Latest issue: August 6, 2020

In the issue

Abstract

Macrolide antibiotics exert antiinflammatory effects; however, little is known regarding their immunomodulatory mechanisms. In this study, using 2 distinct mouse models of mucosal inflammatory disease (LPS-induced acute lung injury and ligature-induced periodontitis), we demonstrated that the antiinflammatory action of erythromycin (ERM) is mediated through upregulation of the secreted homeostatic protein developmental endothelial locus-1 (DEL-1). Consistent with the anti–neutrophil recruitment action of endothelial cell–derived DEL-1, ERM inhibited neutrophil infiltration in the lungs and the periodontium in a DEL-1–dependent manner. Whereas ERM (but not other antibiotics, such as josamycin and penicillin) protected against lethal pulmonary inflammation and inflammatory periodontal bone loss, these protective effects of ERM were abolished in Del1-deficient mice. By interacting with the growth hormone secretagogue receptor and activating JAK2 in human lung microvascular endothelial cells, ERM induced DEL-1 transcription that was mediated by MAPK p38 and was CCAAT/enhancer binding protein–β dependent. Moreover, ERM reversed IL-17–induced inhibition of DEL-1 transcription, in a manner that was dependent not only on JAK2 but also on PI3K/AKT signaling. Because DEL-1 levels are severely reduced in inflammatory conditions and with aging, the ability of ERM to upregulate DEL-1 may lead to a novel approach for the treatment of inflammatory and aging-related diseases.

Authors

Tomoki Maekawa, Hikaru Tamura, Hisanori Domon, Takumi Hiyoshi, Toshihito Isono, Daisuke Yonezawa, Naoki Hayashi, Naoki Takahashi, Koichi Tabeta, Takeyasu Maeda, Masataka Oda, Athanasios Ziogas, Vasileia Ismini Alexaki, Triantafyllos Chavakis, Yutaka Terao, George Hajishengallis

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Abstract

Noonan syndrome with multiple lentigines (NSML) is a rare autosomal dominant disorder that presents with cardio-cutaneous-craniofacial defects. Hypertrophic cardiomyopathy (HCM) represents the major life-threatening presentation in NSML. Mutations in the PTPN11 gene that encodes for the protein tyrosine phosphatase (PTP), SHP2, represents the predominant cause of HCM in NSML. NSML-associated PTPN11 mutations render SHP2 catalytically inactive with an “open” conformation. NSML-associated PTPN11 mutations cause hypertyrosyl phosphorylation of the transmembrane glycoprotein, protein zero-related (PZR), resulting in increased SHP2 binding. Here we show that NSML mice harboring a tyrosyl phosphorylation–defective mutant of PZR (NSML/PZRY242F) that is defective for SHP2 binding fail to develop HCM. Enhanced AKT/S6 kinase signaling in heart lysates of NSML mice was reversed in NSML/PZRY242F mice, demonstrating that PZR/SHP2 interactions promote aberrant AKT/S6 kinase activity in NSML. Enhanced PZR tyrosyl phosphorylation in the hearts of NSML mice was found to drive myocardial fibrosis by engaging an Src/NF-κB pathway, resulting in increased activation of IL-6. Increased expression of IL-6 in the hearts of NSML mice was reversed in NSML/PZRY242F mice, and PZRY242F mutant fibroblasts were defective for IL-6 secretion and STAT3-mediated fibrogenesis. These results demonstrate that NSML-associated PTPN11 mutations that induce PZR hypertyrosyl phosphorylation trigger pathophysiological signaling that promotes HCM and cardiac fibrosis.

Authors

Jae-Sung Yi, Sravan Perla, Liz Enyenihi, Anton M. Bennett

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Abstract

Triple-negative breast cancers (TNBCs) are heterogeneous and aggressive, with high mortality rates. TNBCs frequently respond to chemotherapy, yet many patients develop chemoresistance. The molecular basis and roles for tumor cell–stromal crosstalk in establishing chemoresistance are complex and largely unclear. Here we report molecular studies of paired TNBC patient–derived xenografts (PDXs) established before and after the development of chemoresistance. Interestingly, the chemoresistant model acquired a distinct KRASQ61R mutation that activates K-Ras. The chemoresistant KRAS-mutant model showed gene expression and proteomic changes indicative of altered tumor cell metabolism. Specifically, KRAS-mutant PDXs exhibited increased redox ratios and decreased activation of AMPK, a protein involved in responding to metabolic homeostasis. Additionally, the chemoresistant model exhibited increased immunosuppression, including expression of CXCL1 and CXCL2, cytokines responsible for recruiting immunosuppressive leukocytes to tumors. Notably, chemoresistant KRAS-mutant tumors harbored increased numbers of granulocytic myeloid-derived suppressor cells (gMDSCs). Interestingly, previously established Ras/MAPK-associated gene expression signatures correlated with myeloid/neutrophil-recruiting CXCL1/2 expression and negatively with T cell–recruiting chemokines (CXCL9/10/11) across patients with TNBC, even in the absence of KRAS mutations. MEK inhibition induced tumor suppression in mice while reversing metabolic and immunosuppressive phenotypes, including chemokine production and gMDSC tumor recruitment in the chemoresistant KRAS-mutant tumors. These results suggest that Ras/MAPK pathway inhibitors may be effective in some breast cancer patients to reverse Ras/MAPK-driven tumor metabolism and immunosuppression, particularly in the setting of chemoresistance.

