Chromatin remodeler HELLS maintains glioma stem cells through E2F3 and MYC

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Abstract

Glioblastoma represent universally lethal cancers, containing stem cell-like glioblastoma stem cells (GSCs). While neural stem cells (NSCs) are usually quiescent, single-cell studies suggest that proliferating glioblastoma cells reside in the GSC population. Interrogating in silico glioma databases for epigenetic regulators that correlate with cell cycle regulation, we identified the chromatin remodeler, HELLS, as a potential target in glioblastoma. GSCs preferentially expressed HELLS compared to their differentiated tumor progeny and non-malignant brain cells. Targeting HELLS disrupted GSC proliferation, survival, and self-renewal with induction of replication stress and DNA damage. Investigating potential molecular mechanisms downstream of HELLS revealed that HELLS interacted with the core oncogenic transcription factors, E2F3 and MYC, to regulate gene expression critical to GSC proliferation and maintenance. Supporting the interaction, HELLS expression strongly correlated with targets of E2F3 and MYC transcriptional activity in glioblastoma patients. Potential clinical significance of HELLS was reinforced by improved survival of tumor-bearing mice upon targeting HELLS and poor prognosis of glioma patients with elevated HELLS expression. Collectively, targeting HELLS may permit the functional disruption of the relatively undruggable MYC and E2F3 transcription factors and serve as a novel therapeutic paradigm for glioblastoma.

Authors

Guoxin Zhang, Zhen Dong, Briana C. Prager, Leo J. Y. Kim, Qiulian Wu, Ryan C. Gimple, Xiuxing Wang, Shideng Bao, Petra Hamerlik, Jeremy N. Rich

Iron deficiency anemia reduces cardiac contraction by downregulating RyR2 channels and suppressing SERCA pump activity

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Abstract

Iron deficiency is present in approximately 50% of heart failure (HF) patients. Large multi-center trials have shown that treatment of iron deficiency with intravenous iron benefits HF patients, but the underlying mechanisms are not known. To investigate the actions of iron deficiency on the heart, mice were fed an iron-depleted diet and some received intravenous ferric carboxymaltose (FCM), an iron supplementation used clinically. Iron-deficient animals became anemic and had reduced ventricular ejection fraction measured by magnetic resonance imaging. Ca2+ signaling, a pathway linked to the contractile deficit in failing hearts, was also significantly affected. Ventricular myocytes isolated from iron-deficient animals produced smaller Ca2+ transients from an elevated diastolic baseline, but had unchanged sarcoplasmic reticulum (SR) Ca2+-load, trigger L-type Ca2+ current or cytoplasmic Ca2+ buffering. Reduced fractional release from the SR was due to downregulated RyR2 channels, detected at protein and message level. The constancy of diastolic SR Ca2+-load is explained by reduced RyR2 permeability in combination with right-shifted SERCA activity due dephosphorylation of its regulator phospholamban. Supplementing iron levels with FCM restored normal Ca2+ signaling and ejection fraction. Thus, two Ca2+-handling proteins previously implicated in HF become functionally impaired in iron-deficiency anemia, but their activity is rescued by intravenous iron supplementation.

Authors

Yu Jin Chung, Antao Luo, Kyung Chan Park, Aminah Loonat, Samira Lakhal-Littleton, Peter A. Robbins, Pawel Swietach

IL-33-mediated IL-13 secretion by ST2⁺ Treg controls inflammation after lung injury

