A mitochondrial bioenergetic etiology of disease

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Abstract

The classical Mendelian genetic perspective has failed to adequately explain the biology and genetics of common metabolic and degenerative diseases. This is because these diseases are primarily systemic bioenergetic diseases, and the most important energy genes are located in the cytoplasmic mitochondrial DNA (mtDNA). Therefore, to understand these “complex” diseases, we must investigate their bioenergetic pathophysiology and consider the genetics of the thousands of copies of maternally inherited mtDNA, the more than 1,000 nuclear DNA (nDNA) bioenergetic genes, and the epigenomic and signal transduction systems that coordinate these dispersed elements of the mitochondrial genome.

Authors

Douglas C. Wallace

Bruce Beutler wins the 2013 ASCI/Stanley J. Korsmeyer Award

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Abstract

Authors

Kathryn Claiborn

IL-6 polymorphisms: a useful genetic tool for inflammation research?

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In 1998, we described a novel polymorphism in the promoter (G>C, rs1800795) of the IL-6 (IL6) gene. The common allele, G, exhibited higher transcriptional activity in gene reporter assays and was associated with higher serum IL-6 levels in a small cohort of healthy subjects. We explored the ethnic distribution of these alleles and found significant differences among people of mixed European descent, Africans, and Gujarati Asians. Disease association was established in a cohort of 92 children of mixed European descent from the United Kingdom with systemic juvenile idiopathic arthritis (sJIA), with the GG genotype being significantly increased in sJIA cases compared with that in 383 controls, especially in those under 6 years old (P = 0.01). This polymorphism has since been used as a functional variant to explore the role of elevated IL-6 levels in many common disease states, confirming the key causal role of IL-6 in human health and disease.

Authors

Patricia Woo, Steve E. Humphries

Sudden death in epilepsy: of mice and men

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A 20-year-old man with intellectual disability and intractable multifocal epilepsy presented to a neurologist for further evaluation and management. His seizures began at 4 months, the night after his first DPT vaccine, and he continued to have frequent tonic-clonic seizures throughout his life. Several weeks after his visit, he was found facedown on the floor, dead, by his family. His autopsy was unremarkable, but genetic testing revealed a frame shift mutation in SCN1A, consistent with severe myoclonic epilepsy of infancy (Dravet syndrome).

Authors

Daniel Friedman, Janice Chyou, Orrin Devinsky

Lymphatic vessels clean up your arteries

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Reverse cholesterol transport (RCT) is the pathway by which cholesterol accumulated in peripheral tissues, including the artery wall, is transported to the liver for excretion. There is strong evidence suggesting that interventions that increase macrophage cholesterol efflux and RCT would be antiatherogenic. In this issue of the JCI, Martel et al. investigate the contribution of lymphatic vasculature to RCT. Their results support the concept that the lymphatic vessel route is critical for RCT from atherosclerotic plaques. Therefore, strategies to improve lymphatic transport might be useful for treating atherosclerotic vascular disease.

Authors

Carlos Fernández-Hernando

Desmoglein-1, differentiation, and disease

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Desmoglein-1 (DSG1), a desmosomal protein, maintains the structure of epidermis through its adhesive function. However, heterozygous mutations in DSG1 in humans result in abnormal differentiation, as does downregulation of DSG1 in human skin organ culture, suggesting that it may have important signaling functions. In this issue of the JCI, Harmon et al. elucidate how the binding of the DSG1 cytoplasmic tail to the scaffolding protein Erbin decreases signaling through the Ras-Raf pathway to promote stratification and differentiation of keratinocytes in the epidermis.

Authors

Christoph M. Hammers, John R. Stanley

WNT signaling in stem cell differentiation and tumor formation

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Abstract

Embryonic stem cells (ESCs) hold great therapeutic promise for the regeneration of functional cell types and clinical applications. However, tumorigenic potential of stem cells in a transplanted host remains a major obstacle. In this issue of the JCI, Cui and colleagues identified TCF7-mediated canonical WNT signaling as a critical determinant of both the tumorigenicity and therapeutic function of ESC-derived retinal progenitor cells (ESC-RPCs). Their findings suggested that addressing key extracellular signaling and related intrinsic factors will be essential for the successful use of ESC-derived progenitor transplantation.

