Research
Abstract
Altered islet architecture is associated with β cell dysfunction and Type 2 Diabetes (T2D) progression, but molecular effectors of islet spatial organization remain mostly unknown. Although Notch signaling is known to regulate pancreatic development, we observed “re-activated” β cell Notch activity in obese mouse models. To test the repercussions and reversibility of Notch effects, we generated doxycycline-dependent, β cell-specific Notch gain-of-function mice. As predicted, we found that Notch activation in post-natal β cells impaired glucose stimulated insulin secretion (GSIS) and glucose intolerance, but we observed a surprising remnant glucose intolerance after doxycycline withdrawal and cessation of Notch activity, associated with a marked disruption of normal islet architecture. Transcriptomic screening of Notch-active islets revealed increased Ephrin signaling. Commensurately, exposure to Ephrin ligands increased β cell repulsion, and impaired murine and human pseudo-islet formation. Consistent with our mouse data, Notch and Ephrin signaling are increased in metabolically-inflexible β cells in patients with T2D. These studies suggest than islet architecture can be permanently altered by β cell Notch/Ephrin signaling during a morphogenetic window in early life.
Authors
Alberto Bartolomé, Nina Suda, Junjie Yu, Changyu Zhu, Jinsook Son, Hongxu Ding, Andrea Califano, Domenico Accili, Utpal B. Pajvani
Abstract
Standard-of-care treatment for advanced HER2+ breast cancers (BC) is comprised of two HER2-specific monoclonal antibodies (mAb), Trastuzumab (T) and Pertuzumab (P) with chemotherapy. While this combination (T+P) is highly effective, its synergistic mechanism of action (MOA) is not completely known. Initial studies had demonstrated that Pertuzumab suppressed HER2 hetero-dimerization as the potential therapeutic MOA, thus the improved outcome associated with the T+P combination MOA compared to Trastuzumab alone has been widely reported as being due to Pertuzumab-mediated suppression of HER2 signaling in combination with Trastuzumab-mediated induction of anti-tumor immunity. Unraveling this MOA may be critical to extend this combination strategy to other antigens or other cancers, as well as improving this current treatment modality. Using novel murine and human versions of Pertuzumab, we found it induced both Antibody-Dependent-Cellular-Phagocytosis (ADCP) by tumor-associated macrophages and suppression of HER2 oncogenic signaling. Most significantly, we identified that only T+P combination therapy, but not when either antibody used in isolation, allows for the activation of the classical complement pathway, resulting in both direct complement-dependent cytotoxicity (CDC) as well as complement-dependent cellular phagocytosis (CDCP) of HER2+ BC cells. Notably, we show that tumor expression of C1q was positively associated with survival outcome in HER2+ BC patients, whereas expression of complement regulators CD55 and CD59 were inversely correlated, suggesting the importance of complement activity in clinical outcomes. Accordingly, inhibition of C1 activity in mice abolished the synergistic therapeutic activity of T+P therapy, whereas knockdown of CD55 and CD59 expression enhanced T+P efficacy. In summary, our study identifies classical complement activation as a significant anti-tumor MOA for T+P therapy that may be functionally enhanced to augment therapeutic efficacy in the clinic.
Authors
Li-Chung Tsao, Erika J. Crosby, Timothy N. Trotter, Junping Wei, Tao Wang, Xiao Yang, Amanda N. Summers, Gangjun Lei, Christopher A. Rabiola, Lewis A. Chodosh, William J. Muller, Herbert Kim Lyerly, Zachary C. Hartman
Abstract
Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by the accumulation of myofibroblasts and progressive lung scarring. To identify transcriptional gene programs driving persistent lung fibrosis in aging, we performed RNA-seq on lung fibroblasts isolated from young and aged mice during the early resolution phase post-bleomycin injury. We discovered that relative to injured young fibroblasts, injured aged fibroblasts exhibited a pro-fibrotic state characterized by elevated expression of genes implicated in inflammation, matrix remodeling, and cell survival. We identified pro-viral integration site of Moloney murine leukemia virus 1 (PIM1) and its target Nuclear Factor of Activated T Cells-1 (NFATc1) as putative drivers of the sustained pro-fibrotic gene signatures in injured aged fibroblasts. PIM1 and NFATc1 transcripts were enriched in a pathogenic fibroblast population recently discovered in IPF lungs, and their protein expression was abundant in fibroblastic foci. Overexpression of PIM1 in normal human lung fibroblasts in vitro potentiated their fibrogenic activation in a NFATc1-dependent manner. Pharmacological inhibition of PIM1 attenuated IPF fibroblast activation and sensitized them to apoptotic stimuli. Inhibition of PIM1 signaling in IPF lung explants ex vivo inhibited pro-survival gene expression and collagen secretion, suggesting that targeting this pathway may represent a therapeutic strategy to block IPF progression.