Authors

Derek A. Franklin, Joe T. Sharick, Paula I. Ericsson-Gonzalez, Violeta Sanchez, Phillip T. Dean, Susan R. Opalenik, Stefano Cairo, Jean-Gabriel Judde, Michael T. Lewis, Jenny C. Chang, Melinda E. Sanders, Rebecca S. Cook, Melissa C. Skala, Jennifer Bordeaux, Jehovana Orozco Bender, Christine Vaupel, Gary Geiss, Douglas Hinerfeld, Justin M. Balko

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Abstract

Acute rejection (AR) in renal transplantation is an established risk factor for reduced allograft survival. Molecules with regulatory control among immune pathways of AR that are inadequately suppressed, despite standard-of-care immunosuppression, could serve as important targets for therapeutic manipulation to prevent rejection. Here, an integrative, network-based computational strategy incorporating gene expression and genotype data of human renal allograft biopsy tissue was applied, to identify the master regulators — the key driver genes (KDGs) — within dysregulated AR pathways. A 982–meta-gene signature with differential expression in AR versus non-AR was identified from a meta-analysis of microarray data from 735 human kidney allograft biopsy samples across 7 data sets. Fourteen KDGs were derived from this signature. Interrogation of 2 publicly available databases identified compounds with predicted efficacy against individual KDGs or a key driver–based gene set, respectively, which could be repurposed for AR prevention. Minocycline, a tetracycline antibiotic, was chosen for experimental validation in a murine cardiac allograft model of AR. Minocycline attenuated the inflammatory profile of AR compared with controls and when coadministered with immunosuppression prolonged graft survival. This study demonstrates that a network-based strategy, using expression and genotype data to predict KDGs, assists target prioritization for therapeutics in renal allograft rejection.

Authors

Zhengzi Yi, Karen L. Keung, Li Li, Min Hu, Bo Lu, Leigh Nicholson, Elvira Jimenez-Vera, Madhav C. Menon, Chengguo Wei, Stephen Alexander, Barbara Murphy, Philip J. O’Connell, Weijia Zhang

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Abstract

T follicular helper (Tfh) cell migration into germinal centers (GCs) is essential for the generation of GC B cells and antibody responses to T cell–dependent (TD) antigens. This process requires interactions between lymphocyte function–associated antigen 1 (LFA-1) on Tfh cells and ICAMs on B cells. The mechanisms underlying defective antibody responses to TD antigens in DOCK8 deficiency are incompletely understood. We show that mice selectively lacking DOCK8 in T cells had impaired IgG antibody responses to TD antigens, decreased GC size, and reduced numbers of GC B cells. However, they developed normal numbers of Tfh cells with intact capacity for driving B cell differentiation into a GC phenotype in vitro. Notably, migration of DOCK8-deficient T cells into GCs was defective. Following T cell receptor (TCR)/CD3 ligation, DOCK8-deficient T cells had impaired LFA-1 activation and reduced binding to ICAM-1. Our results therefore indicate that DOCK8 is important for LFA-1–dependent positioning of Tfh cells in GCs, and thereby the generation of GC B cells and IgG antibody responses to TD antigen.

Authors

Erin Janssen, Mira Tohme, Jordan Butts, Sophie Giguere, Peter T. Sage, Francisco E. Velázquez, Christy Kam, Elena Milin, Mrinmoy Das, Ali Sobh, Salem Al-Tamemi, Francis W. Luscinskas, Facundo Batista, Raif S. Geha

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Abstract

We identified a potentially novel homozygous duplication involving the promoter region and exons 1–4 of the gene encoding type 2 cardiac ryanodine receptor (RYR2) that is responsible for highly penetrant, exertion-related sudden deaths/cardiac arrests in the Amish community without an overt phenotype to suggest RYR2-mediated catecholaminergic polymorphic ventricular tachycardia (CPVT). Homozygous RYR2 duplication (RYR2-DUP) induced pluripotent stem cell cardiomyocytes (iPSC-CMs) were generated from 2 unrelated patients. There was no difference in baseline Ca2+ handling measurements between WT-iPSC-CM and RYR2-DUP-iPSC-CM lines. However, compared with WT-iPSC-CMs, both patient lines demonstrated a dramatic reduction in caffeine-stimulated and isoproterenol-stimulated (ISO-stimulated) Ca2+ transient amplitude, suggesting RyR2 loss of function. There was a greater than 50% reduction in RYR2 transcript/RyR2 protein expression in both patient iPSC-CMs compared with WT. Delayed afterdepolarization was observed in the RYR2-DUP-iPSC-CMs but not in the WT-iPSC-CMs. Compared with WT-iPSC-CMs, there was significantly elevated arrhythmic activity in the RYR2-DUP-iPSC-CMs in response to ISO. Nadolol, propranolol, and flecainide reduced erratic activity by 8.5-fold, 6.8-fold, and 2.4-fold, respectively, from ISO challenge. Unlike the gain-of-function mechanism observed in RYR2-mediated CPVT, the homozygous multiexon duplication precipitated a dramatic reduction in RYR2 transcription and RyR2 protein translation, a loss of function in calcium handling, and a calcium-induced calcium release apparatus that is insensitive to catecholamines and caffeine.

