Abstract
STING gain-of-function mutations cause STING-associated vasculopathy with onset in infancy (SAVI) in humans, a disease characterized by spontaneous lung inflammation and fibrosis. Mice with STING gain-of-function mutations (SAVI mice) develop αβ T cell–dependent lung disease and also lack lymph nodes. Although SAVI has been regarded as a type I interferonopathy, the relative contributions of the three interferon receptors are incompletely understood. Here, we show that STING gain of function led to upregulation of IFN-γ–induced chemokines in the lungs of SAVI mice and that deletion of the type II IFN receptor (IFNGR1), but not the type I IFN receptor (IFNAR1) or type III IFN receptor (IFNλR1), ameliorated lung disease and restored lymph node development in SAVI mice. Furthermore, deletion of IFNGR1, but not IFNAR1 or IFNλR1, corrected the ratio of effector to Tregs in SAVI mice and in mixed bone marrow chimeric mice. Finally, cultured SAVI mouse macrophages were hyperresponsive to IFN-γ, but not IFN-β, in terms of Cxcl9 upregulation and cell activation. These results demonstrate that IFNGR1 plays a major role in autoinflammation and immune dysregulation mediated by STING gain of function.
Authors
W. Alexander Stinson, Cathrine A. Miner, Fang R. Zhao, Annena Jane Lundgren, Subhajit Poddar, Jonathan J. Miner
Abstract
Osteosarcoma (OS) is a lethal disease with few known targeted therapies. Here, we show that decreased ATRX expression is associated with more aggressive tumor cell phenotypes, including increased growth, migration, invasion, and metastasis. These phenotypic changes correspond with activation of NF-κB signaling, extracellular matrix remodeling, increased integrin αvβ3 expression, and ETS family transcription factor binding. Here, we characterize these changes in vitro, in vivo, and in a data set of human OS patients. This increased aggression substantially sensitizes ATRX-deficient OS cells to integrin signaling inhibition. Thus, ATRX plays an important tumor-suppression role in OS, and loss of function of this gene may underlie new therapeutic vulnerabilities. The relationship between ATRX expression and integrin binding, NF-κB activation, and ETS family transcription factor binding has not been described in previous studies and may impact the pathophysiology of other diseases with ATRX loss, including other cancers and the ATR-X α thalassemia intellectual disability syndrome.
Authors
Suzanne Bartholf DeWitt, Sarah Hoskinson Plumlee, Hailey E. Brighton, Dharshan Sivaraj, E.J. Martz, Maryam Zand, Vardhman Kumar, Maya U. Sheth, Warren Floyd, Jacob V. Spruance, Nathan Hawkey, Shyni Varghese, Jianhua Ruan, David G. Kirsch, Jason A. Somarelli, Ben Alman, William C. Eward
Abstract
Heterozygous mutations in FLT3ITD, TET2, and DNMT3A are associated with hematologic malignancies in humans. In patients, cooccurrence of mutations in FLT3ITD combined with TET2 (TF) or FLT3ITD combined with DNMT3A (DF) are frequent. However, in some rare complex acute myeloid leukemia (AML), all 3 mutations cooccur — i.e., FLT3ITD, TET2, and DNMT3A (TFD). Whether the presence of these mutations in combination result in quantitative or qualitative differences in disease manifestation has not been investigated. We generated mice expressing heterozygous Flt3ITD and concomitant for either heterozygous loss of Tet2 (TF) or Dnmt3a (DF) or both (TFD). TF and DF mice did not induce disease early on, in spite of similar changes in gene expression; during the same time frame, an aggressive form of transplantable leukemia was observed in TFD mice, which was mostly associated with quantitative but not qualitative differences in gene expression relative to TF or DF mice. The gene expression signature of TFD mice showed remarkable similarity to the human TFD gene signature at the single-cell RNA level. Importantly, TFD-driven AML responded to a combination of drugs that target Flt3ITD, inflammation, and methylation in a mouse model, as well as in a PDX model of AML bearing 3 mutations.