Authors

Hong Ouyang, Yehong Zhuo, Kang Zhang

Striking the target in iron overload disorders

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The liver, a major site of body iron stores, mediates key responses that preserve systemic iron homeostasis. In this issue of the JCI, Guo et al. demonstrate that administration of antisense oligonucleotides that reduce expression of Tmprss6, a hepatic protein that plays an essential role in maintaining iron balance, can attenuate disease severity in mouse models of human iron overload disorders. These data reveal the potential of novel TMPRSS6-targeted therapies for the treatment of clinical conditions such as hereditary hemochromatosis and β-thalassemia.

Authors

Karin E. Finberg

Tumor fibroblast–derived epiregulin promotes growth of colitis-associated neoplasms through ERK

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Molecular mechanisms specific to colitis-associated cancers have been poorly characterized. Using comparative whole-genome expression profiling, we observed differential expression of epiregulin (EREG) in mouse models of colitis-associated, but not sporadic, colorectal cancer. Similarly, EREG expression was significantly upregulated in cohorts of patients with colitis-associated cancer. Furthermore, tumor-associated fibroblasts were identified as a major source of EREG in colitis-associated neoplasms. Functional studies showed that Ereg-deficient mice, although more prone to colitis, were strongly protected from colitis-associated tumors. Serial endoscopic studies revealed that EREG promoted tumor growth rather than initiation. Additionally, we demonstrated that fibroblast-derived EREG requires ERK activation to induce proliferation of intestinal epithelial cells (IEC) and tumor development in vivo. To demonstrate the functional relevance of EREG-producing tumor-associated fibroblasts, we developed a novel system for adoptive transfer of these cells via mini-endoscopic local injection. It was found that transfer of EREG-producing, but not Ereg-deficient, fibroblasts from tumors significantly augmented growth of colitis-associated neoplasms in vivo. In conclusion, our data indicate that EREG and tumor-associated fibroblasts play a crucial role in controlling tumor growth in colitis-associated neoplasms.

Authors

Clemens Neufert, Christoph Becker, Özlem Türeci, Maximilian J. Waldner, Ingo Backert, Katharina Floh, Imke Atreya, Moritz Leppkes, Andre Jefremow, Michael Vieth, Regine Schneider-Stock, Patricia Klinger, Florian R. Greten, David W. Threadgill, Ugur Sahin, Markus F. Neurath

Liver-resident NK cells confer adaptive immunity in skin-contact inflammation

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Liver natural killer (NK) cells were recently reported to possess memory-like properties in contact hypersensitivity (CHS) models. However, the phenotype and origin of these “memory” NK cells cannot be distinguished from other NK cell subpopulations. Here, we define the transcriptional, phenotypic, and functional features of liver NK cell subsets and their roles in mediating CHS. Liver NK cells can be divided into two distinct subsets: CD49a⁺DX5^– and CD49a^–DX5⁺. Substantial transcriptional and phenotypic differences existed between liver CD49a⁺DX5^– NK cells and other NK cell subsets. CD49a⁺DX5^– NK cells possessed memory potential and conferred hapten-specific CHS responses upon hapten challenge. Importantly, CD49a⁺DX5^– NK cells were liver resident and were present in the liver sinusoidal blood, but not the afferent and efferent blood of the liver. Moreover, they appeared to originate from hepatic hematopoietic progenitor/stem cells (HPCs/HSCs) but not from the bone marrow, and maintained their phenotypes in the steady state. Our findings of liver-resident NK cells shed new light on the acquisition of memory-like properties of NK cells.