Authors
Tho X. Pham, Jisu Lee, Jiazhen Guan, Nunzia Caporarello, Jeffrey A. Meridew, Dakota L. Jones, Qi Tan, Steven K. Huang, Daniel J. Tschumperlin, Giovanni Ligresti
Abstract
Kawasaki disease (KD) is the leading cause of non-congenital heart disease in children. Studies in mice and humans propound the NLRP3-IL-1β pathway as the principal driver of KD pathophysiology. Endoplasmic reticulum (ER) stress can activate the NLRP3 inflammasome, but the potential implication of ER stress in KD pathophysiology has not been investigated. We used human patient data and the Lactobacillus casei cell wall extract (LCWE) murine model of KD vasculitis to characterize the impact of ER stress on the development of cardiovascular lesions. KD patient transcriptomics and single-cell RNA sequencing of the abdominal aorta from LCWE-injected mice revealed changes in the expression of ER stress genes. Alleviating ER stress genetically, by conditional deletion of Inositol Requiring Enzyme-1 (IRE1) in myeloid cells, or pharmacologically, by inhibition of IRE1 endoribonuclease (RNase) activity, led to significant reduction of LCWE-induced cardiovascular lesion formation as well as reduced caspase-1 activity and IL-1β secretion. These results demonstrate the causal relationship of ER stress to KD pathogenesis, and highlight IRE1 RNase activity as a potential new therapeutic target.
Authors
Stefanie Marek-Iannucci, Asli D. Yildirim, Syed M. Hamid, Asli B. Ozdemir, Angela C. Gomez, Begüm Kocatürk, Rebecca A. Porritt, Michael C. Fishbein, Takao Iwawaki, Magali Noval Rivas, Ebru Erbay, Moshe Arditi
Abstract
Sporozoite-based approaches currently represent the most effective vaccine strategies for induction of sterile protection against Plasmodium falciparum (Pf) malaria. Clinical development of sub-unit vaccines is almost exclusively centered around the Circum-sporozoite Protein (CSP) an abundantly expressed protein on the sporozoite membrane. Anti-CSP antibodies are able to block sporozoite invasion and development in human hepatocytes and subsequently prevent clinical malaria. Here we investigated whether sporozoite-induced human antibodies with specificities different from CSP can reduce Pf-liver stage development. IgG preparations were obtained from 12 volunteers inoculated with a protective immunization regime of whole-sporozoites under chloroquine prophylaxis. These IgGs were depleted for CSP-specificity by affinity chromatography. Recovered non-CSP antibodies were tested for sporozoite membrane binding and for functional inhibition of sporozoite invasion of a human hepatoma cell line and hepatocytes both in vitro and in vivo. Post-immunization IgGs depleted for CSP-specificity of 9 out of 12 donors recognized sporozoite surface antigens. Samples from 5 out of 12 donors functionally reduced parasite-liver cell invasion or development using the hepatoma cell line HC-04 and FRG-huHep mice containing human liver cells. The combined data provide clear evidence that non-CSP proteins as yet undefined do represent antibody targets for functional immunity against Plasmodium falciparum parasites responsible for malaria.