Authors

David J. Tester, CS John Kim, Samantha K. Hamrick, Dan Ye, Bailey J. O’Hare, Hannah M. Bombei, Kristi K. Fitzgerald, Carla M. Haglund-Turnquist, Dianne L. Atkins, Luis A. Ochoa Nunez, Ian Law, Joel Temple, Michael J. Ackerman

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Abstract

M3 muscarinic acetylcholine receptor (M3R) is one of the autoantigens associated with Sjögren’s syndrome (SS) and is localized in exocrine glands where disease-specific inflammation occurs. The inflammatory lesion is characterized by infiltration of CD4+ T cells, including clonally expanded Th17 cells. We undertook this study to identify circulating M3R-specific Th17 cells and to determine functional properties of those cells. Using the enzyme-linked immunospot assay (ELISpot) method, we identified M3R-reactive Th17 cells in the peripheral blood of patients with primary SS (pSS). Among 10 examined pSS patients, 10 healthy subjects (HS), and 5 IgG4-related disease (IgG4-RD) patients, M3R-reactive IL-17 secreting cells were significantly increased in 5 pSS patients specifically. The most common T cell epitope, which was analyzed and confirmed by coculture of isolated CD4+ T cells with antigen presenting cells plus M3R peptides in vitro, was peptide 83-95 of M3R. Peptide recognition was partly in an HLA-DR–restricted manner, confirmed by blocking assay. M3R-reactive Th17 cells positivity correlated with higher titers of anti-M3R antibodies, whose systemic disease activity score tended to be higher. Our studies highlight the role of tissue-specific autoantigen–derived circulating Th17 cells in pSS, for which further work might lead to antigen-specific targeted therapy.

Authors

Saori Abe, Hiroto Tsuboi, Hanae Kudo, Hiromitsu Asashima, Yuko Ono, Fumika Honda, Hiroyuki Takahashi, Mizuki Yagishita, Shinya Hagiwara, Yuya Kondo, Isao Matsumoto, Takayuki Sumida

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Abstract

Arrestin domain containing 3 (ARRDC3) represents a newly discovered α-arrestin involved in obesity, inflammation, and cancer. Here, we demonstrate a proinflammation role of ARRDC3 in Helicobacter pylori–associated gastritis. Increased ARRDC3 was detected in gastric mucosa of patients and mice infected with H. pylori. ARRDC3 in gastric epithelial cells (GECs) was induced by H. pylori, regulated by ERK and PI3K-AKT pathways in a cagA-dependent manner. Human gastric ARRDC3 correlated with the severity of gastritis, and mouse ARRDC3 from non-BM–derived cells promoted gastric inflammation. This inflammation was characterized by the CXCR2-dependent influx of CD45+CD11b+Ly6C–Ly6G+ neutrophils, whose migration was induced via the ARRDC3-dependent production of CXCL2 by GECs. Importantly, gastric inflammation was attenuated in Arrdc3–/– mice but increased in protease-activated receptor 1–/– (Par1–/–) mice. Mechanistically, ARRDC3 in GECs directly interacted with PAR1 and negatively regulated PAR1 via ARRDC3-mediated lysosomal degradation, which abrogated the suppression of CXCL2 production and following neutrophil chemotaxis by PAR1, thereby contributing to the development of H. pylori–associated gastritis. This study identifies a regulatory network involving H. pylori, GECs, ARRDC3, PAR1, and neutrophils, which collectively exert a proinflammatory effect within the gastric microenvironment. Efforts to inhibit this ARRDC3-dependent pathway may provide valuable strategies in treating of H. pylori–associated gastritis.

Authors

Yu-gang Liu, Yong-sheng Teng, Zhi-guo Shan, Ping Cheng, Chuan-jie Hao, Yi-pin Lv, Fang-yuan Mao, Shi-ming Yang, Weisan Chen, Yong-liang Zhao, Nan You, Quan-ming Zou, Yuan Zhuang

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Abstract

Kidney disease is one of the most devastating complications of diabetes, and tubular atrophy predicts diabetic kidney disease (DKD) progression to end-stage renal disease. We have proposed that fatty acids bound to albumin contribute to tubular atrophy by inducing lipotoxicity, after filtration across damaged glomeruli, and subsequent proximal tubule reabsorption by a fatty acid transport protein-2–dependent (FATP2-dependent) mechanism. To address this possibility, genetic (Leprdb/db eNOS–/–) and induced (high-fat diet plus low-dose streptozotocin) mouse models of obesity and DKD were bred with global FATP2 gene–deleted mice (Slc27a2) and then phenotyped. DKD-prone mice with the Slc27a2–/– genotype demonstrated normalization of glomerular filtration rate, reduced albuminuria, improved kidney histopathology, and longer life span compared with diabetic Slc27a2+/+ mice. Genetic and induced DKD-prone Slc27a2–/– mice also exhibited markedly reduced fasting plasma glucose, with mean values approaching euglycemia, despite increased obesity and decreased physical activity. Glucose lowering in DKD-prone Slc27a2–/– mice was accompanied by β cell hyperplasia and sustained insulin secretion. Together, our data indicate that FATP2 regulates DKD pathogenesis by a combined lipotoxicity and glucotoxicity (glucolipotoxicity) mechanism.

