Research
Abstract
Increased fibrosis is a characteristic remodeling response to biomechanical and neurohumoral stress and a determinant of cardiac mechanical and electrical dysfunction in disease. Stress-induced activation of cardiac fibroblasts (CF) is a critical step in the fibrotic response, although the precise sequence of events underlying activation of these critical cells in vivo remain unclear. Here, we test the hypothesis that a βIV-spectrin/STAT3 complex is essential for maintenance of a quiescent phenotype (basal non-activated state) in CFs. We report increased fibrosis, decreased cardiac function, and electrical impulse conduction defects in genetic and acquired mouse models of βIV-spectrin deficiency. Loss of betaIV-spectrin function promotes STAT3 nuclear accumulation and transcriptional activity, altered gene expression and CF activation. Furthermore, we demonstrate that a quiescent phenotype may be restored in βIV-spectrin deficient fibroblasts by expressing a βIV-spectrin fragment including the STAT3-binding domain or through pharmacological STAT3 inhibition. We find that in vivo STAT3 inhibition abrogates fibrosis and cardiac dysfunction in the setting of global βIV-spectrin deficiency. Finally, we demonstrate that fibroblast-specific deletion of βIV-spectrin is sufficient to induce fibrosis and decreased cardiac function. We propose that the βIV-spectrin/STAT3 complex is a determinant of fibroblast phenotype and fibrosis, with implications for remodeling response in cardiovascular disease.
Authors
Nehal J. Patel, Drew M. Nassal, Amara D. Greer-Short, Sathya D. Unudurthi, Benjamin W. Scandling, Daniel Gratz, Xianyao Xu, Anuradha Kalyanasundaram, Vadim V. Fedorov, Federica Accornero, Peter J. Mohler, Keith J. Gooch, Thomas J. Hund
Abstract
Filoviruses of the genus Ebolavirus include five species with marked differences in their ability to cause disease in humans. From the highly virulent Ebola virus to the seemingly nonpathogenic Reston virus, case-fatality rates can range between 0-90%. In order to understand the molecular basis of these differences it is imperative to establish disease models that recapitulate human disease as faithfully as possible. Non-human primates are the gold-standard models for filovirus pathogenesis, but comparative studies are skewed by the fact that Reston virus infection can be lethal for NHP. Here we have used HLA-A2 transgenic, NOD-scid-interleukin 2γ receptor knockout (NSG-A2) mice reconstituted with human hematopoiesis to compare Ebola virus and Reston virus pathogenesis in a human-like environment. While significantly less pathogenic than Ebola virus, Reston virus killed 20% of infected mice, a finding that was linked to exacerbated inflammation and viral replication in the liver. In addition, ‘humanized’ mice recapitulated the case-fatality ratios of different Ebolavirus species in humans. Our findings point out at humanized mice as a putative model to test the pathogenicity of newly discovered filoviruses, and warrants further investigations on Reston virus pathogenesis in humans.
Authors
Beatriz Escudero-Pérez, Paula Ruibal, Monika Rottstegge, Anja Lüdtke, Julia R. Port, Kristin Hartmann, Sergio Gómez-Medina, Juergen Müller-Guhl, Emily V. Nelson, Susanne Krasemann, Estefanía Rodríguez, César Muñoz-Fontela
Abstract
Severe asthma with fungal sensitization (SAFS) defines a subset of human asthmatics with allergy to one or more fungal species and difficult to control asthma. We have reported that human asthmatics sensitized to fungi have worse lung function and a higher degree of atopy, which was associated with higher IL-1RA levels in bronchoalveolar lavage fluid. IL-1RA further demonstrated a significant negative association with bronchial hyperresponsiveness to methacholine. Here, we show that IL-1α and IL-1β are elevated in both bronchoalveolar lavage fluid and sputum from human asthmatics sensitized to fungi, implicating an association with IL-1α, IL-1β or IL-1RA in fungal asthma severity. In an experimental model of fungal-associated allergic airway inflammation, we demonstrate that IL-1R1 signaling promotes type 1 (IFN-γ, CXCL9, CXCL10) and type 17 (IL-17A, IL-22) responses that were associated with neutrophilic inflammation and increased airway hyperreactivity. Each of these were exacerbated in the absence of IL-1RA. Administration of human recombinant IL-1RA (Kineret/Anakinra) during fungal-associated allergic airway inflammation improved airway hyperreactivity and lowered type 1 and type 17 responses. Taken together, these data suggest that IL-1 receptor signaling contributes to fungal asthma severity via immunopathogenic type 1 and type 17 responses and can be targeted for improving allergic asthma severity.
