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
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
Biomechanical forces and endothelial-to-mesenchymal transition (EndoMT) are known to mediate valvulogenesis. However, the relative contributions of myocardial contractile and hemodynamic shear forces remain poorly understood. We integrated 4-D light-sheet imaging of transgenic zebrafish models with moving-domain computational fluid dynamics to determine effects of changes in contractile forces and fluid wall shear stress (WSS) on ventriculobulbar (VB) valve development. Augmentation of myocardial contractility with isoproterenol increased both WSS and Notch1b activity in the developing outflow tract (OFT) and resulted in VB valve hyperplasia. Increasing WSS in the OFT, achieved by increasing blood viscosity through EPO mRNA injection, also resulted in VB valve hyperplasia. Conversely, decreasing myocardial contractility by Tnnt2a morpholino oligonucleotide (MO) administration, 2,3-butanedione monoxime treatment, or Plcγ1 inhibition completely blocked VB valve formation, which could not be rescued by increasing WSS or activating Notch. Decreasing WSS in the OFT, achieved by slowing heart rate with metoprolol or reducing viscosity with Gata1a MO, did not affect VB valve formation. Immunofluorescent staining with the mesenchymal marker, DM-GRASP, revealed that biomechanical force-mediated Notch1b activity is implicated in EndoMT to modulate valve morphology. Altogether, increases in WSS result in Notch1b- EndoMT-mediated VB valve hyperplasia, whereas decreases in contractility result in reduced Notch1b activity, absence of EndoMT, and VB valve underdevelopment. Thus, we provide developmental mechanotransduction mechanisms underlying Notch1b-mediated EndoMT in the OFT.
Jeffrey J. Hsu, Vijay Vedula, Kyung In Baek, Cynthia Chen, Junjie Chen, Man In Chou, Jeffrey Lam, Shivani Subhedar, Jennifer Wang, Yichen Ding, Chih-Chiang Chang, Juhyun Lee, Linda L. Demer, Yin Tintut, Alison L. Marsden, Tzung K. Hsiai
Breast cancer bone metastases often cause a debilitating non-curable condition with osteolytic lesions, muscle weakness and a high mortality. Current treatment comprises chemotherapy, irradiation, surgery and anti-resorptive drugs that restrict but do not revert bone destruction. In metastatic breast cancer cells, we determined the expression of sclerostin, a soluble Wnt inhibitor that represses osteoblast differentiation and bone formation. In mice with breast cancer bone metastases, pharmacological inhibition of sclerostin using an anti-sclerostin antibody (Scl-Ab) reduced metastases without tumor cell dissemination to other distant sites. Sclerostin inhibition prevented the cancer-induced bone destruction by augmenting osteoblast-mediated bone formation and reducing osteoclast-dependent bone resorption. During advanced disease, NF-κB and p38 signaling was increased in muscles in a TGF-β1-dependent manner, causing muscle fiber atrophy, muscle weakness and tissue regeneration with an increase in Pax7-positive satellite cells. Scl-Ab treatment restored NF-κB and p38 signaling, the abundance of Pax7-positive cells and ultimately muscle function. These effects improved the overall health condition and expanded the life span of cancer-bearing mice. Together, these results demonstrate that pharmacological inhibition of sclerostin reduces bone metastatic burden and muscle weakness with a prolongation of the survival time. This might provide novel options for treating musculoskeletal complications in breast cancer patients.
Eric Hesse, Saskia Schröder, Diana Brandt, Jenny Pamperin, Hiroaki Saito, Hanna Taipaleenmäki
Imatinib (Gleevec) reverses type 1 diabetes (T1D) in NOD mice and is currently in clinical trials in individuals with recent-onset disease. While research has demonstrated that imatinib protects islet β cells from the harmful effects of ER stress, the role the immune system plays in its reversal of T1D has been less well understood, and specific cellular immune targets have not been identified. In this study, we demonstrate that B lymphocytes, an immune subset that normally drives diabetes pathology, are unexpectedly required for reversal of hyperglycemia in NOD mice treated with imatinib. In the presence of B lymphocytes, reversal was linked to an increase in serum insulin concentration, but not an increase in islet β cell mass or proliferation. However, improved β cell function was reflected by a partial recovery of MafA transcription factor expression, a sensitive marker of islet β cell stress that is important to adult β cell function. Imatinib treatment was found to increase the antioxidant capacity of B lymphocytes, improving reactive oxygen species (ROS) handling in NOD islets. This study reveals a novel mechanism through which imatinib enables B lymphocytes to orchestrate functional recovery of T1D β cells.
Christopher S. Wilson, Jason M. Spaeth, Jay Karp, Blair T. Stocks, Emilee M. Hoopes, Roland W. Stein, Daniel J. Moore
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