Authors
Amanda Fabra-García, Annie S.P. Yang, Marije C. Behet, Xi Zen Yap, Youri van Waardenburg, Swarnendu Kaviraj, Kjerstin Lanke, Geert-Jan van Gemert, Matthijs M. Jore, Teun Bousema, Robert W. Sauerwein
Abstract
Ca2+ is critical for cardiac electrical conduction and contractility, and aberrant Ca2+ homeostasis causes arrhythmia and heart failure. Chromatin remodeling modulates gene expression involved in cardiac sarcomere assembly and postnatal heart function. However, the chromatin remodeling-regulatory cardiac Ca2+ homeostasis is unknown. Here, we found that Znhit1, a core subunit of the SRCAP remodeling complex, was essential for heart function. Deletion of Znhit1 in postnatal heart of mice resulted in arrhythmia, idiopathic vacuolar cardiomyopathy, rapid heart failure and premature sudden death. In addition, the level of Casq1, a sarcoplasmic reticulum (SR) Ca2+ regulatory protein, was massively elevated while SERCA2a showed reduced protein level. Mechanistically, the Znhit1 modulated the expression of Casq1 and SERCA2a through depositing H2A.Z at their promoters. Deletion of Casq1 could substantially alleviate the vacuolar formation in Znhit1 cKO mice. These findings have demonstrated that Znhit1 is required for post-natal heart function and maintains cardiac Ca2+ homeostasis, and accumulation of Casq1 might be a causative factor for vacuolar cardiomyopathy.
Authors
Yingchao Shi, Wenli Fan, Mingjie Xu, Xinhua Lin, Wukui Zhao, Zhongzhou Yang
Abstract
Cilia, microtubule-based organelles that project from the apical luminal surface of endothelial cells (ECs), are widely regarded as a low flow-sensors. Previous reports suggest that upon high shear stress, cilia on the EC surface are lost, and more recent evidence suggests that deciliation - the physical removal of cilia from the cell surface - is a predominant mechanism for cilia loss in mammalian cells. Thus, we hypothesized that EC deciliation facilitated by changes in shear stress will manifest in increased abundance of cilia-related proteins in circulation. To test this hypothesis, we performed shear stress experiments that mimicked flow conditions from low to high shear stress in human primary cells and a zebrafish model system. In the primary cells, we showed that upon shear stress induction, indeed, ciliary fragments were observed in the effluent in vitro and effluents contained ciliary proteins normally expressed in both endothelial and epithelial cells. In zebrafish, upon shear stress induction, fewer ciliary-expressing ECs were observed. To test the translational relevance of these findings, we investigated our hypothesis using patient blood samples from sickle cell disease and found that plasma levels of ciliary proteins were elevated compared to healthy controls. Further, sickled red blood cells demonstrated high levels of ciliary protein (Arl13b) on their surface post-adhesion to brain ECs. Brain ECs post interaction with sickle RBCs show high reactive oxygen species (ROS) levels. Attenuating ROS levels in brain ECs decreases cilia protein levels on RBCs and rescues ciliary protein levels in brain ECs. Collectively, these data suggest that cilia and ciliary proteins in circulation are detectable under various altered flow conditions, which could serve as a surrogate biomarker of the damaged endothelium.
Authors
Ankan Gupta, Karthikeyan Thirugnanam, Madhan Thamilarasan, Ashraf M. Mohieldin, Hadeel T. Zedan, Shubhangi Prabhudesai, Meghan R. Griffin, Andrew D. Spearman, Amy Pan, Sean P. Palecek, Huseyin C. Yalcin, Surya M. Nauli, Kevin R. Rarick, Rahima Zennadi, Ramani Ramchandran
Abstract
Mucosal healing is a key treatment goal for inflammatory bowel disease, and adequate epithelial regeneration is required for an intact gut epithelium. However, the underlying mechanism is unclear. Long non-coding RNAs (lncRNAs) have been reported to be involved in the development of inflammatory bowel disease. Here, we report that a lncRNA named Gm31629, decreases in intestinal epithelial cells in response to inflammatory stimulation. Gm31629 deficiency leads to exacerbated intestinal inflammation and delayed epithelial regeneration in dextran sulfate sodium (DSS) -induced colitis model. Mechanistically, Gm31629 promotes E2F pathways and cell proliferation by stabilizing Y-box protein 1 (YB-1), thus facilitating epithelial regeneration. Genetic overexpression of Gm31629 protects against DSS-induced colitis in vivo. Theaflavin 3-gallate, a natural compound mimicking Gm31629, alleviates DSS-induced epithelial inflammation and mucosal damage. These results demonstrate an essential role of lncRNA Gm31629 in linking intestinal inflammation and epithelial cell proliferation, providing a potential therapeutic approach to inflammatory bowel disease.
