Research Article
Abstract
Chronic obstructive pulmonary disease (COPD) is an incurable inflammatory lung disease that afflicts millions of people worldwide, and it is the fourth leading cause of death. Systemic comorbidities affecting the heart, skeletal muscle, bone, and metabolism are major contributors to morbidity and mortality. Given the surprising finding in large prospective clinical biomarker studies that peripheral white blood cell count is more closely associated with disease than inflammatory biomarkers, we probed the role of blood growth factors. Using the SHIP-1–deficient COPD mouse model, which manifests a syndrome of destructive lung disease and a complex of comorbid pathologies, we have identified a critical and unexpected role for granulocyte-CSF (G-CSF) in linking these conditions. Deletion of G-CSF greatly reduced airway inflammation and lung tissue destruction, and attenuated systemic inflammation, right heart hypertrophy, loss of fat reserves, and bone osteoporosis. In human clinical translational studies, bronchoalveolar lavage fluid of patients with COPD demonstrated elevated G-CSF levels. These studies suggest that G-CSF may play a central and unforeseen pathogenic role in COPD and its complex comorbidities, and identify G-CSF and its regulators as potential therapeutic targets.
Authors
Evelyn Tsantikos, Maverick Lau, Cassandra M.N. Castelino, Mhairi J. Maxwell, Samantha L. Passey, Michelle J. Hansen, Narelle E. McGregor, Natalie A. Sims, Daniel P. Steinfort, Louis B. Irving, Gary P. Anderson, Margaret L. Hibbs
Abstract
Tyro3, Axl, Mer (TAM) receptor tyrosine kinases reduce inflammatory, innate immune responses. We demonstrate that tumor-secreted protein S (Pros1), a Mer/Tyro3 ligand, decreased macrophage M1 cytokine expression in vitro and in vivo. In contrast, tumor cells with CRISPR-based deletion of Pros1 failed to inhibit M1 polarization. Tumor cell–associated Pros1 action was abrogated in macrophages from Mer- and Tyro3- but not Axl-KO mice. In addition, several other murine and human tumor cell lines suppressed macrophage M1 cytokine expression induced by IFN-γ and LPS. Investigation of the suppressive pathway demonstrated a role for PTP1b complexing with Mer. Substantiating the role of PTP1b, M1 cytokine suppression was also lost in macrophages from PTP1b-KO mice. Mice bearing Pros1-deficient tumors showed increased innate and adaptive immune infiltration, as well as increased median survival. TAM activation can also inhibit TLR-mediated M1 polarization. Treatment with resiquimod, a TLR7/8 agonist, did not improve survival in mice bearing Pros1-secreting tumors but doubled survival for Pros1-deleted tumors. The tumor-derived Pros1 immune suppressive system, like PD-L1, was cytokine responsive, with IFN-γ inducing Pros1 transcription and secretion. Inhibition of Pros1/TAM interaction represents a potential novel strategy to block tumor-derived immune suppression.
Authors
Eric Ubil, Laura Caskey, Alisha Holtzhausen, Debra Hunter, Charlotte Story, H. Shelton Earp
DNA methyltransferase expression in triple-negative breast cancer predicts sensitivity to decitabine
Abstract
Triple-negative breast cancer (TNBC) is a heterogeneous disease with poor prognosis that lacks targeted therapies, especially in patients with chemotherapy-resistant disease. Since DNA methylation-induced silencing of tumor suppressors is common in cancer, reversal of promoter DNA hypermethylation by 5-aza-2′-deoxycytidine (decitabine), an FDA-approved DNA methyltransferase (DNMT) inhibitor, has proven effective in treating hematological neoplasms. However, its antitumor effect varies in solid tumors, stressing the importance of identifying biomarkers predictive of therapeutic response. Here, we focused on the identification of biomarkers to select decitabine-sensitive TNBC through increasing our understanding of the mechanism of decitabine action. We showed that protein levels of DNMTs correlated with response to decitabine in patient-derived xenograft (PDX) organoids originating from chemotherapy-sensitive and -resistant TNBCs, suggesting DNMT levels as potential biomarkers of response. Furthermore, all 3 methytransferases, DNMT1, DNMT3A, and DNMT3B, were degraded following low-concentration, long-term decitabine treatment both in vitro and in vivo. The DNMT proteins could be ubiquitinated by the E3 ligase, TNF receptor–associated factor 6 (TRAF6), leading to lysosome-dependent protein degradation. Depletion of TRAF6 blocked decitabine-induced DNMT degradation, conferring resistance to decitabine. Our study suggests a potential mechanism of regulating DNMT protein degradation and DNMT levels as response biomarkers for DNMT inhibitors in TNBCs.
