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Abstract
Cells sense extracellular environment and mechanical stimuli and translate these signals into intracellular responses through mechanotransduction and alters cell maintenance, proliferation, and differentiation. Here we use a mouse model of trauma induced heterotopic ossification (HO) to examine how cell-extrinsic forces impact MPC fate. After injury, single cell (sc) RNA sequencing of the injury site reveals an early increase in MPC genes associated with pathways of cell adhesion and ECM-receptor interactions, and MPC trajectories to cartilage and bone. Immunostaining uncovers active mechanotransduction after injury with increased focal adhesion kinase signaling and nuclear translocation of transcriptional co-activator TAZ, inhibition of which mitigates HO. Similarly, joint immobilization decreases mechanotransductive signaling, and completely inhibits HO. Joint immobilization decreases collagen alignment and increases adipogenesis. Further, scRNA sequencing of the HO site after injury with or without immobilization identifies gene signatures in mobile MPCs correlating with osteogenesis, while signatures from immobile MPCs with adipogenesis. scATAC-seq in these same MPCs confirm that in mobile MPCs, chromatin regions around osteogenic genes are open, while in immobile MPCs, regions around adipogenic genes are open. Together these data suggest that joint immobilization after injury results in decreased ECM alignment, altered MPC mechanotransduction, and changes in genomic architecture favoring adipogenesis over osteogenesis, resulting in decreased formation of HO.
Authors
Amanda K. Huber, Nicole Patel, Chase A. Pagani, Simone Marini, Karthik Padmanabhan, Daniel L. Matera, Mohamed Said, Charles Hwang, Ginny Ching-Yun Hsu, Andrea A. Poli, Amy L. Strong, Noelle D. Visser, Joseph A. Greenstein, Reagan Nelson, Shuli Li, Michael T. Longaker, Yi Tang, Stephen J. Weiss, Brendon M. Baker, Aaron W. James, Benjamin Levi
Abstract
To improve the clinical outcome of adoptive NK cell therapy in patients with solid tumors, NK cells need to persist within the tumor microenvironment (TME) in which the abundance of reactive oxygen species (ROS) could dampen anti-tumor immune responses. In the present study, we demonstrated that IL-15 primed NK cells acquire resistance against oxidative stress through thioredoxin system activated by mTOR. Mechanistically, the activation of thioredoxin showed dependence on localization of thioredoxin-interacting protein. For the first time, we showed that NK cells residing in the tumor core expressed higher thiol density which could aid to protect other lymphocytes against ROS within the TME. Furthermore, the prognostic value of IL15 and NK cell gene signature in tumors may be influenced by tobacco smoking history in NSCLC patients. Collectively, the levels of reducing antioxidants in NK cells may not only predict for better tumor penetrance but even potentially response to immune therapy.
Authors
Ying Yang, Shi Yong Neo, Ziqing Chen, Weiyingqi Cui, Yi Chen, Min Guo, Yongfang Wang, Haiyan Xu, Annina Kurzay, Evren Alici, Lars Holmgren, Felix Haglund, Kai Wang, Andreas Lundqvist
Abstract
Drivers of sporadic benign pituitary adenoma growth are largely unknown. Whole exome sequencing of 159 prospectively resected pituitary adenomas showed somatic copy number alteration (SCNA) rather than mutation is a hallmark of hormone-secreting adenomas and that SCNA correlate with adenoma phenotype. Using single-gene SCNA pathway analysis, we observed cAMP and Fanconi anemia DNA damage repair pathways both affected by SCNA in growth hormone (GH)-secreting somatotroph adenomas. As somatotroph differentiation and GH secretion is dependent on cAMP activation and we previously showed DNA damage, aneuploidy, and senescence in somatotroph adenomas, we studied links between cAMP signaling and DNA damage. Stimulation of cAMP in C57Bl/6 mouse primary pituitary cultures using forskolin or long-acting GH releasing hormone (GHRH) analogue increased GH production and DNA damage measured by phosphorylated H2AX and Comet assay. Octreotide, a somatostatin receptor ligand that targets somatotroph adenoma GH secretion in patients with acromegaly, inhibited cAMP and GH and reversed DNA damage induction. In vivo long-acting GHRH treatment also induced mouse pituitary DNA damage. We conclude that cAMP, which induces somatotroph proliferation and GH secretion, may concomitantly induce DNA damage, potentially linking hormone hypersecretion to SCNA and genome instability. These results elucidating somatotroph adenoma pathophysiology identify pathways for treatment targeting.
Authors
Anat Ben-Shlomo, Nan Deng, Evelyn Ding, Masaaki Yamamoto, Adam Mamelak, Vera Chesnokova, Artak Labadzhyan, Shlomo Melmed
Abstract
Chronic inflammation is deeply involved in various human disorders, such as cancer, neurodegenerative disorders, and metabolic disorders. Induction of epigenetic alterations, especially aberrant DNA methylation, is one of the major mechanisms, but how it is induced is still unclear. Here, we found that expression of TET genes, methylation erasers, was down-regulated in inflamed mouse and human tissues, and that this was caused by up-regulation of TET-targeting miRNAs, such as MIR20A, MIR26B, and MIR29C, likely due to activation of NF-kB signaling, downstream of IL-1b and TNF-a. However, TET knockdown induced only mild aberrant methylation. Nitric oxide (NO), produced by NOS2, enhanced enzymatic activity of DNMTs, methylation writers, and NO exposure induced minimal aberrant methylation. In contrast, a combination of TET knockdown and NO exposure synergistically induced aberrant methylation, involving genomic regions not methylated by either alone. The results showed that a vicious combination of TET repression, due to NF-kB activation, and DNMT activation, due to NO production, is responsible for aberrant methylation induction in human tissues.