Authors
Matthew S. Godwin, Kristen M. Reeder, Jaleesa M. Garth, Jonathan P. Blackburn, MaryJane Jones, Zhihong Yu, Sadis Matalon, Annette T. Hastie, Deborah A. Meyers, Chad Steele
Abstract
Lymphatic malformations (LMs) are congenital, non-neoplastic vascular malformations associated with post-zygotic activating PIK3CA mutations. The mutation spectrum within LMs is narrow, with the majority having one of three “hotspot” mutations. Despite this relative genetic homogeneity, clinical presentations differ dramatically. We used molecular inversion probes and droplet digital polymerase chain reaction to perform deep, targeted sequencing of PIK3CA in 271 affected and unaffected tissue samples from 81 individuals with isolated LMs and retrospectively collected clinical data. Pathogenic PIK3CA mutations were identified in affected LM tissue in 64 individuals (79%) with isolated LMs, with variant allele fractions (VAFs) ranging from 0.1 to 13%. Initial analyses revealed no correlation between VAF and phenotype variables. Recognizing that different mutations activate PI3K to varying degrees, we developed a metric, the genotype-adjusted VAF (GVAF), to account for differences in mutation strength, and found significantly higher GVAFs in LMs with more severe clinical characteristics including orofacial location or microcystic structure. In addition to providing insight into LM pathogenesis, we believe GVAF may have broad applicability for genotype-phenotype analyses in mosaic disorders.
Authors
Kaitlyn Zenner, Chi Vicky Cheng, Dana M. Jensen, Andrew E. Timms, Giridhar Shivaram, Randall Bly, Sheila Ganti, Kathryn B. Whitlock, William B. Dobyns, Jonathan Perkins, James T. Bennett
Abstract
miR-511-3p, encoded by CD206/Mrc1, was demonstrated to reduce allergic inflammation and promote alternative (M2) macrophage polarization. Here, we sought to elucidate the fundamental mechanism by which miR-511-3p attenuates allergic inflammation and promotes macrophage polarization. Compared with wild-type mice, the allergen-challenged Mrc1-/- mice showed increased airway hyper-responsiveness (AHR) and inflammation. However, this increased AHR and inflammation were significantly attenuated when these mice were pre-transduced with adeno-associated virus (AAV)-miR-511-3p. Gene expression profiling of macrophages identified Ccl2 as one of the major genes that was highly expressed in M2 macrophages but antagonized by miR-511-3p. The interaction between miR-511-3p and Ccl2 was confirmed by in silico analysis and mRNA-miRNA pull-down assay. Further evidence for the inhibition of Ccl2 by miR-511-3p was given by reduced levels of Ccl2 in supernatants of miR-511-3p transduced macrophages and in bronchoalveolar lavage fluids of AAV-miR-511-3p-infected Mrc1-/- mice. Mechanistically, we demonstrated that Ccl2 promotes M1 macrophage polarization by activating RhoA signaling through Ccr2. The interaction between Ccr2 and RhoA was also supported by co-immunoprecipitation assay. Importantly, inhibition of RhoA signaling suppressed cockroach allergen-induced AHR and lung inflammation. These findings suggest a novel mechanism by which miR-511-3p regulates allergic inflammation and macrophage polarization by targeting Ccl2 and its downstream Ccr2/RhoA axis.
