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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 sequencing 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
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) 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, 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
TGF-β is a central driver of kidney fibrosis, a common pathological hallmark of chronic kidney disease (CKD). Initiation of TGF-β signaling requires not only its synthesis but also the conversion of latent TGF-β to its bioactive form. However, the mechanisms governing TGF-β activation in the kidney and their contribution to kidney fibrosis remain poorly understood. Glycoprotein A repetitions predominant (GARP) anchors latent TGF-β on the cell surface and facilitates its bioactive release. Here, we show that GARP-mediated TGF‐β activation promotes kidney fibrosis. GARP was upregulated in both human and mouse CKD kidneys, predominantly in fibroblasts, and was induced by TNF in an NF-kB-dependent fashion. In multiple mouse models of kidney fibrosis, either global or fibroblast-specific deletion of GARP significantly reduced fibrosis. Mechanistically, GARP enables sustained production of active TGF-β, thereby amplifying fibroblast stimulation. Deletion of GARP in kidney fibroblasts lowered active TGF-β levels and attenuated fibroblast activation, whereas GARP overexpression enhanced TGF-β signaling. Notably, tamoxifen-induced deletion of GARP after fibrosis onset attenuated kidney fibrosis. Together, our findings identify GARP-mediated release of active TGF‐β as a critical step in sustaining fibroblast activation during kidney fibrosis and highlight GARP as a promising therapeutic target for CKD.
Authors
Yintong Chen, Weiwei Xu, Jieli Yu, Pei Deng, Nianping Liu, Yinyin Li, Hui Zhou, Hong Zhou, Jianchuan Wang, Bo Zhao, Florian Winau, Fan Fan Hou, Yu Hu
Abstract
The adult mammalian heart has a limited ability to regenerate lost myocardium following myocardial infarction (MI), largely due to the poor proliferative capacity of cardiomyocytes (CMs). Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a known regulator of cell quiescence, though the mechanisms underlying its function remain unclear. Previous studies have shown that pharmacological inhibition of DYRK1A using harmine induces CM cell cycle re-entry after ischemia/reperfusion (I/R) MI. Here, we developed a computational network model of DYRK1A-mediated regulation of the cell cycle, which predicts how DYRK1A inhibition promotes CM re-entry. To validate these predictions, we tested selective DYRK1A inhibitors and observed robust induction of cell cycle activity in neonatal rat cardiomyocytes (NRCMs). Integrating our network model with bulk RNA-sequencing data from DYRK1A inhibitor-treated NRCMs, we identified E2F1 as a key transcriptional driver of cell cycle gene expression. Finally, we demonstrate that both pharmacological and post-developmental inhibition of DYRK1A enhances heart function and increases CM cycling following I/R MI. Our findings suggest that functional recovery induced by small molecule inhibitor of DYRK1A is mediated by the induction of cycling CMs.
Authors
Bryce C. Murillo, Alexander Young, Kaitlyn L. Wintruba, Alexander J. Eichert, Klara Siejda, Dennon Hoernig, Leigh A. Bradley, Bryana N. Harris, Catherine Zhao, MIchelle Wu, Emmanuel Deau, Mattias F. Lindberg, Laurent Meijer, Jeffrey J. Saucerman, Matthew J. Wolf
Abstract
Tregs play an essential role in immune tolerance, and Treg-promoting therapies are in development for the treatment of many inflammatory disorders. Interleukin-2 (IL-2)-based therapies increase Treg frequency, but little is known about impacts on Treg heterogeneity and function. We extended analyses of an IL-2 mutein (MK-6194) single–ascending-dose trial in healthy human participants by comprehensively defining Treg subsets and gene expression changes in vitro and in vivo. We found highly specific and dose-dependent activation and expansion of Tregs in clinical and pre-clinical studies. Following a single subcutaneous dose in humans, thymic-derived Tregs were selectively activated and expanded, while peripherally induced Tregs were unaffected. Expanded Tregs had increased expression of genes and proteins consistent with activation, suppressor function, and homing to non-lymphoid tissue, as well as increased transendocytosis activity, as measured by CTLA-4–dependent capture of CD80 and CD86 from non-Tregs. These results shed light onto underlying mechanisms by which Treg-targeted therapy may promote immune tolerance.