Authors

Shenaz Khan, Robert Gaivin, Caroline Abramovich, Michael Boylan, Jorge Calles, Jeffrey R. Schelling

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Abstract

DCs are a critical component of immune responses in cancer primarily due to their ability to cross-present tumor-associated antigens. Cross-presentation by DCs in cancer is impaired, which may represent one of the obstacles for the success of cancer immunotherapies. Here, we report that polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) blocked cross-presentation by DCs without affecting direct presentation of antigens by these cells. This effect did not require direct cell-cell contact and was associated with transfer of lipids. Neutrophils (PMN) and PMN-MDSC transferred lipid to DCs equally well; however, PMN did not affect DC cross-presentation. PMN-MDSC generate oxidatively truncated lipids previously shown to be involved in impaired cross-presentation by DCs. Accumulation of oxidized lipids in PMN-MDSC was dependent on myeloperoxidase (MPO). MPO-deficient PMN-MDSC did not affect cross-presentation by DCs. Cross-presentation of tumor-associated antigens in vivo by DCs was improved in MDSC-depleted or tumor-bearing MPO-KO mice. Pharmacological inhibition of MPO in combination with checkpoint blockade reduced tumor progression in different tumor models. These data suggest MPO-driven lipid peroxidation in PMN-MDSC as a possible non–cell autonomous mechanism of inhibition of antigen cross-presentation by DCs and propose MPO as potential therapeutic target to enhance the efficacy of current immunotherapies for patients with cancer.

Authors

Alessio Ugolini, Vladimir A. Tyurin, Yulia Y. Tyurina, Evgenii N. Tcyganov, Laxminarasimha Donthireddy, Valerian E. Kagan, Dmitry I. Gabrilovich, Filippo Veglia

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Abstract

One of the major challenges in using pancreatic cancer patient–derived organoids (PDOs) in precision oncology is the time from biopsy to functional characterization. This is particularly true for endoscopic ultrasound-guided fine-needle aspiration biopsies, typically resulting in specimens with limited tumor cell yield. Here, we tested conditioned media of individual PDOs for cell-free DNA to detect driver mutations already early on during the expansion process to accelerate the genetic characterization of PDOs as well as subsequent functional testing. Importantly, genetic alterations detected in the PDO supernatant, collected as early as 72 hours after biopsy, recapitulate the mutational profile of the primary tumor, indicating suitability of this approach to subject PDOs to drug testing in a reduced time frame. In addition, we demonstrated that this workflow was practicable, even in patients for whom the amount of tumor material was not sufficient for molecular characterization by established means. Together, our findings demonstrate that generating PDOs from very limited biopsy material permits molecular profiling and drug testing. With our approach, this can be achieved in a rapid and feasible fashion with broad implications in clinical practice.

Authors

Zahra Dantes, Hsi-Yu Yen, Nicole Pfarr, Christof Winter, Katja Steiger, Alexander Muckenhuber, Alexander Hennig, Sebastian Lange, Thomas Engleitner, Rupert Öllinger, Roman Maresch, Felix Orben, Irina Heid, Georgios Kaissis, Kuangyu Shi, Geoffrey Topping, Fabian Stögbauer, Matthias Wirth, Katja Peschke, Aristeidis Papargyriou, Massoud Rezaee-Oghazi, Karin Feldmann, Arlett P.G. Schäfer, Raphela Ranjan, Clara Lubeseder-Martellato, Daniel E. Stange, Thilo Welsch, Marc Martignoni, Güralp O. Ceyhan, Helmut Friess, Alexander Herner, Lucia Liotta, Matthias Treiber, Guido von Figura, Mohamed Abdelhafez, Peter Klare, Christoph Schlag, Hana Algül, Jens Siveke, Rickmer Braren, Gregor Weirich, Wilko Weichert, Dieter Saur, Roland Rad, Roland M. Schmid, Günter Schneider, Maximilian Reichert

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Abstract

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a relative paucity of cancer cells that are surrounded by an abundance of nontumor cells and extracellular matrix, known as stroma. The interaction between stroma and cancer cells contributes to poor outcome, but how proteins from these individual compartments drive aggressive tumor behavior is not known. Here, we report the proteomic analysis of laser-capture microdissected (LCM) PDAC samples. We isolated stroma, tumor, and bulk samples from a cohort with long- and short-term survivors. Compartment-specific proteins were measured by mass spectrometry, yielding what we believe to be the largest PDAC proteome landscape to date. These analyses revealed that, in bulk analysis, tumor-derived proteins were typically masked and that LCM was required to reveal biology and prognostic markers. We validated tumor CALB2 and stromal COL11A1 expression as compartment-specific prognostic markers. We identified and functionally addressed the contributions of the tumor cell receptor EPHA2 to tumor cell viability and motility, underscoring the value of compartment-specific protein analysis in PDAC.

Authors

Tessa Y.S. Le Large, Giulia Mantini, Laura L. Meijer, Thang V. Pham, Niccola Funel, Nicole C.T. van Grieken, Bart Kok, Jaco Knol, Hanneke W.M. van Laarhoven, Sander R. Piersma, Connie R. Jimenez, G. Kazemier, Elisa Giovannetti, Maarten F. Bijlsma