Authors
Hideyuki Takeshima, Tohru Niwa, Satoshi Yamashita, Takeji Takamura-Enya, Naoko Iida, Mika Wakabayashi, Sohachi Nanjo, Masanobu Abe, Toshiro Sugiyama, Young-Joon Kim, Toshikazu Ushijima
Abstract
Tissue factor (TF) is the primary initiator of blood coagulation in vivo and the only blood coagulation factor for which a human genetic defect has not been described. As there are no routine clinical assays that capture the contribution of endogenous TF to coagulation initiation, the extent to which reduced TF activity contributes to unexplained bleeding is unknown. Using whole genome sequencing, we identified a heterozygous frameshift variant (p.Ser117HisfsTer10) in F3, the gene encoding TF, causing premature termination of TF ("TFshort") in a woman with unexplained bleeding. Routine hematological laboratory evaluation of the proposita was normal. CRISPR-edited human induced pluripotent stem cells recapitulating the variant were differentiated into vascular smooth muscle and endothelial cells that demonstrated haploinsufficiency of TF. The variant F3 transcript is eliminated by nonsense-mediated decay. Neither overexpression nor addition of exogenous recombinant TFshort inhibited factor Xa or thrombin generation, excluding a dominant negative mechanism. F3+/- mice provide an animal model of TF haploinsufficiency and exhibited prolonged bleeding times, impaired thrombus formation, and reduced survival following major injury. Heterozygous TF deficiency is present in at least 1 in 25,000 individuals and could limit coagulation initiation in undiagnosed individuals with abnormal bleeding but a normal routine laboratory evaluation.
Authors
Sol Schulman, Emale El-Darzi, Mary HC Florido, Max Friesen, Glenn Merrill-Skoloff, Marisa A. Brake, Calvin R. Schuster, Lin Lin, Randal J. Westrick, Chad A. Cowan, Robert Flaumenhaft, NIHR BioResource, Willem H. Ouwehand, Kathelijne Peerlinck, Kathleen Freson, Ernest Turro, Bruce Furie
Abstract
During hemolysis, macrophages in the liver phagocytose damaged erythrocytes to prevent the toxic effects of cell-free hemoglobin and heme. It remains unclear how this homeostatic process modulates phagocyte functions in inflammatory diseases. Using a genetic mouse model of spherocytosis and single-cell RNA sequencing, we found that erythrophagocytosis skewed liver macrophages into a unique anti-inflammatory phenotype that we defined as Marcohigh/Hmoxhigh/MHC-class IIlow erythrophagocytes. This phenotype transformation profoundly mitigated disease expression in a model of an anti-CD40-induced hyperinflammatory syndrome with necrotic hepatitis and in a non-alcoholic steatohepatitis model, representing two macrophage-driven sterile inflammatory diseases. We reproduced the anti-inflammatory erythrophagocyte transformation in vitro by heme-exposure of mouse and human macrophages, yielding a distinctive transcriptional signature that segregated heme-polarized from M1- and M2-polarized cells. Mapping transposase-accessible chromatin in single cells by sequencing (scATAC-seq) defined the transcription factor NFE2L2/NRF2 as a critical driver of erythrophagocytes, and Nfe2l2/Nrf2-deficiency restored heme-suppressed inflammation. Our findings point to a pathway that regulates macrophage functions to link erythrocyte homeostasis with innate immunity.
Authors
Marc Pfefferlé, Giada Ingoglia, Christian A. Schaer, Ayla Yalamanoglu, Raphael M. Buzzi, Irina L. Dubach, Ge Tan, Emilio Y. López-Cano, Nadja Schulthess, Kerstin Hansen, Rok Humar, Dominik J. Schaer, Florence Vallelian
Abstract
Immune checkpoint blockade (ICB) has revolutionized cancer therapeutics. Desmoplastic malignancies such as cholangiocarcinoma (CCA) have an abundant tumor immune microenvironment (TIME). However, to date ICB monotherapy in such malignancies has been ineffective. Herein, we identify that tumor-associated macrophages (TAMs) are the primary source of PD-L1 in human and murine CCA. In a murine model of CCA, recruited PD-L1+ TAMs facilitate CCA progression. However, TAM blockade failed to decrease tumor progression due to a compensatory emergence of granulocytic-myeloid-derived suppressor cells (G-MDSCs) that mediated immune escape by impairing T-cell response. Single-cell RNA sequencing (scRNA-seq) of murine tumor G-MDSCs highlighted a novel ApoE G-MDSC subset enriched with TAM blockade; further analysis of a human scRNA-seq dataset demonstrated the presence of a similar G-MDSC subset in human CCA. Finally, dual inhibition of TAMs and G-MDSCs potentiated ICB. In summary, our findings highlight the therapeutic potential of coupling ICB with immunotherapies targeting immunosuppressive myeloid cells in CCA.