Authors
Laura A. Cooney, Mitch Fahning, Liliane Khoryati, Anna Kus, Sheila Scheiding, Lori Blanchfield, Matthew Lawrance, Basilin Benson, Kristina M. Harris, Gretchen A. Baltus, Shiuli Agarwal, Richard Wnek, Johannes F. Scheid, Kiki Cunningham-Bussel, Nancy D. Kim, S. Aubrey Stoch, Jyothsna Visweswaraiah, Nathan Higginson-Scott, Katalin Kis-Toth, Joanne L. Viney, Kevin L. Otipoby, Erik Sampson, Bridget Larkin, Daniel J. Campbell, S. Alice Long
Abstract
Authors
Jianfei Ji, Wenjing You, Xiaoli Sun, Peng Zhao
Abstract
Cell metabolic rewiring is associated with resistance to venetoclax-azacitidine (Ven-Aza) combination therapy and relapse in acute myeloid leukemia (AML) patients. Drug-resistant cells exhibit an enhanced reliance on oxidative phosphorylation (OXPHOS) for energy production. Therefore, impairing mitochondrial metabolism represents an exciting strategy to face this unmet clinical need. We recently demonstrated that the specific activation of the phosphatase PP2A-B56α enhances the pro-apoptotic efficacy of venetoclax in AML. Here, through leveraging unbiased multi-omics-based approaches and using both genetic and pharmacological tools, we define key roles for the tumor suppressor PP2A-B56α complex in OXPHOS regulation and treatment response in disease-relevant AML models. From a translational perspective, the specific stabilization of PP2A-B56α heterocomplex with the novel PP2A molecular glue activator, RPT04402, reduces OXPHOS levels in treatment-resistant AML cells and improves treatment response in both Ven-Aza-sensitive and -resistant AML cell lines, primary cells, and in vivo models. Together, our work supports further research on targeted combination therapy approaches based on PP2A-B56α stabilization to counteract OXPHOS-related treatment resistance and improve AML responses in a patient population with historically poor outcomes.
Authors
Silvia Romero-Murillo, Irene Peris, Anna Maria Lucianò, Nerea Marcotegui, Carmen Vicente, Brian Tran, Kelsey Barrie, Caitlin M. O'Connor, Andrea Torres-López, Maria C. Mateos, Maria L. Cayuela, Victoriano Mulero, Joaquín Fernández-Irigoyen, Enrique Santamaría, Maria D. Odero, Goutham Narla
Abstract
Despite advances in treatment approaches for lung cancer, the morbidity and survival of lung cancer patients with malignant pleural effusions (MPE) remain poor. This is in part due to gaps in understanding the role of immune cells in the pleural fluid microenvironment. We performed single cell analysis with flow cytometry validation of CD45+ cells in eight malignant and five benign pleural fluid (BPE) specimens to identify changes in the transcriptomic landscape of immune cells across disease states. We found upregulation of pro-inflammatory signaling pathways, including interferon and TNF signaling, in T cells, B cells, and macrophages in benign compared to malignant pleural effusions. Pro-inflammatory HLA-DR+ macrophages were associated with good survival outcomes while pro-tumorigenic HLA-DR- macrophages with upregulation of angiogenesis, TGFβ, and fibronectin signaling were associated with poor survival outcomes in patients with MPE. We also validated these findings with macrophage cell surface expression markers using flow cytometry in 14 MPE and 7 BPE specimens. Finally, we performed multiplex cytokine analysis which showed enrichment of the type 3 inflammatory cytokine, IL17A, in MPE as a putative mechanism for macrophage reprogramming. These data provide a rich resource for interrogating the immune cell types and states present across the spectrum of pleural disease. They offer not only prognostic value for patient outcomes at the time of pleural fluid collection, but also insights into novel immunotherapy targets.
