Epidemiological findings indicate that coinfection with influenza viruses is associated with an increased risk of death in patients suffering from tuberculosis but the underlying pathomechanisms are not well understood. In this study, we demonstrate that influenza A virus (IAV) coinfection rapidly impairs control of Mycobacterium tuberculosis (Mtb) in C57BL/6 mice. IAV coinfection was associated with significantly increased bacterial loads, reduced survival and a substantial modulation of innate and adaptive immune defenses including an impaired onset and development of Mtb-specific CD4+ T cell responses and the accumulation of macrophages with increased arginase-1 production in the lungs. Our findings strongly indicate that IAV coinfection compromises the host’s ability to control Mtb infection via the production of IL-10 which was rapidly induced upon viral infection. The blockade of IL-10 receptor signaling reduced the bacterial load in coinfected mice to a level comparable with that in Mtb-only-infected animals. Taken together, our data suggest that IL-10 signaling constitutes a major pathway that enhances susceptibility to Mtb during concurrent IAV infection.
Sarah Ring, Lars Eggers, Jochen Behrends, Adam Wutkowski, Dominik Schwudke, Andrea Kröger, Alexandra Maximiliane Hierweger, Christoph Hölscher, Gülsah Gabriel, Bianca Schneider
Gain of the long arm of chromosome 17 (17q) is a cytogenetic hallmark of high-risk neuroblastoma, yet its contribution to neuroblastoma pathogenesis remains incompletely understood. Combining whole-genome and RNA sequencing of neuroblastomas, we identified the prohibitin (PHB) gene as highly expressed in tumors with 17q gain. High PHB expression correlated with poor prognosis and was associated with loss of gene expression programs promoting neuronal development and differentiation. PHB depletion induced differentiation and apoptosis and slowed cell cycle progression of neuroblastoma cells, at least in part through impaired ERK1/2 activation. Conversely, ectopic expression of PHB was sufficient to increase proliferation of neuroblastoma cells and was associated with suppression of markers associated with neuronal differentiation and favorable neuroblastoma outcome. Thus, PHB is a 17q oncogene in neuroblastoma that promotes tumor cell proliferation, and de-differentiation.
Ian C. MacArthur, Yi Bei, Heathcliff Dorado García, Michael V. Ortiz, Joern Toedling, Filippos Klironomos, Jana Rolff, Angelika Eggert, Johannes H. Schulte, Alex Kentsis, Anton G. Henssen
The bone marrow microenvironment (BMME) contributes to the regulation of hematopoietic stem cell (HSC) function, though its role in age-associated lineage skewing is poorly understood. Here we show that dysfunction of aged marrow macrophages (Mφs) directs HSC platelet-bias. Mφs from the marrow of aged mice and humans exhibited an activated phenotype, with increased expression of inflammatory signals. Aged marrow Mφs also displayed decreased phagocytic function. Senescent neutrophils, typically cleared by marrow Mφs, were markedly increased in aged mice, consistent with functional defects in Mφ phagocytosis and efferocytosis. In aged mice, Interleukin 1B (IL1B) was elevated in the bone marrow and caspase 1 activity, which can process pro-IL1B, was increased in marrow Mφs and neutrophils. Mechanistically, IL1B signaling was necessary and sufficient to induce a platelet bias in HSCs. In young mice, depletion of phagocytic cell populations or loss of the efferocytic receptor Axl expanded platelet-biased HSCs. Our data support a model wherein increased inflammatory signals and decreased phagocytic function of aged marrow Mφs induce the acquisition of platelet bias in aged HSCs. This work highlights the instructive role of Mφs and IL1B in the age-associated lineage-skewing of HSCs, and reveals the therapeutic potential of their manipulation as antigeronic targets.
Benjamin J. Frisch, Corey M. Hoffman, Sarah E. Latchney, Mark W. LaMere, Jason Myers, John Ashton, Allison J. Li, Jerry Saunders, James Palis, Archibald S. Perkins, Amanda McCabe, Julianne N. Smith, Kathleen E. McGrath, Fatima Rivera-Escalera, Andrew McDavid, Jane L. Liesveld, Vyacheslav A. Korshunov, Michael R. Elliott, Katherine C. MacNamara, Michael W. Becker, Laura M. Calvi
Children with trisomy 21 (Down syndrome [DS]) have a 130-fold increased incidence of Hirschsprung Disease (HSCR), a developmental defect where the enteric nervous system (ENS) is missing from distal bowel (i.e., distal bowel is aganglionic). Treatment for HSCR is surgical resection of aganglionic bowel, but many children have bowel problems after surgery. Post-surgical problems like enterocolitis and soiling are especially common in children with DS. To determine how trisomy 21 affects ENS development, we evaluated the ENS in two DS mouse models, Ts65Dn and Tc1. These mice are trisomic for many chromosome 21 homologous genes, including Dscam and Dyrk1a, which are hypothesized to contribute to HSCR risk. Ts65Dn and Tc1 mice have normal ENS precursor migration at E12.5 and almost normal myenteric plexus structure as adults. However, Ts65Dn and Tc1 mice have markedly reduced submucosal plexus neuron density throughout the bowel. Surprisingly, the submucosal neuron defect in Ts65Dn mice is not due to excess Dscam or Dyrk1a, since normalizing copy number for these genes does not rescue the defect. These findings suggest the possibility that the high frequency of bowel problems in children with DS and HSCR may occur because of additional unrecognized problems with ENS structure.