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Abstract

Acute respiratory distress syndrome is an often fatal disease that develops after acute lung injury and trauma. How released tissue damage signals, or alarmins, orchestrate early inflammatory events is poorly understood. Herein we reveal that IL-33, an alarmin sequestered in the lung epithelium, is required to limit inflammation after injury due to an unappreciated capacity to mediate Foxp3+ Treg control of local cytokines and myeloid populations. Specifically, Il33–/– mice are more susceptible to lung damage-associated morbidity and mortality that is typified by augmented levels of the proinflammatory cytokines and Ly6Chi monocytes in the bronchoalveolar lavage fluid. Local delivery of IL-33 at the time of injury is protective, but requires the presence of Treg cells. IL-33 stimulates both mouse and human Treg to secrete IL-13. Using Foxp3Cre x Il4/Il13fl/fl mice, we show that Treg expression of IL-13 is required to prevent mortality after acute lung injury by controlling local levels of G-CSF, IL-6, and MCP-1 and inhibiting accumulation of Ly6Chi monocytes. Our study identifies a new regulatory mechanism involving IL-33 and Treg secretion of IL-13 in response to tissue damage that is instrumental in limiting local inflammatory responses and may shape the myeloid compartment after lung injury.

Authors

Quan Liu, Gaelen K. Dwyer, Yifei Zhao, Huihua Li, Lisa R. Mathews, Anish Bhaswanth Chakka, Uma R. Chandran, Jake A. Demetris, John F. Alcorn, Keven M. Robinson, Luis A. Ortiz, Bruce Pitt, Angus W. Thomson, Ming-Hui Fan, Timothy R. Billiar, Heth R. Turnquist

CXCR3 regulates CD4⁺ T cell cardiotropism in pressure overload induced cardiac dysfunction

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Abstract

Heart failure (HF) is associated in humans and mice with increased circulating levels of CXCL9 and CXCL10, chemokine ligands of the CXCR3 receptor, predominantly expressed on CD4+ T helper type 1 (Th1) cells. Chemokine engagement of receptors is required for T cell integrin activation and recruitment to sites of inflammation. Th1 cells drive adverse cardiac remodeling in pressure overload induced cardiac dysfunction, and mice lacking the integrin ligand ICAM-1 show defective T cell recruitment to the heart. Here, we show that CXCR3+ T cells infiltrate the heart in humans and mice with pressure overload induced cardiac dysfunction. Genetic deletion of CXCR3 disrupts CD4+ T cell heart infiltration and prevents adverse cardiac remodeling. We demonstrate that cardiac myeloid cells that include resident and infiltrated macrophages, and cardiac fibroblasts are the source of CXCL9 and CXCL10; which, mechanistically promote Th1 cell adhesion to ICAM-1 under shear conditions in a CXCR3-dependent manner. Our findings identify a previously unrecognized role for CXCR3 in Th1 cell recruitment into the heart in pressure overload induced cardiac dysfunction.

Authors

Njabulo Ngwenyama, Ane M. Salvador, Francisco Velázquez, Tania Nevers, Alexander Levy, Mark J. Aronovitz, Andrew D. Luster, Gordon S. Huggins, Pilar Alcaide

Targeting MRTF/SRF in CAP2-dependent dilated cardiomyopathy delays disease onset

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Abstract

About one-third of dilated cardiomyopathy (DCM) cases are caused by mutations in sarcomere or cytoskeletal proteins. Yet treating the cytoskeleton directly is not possible because drugs that bind to actin are not well tolerated. Mutations in the actin binding protein CAP2 can cause DCM and knockout mice, either whole body (CAP2 KO) or cardiomyocyte-specific knockouts (CAP2 CKO), develop DCM with cardiac conduction disease. RNA-seq analysis of CAP2 KO hearts and isolated cardiomyocytes revealed over-activation of fetal genes including serum response factor (SRF) regulated genes such as Myl9 and Acta2 prior to the emergence of cardiac disease. To test if we could treat CAP2 KO mice, we synthesized and tested the SRF inhibitor CCG-1423-8u. CCG-1423-8u reduced expression of the SRF targets Myl9 and Acta2, as well as the biomarker of heart failure, Nppa. The median survival of CAP2 CKO mice was 98 days, while CCG-1423-8u treated CKO mice survived for 116 days and also maintain normal cardiac function longer. These results suggest that some forms of sudden cardiac death and cardiac conduction disease are under cytoskeletal stress and that inhibiting signaling through SRF may benefit DCM by reducing cytoskeletal stress.