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Abstract

BACKGROUND Our objective was to investigate whether primary Sjögren’s syndrome (pSS) is associated with multiple system atrophy (MSA).METHODS We performed a retrospective cohort study assessing (a) rates of MSA in a cohort of patients with pSS and (b) rates of pSS in a cohort of patients with MSA. These data were compared with rates in respective control groups. We additionally reviewed the neuropathologic findings in 2 patients with pSS, cerebellar degeneration, parkinsonism, and autonomic dysfunction.RESULTS Our cohort of 308 patients with pSS had a greater incidence of MSA compared with 4 large population-based studies and had a significantly higher prevalence of at least probable MSA (1% vs. 0%, P = 0.02) compared with 776 patients in a control cohort of patients with other autoimmune disorders. Our cohort of 26 autopsy-proven patients with MSA had a significantly higher prevalence of pSS compared with a cohort of 115 patients with other autopsy-proven neurodegenerative disorders (8% vs. 0%, P = 0.03). The 2 patients we described with pSS and progressive neurodegenerative disease showed classic MSA pathology at autopsy.CONCLUSION Our findings provide evidence for an association between MSA and pSS that is specific to both pSS, among autoimmune disorders, and MSA, among neurodegenerative disorders. The 2 cases we describe of autopsy-proven MSA support that MSA pathology can explain neurologic disease in a subset of patients with pSS. These findings together support the hypothesis that systemic autoimmune disease plays a role in neurodegeneration.FUNDING The Michigan Brain Bank is supported in part through NIH grant P30AG053760.

Authors

Kyle S. Conway, Sandra Camelo-Piragua, Amanda Fisher-Hubbard, William R. Perry, Vikram G. Shakkottai, Sriram Venneti

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Abstract

Coronavirus disease 2019 (COVID-19), the disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has resulted in a global pandemic and a disruptive health crisis. COVID-19–related morbidity and mortality have been attributed to an exaggerated immune response. The role of complement activation and its contribution to illness severity is being increasingly recognized. Here, we summarize current knowledge about the interaction of coronaviruses with the complement system. We posit that (a) coronaviruses activate multiple complement pathways; (b) severe COVID-19 clinical features often resemble complementopathies; (c) the combined effects of complement activation, dysregulated neutrophilia, endothelial injury, and hypercoagulability appear to be intertwined to drive the severe features of COVID-19; (d) a subset of patients with COVID-19 may have a genetic predisposition associated with complement dysregulation; and (e) these observations create a basis for clinical trials of complement inhibitors in life-threatening illness.

Authors

Anuja Java, Anthony J. Apicelli, M. Kathryn Liszewski, Ariella Coler-Reilly, John P. Atkinson, Alfred H.J. Kim, Hrishikesh S. Kulkarni

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Abstract

The bromodomain and extraterminal (BET) family comprises epigenetic reader proteins that are important regulators of inflammatory and hypertrophic gene expression in the heart. We previously identified the activation of proinflammatory gene networks as a key early driver of dilated cardiomyopathy (DCM) in transgenic mice expressing a mutant form of phospholamban (PLNR9C) — a genetic cause of DCM in humans. We hypothesized that BETs coactivate this inflammatory process, representing a critical node in the progression of DCM. To test this hypothesis, we treated PLNR9C or age-matched WT mice longitudinally with the small molecule BET bromodomain inhibitor JQ1 or vehicle. BET inhibition abrogated adverse cardiac remodeling, reduced cardiac fibrosis, and prolonged survival in PLNR9C mice by inhibiting expression of proinflammatory gene networks at all stages of disease. Specifically, JQ1 had profound effects on proinflammatory gene network expression in cardiac fibroblasts, while having little effect on gene expression in cardiomyocytes. Cardiac fibroblast proliferation was also substantially reduced by JQ1. Mechanistically, we demonstrated that BRD4 serves as a direct and essential regulator of NF-κB–mediated proinflammatory gene expression in cardiac fibroblasts. Suppressing proinflammatory gene expression via BET bromodomain inhibition could be a novel therapeutic strategy for chronic DCM in humans.

Authors

Andrew Antolic, Hiroko Wakimoto, Zhe Jiao, Joshua M. Gorham, Steven R. DePalma, Madeleine E. Lemieux, David A. Conner, Da Young Lee, Jun Qi, Jonathan G. Seidman, James E. Bradner, Jonathan D. Brown, Saptarsi M. Haldar, Christine E. Seidman, Michael A. Burke

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Abstract

Critical illness is accompanied by the release of large amounts of the anaphylotoxin, C5a. C5a suppresses antimicrobial functions of neutrophils which is associated with adverse outcomes. The signaling pathways that mediate C5a-induced neutrophil dysfunction are incompletely understood. Healthy donor neutrophils exposed to purified C5a demonstrated a prolonged defect (7 hours) in phagocytosis of Staphylococcus aureus. Phosphoproteomic profiling of 2712 phosphoproteins identified persistent C5a signaling and selective impairment of phagosomal protein phosphorylation on exposure to S. aureus. Notable proteins included early endosomal marker ZFYVE16 and V-ATPase proton channel component ATPV1G1. An assay of phagosomal acidification demonstrated C5a-induced impairment of phagosomal acidification, which was recapitulated in neutrophils from critically ill patients. Examination of the C5a-impaired protein phosphorylation indicated a role for the PI3K VPS34 in phagosomal maturation. Inhibition of VPS34 impaired neutrophil phagosomal acidification and killing of S. aureus. This study provides a phosphoproteomic assessment of human neutrophil signaling in response to S. aureus and its disruption by C5a, identifying a defect in phagosomal maturation and mechanisms of immune failure in critical illness.