Ellen M. Schill, Christina M. Wright, Alisha Jamil, Jonathan M. LaCombe, Randall J. Roper, Robert O. Heuckeroth
Background: Current dosing of intrapleural fibrinolytic therapy (IPFT) in adults with complicated parapneumonic effusion (CPE) / empyema is empiric, as dose-escalation trials have not previously been conducted. We hypothesized that LTI-01 (scuPA), which is relatively resistant to PA inhibitor-1 (PAI-1), would be well-tolerated. Methods: This was an open-label, dose-escalation trial of LTI-01 IPFT at 50,000-800,000 IU daily for up to 3 days in adults with loculated CPE/empyema and failed pleural drainage. The primary objective was to evaluate safety and tolerability, and secondary objectives included assessments of processing and bioactivity of scuPA in blood and pleural fluid (PF), and early efficacy. Results: LTI-01 was well tolerated with no bleeding, treatment-emergent adverse events or surgical referrals (n=14 subjects). uPA antigen increased in PFs at 3 hours after LTI-01 (p<0.01) but not in plasma. PF saturated active PAI-1, generated PAI-1-resistant bioactive complexes, increased PA and fibrinolytic activities and D-dimers. There was no systemic fibrinogenolysis, nor increments in plasma D-dimer. Decreased pleural opacities occurred in all but one subject. Both subjects receiving 800,000 IU required two doses to relieve pleural sepsis, with two other subjects similarly responding at lower doses. Conclusion: LTI-01 IPFT was well-tolerated at these doses with no safety concerns. Bioactivity of LTI-01 IPFT was confirmed, limited to PFs where its processing simulated that previously reported in preclinical studies. Preliminary efficacy signals including reduction of pleural opacity were observed.
Lutz Beckert, Ben Brockway, Graham Simpson, Anne Marie Southcott, Y.C. Gary Lee, Najib Rahman, Richard W. Light, Steven Shoemaker, John Gillies, Andrey A. Komissarov, Galina Florova, Timothy Ochran, William Bradley, Harrison Ndetan, Karan P. Singh, Krishna Sarva, Steven Idell
The E3 ubiquitin ligase Parkin plays an important role in regulating clearance of dysfunctional or unwanted mitochondria in tissues, including the heart. However, whether Parkin also functions to prevent cardiac aging by maintaining a healthy population of mitochondria is still unclear. Here, we have examined the role of Parkin in the context of mtDNA damage and myocardial aging using a mouse model carrying a proofreading defective mitochondrial DNA polymerase gamma (POLG). We observed both decreased Parkin protein levels and development of cardiac hypertrophy in POLG hearts with age; however, cardiac hypertrophy in POLG mice was neither rescued, nor worsened by cardiac specific overexpression or global deletion of Parkin, respectively. Unexpectedly, mitochondrial fitness did not substantially decline with age in POLG mice when compared to WT. We found that baseline mitophagy receptor-mediated mitochondrial turnover and biogenesis were enhanced in aged POLG hearts. We also observed the presence of megamitochondria in aged POLG hearts. Thus, these processes may limit the accumulation of dysfunctional mitochondria as well as the degree of cardiac functional impairment in the aging POLG heart. Overall, our results demonstrate that Parkin is dispensable for constitutive mitochondrial quality control in a mtDNA mutation model of cardiac aging.
Benjamin P. Woodall, Amabel M. Orogo, Rita H. Najor, Melissa Q. Cortez, Eileen R. Moreno, Hongxia Wang, Ajit S. Divakaruni, Anne N. Murphy, Asa B. Gustafsson
Adipose tissue macrophages (ATM) are crucial for maintaining adipose tissue homeostasis and mediating obesity-induced metabolic abnormalities, including prediabetic conditions and type 2 diabetes mellitus. Despite their key functions in regulating adipose tissue metabolic and immunologic homeostasis under normal and obese conditions, a high-resolution transcriptome annotation system that can capture ATM multifaceted activation profiles has not yet been developed. This is primarily attributed to the complexity of their differentiation/activation process in adipose tissue and their diverse activation profiles in response to microenvironmental cues. Although the concept of multifaceted macrophage action is well-accepted, no current model precisely depicts their dynamically regulated in vivo features. To address this knowledge gap, we generated single-cell transcriptome data from primary bone marrow-derived macrophages under polarizing and non-polarizing conditions to develop new high-resolution algorithms. The outcome was creation of a two-index platform, MacSpectrum (https://macspectrum.uconn.edu), that enables comprehensive high-resolution mapping of macrophage activation states from diverse mixed cell populations. MacSpectrum captured dynamic transitions of macrophage subpopulations under both in vitro and in vivo conditions. Importantly, MacSpectrum revealed unique “signature” gene sets in ATMs and circulating monocytes that displayed significant correlation with BMI and homeostasis model assessment of insulin resistance (HOMA-IR) in obese human patients. Thus, MacSpectrum provides unprecedented resolution to decode macrophage heterogeneity and will open new areas of clinical translation.