Authors

Hui Peng, Xiaojun Jiang, Yonglin Chen, Dorothy K. Sojka, Haiming Wei, Xiang Gao, Rui Sun, Wayne M. Yokoyama, Zhigang Tian

IgG4 subclass antibodies impair antitumor immunity in melanoma

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Host-induced antibodies and their contributions to cancer inflammation are largely unexplored. IgG4 subclass antibodies are present in IL-10–driven Th2 immune responses in some inflammatory conditions. Since Th2-biased inflammation is a hallmark of tumor microenvironments, we investigated the presence and functional implications of IgG4 in malignant melanoma. Consistent with Th2 inflammation, CD22⁺ B cells and IgG4⁺-infiltrating cells accumulated in tumors, and IL-10, IL-4, and tumor-reactive IgG4 were expressed in situ. When compared with B cells from patient lymph nodes and blood, tumor-associated B cells were polarized to produce IgG4. Secreted B cells increased VEGF and IgG4, and tumor cells enhanced IL-10 secretion in cocultures. Unlike IgG1, an engineered tumor antigen-specific IgG4 was ineffective in triggering effector cell–mediated tumor killing in vitro. Antigen-specific and nonspecific IgG4 inhibited IgG1-mediated tumoricidal functions. IgG4 blockade was mediated through reduction of FcγRI activation. Additionally, IgG4 significantly impaired the potency of tumoricidal IgG1 in a human melanoma xenograft mouse model. Furthermore, serum IgG4 was inversely correlated with patient survival. These findings suggest that IgG4 promoted by tumor-induced Th2-biased inflammation may restrict effector cell functions against tumors, providing a previously unexplored aspect of tumor-induced immune escape and a basis for biomarker development and patient-specific therapeutic approaches.

Authors

Panagiotis Karagiannis, Amy E. Gilbert, Debra H. Josephs, Niwa Ali, Tihomir Dodev, Louise Saul, Isabel Correa, Luke Roberts, Emma Beddowes, Alexander Koers, Carl Hobbs, Silvia Ferreira, Jenny L.C. Geh, Ciaran Healy, Mark Harries, Katharine M. Acland, Philip J. Blower, Tracey Mitchell, David J. Fear, James F. Spicer, Katie E. Lacy, Frank O. Nestle, Sophia N. Karagiannis

The ubiquitin ligase Mindbomb 1 coordinates gastrointestinal secretory cell maturation

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After cell fate specification, differentiating cells must amplify the specific subcellular features required for their specialized function. How cells regulate such subcellular scaling is a fundamental unanswered question. Here, we show that the E3 ubiquitin ligase Mindbomb 1 (MIB1) is required for the apical secretory apparatus established by gastric zymogenic cells as they differentiate from their progenitors. When Mib1 was deleted, death-associated protein kinase–1 (DAPK1) was rerouted to the cell base, microtubule-associated protein 1B (MAP1B) was dephosphorylated, and the apical vesicles that normally support mature secretory granules were dispersed. Consequently, secretory granules did not mature. The transcription factor MIST1 bound the first intron of Mib1 and regulated its expression. We further showed that loss of MIB1 and dismantling of the apical secretory apparatus was the earliest quantifiable aberration in zymogenic cells undergoing transition to a precancerous metaplastic state in mouse and human stomach. Our results reveal a mechanistic pathway by which cells can scale up a specific, specialized subcellular compartment to alter function during differentiation and scale it down during disease.

Authors

Benjamin J. Capoccia, Ramon U. Jin, Young-Yun Kong, Richard M. Peek Jr., Matteo Fassan, Massimo Rugge, Jason C. Mills

Coordination of hypothalamic and pituitary T3 production regulates TSH expression

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Type II deiodinase (D2) activates thyroid hormone by converting thyroxine (T4) to 3,5,3′-triiodothyronine (T3). This allows plasma T4 to signal a negative feedback loop that inhibits production of thyrotropin-releasing hormone (TRH) in the mediobasal hypothalamus (MBH) and thyroid-stimulating hormone (TSH) in the pituitary. To determine the relative contributions of these D2 pathways in the feedback loop, we developed 2 mouse strains with pituitary- and astrocyte-specific D2 knockdown (pit-D2 KO and astro-D2 KO mice, respectively). The pit-D2 KO mice had normal serum T3 and were systemically euthyroid, but exhibited an approximately 3-fold elevation in serum TSH levels and a 40% reduction in biological activity. This was the result of elevated serum T4 that increased D2-mediated T3 production in the MBH, thus decreasing Trh mRNA. That tanycytes, not astrocytes, are the cells within the MBH that mediate T4-to-T3 conversion was defined by studies using the astro-D2 KO mice. Despite near-complete loss of brain D2, tanycyte D2 was preserved in astro-D2 KO mice at levels that were sufficient to maintain both the T4-dependent negative feedback loop and thyroid economy. Taken together, these data demonstrated that the hypothalamic-thyroid axis is wired to maintain normal plasma T3 levels, which is achieved through coordination of T4-to-T3 conversion between thyrotrophs and tanycytes.