Authors

Alexander J.T. Wood, Arlette M. Vassallo, Marie-Hélène Ruchaud-Sparagano, Jonathan Scott, Carmelo Zinnato, Carmen Gonzalez-Tejedo, Kamal Kishore, Clive S. D’Santos, A. John Simpson, David K. Menon, Charlotte Summers, Edwin R. Chilvers, Klaus Okkenhaug, Andrew Conway Morris

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Abstract

BACKGROUND A treatment option for autosomal dominant polycystic kidney disease (ADPKD) has highlighted the need to identify rapidly progressive patients. Kidney size/age and genotype have predictive power for renal outcomes, but their relative and additive value, plus associated trajectories of disease progression, are not well defined.METHODS The value of genotypic and/or kidney imaging data (Mayo Imaging Class; MIC) to predict the time to functional (end-stage kidney disease [ESKD] or decline in estimated glomerular filtration rate [eGFR]) or structural (increase in height-adjusted total kidney volume [htTKV]) outcomes were evaluated in a Mayo Clinic PKD1/PKD2 population, and eGFR and htTKV trajectories from 20–65 years of age were modeled and independently validated in similarly defined CRISP and HALT PKD patients.RESULTS Both genotypic and imaging groups strongly predicted ESKD and eGFR endpoints, with genotype improving the imaging predictions and vice versa; a multivariate model had strong discriminatory power (C-index = 0.845). However, imaging but not genotypic groups predicted htTKV growth, although more severe genotypic and imaging groups had larger kidneys at a young age. The trajectory of eGFR decline was linear from baseline in the most severe genotypic and imaging groups, but it was curvilinear in milder groups. Imaging class trajectories differentiated htTKV growth rates; severe classes had rapid early growth and large kidneys, but growth later slowed.CONCLUSION The value of imaging, genotypic, and combined data to identify rapidly progressive patients was demonstrated, and reference values for clinical trials were provided. Our data indicate that differences in kidney growth rates before adulthood significantly define patients with severe disease.FUNDING NIDDK grants: Mayo DK058816 and DK090728; CRISP DK056943, DK056956, DK056957, and DK056961; and HALT PKD DK062410, DK062408, DK062402, DK082230, DK062411, and DK062401.

Authors

Sravanthi Lavu, Lisa E. Vaughan, Sarah R. Senum, Timothy L. Kline, Arlene B. Chapman, Ronald D. Perrone, Michal Mrug, William E. Braun, Theodore I. Steinman, Frederic F. Rahbari-Oskoui, Godela M. Brosnahan, Kyongtae T. Bae, Douglas Landsittel, Fouad T. Chebib, Alan S.L. Yu, Vicente E. Torres, the HALT PKD and CRISP Study Investigators, Peter C. Harris

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Abstract

Stem cell transplantation has emerged as a promising strategy in regenerative medicine. However, the poor survival and persistence of the transplanted cells, including mesenchymal stem cells (MSCs), in the hostile ischemic microenvironments represents a major therapeutic barrier. Here we report that plasminogen (Plg) stimulated MSC functions and promoted MSC survival during tissue repair after ischemia. Genetic Plg ablation abolished MSC survival, migration, and proliferation in mouse ischemic limbs, and abrogated MSC-mediated blood reperfusion, neovascularization, and tissue repair after ischemia, suggesting a critical role for Plg in MSC-mediated tissue repair. Furthermore, multiplex cytokine array analysis identified that Plg cleaved and activated cysteine-rich protein 61 (Cyr61), an ECM-associated growth factor, to stimulate MSC survival and migration. Overexpression with truncated Cyr61 in MSCs rescued blood reperfusion after hind limb ischemia in Plg-deficient mice. Finally, Plg-mediated Cyr61 cleavage promoted endothelial cell migration and neovascularization in vitro and in vivo. Our study reveals that Plg promotes MSC survival, persistence, and paracrine effects and improves postischemic neovascularization and tissue repair through Cyr61 cleavage and activation. Thus, targeting Plg/Cyr61 may offer exciting therapeutic opportunities for strengthening MSC therapy in ischemic diseases.

Authors

Hao Duan, Zhenqiang He, Maohuan Lin, Yanling Wang, Fan Yang, R. Alan Mitteer, Hyun-Jun Kim, Eujing Yeo, Hongyu Han, Ling Qin, Yi Fan, Yanqing Gong

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Abstract

Alpha 1-antitrypsin (AAT) deficiency, a hereditary disorder characterized by low serum levels of functional AAT, is associated with early development of panacinar emphysema. AAT inhibits serine proteases, including neutrophil elastase, protecting the lung from proteolytic destruction. Cigarette smoke, pollution, and inflammatory cell–mediated oxidation of methionine (M) 351 and 358 inactivates AAT, limiting lung protection. In vitro studies using amino acid substitutions demonstrated that replacing M351 with valine (V) and M358 with leucine (L) on a normal M1 alanine (A) 213 background provided maximum antiprotease protection despite oxidant stress. We hypothesized that a onetime administration of a serotype 8 adeno-associated virus (AAV8) gene transfer vector coding for the oxidation-resistant variant AAT (A213/V351/L358; 8/AVL) would maintain antiprotease activity under oxidant stress compared with normal AAT (A213/M351/M358; 8/AMM). 8/AVL was administered via intravenous (IV) and intrapleural (IPL) routes to C57BL/6 mice. High, dose-dependent AAT levels were found in the serum and lung epithelial lining fluid (ELF) of mice administered 8/AVL or 8/AMM by IV or IPL. 8/AVL serum and ELF retained serine protease–inhibitory activity despite oxidant stress while 8/AMM function was abolished. 8/AVL represents a second-generation gene therapy for AAT deficiency providing effective antiprotease protection even with oxidant stress.