Authors
Jia Yu, Bo Qin, Ann M. Moyer, Somaira Nowsheen, Tongzheng Liu, Sisi Qin, Yongxian Zhuang, Duan Liu, Shijia W. Lu, Krishna R. Kalari, Daniel W. Visscher, John A. Copland, Sarah A. McLaughlin, Alvaro Moreno-Aspitia, Donald W. Northfelt, Richard J. Gray, Zhenkun Lou, Vera J. Suman, Richard Weinshilboum, Judy C. Boughey, Matthew P. Goetz, Liewei Wang
Abstract
The ability of the Cav1 channel inhibitor isradipine to slow the loss of substantia nigra pars compacta (SNc) dopaminergic (DA) neurons and the progression of Parkinson’s disease (PD) is being tested in a phase 3 human clinical trial. But it is unclear whether and how chronic isradipine treatment will benefit SNc DA neurons in vivo. To pursue this question, isradipine was given systemically to mice at doses that achieved low nanomolar concentrations in plasma, near those achieved in patients. This treatment diminished cytosolic Ca2+ oscillations in SNc DA neurons without altering autonomous spiking or expression of Ca2+ channels, an effect mimicked by selectively knocking down expression of Cav1.3 channel subunits. Treatment also lowered mitochondrial oxidant stress, reduced a high basal rate of mitophagy, and normalized mitochondrial mass — demonstrating that Cav1 channels drive mitochondrial oxidant stress and turnover in vivo. Thus, chronic isradipine treatment remodeled SNc DA neurons in a way that should not only diminish their vulnerability to mitochondrial challenges, but to autophagic stress as well.
Authors
Jaime N. Guzman, Ema Ilijic, Ben Yang, Javier Sanchez-Padilla, David Wokosin, Dan Galtieri, Jyothisri Kondapalli, Paul T. Schumacker, D. James Surmeier
Abstract
The superoxide-generating enzyme Nox2 contributes to hypertension and cardiovascular remodeling triggered by activation of the renin-angiotensin system. Multiple Nox2-expressing cells are implicated in angiotensin II (AngII)-induced pathophysiology, but the importance of Nox2 in leukocyte subsets is poorly understood. Here, we investigated the role of Nox2 in T cells, particularly Tregs. Mice globally deficient in Nox2 displayed increased numbers of Tregs in the heart at baseline whereas AngII-induced T-effector cell (Teffs) infiltration was inhibited. To investigate the role of Treg Nox2, we generated a mouse line with CD4-targeted Nox2 deficiency (Nox2fl/flCD4Cre+). These animals showed inhibition of AngII-induced hypertension and cardiac remodeling related to increased tissue-resident Tregs and reduction in infiltrating Teffs, including Th17 cells. The protection in Nox2fl/flCD4Cre+ mice was reversed by anti-CD25 Ab-depletion of Tregs. Mechanistically, Nox2–/y Tregs showed higher in vitro suppression of Teffs proliferation than WT Tregs, increased nuclear levels of FoxP3 and NF-κB, and enhanced transcription of CD25, CD39, and CD73. Adoptive transfer of Tregs confirmed that Nox2-deficient cells had greater inhibitory effects on AngII-induced heart remodeling than WT cells. These results identify a previously unrecognized role of Nox2 in modulating suppression of Tregs, which acts to enhance hypertension and cardiac remodeling.