Authors

Tatiana L. Fonseca, Mayrin Correa-Medina, Maira P.O. Campos, Gabor Wittmann, Joao P. Werneck-de-Castro, Rafael Arrojo e Drigo, Magda Mora-Garzon, Cintia Bagne Ueta, Alejandro Caicedo, Csaba Fekete, Balazs Gereben, Ronald M. Lechan, Antonio C. Bianco

Mycolactone activation of Wiskott-Aldrich syndrome proteins underpins Buruli ulcer formation

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Mycolactone is a diffusible lipid secreted by the human pathogen Mycobacterium ulcerans, which induces the formation of open skin lesions referred to as Buruli ulcers. Here, we show that mycolactone operates by hijacking the Wiskott-Aldrich syndrome protein (WASP) family of actin-nucleating factors. By disrupting WASP autoinhibition, mycolactone leads to uncontrolled activation of ARP2/3-mediated assembly of actin in the cytoplasm. In epithelial cells, mycolactone-induced stimulation of ARP2/3 concentrated in the perinuclear region, resulting in defective cell adhesion and directional migration. In vivo injection of mycolactone into mouse ears consistently altered the junctional organization and stratification of keratinocytes, leading to epidermal thinning, followed by rupture. This degradation process was efficiently suppressed by coadministration of the N-WASP inhibitor wiskostatin. These results elucidate the molecular basis of mycolactone activity and provide a mechanism for Buruli ulcer pathogenesis. Our findings should allow for the rationale design of competitive inhibitors of mycolactone binding to N-WASP, with anti–Buruli ulcer therapeutic potential.

Authors

Laure Guenin-Macé, Romain Veyron-Churlet, Maria-Isabel Thoulouze, Guillaume Romet-Lemonne, Hui Hong, Peter F. Leadlay, Anne Danckaert, Marie-Thérèse Ruf, Serge Mostowy, Chiara Zurzolo, Philippe Bousso, Fabrice Chrétien, Marie-France Carlier, Caroline Demangel

The TGR5 receptor mediates bile acid–induced itch and analgesia

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Abstract

Patients with cholestatic disease exhibit pruritus and analgesia, but the mechanisms underlying these symptoms are unknown. We report that bile acids, which are elevated in the circulation and tissues during cholestasis, cause itch and analgesia by activating the GPCR TGR5. TGR5 was detected in peptidergic neurons of mouse dorsal root ganglia and spinal cord that transmit itch and pain, and in dermal macrophages that contain opioids. Bile acids and a TGR5-selective agonist induced hyperexcitability of dorsal root ganglia neurons and stimulated the release of the itch and analgesia transmitters gastrin-releasing peptide and leucine-enkephalin. Intradermal injection of bile acids and a TGR5-selective agonist stimulated scratching behavior by gastrin-releasing peptide– and opioid-dependent mechanisms in mice. Scratching was attenuated in Tgr5-KO mice but exacerbated in Tgr5-Tg mice (overexpressing mouse TGR5), which exhibited spontaneous pruritus. Intraplantar and intrathecal injection of bile acids caused analgesia to mechanical stimulation of the paw by an opioid-dependent mechanism. Both peripheral and central mechanisms of analgesia were absent from Tgr5-KO mice. Thus, bile acids activate TGR5 on sensory nerves, stimulating the release of neuropeptides in the spinal cord that transmit itch and analgesia. These mechanisms could contribute to pruritus and painless jaundice that occur during cholestatic liver diseases.