Authors
Baskar Ramdas, Palam Lakshmi Reddy, Raghuveer Singh Mali, Santhosh Kumar Pasupuleti, Ji Zhang, Mark R. Kelley, Sophie Paczesny, Chi Zhang, Reuben Kapur
Abstract
Adaptation to increased insulin demand is mediated by β cell proliferation and neogenesis, among other mechanisms. Although it is known that pancreatic β cells can arise from ductal progenitors, these observations have been limited mostly to the neonatal period. We have recently reported that the duct is a source of insulin-secreting cells in adult insulin-resistant states. To further explore the signaling pathways underlying the dynamic β cell reserve during insulin resistance, we undertook human islet and duct transplantations under the kidney capsule of immunodeficient NOD/SCID-γ (NSG) mouse models that were pregnant, were insulin-resistant, or had insulin resistance superimposed upon pregnancy (insulin resistance + pregnancy), followed by single-nucleus RNA-Seq (snRNA-Seq) on snap-frozen graft samples. We observed an upregulation of proliferation markers (e.g., NEAT1) and expression of islet endocrine cell markers (e.g., GCG and PPY), as well as mature β cell markers (e.g., INS), in transplanted human duct grafts in response to high insulin demand. We also noted downregulation of ductal cell identity genes (e.g., KRT19 and ONECUT2) coupled with upregulation of β cell development and insulin signaling pathways. These results indicate that subsets of ductal cells are able to gain β cell identity and reflect a form of compensation during the adaptation to insulin resistance in both physiological and pathological states.
Authors
Ercument Dirice, Giorgio Basile, Sevim Kahraman, Danielle Diegisser, Jiang Hu, Rohit N. Kulkarni
Abstract
Key molecular regulators of acquired radiation resistance in recurrent glioblastoma (GBM) are largely unknown, with a dearth of accurate preclinical models. To address this, we generated 8 GBM patient-derived xenograft (PDX) models of acquired radiation therapy–selected (RTS) resistance compared with same-patient, treatment-naive (radiation-sensitive, unselected; RTU) PDXs. These likely unique models mimic the longitudinal evolution of patient recurrent tumors following serial radiation therapy. Indeed, while whole-exome sequencing showed retention of major genomic alterations in the RTS lines, we did detect a chromosome 12q14 amplification that was associated with clinical GBM recurrence in 2 RTS models. A potentially novel bioinformatics pipeline was applied to analyze phenotypic, transcriptomic, and kinomic alterations, which identified long noncoding RNAs (lncRNAs) and targetable, PDX-specific kinases. We observed differential transcriptional enrichment of DNA damage repair pathways in our RTS models, which correlated with several lncRNAs. Global kinomic profiling separated RTU and RTS models, but pairwise analyses indicated that there are multiple molecular routes to acquired radiation resistance. RTS model–specific kinases were identified and targeted with clinically relevant small molecule inhibitors. This cohort of in vivo RTS patient-derived models will enable future preclinical therapeutic testing to help overcome the treatment resistance seen in patients with GBM.
Authors
Christian T. Stackhouse, Joshua C. Anderson, Zongliang Yue, Thanh Nguyen, Nicholas J. Eustace, Catherine P. Langford, Jelai Wang, James R. Rowland IV, Chuan Xing, Fady M. Mikhail, Xiangqin Cui, Hasan Alrefai, Ryan E. Bash, Kevin J. Lee, Eddy S. Yang, Anita B. Hjelmeland, C. Ryan Miller, Jake Y. Chen, G. Yancey Gillespie, Christopher D. Willey
Abstract
Expression of the tight junction–associated protein junctional adhesion molecule-A (JAM-A) is increased in sepsis, although the significance of this is unknown. Here, we show that septic JAM-A –/– mice have increased gut permeability, yet paradoxically have decreased bacteremia and systemic TNF and IL-1β expression. Survival is improved in JAM-A–/– mice. However, intestine-specific JAM-A–/– deletion does not alter mortality, suggesting that the mortality benefit conferred in mice lacking JAM-A is independent of the intestine. Septic JAM-A–/– mice have increased numbers of splenic CD44hiCD4+ T cells, decreased frequency of TNF+CD4+ cells, and elevated frequency of IL-2+CD4+ cells. Septic JAM-A–/– mice have increased numbers of B cells in mesenteric lymph nodes with elevated serum IgA and intraepithelial lymphocyte IgA production. JAM-A–/– × RAG–/– mice have improved survival compared with RAG–/– mice and identical mortality as WT mice. Gut neutrophil infiltration and neutrophil phagocytosis are increased in JAM-A–/– mice, while septic JAM-A–/– mice depleted of neutrophils lose their survival advantage. Therefore, increased bacterial clearance via neutrophils and an altered systemic inflammatory response with increased opsonizing IgA produced through the adaptive immune system results in improved survival in septic JAM-A–/– mice. JAM-A may be a therapeutic target in sepsis via immune mechanisms not related to its role in permeability.