Authors
Amber Emmerson, Silvia Cellone Trevelin, Heloise Mongue-Din, Pablo D. Becker, Carla Ortiz, Lesley A. Smyth, Qi Peng, Raul Elgueta, Greta Sawyer, Aleksandar Ivetic, Robert I. Lechler, Giovanna Lombardi, Ajay M. Shah
Blocking p62/SQSTM1-dependent SMN degradation ameliorates Spinal Muscular Atrophy disease phenotypes
Abstract
Spinal muscular atrophy (SMA), a degenerative motor neuron (MN) disease caused by loss of functional SMN protein due to SMN1 gene mutations, is a leading cause of infant mortality. Increasing SMN levels ameliorates the disease phenotype and is unanimously accepted as a therapeutic approach for SMA patients. The ubiquitin/proteasome system is known to regulate SMN protein levels; however whether autophagy controls SMN levels remains poorly explored. Here we show that SMN protein is degraded by autophagy. Pharmacological and genetic inhibition of autophagy increase SMN levels, while induction of autophagy decreases SMN. SMN degradation occurs via its interaction with the autophagy adapter p62/SQSTM1. We also show that SMA neurons display reduced autophagosome clearance, increased p62/ubiquitinated protein levels, and hyperactivated mTORC1 signaling. Importantly, reducing p62 levels markedly increases SMN and its binding partner gemin2, promotes MN survival and extends lifespan in fly and mouse SMA models revealing p62 as a new potential therapeutic target to treat SMA.
Authors
Natalia Rodriguez-Muela, Andrey Parkhitko, Tobias Grass, Rebecca M. Gibbs, Erika M. Norabuena, Norbert Perrimon, Rajat Singh, Lee L. Rubin
Abstract
Progression of chronic kidney disease associated with progressive fibrosis and impaired tubular epithelial regeneration is still an unmet biomedical challenge, because once chronic lesions have manifested, no effective therapies are available as of yet for clinical use. Prompted by various studies across multiple organs demonstrating that preconditioning regimens to induce endogenous regenerative mechanisms protect various organs from later incurring acute injuries, we here aimed to gain insights into the molecular mechanisms underlying successful protection and to explore whether such pathways could be utilized to inhibit progression of chronic organ injury. We identified a protective mechanism that is controlled by the transcription factor ARNT, which effectively inhibits progression of chronic kidney injury by transcriptional induction of ALK3, the principal mediator of anti-fibrotic and pro-regenerative BMP signaling responses. We further report that ARNT expression itself is controlled by the FKBP12/YY1 transcriptional repressor complex, and that disruption of such FKBP12/YY1 complexes by picomolar FK506 at sub-immunosuppressive doses increases ARNT expression, subsequently leading to homodimeric ARNT-induced ALK3 transcription. Direct targeting of FKBP12/YY1 with in vivo-morpholino approaches or small molecule inhibitors including GPI-1046 were equally effective to induce ARNT expression with subsequent activation of ALK3-dependent canonical BMP signaling responses and attenuated chronic organ failure in models of chronic kidney, but also cardiac and liver injuries. In summary, we report an organ protective mechanism, which can be pharmacologically modulated by immunophilin ligands FK506, GPI-1046 or therapeutically targeted by in vivo-morpholino approaches.
Authors
Björn Tampe, Désirée Tampe, Gunsmaa Nyamsuren, Friederike Klöpper, Gregor Rapp, Anne Kauffels, Thomas Lorf, Elisabeth M. Zeisberg, Gerhard A. Müller, Raghu Kalluri, Samy Hakroush, Michael Zeisberg
Abstract
Complications of diabetes affect tissues throughout body, including central nervous system. Epidemiological studies show that diabetic patients have increased risk of depression, anxiety, age-related cognitive decline and Alzheimer’s disease. Mice lacking insulin receptor in brain or on hypothalamic neurons display an array of metabolic abnormalities, however, the role of insulin action on astrocytes and neurobehaviors remains less well-studied. Here, we demonstrate that astrocytes are a direct insulin target in the brain and that knockout of IR on astrocytes causes increased anxiety and depressive-like behaviors in mice. This can be reproduced in part by deletion of IR on astrocytes in the nucleus accumbens. At a molecular level, loss of insulin signaling in astrocytes impaired tyrosine phosphorylation of Munc18c. This led to decreased exocytosis of ATP from astrocytes, resulting in decreased purinergic signaling on dopaminergic neurons. These reductions contributed to decreased dopamine release from brain slices. Central administration of ATP analogues could reverse depressive-like behaviors in mice with astrocyte IR knockout. Thus, astrocytic insulin signaling plays an important role in dopaminergic signaling, providing a potential mechanism by which astrocytic insulin action may contribute to increased rates of depression in people with diabetes, obesity and other insulin resistant states.
