Abdominal aortic aneurysm (AAA) is a life-threatening degenerative vascular disease. Endothelial cell (EC) dysfunction is implicated in AAA. Our group recently demonstrated that Krüppel-like factor 11 (KLF11) plays an essential role in maintaining vascular homeostasis, at least partially through inhibition of EC inflammatory activation. However, the functions of endothelial KLF11 in AAA remain unknown. Here we found that endothelial KLF11 expression was reduced in the ECs from human aneurysms and was time-dependently decreased in the aneurysmal endothelium from both elastase- and Pcsk9/AngII-induced AAA mouse models. KLF11 deficiency in ECs markedly aggravated AAA formation, whereas EC-selective KLF11 overexpression significantly inhibited AAA formation. Mechanistically, KLF11 not only inhibited the EC inflammatory response but also diminished MMP9 expression and activity and reduced NADPH oxidase 2-mediated production of reactive oxygen species in ECs. In addition, KLF11-deficient ECs induce smooth muscle cell dedifferentiation and apoptosis. Overall, we established endothelial KLF11 as a novel factor protecting against AAA and a potential target for intervention in aortic aneurysms.
Guizhen Zhao, Ziyi Chang, Yang Zhao, Yanhong Guo, Haocheng Lu, Wenying Liang, Oren Rom, Huilun Wang, Jinjian Sun, Tianqing Zhu, Yanbo Fan, Lin Chang, Bo Yang, Minerva Garcia-Barrio, Eugene Chen, Jifeng Zhang
Myotonic dystrophy type 1 (DM1) is caused by a CTG-repeat expansion in the DMPK gene. Expression of pathogenic expanded CUG-repeat (CUGexp) RNA causes multisystemic disease by perturbing the functions of RNA binding proteins, resulting in expression of fetal protein isoforms in adult tissues. Cardiac involvement affects 50% of individuals with DM1 and causes 25% of disease-related deaths. We developed a transgenic mouse model for tetracycline-inducible and heart-specific expression of human DMPK mRNA containing 960 CUG repeats. CUGexp RNA is expressed in atria and ventricles and induced mice exhibit electrophysiological and molecular features of DM1 disease including cardiac conduction delays, supraventricular arrhythmias, nuclear RNA foci with Muscleblind protein colocalization and alternative splicing defects. Importantly, these phenotypes were rescued upon loss of CUGexp RNA expression. Transcriptome analysis revealed gene expression and alternative splicing changes in ion transport genes that are associated with inherited cardiac conduction diseases, including a subset of genes involved in calcium handling. Consistent with RNA-seq results, calcium handling defects were identified in atrial cardiomyocytes isolated from mice expressing CUGexp RNA. These results identify potential tissue-specific mechanisms contributing to cardiac pathogenesis in DM1 and demonstrate the utility of reversible phenotypes in our model to facilitate development of targeted therapeutic approaches.
Ashish N. Rao, Hannah M. Campbell, Xiangnan Guan, Tarah A. Word, Xander H.T. Wehrens, Zheng Xia, Thomas A. Cooper
Limitations of checkpoint inhibitor cancer immunotherapy include induction of autoimmune syndromes and resistance of many cancers. Since CD318, a novel CD6 ligand, is associated with aggressiveness and metastatic potential of human cancers, we tested the effect of an anti-CD6 monoclonal antibody, UMCD6, on killing of cancer cells by human lymphocytes. UMCD6 augmented killing of breast, lung or prostate cancer cells through direct effects on both CD8+ T cells and natural killer (NK) cells, increasing cancer cell death and lowering cancer cell survival in vitro more robustly than monoclonal antibody checkpoint inhibitors that interrupt the PD-1/PD-L1 axis. UMCD6 also augmented in vivo killing by human peripheral blood lymphocytes of a human breast cancer line xeno-transplanted into immunodeficient mice. Mechanistically, UMCD6 upregulated the expression of the activating receptor NKG2D and down-regulated expression of the inhibitory receptor NKG2A on both NK cells and CD8+ T cells, with concurrent increases in perforin and granzyme-B production. The combined capabilities of an anti-CD6 monoclonal antibody to control autoimmunity through effects on CD4+ lymphocyte differentiation, while enhancing killing of cancer cells through distinct effects on CD8+ and NK cells, opens a potential new approach to cancer immunotherapy that would suppress rather than instigate autoimmunity.