Authors
Nathan J. Klingensmith, Katherine T. Fay, David A. Swift, Julia M.R. Bazzano, John D. Lyons, Ching-wen Chen, Mei Meng, Kimberly M. Ramonell, Zhe Liang, Eileen M. Burd, Charles A. Parkos, Mandy L. Ford, Craig M. Coopersmith
Abstract
Usual interstitial pneumonia (UIP) is a histological pattern characteristic of idiopathic pulmonary fibrosis (IPF). The UIP pattern is patchy with histologically normal lung adjacent to dense fibrotic tissue. At this interface, fibroblastic foci (FF) are present and are sites where myofibroblasts and extracellular matrix (ECM) accumulate. Utilizing laser capture microdissection-coupled mass spectrometry, we interrogated the FF, adjacent mature scar, and adjacent alveoli in 6 fibrotic (UIP/IPF) specimens plus 6 nonfibrotic alveolar specimens as controls. The data were subjected to qualitative and quantitative analysis and histologically validated. We found that the fibrotic alveoli protein signature is defined by immune deregulation as the strongest category. The fibrotic mature scar classified as end-stage fibrosis whereas the FF contained an overabundance of a distinctive ECM compared with the nonfibrotic control. Furthermore, FF were positive for both TGFB1 and TGFB3, whereas the aberrant basaloid cell lining of FF was predominantly positive for TGFB2. In conclusion, spatial proteomics demonstrated distinct protein compositions in the histologically defined regions of UIP/IPF tissue. These data revealed that FF are the main site of collagen biosynthesis and that the adjacent alveoli are abnormal. This essential information will inform future mechanistic studies on fibrosis progression.
Authors
Jeremy A. Herrera, Lewis Dingle, M. Angeles Montero, Rajamiyer V. Venkateswaran, John F. Blaikley, Craig Lawless, Martin A. Schwartz
Abstract
Recent clinical trials have shown promising results for the next-generation Bruton’s tyrosine kinase (BTK) inhibitor evobrutinib in the treatment of multiple sclerosis (MS). BTK has a central role in signaling pathways that govern the development of B cells. Whether and how BTK activity shapes B cells as key drivers of MS is currently unclear. Compared with levels of BTK protein, we found higher levels of phospho-BTK in ex vivo blood memory B cells from patients with relapsing-remitting MS and secondary progressive MS compared with controls. In these MS groups, BTK activity was induced to a lesser extent after anti-IgM stimulation. BTK positively correlated with CXCR3 expression, both of which were increased in blood B cells from clinical responders to natalizumab (anti–VLA-4 antibody) treatment. Under in vitro T follicular helper–like conditions, BTK phosphorylation was enhanced by T-bet–inducing stimuli, IFN-γ and CpG-ODN, while the expression of T-bet and T-bet–associated molecules CXCR3, CD21, and CD11c was affected by evobrutinib. Furthermore, evobrutinib interfered with in vitro class switching, as well as memory recall responses, and disturbed CXCL10-mediated migration of CXCR3+ switched B cells through human brain endothelial monolayers. These findings demonstrate a functional link between BTK activity and disease-relevant B cells and offer valuable insights into how next-generation BTK inhibitors could modulate the clinical course of patients with MS.