Authors
Aaditya Khatri, Huimin Wang, Zhicheng Ji, Prekshaben Patel, Smita K. Nair, Javid P. Mohammed, Beth H. Shaz, Andrew B. Nixon, Scott M. Palmer, Kamran Mahmood
Abstract
Activation of the mechanistic target of rapamycin (mTOR) complex1 (mTORC1) promotes muscle protein synthesis, mass, and function. Muscle mTORC1 can be activated by feeding and contraction. Here, muscle mTORC1 signaling, protein synthesis, mass, and function are characterized in a genetic mouse model that separates these two major modes of muscle mTORC1 regulation. AKT signaling is required for feeding-induced muscle mTORC1 signaling and protein synthesis, and mice expressing a mutant of tuberous sclerosis complex 2 (TSC2) that cannot be phosphorylated by AKT specifically in skeletal muscle (SkM-TSC2-5A) attenuate these effects of feeding. Despite this loss of postprandial protein synthesis, SkM-TSC2-5A mice have similar muscle and myofiber size compared to SkM-TSC2-WT mice. SkM-TSC2-5A mice maintain normal muscle mTORC1 activation in response to contraction and exhibit no differences in atrophy-related gene expression or ribosomal content. SkM-TSC2-5A mice exhibit improved maximal endurance capacity without changes in muscle contractile function. This phenotype occurs without alterations in muscle glycogen content or myofiber type but does coincide with a modest increase in muscle mitochondrial content. Therefore, AKT-mediated phosphorylation of TSC2 is required for postprandial mTORC1 activation and the induction of protein synthesis; however, these are dispensable for the development and maintenance of muscle mass in sedentary mice.
Authors
Samuel C. Lapp, Krystle C. Kalafut, Madi Y. Cissé, Khaled Tighanimine, Dean M. Rosenthal, Will Doxsey, Sheng Hui, Karen E. Inouye, Claire E. Morrow, Yann Cormerais, Brendan D. Manning
Endothelial cell cycle inhibition enables blood vessel maturation to normalize the tumor vasculature
Abstract
Dysfunctional tumor vessels promote disease progression, whereas improved function enhances therapeutic delivery. However, current approaches to normalize tumor vasculature have limited efficacy. In vascular malformations, vessels are similarly dysfunctional, with endothelial cell (EC) hyperproliferation impairing arterial-venous specification. These defects are corrected with palbociclib, a cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) that has beneficial effects on tumor and immune cells, but the effects on tumor vasculature are not well characterized. In our studies, murine mammary tumor ECs (TECs) exhibited disrupted cell cycle and specification, and CDK4/6i promoted TEC cycle control, enabling improved tumor vascular function. To investigate transcriptomic changes, we performed single-cell RNA sequencing (scRNAseq) of treated and untreated tumors, and healthy tissues. CDK4/6i-mediated TEC cycle arrest promoted arterial-venous specification, cellular junctions, and pericyte association, and suppressed glycolytic and immunosuppressive gene expression. These effects were associated with increased vessel perfusion, decreased tumor hypoxia, and a more favorable immune landscape with immunotherapy. In scRNAseq datasets from patients treated long-term with CDK4/6i, TECs exhibited similar transcriptomic changes associated with arterial-venous specification, pericyte recruitment, and immune signaling. Thus, in contrast to current strategies, CDK4/6i-mediated vascular changes may be maintained with continued treatment, highlighting the relevance of modulating TEC cycle to improve vessel maturation/function.
Authors
Shelby R. Cain, Gael Genet, Nafiisha Genet, Jordon W. Aragon, Madeline G. Jackson, Victoria M. Milosek, Mark R. Schwartz, Umadevi Paila, Aleksandra Cwiek, Zaneta Markowska, Nicholas W. Chavkin, Richard J. Price, Andrew C. Dudley, Karen K. Hirschi
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