Authors
Danh C. Do, Jie Mu, Xia Ke, Karan Sachdeva, Zili Qin, Mei Wan, Faoud T. Ishmael, Peisong Gao
Abstract
The glucagon-like peptide 1 receptor agonist exenatide improves glycemic control by several and not completely understood mechanisms. Herein, we examined the effects of chronic intravenous exenatide infusion on insulin sensitivity, β- and α-cell function and relative volumes, islet cell apoptosis and replication in nondiabetic non-human primates (baboons). At baseline, baboons received a 2-step hyperglycemic clamp followed by an L-arginine bolus (HC/A). After HC/A, baboons underwent a partial pancreatectomy (tail removal) and received a continuous exenatide (n = 12) or saline (n = 12) infusion for 13 weeks. At the end of treatment, HC/A was repeated and the remnant pancreas (head-body) harvested. Insulin sensitivity increased dramatically after exenatide treatment and was accompanied by a decrease in insulin and C-peptide secretion, while the insulin secretion/insulin resistance (disposition) index increased by approximately 2-fold. β-, α-, and δ-cell relative volumes in exenatide-treated baboons were significantly increased compared to saline-treated controls, primarily as the result of increased islet cell replication. Features of cellular stress and secretory dysfunction were present in islets of saline-treated baboons and absent in islets of exenatide-treated baboons. In conclusion, chronic administration of exenatide exerts proliferative and cytoprotective effects on β-, α-, and δ-cells and produces a robust increase in insulin sensitivity in non-human primates.
Authors
Teresa Vanessa Fiorentino, Francesca Casiraghi, Alberto M. Davalli, Giovanna Finzi, Stefano La Rosa, Paul B. Higgins, Gregory A. Abrahamian, Alessandro Marando, Fausto Sessa, Carla Perego, Rodolfo Guardado- Mendoza, Subhash Kamath, Andrea Ricotti, Paolo Fiorina, Giuseppe Daniele, Ana M. Paez, Francesco Andreozzi, Raul A. Bastarrachea, Anthony G. Comuzzie, Amalia Gastaldelli, Alberto O. Chavez, Eliana S. Di Cairano, Patrice A. Frost, Livio Luzi, Edward J. Dick, Jr., Glenn A. Halff, Ralph A. DeFronzo, Franco Folli
Abstract
Itch induces scratching that removes irritants from the skin, whereas pain initiates withdrawal or avoidance of tissue damage. Whilst pain arises from both the skin and viscera, we investigated whether pruritogenic irritant mechanisms also function within visceral pathways. We show that subsets of colon-innervating sensory neurons in mice express, either individually or in combination, the pruritogenic receptors Tgr5 and the Mas-gene-related G protein-coupled receptors, Mrgpra3 and Mrgpra11. Agonists of these receptors activated subsets of colonic sensory neurons and evoked colonic afferent mechanical hypersensitivity via a TRPA1-dependent mechanism. In vivo intra-colonic administration of individual TGR5, MRGPRA3, or MRGPRC11 agonists induced pronounced visceral hypersensitivity to colorectal distension. Co-administration of these agonists as an ‘itch cocktail’ augmented hypersensitivity to colorectal distension and changed mouse behaviour. These irritant mechanisms were maintained and enhanced in a model of chronic visceral hypersensitivity relevant to irritable bowel syndrome. Neurons from human dorsal root ganglia also expressed TGR5 as well as the human ortholog MRGPRX1 and showed increased responsiveness to pruritogenic agonists in pathological states. These data support the existence of an irritant-sensing system in the colon that is a visceral representation of the itch pathways found in skin, thereby contributing to sensory disturbances accompanying common intestinal disorders.
Authors
Joel Castro, Andrea M. Harrington, TinaMarie Lieu, Sonia Garcia-Caraballo, Jessica Maddern, Gudrun Schober, Tracey O'Donnell, Luke Grundy, Amanda L. Lumsden, Paul E. Miller, Andre Ghetti, Martin S. Steinhoff, Daniel P. Poole, Xinzhong Dong, Lin Chang, Nigel W. Bunnett, Stuart M. Brierley
Efficient ADCC- killing of meningioma by avelumab and a high-affinity natural killer cell line, haNK
Abstract
Meningiomas are the most common adult primary tumor of the central nervous system, but there are no known effective medical therapies for recurrent meningioma, particularly for WHO grade II and III tumors. Meningiomas arise from the meninges, located outside the blood-brain barrier, and therefore may be directly targeted by antibody-mediated immunotherapy. We found that PD-L1 was highly expressed in multiple human malignant meningioma cell lines and patient tumor samples. PD-L1 was targeted with the anti-PD-L1 antibody avelumab and directed natural killer cells to mediate antibody-dependent cellular cytotoxicity (ADCC) of PD-L1-expressing meningioma tumors both in vitro and in vivo. ADCC of meningioma cells was significantly increased in target cells that upregulated PD-L1 expression and, conversely, abrogated in tumor cells that were depleted of PD-L1. Additionally, the high-affinity natural killer cell line, haNK, outperformed healthy donor NK cells in meningioma ADCC. Together, these data support a clinical trial designed to target PD-L1 with avelumab and haNK cells, potentially offering a novel immunotherapeutic approach for patients with malignant meningioma.