Authors
Emilien Loeuillard, Jingchun Yang, EeeLN Buckarma, Juan Wang, Yuanhang Liu, Caitlin B. Conboy, Kevin D. Pavelko, Ying Li, Daniel O'Brien, Chen Wang, Rondell P. Graham, Rory L. Smoot, Haidong Dong, Sumera Rizvi
Abstract
Tumor immunosuppression is a limiting factor for successful cancer therapy. The lipid Sphingosine-1-phosphate (S1P), which signals through five distinct G-protein-coupled receptors (S1PR1-5), emerged as an important regulator of carcinogenesis. However, the utility of targeting S1P in tumors is hindered by its impact on immune cell trafficking. Here we report that ablation of the immune cell-specific receptor S1PR4, which plays a minor role in immune cell trafficking, delayed tumor development and improved therapy success in murine models of mammary and colitis-associated colorectal cancer due to an increased CD8+ T cell abundance. Transcriptome analysis revealed that S1PR4 affected proliferation and survival of CD8+ T cells in a cell-intrinsic manner via the expression of Pik3ap1 and Lta4h. Accordingly, PIK3AP1 expression was connected to increased CD8+ T cell proliferation and clinical parameters in human breast and colon cancer. Our data indicate a so far unappreciated tumor-promoting role of S1P by restricting CD8+ T cell expansion via S1PR4.
Authors
Catherine Olesch, Evelyn Sirait-Fischer, Matthias Berkefeld, Annika F. Fink, Rosa Martha Susen, Birgit Ritter, Birgitta E. Michels, Dieter Steinhilber, Florian R. Greten, Rajkumar Savai, Kazuhiko Takeda, Bernhard Brüne, Andreas Weigert
Abstract
The development of broadly neutralizing antibodies (BNAbs) in HIV infection is a result of long-term co-evolutionary interaction between viruses and antibodies. Understanding how this interaction promotes the increase of neutralization breadth during infection will improve the way in which we design AIDS vaccine strategies. In this paper, we used SIV-infected rhesus macaques as a model to study the development of neutralization breadth by infecting rhesus macaques with longitudinal NAb escape variants and evaluating the kinetics of NAb response and viral evolution. We found that the infected macaques developed a stepwise NAb response against escape variants and increased neutralization breadth during the course of infection. Furthermore, the increase of neutralization breadth correlated with the duration of infection but was independent of properties of the inoculum, viral loads or viral diversity during infection. These results imply that the duration of infection was the main factor driving the development of BNabs. These data suggest the importance of novel immunization strategies to induce effective NAb response against HIV infection by mimicking long-term infection.
Authors
Fan Wu, Ilnour Ourmanov, Andrea Kirmaier, Sivan Leviyang, Celia LaBranche, Jinghe Huang, Sonya Whitted, Kenta Matsuda, David Montefiori, Vanessa M. Hirsch
Abstract
Alarmins, sequestered self-molecules containing damage-associated molecular patterns, are released during tissue injury to drive innate immune cell pro-inflammatory responses. Whether endogenous negative regulators controlling early immune responses are also released at the site of injury is poorly understood. Herein, we establish that the stromal cell-derived alarmin interleukin-33 (IL-33) is a local factor that directly restricts the pro-inflammatory capacity of graft infiltrating macrophages early after transplantation. By assessing heart transplant recipient samples and using a mouse heart transplant model, we establish that IL-33 is upregulated in allografts to limit chronic rejection. Mouse cardiac transplants lacking IL-33 displayed dramatically accelerated vascular occlusion and subsequent fibrosis, which was not due to altered systemic immune responses. Instead, a lack of graft IL-33 caused local augmentation of pro-inflammatory iNOS+ macrophages that accelerated graft loss. IL-33 facilitated a metabolic program in macrophages associated with reparative and regulatory functions, and local delivery of IL-33 prevented the chronic rejection of IL-33-deficient cardiac transplants. Therefore, IL-33 represents a novel regulatory alarmin in transplantation that limits chronic rejection by restraining the local activation of pro-inflammatory macrophages. The local delivery of IL-33 in extracellular matrix based-based materials may be a promising biologic for chronic rejection prophylaxis.
Authors
Tengfang Li, Zhongqiang Zhang, Joseph Guido Bartolacci, Gaelen K. Dwyer, Quan Liu, Lisa Mathews, Murugesan Velayutham, Anna Roessing, Yoojin C. Lee, Helong Dai, Sruti Shiva, Martin H. Oberbarnscheidt, Jenna L. Dziki, Steven J. Mullett, Stacy G. Wendell, James D. Wilkinson, .Steven A Webber, Michelle A. Wood-Trageser, Simon C. Watkins, Anthony J. Demetris, George S. Hussey, Stephen F. Badylak, Heth R. Turnquist
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Copyright © 2026 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)