Abstract
Reduction/elimination of HIV-1 reservoirs that persist despite combination antiretroviral therapy (cART) will likely require induction of viral expression by residual infected cells and enhanced clearance of these cells. TLR7 agonists have potential to mediate these activities. We evaluated immunologic and virologic effects of repeated doses of the TLR7 agonist GS-9620 in SIV-infected rhesus macaques receiving cART, which was initiated at 13 days after infection and was continued for 75 weeks prior to GS-9620 administration. During cART, GS-9620 induced transient upregulation of IFN-stimulated genes in blood and tissues, increases in plasma cytokines, and changes in immune cell population activation and phenotypes but did not result in measurable increases in plasma viremia or viral RNA–to–viral DNA ratio in PBMCs or tissues nor decreases in viral DNA in PBMC or tissues. SIV-specific CD8+ T cell responses, negligible prior to GS-9620 treatment, were not measurably boosted by treatment; a second course of GS-9620 administration overlapping with later cART discontinuation was associated with increased CD8+ T cell responses during viral recrudescence. These results confirm and extend evidence for GS-9620–mediated enhancement of antiviral immune responses in SIV-infected macaques but suggest that GS-9620–mediated viral induction may depend critically on the timing of initiation and duration of cART and resulting characteristics of viral reservoirs.
Authors
Gregory Q. Del Prete, W. Gregory Alvord, Yuan Li, Claire Deleage, Mukta Nag, Kelli Oswald, James A. Thomas, Cathi Pyle, William J. Bosche, Vicky Coalter, Adam Wiles, Rodney Wiles, Brian Berkemeier, Michael Hull, Elizabeth Chipriano, Lorna Silipino, Randy Fast, Jacob Kiser, Rebecca Kiser, Tyler Malys, Joshua Kramer, Matthew W. Breed, Charles M. Trubey, Jacob D. Estes, Tiffany L. Barnes, Joseph Hesselgesser, Romas Geleziunas, Jeffrey D. Lifson
Abstract
Immune responses to therapeutic transgenes are a potential hurdle to treat monogenic muscle disorders. These responses result from the neutralizing activity of transgene-specific B cells and cytotoxic T cells recruited upon gene transfer. We explored here how dual muscle-liver expression of a foreign transgene allows muscle transgene engraftment after adenoassociated viral vector delivery. We found in particular that induction of transgene-specific tolerance is imposed by concurrent muscle and liver targeting, resulting in the absence of CD8+ T cell responses to the transgene. This tolerance can be temporally decoupled, because transgene engraftment can be achieved in muscle weeks after liver transduction. Importantly, transgene-specific CD8+ T cell tolerance can be established despite preexisting immunity to the transgene. Whenever preexisting, transgene-specific CD4+ and CD8+ memory T cell responses are present, dual muscle-liver transduction turns polyclonal, transgene-specific CD8+ T cells into typically exhausted T cells with high programmed cell death 1 (PD-1) expression and lack of IFN-γ production. Our results demonstrate that successful transduction of muscle tissue can be achieved through liver-mediated control of humoral and cytotoxic T cell responses, even in the presence of preexisting immunity to the muscle-associated transgene.
Authors
Laurent Bartolo, Stéphanie Li Chung Tong, Pascal Chappert, Dominique Urbain, Fanny Collaud, Pasqualina Colella, Isabelle Richard, Giuseppe Ronzitti, Jocelyne Demengeot, David A. Gross, Federico Mingozzi, Jean Davoust
Abstract
Recently, by using conventional and tamoxifen-inducible brain and muscle Arnt-like protein 1–knockout (Bmal1-KO) mice, we found that delaying the loss of circadian rhythms to adulthood attenuates the impact on general integrity and survival at least under 12-hour light/12-hour dark conditions. To understand further the contribution of Bmal1 in postnatal life under conditions of circadian disruption, we subjected inducible-KO and their littermate controls (ctrls) to forced desynchrony protocols, including cycles with non–24-hour periods, randomized light/dark cycles, and jet lag, and monitored their locomotor activity using radiotelemetry. Under these conditions, ctrl 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 3-hour light/3-hour 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 KO mice. Thus, Bmal1 deletion in adult mice facilitates adaptation to new light/dark schedules and protects from insulin resistance induced by circadian disruption.