Jeffrey H. Ruth, Mikel Gurrea-Rubio, Kalana S. Athukorala, Stephanie M. Rasmussen, Daniel Weber, Peggy M. Randon, Rosemary J. Gedert, Matthew E. Lind, Mohammad Asif Amin, Phillip L. Campbell, Pei-Suen Tsou, Yang Mao-Draayer, Qi Wu, Thomas M. Lanigan, Venkateshwar G. Keshamouni, Nora G. Singer, Feng Lin, David A Fox
Cancer is caused primarily by genomic alterations resulting in deregulation of gene regulatory circuits in key growth, apoptosis or DNA repair pathways. Multiple genes associated with the initiation and development of tumors are also regulated at the level of mRNA decay, through the recruitment of RNA binding proteins to AU-rich elements (AREs) located in their 3’-untranslated regions. One of these ARE-binding proteins, tristetraprolin (TTP, encoded by Zfp36) is consistently dysregulated in many human malignancies. Herein, using regulated overexpression or conditional ablation in the context of chemical cutaneous carcinogenesis, we show that TTP represents a critical regulator of skin tumorigenesis. We provide evidence that TTP controls both tumor-associated inflammation and key oncogenic pathways in neoplastic epidermal cells. We identify Areg as a direct target of TTP in keratinocytes, and show that EGFR signaling potentially contributes to exacerbated tumor formation. Finally, single-cell RNA-Sequencing analysis indicates that ZFP36 is downregulated in human malignant keratinocytes. We conclude that TTP expression by epidermal cells plays a major role in the control of skin tumorigenesis.
Assiya Assabban, Ingrid Dubois-Vedrenne, Laurye Van Maele, Rosalba Salcedo, Brittany L. Snyder, Lecong Zhou, Abdulkader Azouz, Bérengère de Toeuf, Gaëlle Lapouge, Caroline La, Maxime Melchior, Muriel Nguyen, Séverine Thomas, Si Fan Wu, Wenqian Hu, Véronique Kruys, Cédric Blanpain, Giorgio Trinchieri, Cyril Gueydan, Perry J. Blackshear, Stanislas Goriely
Dysmorphic pulmonary vascular growth and abnormal endothelial cell (EC) proliferation are paradoxically observed in premature infants with bronchopulmonary dysplasia (BPD) despite vascular pruning. The pentose phosphate pathway (PPP), a metabolic pathway parallel to glycolysis, generates NADPH as a reducing equivalent and ribose 5-phosphate for nucleotide synthesis. It is unknown whether hyperoxia, a known mediator of BPD in rodent models, alters glycolysis and the PPP in lung ECs. We hypothesized that hyperoxia increases glycolysis and the PPP, resulting in abnormal EC proliferation and dysmorphic angiogenesis in neonatal mice. To test this hypothesis, lung ECs and newborn mice were exposed to hyperoxia and allowed to recover in air. Hyperoxia increased glycolysis and the PPP. Increased PPP, but not glycolysis, caused hyperoxia-induced abnormal EC proliferation. Blocking the PPP reduced hyperoxia-induced glucose-derived deoxynucleotide synthesis in cultured ECs. In neonatal mice, hyperoxia-induced abnormal EC proliferation, dysmorphic angiogenesis, and alveolar simplification were augmented by nanoparticle-mediated endothelial overexpression of phosphogluconate dehydrogenase, the second enzyme in the PPP. These effects were attenuated by inhibitors of the PPP. Altogether, neonatal hyperoxia augments the PPP, causing abnormal lung EC proliferation, dysmorphic vascular development, and alveolar simplification. These novel observations provide mechanisms and potential metabolic targets to prevent BPD-associated vascular dysgenesis.