Authors

Farzad Alemi, Edwin Kwon, Daniel P. Poole, TinaMarie Lieu, Victoria Lyo, Fiore Cattaruzza, Ferda Cevikbas, Martin Steinhoff, Romina Nassini, Serena Materazzi, Raquel Guerrero-Alba, Eduardo Valdez-Morales, Graeme S. Cottrell, Kristina Schoonjans, Pierangelo Geppetti, Stephen J. Vanner, Nigel W. Bunnett, Carlos U. Corvera

Reducing TMPRSS6 ameliorates hemochromatosis and β-thalassemia in mice

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β-Thalassemia and HFE-related hemochromatosis are 2 of the most frequently inherited disorders worldwide. Both disorders are characterized by low levels of hepcidin (HAMP), the hormone that regulates iron absorption. As a consequence, patients affected by these disorders exhibit iron overload, which is the main cause of morbidity and mortality. HAMP expression is controlled by activation of the SMAD1,5,8/SMAD4 complex. TMPRSS6 is a serine protease that reduces SMAD activation and blocks HAMP expression. We identified second generation antisense oligonucleotides (ASOs) targeting mouse Tmprss6. ASO treatment in mice affected by hemochromatosis (Hfe^–/–) significantly decreased serum iron, transferrin saturation and liver iron accumulation. Furthermore, ASO treatment of mice affected by β-thalassemia (HBB^th3/+ mice, referred to hereafter as th3/+ mice) decreased the formation of insoluble membrane-bound globins, ROS, and apoptosis, and improved anemia. These animals also exhibited lower erythropoietin levels, a significant amelioration of ineffective erythropoiesis (IE) and splenomegaly, and an increase in total hemoglobin levels. These data suggest that ASOs targeting Tmprss6 could be beneficial in individuals with hemochromatosis, β-thalassemia, and related disorders.

Authors

Shuling Guo, Carla Casu, Sara Gardenghi, Sheri Booten, Mariam Aghajan, Raechel Peralta, Andy Watt, Sue Freier, Brett P. Monia, Stefano Rivella

BM mesenchymal stromal cell–derived exosomes facilitate multiple myeloma progression

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BM mesenchymal stromal cells (BM-MSCs) support multiple myeloma (MM) cell growth, but little is known about the putative mechanisms by which the BM microenvironment plays an oncogenic role in this disease. Cell-cell communication is mediated by exosomes. In this study, we showed that MM BM-MSCs release exosomes that are transferred to MM cells, thereby resulting in modulation of tumor growth in vivo. Exosomal microRNA (miR) content differed between MM and normal BM-MSCs, with a lower content of the tumor suppressor miR-15a. In addition, MM BM-MSC–derived exosomes had higher levels of oncogenic proteins, cytokines, and adhesion molecules compared with exosomes from the cells of origin. Importantly, whereas MM BM-MSC–derived exosomes promoted MM tumor growth, normal BM-MSC exosomes inhibited the growth of MM cells. In summary, these in vitro and in vivo studies demonstrated that exosome transfer from BM-MSCs to clonal plasma cells represents a previously undescribed and unique mechanism that highlights the contribution of BM-MSCs to MM disease progression.

Authors

Aldo M. Roccaro, Antonio Sacco, Patricia Maiso, Abdel Kareem Azab, Yu-Tzu Tai, Michaela Reagan, Feda Azab, Ludmila M. Flores, Federico Campigotto, Edie Weller, Kenneth C. Anderson, David T. Scadden, Irene M. Ghobrial

Desmoglein-1/Erbin interaction suppresses ERK activation to support epidermal differentiation

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Genetic disorders of the Ras/MAPK pathway, termed RASopathies, produce numerous abnormalities, including cutaneous keratodermas. The desmosomal cadherin, desmoglein-1 (DSG1), promotes keratinocyte differentiation by attenuating MAPK/ERK signaling and is linked to striate palmoplantar keratoderma (SPPK). This raises the possibility that cutaneous defects associated with SPPK and RASopathies share certain molecular faults. To identify intermediates responsible for executing the inhibition of ERK by DSG1, we conducted a yeast 2-hybrid screen. The screen revealed that Erbin (also known as ERBB2IP), a known ERK regulator, binds DSG1. Erbin silencing disrupted keratinocyte differentiation in culture, mimicking aspects of DSG1 deficiency. Furthermore, ERK inhibition and the induction of differentiation markers by DSG1 required both Erbin and DSG1 domains that participate in binding Erbin. Erbin blocks ERK signaling by interacting with and disrupting Ras-Raf scaffolds mediated by SHOC2, a protein genetically linked to the RASopathy, Noonan-like syndrome with loose anagen hair (NS/LAH). DSG1 overexpression enhanced this inhibitory function, increasing Erbin-SHOC2 interactions and decreasing Ras-SHOC2 interactions. Conversely, analysis of epidermis from DSG1-deficient patients with SPPK demonstrated increased Ras-SHOC2 colocalization and decreased Erbin-SHOC2 colocalization, offering a possible explanation for the observed epidermal defects. These findings suggest a mechanism by which DSG1 and Erbin cooperate to repress MAPK signaling and promote keratinocyte differentiation.