Chuan Li, Antoine Menoret, Cullen Farragher, Zhengqing Ouyang, Christopher Bonin, Paul Holvoet, Anthony T. Vella, Beiyan Zhou
Obliterative bronchiolitis (OB) is a poorly understood airway disease characterized by the generation of fibrotic bronchiolar occlusions. In the lung transplant setting, OB is a pathological manifestation of bronchiolitis obliterans syndrome (BOS), which is a major impediment to long-term recipient survival. Club cells play a key role in bronchiolar epithelial repair, but whether they promote lung transplant tolerance through preventing OB remains unclear. We determined if OB occurs in mouse orthotopic lung transplants following conditional transgene-targeted club cell depletion. In syngeneic lung transplants club cell depletion leads to transient epithelial injury followed by rapid club cell-mediated repair. In contrast, allogeneic lung transplants develop severe OB lesions and poorly regenerate club cells despite immunosuppression treatment. Lung allograft club cell ablation also triggers the recognition of alloantigens, and pulmonary restricted self-antigens reported associated with BOS development. However, CD8+ T cell depletion restores club cell reparative responses and prevents OB. In addition, ex-vivo analysis reveals a specific role for alloantigen-primed effector CD8+ T cells in preventing club cell proliferation and maintenance. Taken together, we demonstrate a vital role for club cells in maintaining lung transplant tolerance and propose a new model to identify the underlying mechanisms of OB.
Zhiyi Liu, Fuyi Liao, Davide Scozzi, Yuka Furuya, Kaitlyn N. Pugh, Ramsey R. Hachem, Delphine L. Chen, Marlene Cano, Jonathan M. Green, Alexander S. Krupnick, Daniel Kreisel, Anne-Karina T. Perl, Howard J. Huang, Steven L. Brody, Andrew E. Gelman
Nemaline myopathy is a congenital neuromuscular disorder characterized by muscle weakness, fiber atrophy and presence of nemaline bodies within myofibers. However, the understanding of underlying pathomechanisms is lacking. Recently, mutations in KBTBD13, KLHL40 and KLHL41, three substrate adaptors for the E3-ubiquitin ligase Cullin-3, have been associated with early-onset nemaline myopathies. We hypothesized that deregulation of Cullin-3 and its muscle protein substrates may be responsible for the disease development. Using Cullin-3 knockout mice, we identified accumulation of non-muscle alpha-Actinins (ACTN1 and ACTN4) in muscles of these mice, which we also observed in KBTBD13 patients. Our data reveal that proper regulation of Cullin-3 activity and ACTN1 levels is essential for normal muscle and neuromuscular junction development. While ACTN1 is naturally downregulated during myogenesis, its overexpression in C2C12 myoblasts triggered defects in fusion, myogenesis and acetylcholine receptor clustering; features that we characterized in Cullin-3 deficient mice. Taken together, our data highlight the importance for Cullin-3 mediated degradation of ACTN1 for muscle development, and indicate a new pathomechanism for the etiology of myopathies seen in Cullin-3 knockout mice and nemaline myopathy patients.
Jordan Blondelle, Kavya Tallapaka, Jane T. Seto, Majid Ghassemian, Madison Clark, Jenni M. Laitila, Adam Bournazos, Jeffrey D. Singer, Stephan Lange
Recently, by utilizing conventional and tamoxifen inducible Bmal1 (Brain and muscle Arnt-like protein 1) knockout mice, we found that delaying the loss of circadian rhythms to adulthood attenuates the impact on general integrity and survival at least under 12h light/12h dark conditions (LD). To understand further the contribution of Bmal1 in post-natal life under conditions of circadian disruption, we subjected inducible knockout mice (KO) and their littermate controls (Ctrl) to forced desynchrony protocols including cycles with non-24h periods, randomized light/dark cycles, and jet lag, and monitored their locomotor activity using radiotelemetry. Under these conditions, control mice cannot be entrained, as reflected by their maintenance of circadian behavior irrespective of schedules. By contrast, KO mice displayed higher activity levels in the dark phases of most cycles. Under a 3h light/3h dark regime, Ctrls displayed higher activity levels in the dark phases of all cycles although there were still obvious circadian rhythms, suggesting that an ultradian mechanism is also involved. Insulin sensitivity was markedly reduced by disrupted light schedules as expected in Ctrls, but not in the KOs. Thus, Bmal1 deletion in adult mice facilitates adaptation to new light/dark schedules and protects from insulin resistance induced by circadian disruption.
Guangrui Yang, Lihong Chen, Jiayang Zhang, Baoyin Ren, Garret A. FitzGerald
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