Authors

Yao Xiong, Kenneth C. Bedi, Simon Berritt, Thomas G. Brooks, Bennette K. Attipoe, Kevin Wang, Kenneth B. Margulies, Jeffrey Field

PD-L1 on invasive fibroblasts drives fibrosis in a humanized model of idiopathic pulmonary fibrosis

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Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with unremitting extracellular matrix deposition, leading to a distortion of pulmonary architecture and impaired gas exchange. Fibroblasts from IPF patients acquire an invasive phenotype that is essential for progressive fibrosis. Here, we performed RNA-seq analysis on invasive and non-invasive fibroblasts and found that the immune checkpoint ligand CD274 (PD-L1) was up-regulated on invasive lung fibroblasts and was required for the invasive phenotype of lung fibroblasts, is regulated by P53 and FAK, and drives lung fibrosis in a humanized IPF model in mice. Activating CD274 in IPF fibroblasts promoted invasion in vitro and pulmonary fibrosis in vivo. CD274 knockout in IPF fibroblasts and targeting CD274 by FAK inhibition or CD274 neutralizing antibodies blunted invasion and attenuated fibrosis, suggesting that CD274 may be a novel therapeutic target in IPF.

Authors

Yan Geng, Xue Liu, Jiurong Liang, David M. Habiel, Kulur Vrishika, Ana Lucia Coelho, Nan Deng, Ting Xie, Yizhou Wang, Ningshan Liu, Guanling Huang, Adrianne Kurkciyan, Zhenqiu Liu, Jie Tang, Cory M. Hogaboam, Dianhua Jiang, Paul W. Noble

Longitudinally persistent cerebrospinal fluid B-cells can resist treatment in multiple sclerosis

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Abstract

B-cells are key contributors to chronic autoimmune pathology in multiple sclerosis (MS). Clonally related B-cells exist in the cerebrospinal fluid (CSF), meninges, and central nervous system (CNS) parenchyma of MS patients. We sought to investigate the presence of clonally related B-cells over time by performing immunoglobulin heavy chain variable region repertoire sequencing on B-cells from longitudinally collected blood and CSF samples of MS patients (n=10). All patients were untreated at the time of the initial sampling; the majority (n=7) were treated with immune modulating therapies 1.2 (+/-0.3 SD) years later during the second sampling. We found clonal persistence of B-cells in the CSF of five patients; these B-cells were frequently immunoglobulin (Ig) class-switched and CD27+. We identified specific blood B-cell subsets that appear to provide input into CNS repertoires over time. We demonstrate complex patterns of clonal B-cell persistence in CSF and blood, even in patients on immune modulating therapy. Our findings support the concept that peripheral B-cell activation and CNS-compartmentalized immune mechanisms can in part therapy-resistant.

Authors

Ariele L. Greenfield, Ravi Dandekar, Akshaya Ramesh, Erica L. Eggers, Hao Wu, Sarah Laurent, William Harkin, Natalie S. Pierson, Martin S. Weber, Roland G. Henry, Antje Bischof, Bruce A.C. Cree, Stephen L. Hauser, Michael R. Wilson, H.-Christian von Büdingen

Lymphocyte mass cytometry identifies a CD3^-CD4⁺ cells subset with a potential role in psoriasis

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Abstract

Psoriasis (PS) is a systemic, immune-mediated inflammatory disorder. However, the whole lymphocyte compartment and the potential pathologies of PS have not been fully characterized. In the present study, we examined whole lymphocyte subsets and signal transduction proteins using high-dimensional single-cell mass cytometry and a bioinformatics pipeline for an in-depth characterization of the immune cell subsets and protein profiles involved in pathways in the peripheral blood of patients with PS. We identified 15 major immune cell populations in T cell lineages, and characterized various CD3+CD4+T helper and CD3+CD8+T cytotoxic cell populations simultaneously across 24 leukocyte markers and 7 proteins related to the signal transduction pathways. High-dimensional analysis identified three new subsets that are abundant in PS peripheral blood, resembling CD3-CD4+ lymphoid tissue inducer cells, Tc17, and CD8+CXCR3+ Tregs. We confirmed the CD3-CD4+ cells, and their features and functions, in an independent PS cohort. The use of single-cell mass cytometry allows systemic-level characterization of lymphocyte subpopulations and dysregulated signaling pathways in the blood of patients with PS, identifying abnormalities of different immune cell subsets. We validated that the CD3-CD4+ cells had elevated OX40 and decreased FRA2 expression, which were positively associated with the psoriasis area and severity index.