Authors

Meredith L. Sosulski, Katie M. Stiles, Esther Z. Frenk, Fiona M. Hart, Yuki Matsumura, Bishnu P. De, Stephen M. Kaminsky, Ronald G. Crystal

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Abstract

Myelodysplastic syndromes (MDS) are clonal malignant hematopoietic disorders in the elderly characterized by ineffective hematopoiesis. This is accompanied by an altered bone microenvironment, which contributes to MDS progression and higher bone fragility. The underlying mechanisms remain largely unexplored. Here, we show that myelodysplastic NUP98‑HOXD13 (NHD13) transgenic mice display an abnormally high number of osteoblasts, yet a higher fraction of nonmineralized bone, indicating delayed bone mineralization. This was accompanied by high fibroblast growth factor-23 (FGF-23) serum levels, a phosphaturic hormone that inhibits bone mineralization and erythropoiesis. While Fgf23 mRNA expression was low in bone, brain, and kidney of NHD13 mice, its expression was increased in erythroid precursors. Coculturing these precursors with WT osteoblasts induced osteoblast marker gene expression, which was inhibited by blocking FGF-23. Finally, antibody-based neutralization of FGF-23 in myelodysplastic NHD13 mice improved bone mineralization and bone microarchitecture, and it ameliorated anemia. Importantly, higher serum levels of FGF‑23 and an elevated amount of nonmineralized bone in patients with MDS validated the findings. C‑terminal FGF‑23 correlated negatively with hemoglobin levels and positively with the amount of nonmineralized bone. Thus, our study identifies FGF-23 as a link between altered bone structure and ineffective erythropoiesis in MDS with the prospects of a targeted therapeutic intervention.

Authors

Heike Weidner, Ulrike Baschant, Franziska Lademann, Maria G. Ledesma Colunga, Ekaterina Balaian, Christine Hofbauer, Barbara M. Misof, Paul Roschger, Stéphane Blouin, William G. Richards, Uwe Platzbecker, Lorenz C. Hofbauer, Martina Rauner

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Abstract

Angiogenesis is essential for cardiac functional recovery after myocardial infarction (MI). HSPA12B is predominately expressed in endothelial cells and required for angiogenesis. Yes-associated protein (YAP) plays an important role in tumor angiogenesis. This study investigated the cooperative role of HSPA12B and YAP in angiogenesis post-MI. Silencing of either HSPA12B or YAP impairs hypoxia-promoted endothelial cell proliferation and angiogenesis. Deficiency of HSPA12B suppresses YAP expression and nuclear translocation following hypoxia. Knockdown of YAP attenuates hypoxia-stimulated HSPA12B nuclear translocation and abrogates HSPA12B-promoted endothelial cell angiogenesis. Mechanistically, hypoxia induced an interaction between endothelial HSPA12B and YAP. ChIP assay shows that HSPA12B is a target gene of YAP/transcriptional enhanced associated domain4 (TEAD4) and a co-activator in YAP-associated angiogenesis. In vivo studies using the MI model show that endothelial specific deficiency of HSPA12B (eHspa12b-/-) or YAP (eYap-/-) impairs angiogenesis and exacerbates cardiac dysfunction, when compared with wild type (WT) mice. MI increased YAP expression and nuclear translocation in WT hearts, but not in eHspa12b-/- hearts. HSPA12B expression and nuclear translocation were up-regulated in WT MI hearts, but not in eYap-/- MI myocardium. Our data demonstrated that endothelial HSPA12B is a novel target and co-activator for YAP/TEAD4 and cooperates with YAP to regulate endothelial angiogenesis post-MI.

Authors

Min Fan, Kun Yang, Xiaohui Wang, Yana Wang, Fei Tu, Tuanzhu Ha, Li Liu, David L. Williams, Chuanfu Li

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Abstract

Age-associated systemic, chronic inflammation is partially attributed to increased self (auto)-reactivity, resulting from disruption of central tolerance in the aged, involuted thymus. This involution causally results from gradually decreased expression of the transcription factor FOXN1 in thymic epithelial cells (TECs), while exogenous FOXN1 in TECs can partially rescue age-related thymic involution. Given the findings that TECs induced from FOXN1-overexpressing embryonic fibroblasts can generate an ectopic de novo thymus under the kidney capsule and intra-thymically injected naturally young TECs can lead to middle-aged thymus regrowth, we attempted to extend these two findings by combining them as a novel thymic rejuvenation strategy with two types of promoter-driven (Rosa26CreERT and FoxN1Cre) Cre-mediated FOXN1-reprogrammed embryonic fibroblasts (FREFs). We engrafted these two-types of FREFs directly into the aged murine thymus. We found significant regrowth of the native aged thymus with rejuvenated architecture and function in both males and females, exhibiting increased thymopoiesis and reinforced thymocyte negative selection, along with reduced senescent T cells and auto-reactive T cell-mediated inflammation in old mice. Therefore, this strategy has preclinical significance and presents a strategy to potentially rescue decreased thymopoiesis and perturbed negative selection to significantly, albeit partially, restore defective central tolerance and reduce subclinical autoimmune symptoms in the elderly.