Authors
Weikang Cai, Chang Xue, Masaji Sakaguchi, Masahiro Konishi, Alireza Shirazian, Heather A. Ferris, Mengyao Li, Ruichao Yu, Andre Kleinridders, Emmanuel N. Pothos, C. Ronald Kahn
Abstract
Immunotherapy prolongs survival in only a subset of melanoma patients, highlighting the need to better understand the driver tumor microenvironment. We conducted bioinformatic analyses of 703 transcriptomes to probe the immune landscape of primary cutaneous melanomas in a population-ascertained cohort. We identified and validated 6 immunologically distinct subgroups, with the largest having the lowest immune scores and the poorest survival. This poor-prognosis subgroup exhibited expression profiles consistent with β-catenin–mediated failure to recruit CD141+ DCs. A second subgroup displayed an equally bad prognosis when histopathological factors were adjusted for, while 4 others maintained comparable survival profiles. The 6 subgroups were replicated in The Cancer Genome Atlas (TCGA) melanomas, where β-catenin signaling was also associated with low immune scores predominantly related to hypomethylation. The survival benefit of high immune scores was strongest in patients with double-WT tumors for BRAF and NRAS, less strong in BRAF-V600 mutants, and absent in NRAS (codons 12, 13, 61) mutants. In summary, we report evidence for a β-catenin–mediated immune evasion in 42% of melanoma primaries overall and in 73% of those with the worst outcome. We further report evidence for an interaction between oncogenic mutations and host response to melanoma, suggesting that patient stratification will improve immunotherapeutic outcomes.
Authors
Jérémie Nsengimana, Jon Laye, Anastasia Filia, Sally O’Shea, Sathya Muralidhar, Joanna Poźniak, Alastair Droop, May Chan, Christy Walker, Louise Parkinson, Joanne Gascoyne, Tracey Mell, Minttu Polso, Rosalyn Jewell, Juliette Randerson-Moor, Graham P. Cook, D. Timothy Bishop, Julia Newton-Bishop
Abstract
Receptor interacting protein kinase 1 (RIPK1) has important kinase-dependent and kinase-independent scaffolding functions that activate or prevent apoptosis or necroptosis in a cell context–dependent manner. The kinase activity of RIPK1 mediates hypothermia and lethality in a mouse model of TNF-induced shock, reflecting the hyperinflammatory state of systemic inflammatory response syndrome (SIRS), where the proinflammatory “cytokine storm” has long been viewed as detrimental. Here, we demonstrate that cytokine and chemokine levels did not predict survival and, importantly, that kinase-inactive Ripk1D138N/D138N hematopoietic cells afforded little protection from TNF- or TNF/zVAD-induced shock in reconstituted mice. Unexpectedly, RIPK1 kinase–inactive mice transplanted with WT hematopoietic cells remained resistant to TNF-induced shock, revealing that a nonhematopoietic lineage mediated protection. TNF-treated Ripk1D138N/D138N mice exhibited no significant increases in intestinal or vascular permeability, nor did they activate the clotting cascade. We show that TNF administration damaged the liver vascular endothelium and induced phosphorylated mixed lineage kinase domain-like (phospho-MLKL) reactivity in endothelial cells isolated from TNF/zVAD-treated WT, but not Ripk1D138N/D138N, mice. These data reveal that the tissue damage present in this SIRS model is reflected, in part, by breaks in the vasculature due to endothelial cell necroptosis and thereby predict that RIPK1 kinase inhibitors may provide clinical benefit to shock and/or sepsis patients.
Authors
Matija Zelic, Justine E. Roderick, Joanne A. O’Donnell, Jesse Lehman, Sung Eun Lim, Harish P. Janardhan, Chinmay M. Trivedi, Manolis Pasparakis, Michelle A. Kelliher
Copyright © 2018 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)