Jiannan Gong, Zihang Feng, Abigail L. Peterson, Jennifer F. Carr, Xuexin Lu, Haifeng Zhao, Xiangming Ji, You-Yang Zhao, Monique E. De Paepe, Phyllis A. Dennery, Hongwei Yao
Influenza virus infections affect millions of people annually. Current available vaccines provide varying rates of protection. There is a knowledge gap on how the nasal microbiota, particularly established pneumococcal colonization, shapes the response to influenza vaccination. In this study, we inoculated healthy adults with live S. pneumoniae and vaccinated them three days later with either TIV or LAIV. Vaccine-induced immune responses were assessed in nose, blood and lung. Nasal pneumococcal colonization had no impact upon TIV-induced antibody responses to influenza, which manifested in all compartments. However, experimentally-induced pneumococcal colonization dampened LAIV-mediated mucosal antibody responses, primarily IgA in the nose and IgG in the lung. Pulmonary influenza-specific cellular responses were more apparent in the LAIV group compared to either TIV or an unvaccinated group. These results indicate that TIV and LAIV elicit differential immunity to adults and that LAIV immunogenicity is diminished by the nasal presence of S. pneumoniae. Therefore, nasopharyngeal pneumococcal colonization may affect LAIV efficacy.
Beatriz F. Carniel, Fernando Marcon, Jamie Rylance, Esther L. German, Seher Zaidi, Jesus Reine, Edessa Negera, Elissavet Nikolaou, Sherin Pojar, Carla Solórzano, Andrea M. Collins, Victoria Connor, Debby Bogaert, Stephen B. Gordon, Helder I. Nakaya, Daniela M. Ferreira, Simon P. Jochems, Elena Mitsi
Four endemic human coronaviruses (HCoVs) are commonly associated with acute respiratory infection in humans. B cell responses to these “common cold” viruses remain incompletely understood. Here we report a comprehensive analysis of CoV-specific antibody repertoires in 231 children and 1168 adults using phage-immunoprecipitation sequencing. Seroprevalence of antibodies to endemic HCoVs ranged between ~4 and 27% depending on the species and cohort. We identified at least 136 novel linear B cell epitopes. Antibody repertoires against endemic HCoVs were qualitatively different between children and adults in that anti-HCoV IgG specificities more frequently found among children targeted functionally important and structurally conserved regions of the spike, nucleocapsid and matrix proteins. Moreover, antibody specificities targeting the highly conserved fusion peptide region and S2’ cleavage site of the spike protein were broadly cross-reactive with peptides of epidemic human and non-human coronaviruses. In contrast, an acidic tandem repeat in the N-terminal region of the Nsp3 subdomain of the HCoV-HKU1 polyprotein was the predominant target of antibody responses in adult donors. Our findings shed light on the dominant species-specific and pan-CoV target sites of human antibody responses to coronavirus infection, thereby providing important insights for the development of prophylactic or therapeutic monoclonal antibodies and vaccine design.
Taushif Khan, Mahbuba Rahman, Fatima Al Ali, Susie S.Y. Huang, Manar Ata, Qian Zhang, Paul Bastard, Zhiyong Liu, Emmanuelle Jouanguy, Vivien Beziat, Aurélie Cobat, Gheyath K. Nasrallah, Hadi M. Yassine, Maria K. Smatti, Amira Saeed, Isabelle Vandernoot, Jean-Christophe Goffard, Guillaume Smits, Isabelle Migeotte, Filomeen Haerynck, Isabelle Meyts, Laurent Abel, Jean-Laurent Casanova, Mohammad R. Hasan, Nico Marr
Agonistic anti-CD40 monoclonal antibody (mAb) therapy in combination with chemotherapy (chemoimmunotherapy) shows promise for the treatment of pancreatic ductal adenocarcinoma (PDA). To gain insight into immunological mechanisms of response and resistance to chemoimmunotherapy, we analyzed blood samples from patients (n=22) with advanced PDA treated with an anti-CD40 mAb (CP-870,893) in combination with gemcitabine. We found a stereotyped cellular response to chemoimmunotherapy characterized by transient B cell, CD56+CD11c+HLA-DR+CD141+ cell and monocyte depletion and CD4+ T cell activation. However, these cellular pharmacodynamics did not associate with outcomes. In contrast, we identified an inflammatory network in the peripheral blood consisting of neutrophils, cytokines (IL-6 and IL-8) and acute phase reactants (CRP and SAA) that was associated with outcomes. Furthermore, monocytes from patients with elevated plasma IL-6 and IL-8 showed distinct transcriptional profiles, including upregulation of CCR2 and GAS6; genes associated with regulation of leukocyte chemotaxis and response to inflammation. Patients with systemic inflammation, defined by neutrophil-lymphocyte ratio (NLR) >3.1, had a shorter median OS (5.8 vs 12.3mo; p=0.0105) as compared to patients with NLR <3.1. Taken together, our findings identify systemic inflammation as a potential resistance mechanism to a CD40-based chemoimmunotherapy and suggest biomarkers for future studies.