Authors
Guangrui Yang, Lihong Chen, Jiayang Zhang, Baoyin Ren, Garret A. FitzGerald
Abstract
IL-17–producing CD4+ (Th17) cells are pathogenically linked to autoimmunity and, specifically, to autoimmune kidney disease. The newly recognized immunoregulatory functions of erythropoietin (EPO) and its predominant intrarenal source suggested that EPO physiologically regulates Th17 cell differentiation, thereby serving as a barrier to development of autoimmune kidney disease. Using in vitro studies of human and murine cells and in vivo models, we show that EPO ligation of its receptor (EPO-R) on CD4+ T cells directly inhibits Th17 cell generation and promotes transdifferentiation of Th17 cells into IL-17–FOXP3+CD4+ T cells. Mechanistically, EPO/EPO-R ligation abrogates upregulation of SGK1 gene expression and blocks p38 activity to prevent SGK1 phosphorylation, thereby inhibiting RORC-mediated transcription of IL17 and IL23 receptor genes. In a murine model of Th17 cell–dependent aristolochic acid–induced interstitial kidney disease associated with reduced renal EPO production, we demonstrate that transgenic EPO overexpression or recombinant EPO (rEPO) administration limits Th17 cell formation and clinical/histological disease expression. EPO/EPO-R ligations on CD4+ T cells abrogate, while absence of T cell–expressed EPO-R augments, Th17 cell induction and clinical/histological expression of pristane-induced glomerulonephritis (associated with decreased intrarenal EPO). rEPO prevents spontaneous glomerulonephritis and Th17 cell generation in MRL-lpr mice. Together, our findings indicate that EPO physiologically and therapeutically modulates Th17 cells to limit expression of Th17 cell–associated autoimmune kidney disease.
Authors
Chiara Donadei, Andrea Angeletti, Chiara Cantarelli, Vivette D. D’Agati, Gaetano La Manna, Enrico Fiaccadori, Julian K. Horwitz, Huabao Xiong, Chiara Guglielmo, Susan Hartzell, Joren C. Madsen, Umberto Maggiore, Peter S. Heeger, Paolo Cravedi
Abstract
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 dedifferentiation.
Authors
Ian C. MacArthur, Yi Bei, Heathcliff Dorado Garcia, Michael V. Ortiz, Joern Toedling, Filippos Klironomos, Jana Rolff, Angelika Eggert, Johannes H. Schulte, Alex Kentsis, Anton G. Henssen
Abstract
MAPK signaling consists of an array of successively acting kinases. ERK1 and -2 (ERK1/2) are major components of the greater MAPK cascade that transduce growth factor signaling at the cell membrane. Here, we investigated ERK1/2 signaling in skeletal muscle homeostasis and disease. Using mouse genetics, we observed that the muscle-specific expression of a constitutively active MEK1 mutant promotes greater ERK1/2 signaling that mediates fiber-type switching to a slow, oxidative phenotype with type I myosin heavy chain expression. Using a conditional and temporally regulated Cre strategy, as well as Mapk1 (ERK2) and Mapk3 (ERK1) genetically targeted mice, MEK1-ERK2 signaling was shown to underlie this fast-to-slow fiber-type switching in adult skeletal muscle as well as during development. Physiologic assessment of these activated MEK1-ERK1/2 mice showed enhanced metabolic activity and oxygen consumption with greater muscle fatigue resistance. In addition, induction of MEK1-ERK1/2 signaling increased dystrophin and utrophin protein expression in a mouse model of limb-girdle muscle dystrophy and protected myofibers from damage. In summary, sustained MEK1-ERK1/2 activity in skeletal muscle produces a fast-to-slow fiber-type switch that protects from muscular dystrophy, suggesting a therapeutic approach to enhance the metabolic effectiveness of muscle and protect from dystrophic disease.