Authors
Xu Feng, Ye Xiao, Jian He, Mi Yang, Qi Guo, Tian Su, Yan Huang, Jun Yi, Chang-Jun Li, Xiang-Hang Luo, Xiao-Wei Liu, Hai-Yan Zhou
Abstract
Severe acute lung injury has few treatment options and a high mortality rate. Upon injury, neutrophils infiltrate the lungs and form neutrophil extracellular traps (NETs), damaging the lungs and driving an exacerbated immune response. Unfortunately, no drug preventing NET formation has completed clinical development. Here, we report that disulfiram —an FDA-approved drug for alcohol use disorder— dramatically reduced NETs, increased survival, improved blood oxygenation, and reduced lung edema in a transfusion-related acute lung injury (TRALI) mouse model. We then tested whether disulfiram could confer protection in the context of SARS-CoV-2 infection, as NETs are elevated in patients with severe COVID-19. In SARS-CoV-2-infected golden hamsters, disulfiram reduced NETs and perivascular fibrosis in the lungs, and downregulated innate immune and complement/coagulation pathways, suggesting that it could be beneficial for COVID-19 patients. In conclusion, an existing FDA-approved drug can block NET formation and improve disease course in two rodent models of lung injury for which treatment options are limited.
Authors
Jose M. Adrover, Lucia Carrau, Juliane Daßler-Plenker, Yaron Bram, Vasuretha Chandar, Sean Houghton, David Redmond, Joseph R. Merrill, Margaret Shevik, Benjamin R. tenOever, Scott K. Lyons, Robert E. Schwartz, Mikala Egeblad
Abstract
Recovery from pneumococcal pneumonia remodels the pool of alveolar macrophages so that they exhibit new surface marker profiles, transcriptomes, metabolomes, and responses to infection. Mechanisms mediating alveolar macrophage phenotypes after pneumococcal pneumonia have not been delineated. IFNγ and its receptor on alveolar macrophages were essential for aspects but not all of the remodeled alveolar macrophage phenotype. IFNγ was produced by CD4+ T cells plus other cells, and CD4+ cell depletion did not prevent alveolar macrophage remodeling. In mice infected or recovering from pneumococcus, monocytes were recruited to the lungs and the monocyte-derived macrophages developed characteristics of alveolar macrophages. CCR2 mediated the early monocyte recruitment but was not essential to development of the remodeled alveolar macrophage phenotype. Lineage tracing demonstrated that recovery from pneumococcal pneumonias converted the pool of alveolar macrophages from being primarily of embryonic origin to being primarily of adult hematopoietic stem cell origin. Alveolar macrophages of either origin demonstrated similar remodeled phenotypes, suggesting that ontogeny did not dictate phenotype. Altogether, our data reveal that the remodeled alveolar macrophage phenotype in lungs recovered from pneumococcal pneumonia results from a combination of new recruitment plus training of both the original cells and the new recruits.
Authors
Emad I. Arafa, Anukul T. Shenoy, Kimberly A. Barker, Neelou S. Etesami, Ian M.C. Martin, Carolina Lyon De Ana, Elim Na, Christine V. Odom, Wesley N. Goltry, Filiz T. Korkmaz, Alicia K. Wooten, Anna C. Belkina, Antoine Guillon, E. Camilla Forsberg, Matthew R. Jones, Lee J. Quinton, Joseph P. Mizgerd
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