Max M. Wattenberg, Veronica M. Herrera, Michael A. Giannone, Whitney L. Gladney, Erica L. Carpenter, Gregory L. Beatty
The molecular mechanisms that underlie the detrimental effects of particulate matter (PM) on skin barrier function are poorly understood. In this study, the effects of PM2.5 on filaggrin (FLG) and skin barrier function were investigated in vitro and in vivo. The levels of FLG degradation products including pyrrolidone carboxylic acid, urocanic acid (UCA), and cis/trans UCA were significantly decreased in skin tape stripping samples of study subjects when they moved from Denver, an area with low PM2.5, to Seoul, an area with high PM2.5 count. Experimentally, PM2.5 collected in Seoul inhibited FLG, loricrin, keratin-1, desmocollin-1, and corneodesmosin, but did not modulate involucrin or claudin-1 in keratinocyte cultures. Moreover, FLG protein expression was inhibited in human skin equivalents and murine skin treated with PM2.5. We demonstrate that this process was mediated by PM2.5-induced TNF-alpha and was aryl hydrocarbon receptor-dependent. PM2.5 exposure compromised skin barrier function, resulting in increased transepidermal water loss and enhanced the penetration of FITC-dextran in organotypic and mouse skin. PM2.5-induced TNF-alpha causes FLG deficiency in the skin and subsequently induces skin barrier dysfunction. Compromised skin barrier due to PM2.5 exposure may contribute to the development and the exacerbation of allergic diseases such as AD.
Byung Eui Kim, Jihyun Kim, Elena Goleva, Evgeny Berdyshev, Jinyoung Lee, Kathryn A. Vang, Un Ha Lee, SongYi Han, Susan Leung, Clifton F. Hall, Na-Rae Kim, Irina Bronova, Eu Jin Lee, Hye-Ran Yang, Donald Y.M. Leung, Kangmo Ahn
EPAS1, encoding HIF-2α, mutations were previously identified in a syndrome of multiple paragangliomas, somatostatinoma, and polycythemia. HIF-2α, when dimerized with HIF-1β, acts as an angiogenic transcription factor. Patients referred to our institution for new, recurrent, and/or metastatic paraganglioma or pheochromocytoma were confirmed for EPAS1-gain-of-function mutation; imaging was evaluated for vascular malformations. We evaluated the Epas1A529V transgenic syndrome mouse model, corresponding to the mutation initially detected in the patients (EPAS1A530V), for vascular malformations via intravital two photon microscopy of meningeal vessels, terminal vascular perfusion with Microfil silicate polymer and subsequent intact ex vivo 14T MRI and Micro-CT, and histologic sectioning and staining of the brain and identified pathologies. Further, we evaluated retina from corresponding developmental timepoints (P7, P14, and P21) and the adult dura via immunofluorescent labeling of vessels and confocal imaging. We identified a spectrum of vascular malformations in all 9 syndromic patients and in all of our tested mutant mice. Patient vessels had higher variant allele frequency than adjacent normal tissue. Veins of the murine retina and intracranial dura failed to regress normally at the expected developmental timepoints. These findings add vascular malformation as a new clinical feature of EPAS1-gain-of-function syndrome.
Jared S. Rosenblum, Herui Wang, Pauline M. Dmitriev, Anthony J. Cappadona, Panagiotis Mastorakos, Chen Xu, Abhishek Jha, Nancy Edwards, Danielle R. Donahue, Jeeva Munasinghe, Matthew A. Nazari, Russell H. Knutsen, Bruce R. Rosenblum, James G. Smirniotopoulos, Alberto Pappo, Robert F. Spetzler, Alexander Vortmeyer, Mark R. Gilbert, Dorian B. McGavern, Emily Chew, Beth A. Kozel, John D. Heiss, Zhengping Zhuang, Karel Pacak
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