Authors
Amber J. Giles, Shuyu Hao, Michelle R. Padget, Hua Song, Wei Zhang, John Lynes, Victoria E. Sanchez, Yang Liu, Jinkyu Jung, Xiaoyu Cao, Rika Fujii, Randy L. Jensen, David Gillespie, Jeffrey Schlom, Mark R. Gilbert, Edjah K. Nduom, Chunzhang Yang, John H. Lee, Patrick Soon-Shiong, James W. Hodge, Deric M. Park
Abstract
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell-B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here we evaluated the phenotypes of CD4+ T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1hi CXCR5- CD4+ T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1hi CXCR5- CD4+ T cells from SLE patients are T peripheral helper (Tph) cells, a CXCR5- T cell population that stimulates B cell responses via IL-21. The frequency of Tph cells, but not Tfh cells, correlated with both clinical disease activity and the frequency of CD11c+ B cells in SLE patients. PD-1hi CD4+ T cells were found within lupus nephritis kidneys and correlated with B cell numbers in kidney. Both IL-21 neutralization and CRISPR-mediated deletion of MAF abrogated the ability of Tph cells to induce memory B cell differentiation into plasmablasts in vitro. These findings identify Tph cells a highly expanded T cell population in SLE and suggest a key role for Tph cells in stimulating pathologic B cell responses.
Authors
Alexandra V. Bocharnikov, Joshua Keegan, Vanessa S. Wacleche, Ye Cao, Chamith Y. Fonseka, Guoxing Wang, Eric Muise, Kelvin X. Zhang, Arnon Arazi, Gregory Keras, Zhihan J. Li, Yujie Qu, Michael F. Gurish, Michelle Petri, Jill P. Buyon, Chaim Putterman, David Wofsy, Judith A. James, Joel M. Guthridge, Betty Diamond, Jennifer H. Anolik, Matthew F. Mackey, Stephen E. Alves, Peter A. Nigrovic, Karen H. Costenbader, Michael B. Brenner, James A. Lederer, Deepak A. Rao
Abstract
Tissue engineering is a promising approach to address organ shortages currently limiting clinical transplantation. “Off-the-shelf” engineered vascularized organs will likely use allogeneic endothelial cells (ECs) to construct microvessels required for graft perfusion. Vasculogenic ECs can be differentiated from committed progenitors (human endothelial colony forming cells or HECFCs) without risk of mutation or teratoma formation associated with reprogrammed stem cells. Like other ECs, these cells basally express both class I and class II major histocompatibility complex (MHC) molecules, bind donor-specific antibody (DSA), activate alloreactive T effector memory cells, and initiate rejection in the absence of donor leukocytes. We report here that CRISPR/Cas9-mediated dual ablation of β2-microglobulin and CIITA in HECFC-derived ECs eliminates both class I and II MHC expression while retaining EC functions and vasculogenic potential. Importantly, dually ablated ECs no longer bind human DSA or activate allogeneic CD4+ effector memory T cells and are resistant to killing by CD8+ alloreactive cytotoxic T lymphocytes in vitro and in vivo. Despite absent class I MHC molecules, these ECs do not activate or elicit cytotoxic activity from allogeneic natural killer cells. These data suggest that HECFC-derived ECs lacking MHC molecule expression can be utilized for engineering vascularized grafts that evade allorejection.
Authors
Jonathan Merola, Melanie Reschke, Richard W. Pierce, Lingfeng Qin, Susann Spindler, Tania Baltazar, Thomas D. Manes, Francesc Lopez-Giraldez, Guangxin Li, Laura G. Bracaglia, Catherine Xie, Nancy Kirkiles-Smith, W. Mark Saltzman, Gregory T. Tietjen, George Tellides, Jordan S. Pober
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