Authors

Jiyoung Oh, Weikan Wang, Rachel Thomas, Dong-Ming Su

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Abstract

Abstract: S-Nitroso-L-cysteine (L-CSNO) behaves as a ligand. Its soluble guanylate cyclase (sGC)-independent effects are stereoselective - that is, not recapitulated by S-nitroso-D-cysteine (D-CSNO) – and are inhibited by chemical cogeners. However, candidate L-CSNO receptors have never been identified. Here, we have used two complementary affinity chromatography assays - followed by unbiased proteomic analysis - to identify voltage-gated K+ channel (Kv) proteins as binding partners for L-CSNO. Stereoselective L-CSNO-Kv interaction was confirmed structurally and functionally using surface plasmon resonance spectroscopy, hydrogen deuterium exchange and, in Kv1.1/Kv1.2/Kvβ2 overexpressing cells, patch clamp assays. Remarkably, these sGC-independent L-CSNO effects did not involve S-nitrosylation of Kv proteins. In isolated rat and mouse respiratory control (petrosyl) ganglia, L-CSNO stereoselectively inhibited Kv channel function. Genetic ablation of Kv 1.1 prevented this effect. In intact animals, L-CSNO injection at level of the carotid body (CB) dramatically and stereoselectively increased minute ventilation while having no effect on blood pressure; this effect was inhibited by the L-CSNO cogener S-methyl-L-cysteine. Kv proteins are physiologically relevant targets of endogenous L-CSNO. This may be a signaling pathway of broad relevance.

Authors

Benjamin Gaston, Laura A. Smith, Jürgen Bosch, James M. Seckler, Diana L. Kunze, Janna Kiselar, Nadzeya Marozkina, Craig Hodges, Patrick Wintrode, Kellen McGee, Tatiana Morozkina, Spencer T. Burton, Tristan Lewis, Timothy Strassmaier, Paulina Getsy, James N. Bates, Stephen J. Lewis

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Abstract

New strategies are needed to enhance the efficacy of anti-programmed cell death protein (PD-1) antibody (Ab) in cancer. Here, we report that inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of CQ derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti-PD-1 Ab in melanoma. The combination resulted tumor growth impairment and improved survival in mouse models. Genetic suppression of core autophagy genes, but not Ppt1, in cancer cells reduced priming and cytotoxic capacity of primed T cells. Exposure of antigen primed T cells to macrophage conditioned medium derived from macrophages treated with PPT1 inhibitors enhanced melanoma specific killing. Genetic or chemical PPT1 inhibition resulted an M2 to M1 phenotype switching in macrophages. The combination was associated with a reduction in myeloid-derived suppressor cells (MDSCs) in the tumor. Ppt1 inhibition by HCQ, or DC661, induced cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), tank-binding kinase 1 (TBK1) pathway activation and the secretion of interferon β (IFN-β) in macrophages which was a key component for augmented T cell-mediated cytotoxicity. Genetic Ppt1 inhibition produced similar findings. These data provide the rationale for a melanoma clinical trial testing this new immunotherapy combination and may also be effective in other cancers.

Authors

Gaurav Sharma, Rani Ojha, Estela Noguera-Ortega, Vito W. Rebecca, John Attanasio, Shujing Liu, Shengfu Piao, Jennifer J. Lee, Michael C. Nicastri, Sandra L. Harper, Amruta Ronghe, Vaibhav Jain, Jeffrey D. Winkler, David W. Speicher, Jerome Mastio, Phyllis A Gimotty, Xiaowei Xu, E. John Wherry, Dmitry I. Gabrilovich, Ravi K. Amaravadi

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Abstract

Tumor-associated macrophages (TAMs) affect cancer progression and therapy. Ovarian carcinoma often metastasizes to the peritoneal cavity. Here, we found two peritoneal macrophage subsets in mice bearing ID8 ovarian cancer based on the Tim-4 (T-cell immunoglobulin and mucin domain containing 4) expression. Tim-4+ TAMs were embryonically originated and locally sustained while Tim-4- TAMs were replenished from circulating monocytes. Tim-4+ TAMs, but not Tim-4- TAMs, promoted tumor growth in vivo. Relative to Tim-4- TAMs, Tim-4+ TAMs manifested high oxidative phosphorylation and adapted mitophagy to alleviate oxidative stress. High levels of arginase-1 in Tim-4+ TAMs contributed to potent mitophagy activities via weakened mTORC1 activation due to low arginine resultant from arginase-1-mediated metabolism. Furthermore, genetic deficiency of autophagy element FIP200 resulted in Tim-4+ TAM loss via ROS-mediated apoptosis, and elevated T cell-immunity and ID8 tumor inhibition in vivo. Moreover, human ovarian cancer-associated CRIg (complement receptor of the Immunoglobulin superfamily) positive macrophages were transcriptionally, metabolically, and functionally similar to murine Tim-4+ TAMs. Thus, targeting CRIg+ (Tim-4+) TAMs may potentially treat ovarian cancer patients with peritoneal metastasis.

Authors

Houjun Xia, Shasha Li, Xiong Li, Weichao Wang, Yingjie Bian, Shuang Wei, Sara Grove, Weimin Wang, Linda Vatan, J. Rebecca Liu, Karen McLean, Ramandeep Rattan, Adnan R. Munkarah, Jun-Lin Guan, Ilona Kryczek, Weiping Zou

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