Authors

Robert M. Harmon, Cory L. Simpson, Jodi L. Johnson, Jennifer L. Koetsier, Adi D. Dubash, Nicole A. Najor, Ofer Sarig, Eli Sprecher, Kathleen J. Green

Lymphatic vasculature mediates macrophage reverse cholesterol transport in mice

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Reverse cholesterol transport (RCT) refers to the mobilization of cholesterol on HDL particles (HDL-C) from extravascular tissues to plasma, ultimately for fecal excretion. Little is known about how HDL-C leaves peripheral tissues to reach plasma. We first used 2 models of disrupted lymphatic drainage from skin — 1 surgical and the other genetic — to quantitatively track RCT following injection of [³H]-cholesterol–loaded macrophages upstream of blocked or absent lymphatic vessels. Macrophage RCT was markedly impaired in both models, even at sites with a leaky vasculature. Inhibited RCT was downstream of cholesterol efflux from macrophages, since macrophage efflux of a fluorescent cholesterol analog (BODIPY-cholesterol) was not altered by impaired lymphatic drainage. We next addressed whether RCT was mediated by lymphatic vessels from the aortic wall by loading the aortae of donor atherosclerotic Apoe-deficient mice with [²H]₆-labeled cholesterol and surgically transplanting these aortae into recipient Apoe-deficient mice that were treated with anti-VEGFR3 antibody to block lymphatic regrowth or with control antibody to allow such regrowth. [²H]-Cholesterol was retained in aortae of anti–VEGFR3-treated mice. Thus, the lymphatic vessel route is critical for RCT from multiple tissues, including the aortic wall. These results suggest that supporting lymphatic transport function may facilitate cholesterol clearance in therapies aimed at reversing atherosclerosis.

Authors

Catherine Martel, Wenjun Li, Brian Fulp, Andrew M. Platt, Emmanuel L. Gautier, Marit Westerterp, Robert Bittman, Alan R. Tall, Shu-Hsia Chen, Michael J. Thomas, Daniel Kreisel, Melody A. Swartz, Mary G. Sorci-Thomas, Gwendalyn J. Randolph

STAT3 regulates arginase-I in myeloid-derived suppressor cells from cancer patients

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Myeloid-derived suppressor cells (MDSC) play a key immunosuppressive role in various types of cancer, including head and neck squamous cell carcinoma (HNSCC). In this study, we characterized CD14⁺HLA-DR^–/lo cells sorted from the tumors, draining lymph nodes, and peripheral blood of HNSCC patients. CD14⁺HLA-DR^–/lo cells were phenotyped as CD11b⁺, CD33⁺, CD34⁺, arginase-I⁺, and ROS⁺. In all 3 compartments, they suppressed autologous, antigen-independent T cell proliferation in a differential manner. The abundance of MDSC correlated with stage, but did not correlate with previous treatment with radiation or subsites of HNSCC. Interestingly, MDSC from all 3 compartments showed high phosphorylated STAT3 levels that correlated with arginase-I expression levels and activity. Stattic, a STAT3-specific inhibitor, and STAT3-targeted siRNA abrogated MDSC’s suppressive function. Inhibition of STAT3 signaling also resulted in decreased arginase-I activity. Analysis of the human arginase-I promoter region showed multiple STAT3-binding elements, and ChIP demonstrated that phosphorylated STAT3 binds to multiple sites in the arginase-I promoter. Finally, rescue of arginase-I activity after STAT3 blockade restored MDSC’s suppressive function. Taken together, these results demonstrate that the suppressive function of arginase-I in both infiltrating and circulating MDSC is a downstream target of activated STAT3.