Authors

Ruru Guo, Ting Zhang, Xinyu Meng, Zhen Lin, Jinran Lin, Yu Gong, Xuesong Liu, Yuetian Yu, Guilin Zhao, Xianting Ding, Xiaoxiang Chen, Liangjing Lu

A unique androgen excess signature in idiopathic intracranial hypertension is linked to cerebrospinal fluid dynamics

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Abstract

Idiopathic intracranial hypertension (IIH) is a condition of unknown etiology, characterized by elevated intracranial pressure frequently manifesting with chronic headaches and visual loss. Similar to polycystic ovary syndrome (PCOS), IIH predominantly affects obese women of reproductive age. In this study, we comprehensively examined the systemic and cerebrospinal fluid (CSF) androgen metabolome in women with IIH in comparison to sex-, body mass index- and age-matched control groups with either simple obesity and PCOS, i.e. obesity and androgen excess. IIH women showed a pattern of androgen excess distinct to that observed in PCOS and simple obesity, with increased serum testosterone, and increased CSF testosterone and androstenedione. Human choroid plexus expressed the androgen receptor, alongside the androgen-activating enzyme aldoketoreductase type 1C3. We show that in a rat choroid plexus cell line testosterone significantly enhanced the activity of Na+/K+ ATPase, a surrogate of CSF secretion. We demonstrate that IIH patients have a unique signature of androgen excess and provide evidence that androgens can modulate CSF secretion via the choroid plexus. These findings implicate androgen excess as a potential causal driver and therapeutic target in IIH.

Authors

Michael W. O'Reilly, Connar S.J. Westgate, Catherine Hornby, Hannah Botfield, Angela E. Taylor, Keira Markey, James L. Mitchell, William J. Scotton, Susan P. Mollan, Andreas Yiangou, Carl Jenkinson, Lorna C. Gilligan, Mark Sherlock, James Gibney, Jeremy W. Tomlinson, Gareth G. Lavery, David J. Hodson, Wiebke Arlt, Alexandra J. Sinclair

Systematic testing and specificity mapping of alloantigen-specific chimeric antigen receptors in T regulatory cells

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Abstract

Chimeric antigen receptor (CAR) technology can be used to engineer the antigen-specificity of regulatory T cells (Tregs) and improve their potency as an adoptive cell therapy in multiple disease models. As synthetic receptors, CARs carry the risk of immunogenicity, particularly when derived from non-human antibodies. Using an HLA-A*02:01-specific CAR (A2-CAR) encoding a single-chain Fv derived from a mouse antibody, we developed a panel of 20 humanized (h)A2-CARs. Systematic testing demonstrated variations in expression, ability to bind HLA-A*02:01, and stimulate human Treg suppression in vitro. In addition, we developed a new method to comprehensively map the alloantigen-specificity of CARs, revealing that humanization reduced HLA-A cross reactivity. In vivo bioluminescence imaging showed rapid trafficking and persistence of hA2-CAR Tregs in A2-expressing allografts, with eventual migration to draining lymph nodes. Adoptive transfer of hA2-CAR Tregs suppressed HLA-A2+ cell mediated xenogeneic graft-versus-host disease and diminished rejection of human HLA-A2+ skin allografts. These data provide a platform for systematic development and specificity testing of humanized alloantigen-specific CARs which can be used to engineer specificity and homing of therapeutic Tregs.

Authors

Nicholas A.J. Dawson, Caroline Lamarche, Romy E. Hoeppli, Peter Bergqvist, Vivian Fung, Emma McIver, Qing Huang, Jana Gillies, Madeleine Speck, Paul C. Orban, Jonathan W. Bush, Majid Mojibian, Megan K. Levings