Authors
Liza Rijvers, Jamie van Langelaar, Laurens Bogers, Marie-José Melief, Steven C. Koetzier, Katelijn M. Blok, Annet F. Wierenga-Wolf, Helga E. de Vries, Jasper Rip, Odilia B.J. Corneth, Rudi W. Hendriks, Roland Grenningloh, Ursula Boschert, Joost Smolders, Marvin M. van Luijn
Abstract
There is a paucity of information about potential molecular brakes on the activation of fibroblasts that drive tissue fibrosis. The transcription factor Krüppel-like factor 4 (KLF4) is best known as a determinant of cell stemness and a tumor suppressor. We found that its expression was diminished in fibroblasts from fibrotic lung. Gain- and loss-of-function studies showed that KLF4 inhibited fibroblast proliferation, collagen synthesis, and differentiation to myofibroblasts, while restoring their sensitivity to apoptosis. Conditional deletion of KLF4 from fibroblasts potentiated the peak degree of pulmonary fibrosis and abrogated the subsequent spontaneous resolution in a model of transient fibrosis. A small molecule inducer of KLF4 was able to restore its expression in fibrotic fibroblasts and elicit resolution in an experimental model characterized by more clinically relevant persistent pulmonary fibrosis. These data identify KLF4 as a pivotal brake on fibroblast activation whose induction represents a therapeutic approach in fibrosis of the lung and perhaps other organs.
Authors
Loka R. Penke, Jennifer M. Speth, Steven K. Huang, Sean M. Fortier, Jared Baas, Marc Peters-Golden
Abstract
Dysregulation in neutrophil extracellular trap (NET) formation and degradation may play a role in the pathogenesis and severity of COVID-19; however, its role in the pediatric manifestations of this disease, including multisystem inflammatory syndrome in children (MIS-C) and chilblain-like lesions (CLLs), otherwise known as “COVID toes,” remains unclear. Studying multinational cohorts, we found that, in CLLs, NETs were significantly increased in serum and skin. There was geographic variability in the prevalence of increased NETs in MIS-C, in association with disease severity. MIS-C and CLL serum samples displayed decreased NET degradation ability, in association with C1q and G-actin or anti-NET antibodies, respectively, but not with genetic variants of DNases. In adult COVID-19, persistent elevations in NETs after disease diagnosis were detected but did not occur in asymptomatic infection. COVID-19–affected adults displayed significant prevalence of impaired NET degradation, in association with anti-DNase1L3, G-actin, and specific disease manifestations, but not with genetic variants of DNases. NETs were detected in many organs of adult patients who died from COVID-19 complications. Infection with the Omicron variant was associated with decreased NET levels when compared with other SARS-CoV-2 strains. These data support a role for NETs in the pathogenesis and severity of COVID-19 in pediatric and adult patients.
Authors
Carmelo Carmona-Rivera, Yu Zhang, Kerry Dobbs, Tovah E. Markowitz, Clifton L. Dalgard, Andrew J. Oler, Dillon R. Claybaugh, Deborah Draper, Meng Truong, Ottavia M. Delmonte, Francesco Licciardi, Ugo Ramenghi, Nicoletta Crescenzio, Luisa Imberti, Alessandra Sottini, Virginia Quaresima, Chiara Fiorini, Valentina Discepolo, Andrea Lo Vecchio, Alfredo Guarino, Luca Pierri, Andrea Catzola, Andrea Biondi, Paolo Bonfanti, Maria C. Poli Harlowe, Yasmin Espinosa, Camila Astudillo, Emma Rey-Jurado, Cecilia Vial, Javiera de la Cruz, Ricardo Gonzalez, Cecilia Pinera, Jacqueline W. Mays, Ashley Ng, Andrew Platt, NIH COVID Autopsy Consortium, COVID STORM Clinicians, Beth Drolet, John Moon, Edward W. Cowen, Heather Kenney, Sarah E. Weber, Riccardo Castagnoli, Mary Magliocco, Michael A. Stack, Gina Montealegre, Karyl Barron, Danielle L. Fink, Douglas B. Kuhns, Stephen M. Hewitt, Lisa M. Arkin, Daniel S. Chertow, Helen C. Su, Luigi D. Notarangelo, Mariana J. Kaplan
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