Authors
Justin G. Boyer, Vikram Prasad, Taejeong Song, Donghoon Lee, Xing Fu, Kelly M. Grimes, Michelle A. Sargent, Sakthivel Sadayappan, Jeffery D. Molkentin
Abstract
A vast body of literature has established G protein–coupled receptor kinase 2 (GRK2; family: β-adrenergic receptor kinases [βARKs]) as a key player in the development and progression of heart failure. Inhibition of GRK2 improves cardiac function after injury in numerous animal models. In recent years, discovery of several noncanonical GRK2 targets has expanded our view of this kinase. This article describes the exciting finding that cardiac GRK2 activity can regulate whole-body metabolism. Transgenic mice with cardiac-specific expression of a peptide inhibitor of GRK2 (TgβARKct) display an enhanced obesogenic phenotype when fed a high-fat diet (HFD). In contrast, mice with cardiac-specific overexpression of GRK2 (TgGRK2) show resistance to HFD-induced obesity. White adipose tissue (WAT) mass was significantly enhanced in HFD-fed TgβARKct mice. Furthermore, regulators of adipose differentiation were differentially regulated in WAT from mice with gain or loss of GRK2 function. Using complex metabolomics, we found that cardiac GRK2 signaling altered myocardial branched-chain amino acid (BCAA) and endocannabinoid metabolism. In addition, it modulated circulating BCAA and endocannabinoid metabolite profiles on mice fed an HFD. We also found that one of the BCAA metabolites identified here enhances adipocyte differentiation in vitro. These results suggest that metabolic changes in the heart due to GRK2 signaling on mice fed an HFD control whole-body metabolism.
Authors
Benjamin P. Woodall, Kenneth S. Gresham, Meryl A. Woodall, Mesele-Christina Valenti, Alessandro Cannavo, Jessica Pfleger, J. Kurt Chuprun, Konstantinos Drosatos, Walter J. Koch
Abstract
Glucagon, a hormone released from pancreatic α cells, plays a key role in maintaining proper glucose homeostasis and has been implicated in the pathophysiology of diabetes. In vitro studies suggest that intraislet glucagon can modulate the function of pancreatic β cells. However, because of the lack of suitable experimental tools, the in vivo physiological role of this intraislet cross-talk has remained elusive. To address this issue, we generated a mouse model that selectively expressed an inhibitory designer GPCR (Gi DREADD) in α cells only. Drug-induced activation of this inhibitory designer receptor almost completely shut off glucagon secretion in vivo, resulting in markedly impaired insulin secretion, hyperglycemia, and glucose intolerance. Additional studies with mouse and human islets indicated that intraislet glucagon stimulates insulin release primarily by activating β cell GLP-1 receptors. These findings strongly suggest that intraislet glucagon signaling is essential for maintaining proper glucose homeostasis in vivo. Our work may pave the way toward the development of novel classes of antidiabetic drugs that act by modulating intraislet cross-talk between α and β cells.
Authors
Lu Zhu, Diptadip Dattaroy, Jonathan Pham, Lingdi Wang, Luiz F. Barella, Yinghong Cui, Kenneth J. Wilkins, Bryan L. Roth, Ute Hochgeschwender, Franz M. Matschinsky, Klaus H. Kaestner, Nicolai M. Doliba, Jürgen Wess
Abstract
The bone marrow microenvironment 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 (IL-1B) was elevated in the bone marrow, and caspase-1 activity, which can process pro–IL-1B, was increased in marrow Mφs and neutrophils. Mechanistically, IL-1B 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 IL-1B in the age-associated lineage skewing of HSCs, and reveals the therapeutic potential of their manipulation as antigeronic targets.
Authors
Benjamin J. Frisch, Corey M. Hoffman, Sarah E. Latchney, Mark W. LaMere, Jason Myers, John Ashton, Allison J. Li, Jerry Saunders II, James Palis, Archibald S. Perkins, Amanda McCabe, Julianne N.P. 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
Abstract
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 mitochondrial DNA (mtDNA) damage and myocardial aging using a mouse model carrying a proofreading-defective mtDNA polymerase γ (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 with that in WT mice. 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.
Authors
Benjamin P. Woodall, Amabel M. Orogo, Rita H. Najor, Melissa Q. Cortez, Eileen R. Moreno, Hongxia Wang, Ajit S. Divakaruni, Anne N. Murphy, Åsa B. Gustafsson
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