dioxygenase (IDO), IL-10, and TGF-β1. Adoptive transfer of ex vivo–expanded FRCs resulted in their homing to LNs and induced immunosuppressive environments in DLNs to promote heart allograft acceptance. Genetic deletion of LTβR and Cxcl12 in FRCs increased alloreactivity, abrogating the effect of costimulatory blockade in prolonging heart allograft survival. As compared with WT recipients, CXCL12+ FRC–deficient recipients exhibited increased differentiation of CD4+ T cells into Th1 cells. Nano delivery of CXCL12 to DLNs improved allograft survival in heart-transplanted mice. Our study highlights the importance of DLN CXCL12hi FRCs in promoting transplant tolerance.
Authors
Yuta Yamamura, Gianmarco Sabiu, Jing Zhao, Sungwook Jung, Andy J. Seelam, Xiaofei Li, Yang Song, Marina W. Shirkey, Lushen Li, Wenji Piao, Long Wu, Tianshu Zhang, Soyeon Ahn, Pilhan Kim, Vivek Kasinath, Jamil R. Azzi, Jonathan S. Bromberg, Reza Abdi
Abstract
Collagen VI–related disorders (COL6-RDs) are a group of rare muscular dystrophies caused by pathogenic variants in collagen VI genes (COL6A1, COL6A2, and COL6A3). Collagen type VI is a heterotrimeric, microfibrillar component of the muscle extracellular matrix (ECM), predominantly secreted by resident fibroadipogenic precursor cells in skeletal muscle. The absence or mislocalization of collagen VI in the ECM underlies the noncell-autonomous dysfunction and dystrophic changes in skeletal muscle with a yet elusive direct mechanistic link between the ECM and myofiber dysfunction. Here, we conducted a comprehensive natural history and outcome study in a mouse model of COL6-RDs (Col6a2–/– mice) using standardized (TREAT-NMD) functional, histological, and physiological parameters. Notably, we identify a conspicuous dysregulation of the TGF-β pathway early in the disease process and propose that the collagen VI–deficient matrix is not capable of regulating the dynamic TGF-β bioavailability both at baseline and in response to muscle injury. Thus, we propose a new mechanism for pathogenesis of the disease that links the ECM regulation of TGF-β with downstream skeletal muscle abnormalities, paving the way for the development and validation of therapeutics that target this pathway.
Authors
Payam Mohassel, Hailey Hearn, Jachinta Rooney, Yaqun Zou, Kory Johnson, Gina Norato, Matthew A. Nalls, Pomi Yun, Tracy Ogata, Sarah Silverstein, David A. Sleboda, Thomas J. Roberts, Daniel B. Rifkin, Carsten G. Bönnemann
No posts were found with this tag.
Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)