Authors

David Vasquez-Dunddel, Fan Pan, Qi Zeng, Mikhail Gorbounov, Emilia Albesiano, Juan Fu, Richard L. Blosser, Ada J. Tam, Tullia Bruno, Hao Zhang, Drew Pardoll, Young Kim

Blockade of individual Notch ligands and receptors controls graft-versus-host disease

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Graft-versus-host disease (GVHD) is the main complication of allogeneic bone marrow transplantation. Current strategies to control GVHD rely on global immunosuppression. These strategies are incompletely effective and decrease the anticancer activity of the allogeneic graft. We previously identified Notch signaling in T cells as a new therapeutic target for preventing GVHD. Notch-deprived T cells showed markedly decreased production of inflammatory cytokines, but normal in vivo proliferation, increased accumulation of regulatory T cells, and preserved anticancer effects. Here, we report that γ-secretase inhibitors can block all Notch signals in alloreactive T cells, but lead to severe on-target intestinal toxicity. Using newly developed humanized antibodies and conditional genetic models, we demonstrate that Notch1/Notch2 receptors and the Notch ligands Delta-like1/4 mediate all the effects of Notch signaling in T cells during GVHD, with dominant roles for Notch1 and Delta-like4. Notch1 inhibition controlled GVHD, but led to treatment-limiting toxicity. In contrast, Delta-like1/4 inhibition blocked GVHD without limiting adverse effects while preserving substantial anticancer activity. Transient blockade in the peritransplant period provided durable protection. These findings open new perspectives for selective and safe targeting of individual Notch pathway components in GVHD and other T cell–mediated human disorders.

Authors

Ivy T. Tran, Ashley R. Sandy, Alexis J. Carulli, Christen Ebens, Jooho Chung, Gloria T. Shan, Vedran Radojcic, Ann Friedman, Thomas Gridley, Amy Shelton, Pavan Reddy, Linda C. Samuelson, Minhong Yan, Christian W. Siebel, Ivan Maillard

Increased brain uptake and oxidation of acetate in heavy drinkers

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When a person consumes ethanol, the body quickly begins to convert it to acetic acid, which circulates in the blood and can serve as a source of energy for the brain and other organs. This study used ¹³C magnetic resonance spectroscopy to test whether chronic heavy drinking is associated with greater brain uptake and oxidation of acetic acid, providing a potential metabolic reward or adenosinergic effect as a consequence of drinking. Seven heavy drinkers, who regularly consumed at least 8 drinks per week and at least 4 drinks per day at least once per week, and 7 light drinkers, who consumed fewer than 2 drinks per week were recruited. The subjects were administered [2-¹³C]acetate for 2 hours and scanned throughout that time with magnetic resonance spectroscopy of the brain to observe natural ¹³C abundance of N-acetylaspartate (NAA) and the appearance of ¹³C-labeled glutamate, glutamine, and acetate. Heavy drinkers had approximately 2-fold more brain acetate relative to blood and twice as much labeled glutamate and glutamine. The results show that acetate transport and oxidation are faster in heavy drinkers compared with that in light drinkers. Our finding suggests that a new therapeutic approach to supply acetate during alcohol detoxification may be beneficial.

Authors

Lihong Jiang, Barbara Irene Gulanski, Henk M. De Feyter, Stuart A. Weinzimer, Brian Pittman, Elizabeth Guidone, Julia Koretski, Susan Harman, Ismene L. Petrakis, John H. Krystal, Graeme F. Mason

Autocrine production of IL-11 mediates tumorigenicity in hypoxic cancer cells

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IL-11 and its receptor, IL-11Ra, are expressed in human cancers; however, the functional role of IL-11 in tumor progression is not known. We found that IL11 is a hypoxia-inducible, VHL-regulated gene in human cancer cells and that expression of IL11 mRNA was dependent, at least in part, on HIF-1. A cooperative interaction between HIF-1 and AP-1 mediated transcriptional activation of the IL11 promoter. Additionally, we found that human cancer cells expressed a functional IL-11Ra subunit, which triggered signal transduction either by exogenous recombinant human IL-11 or by autocrine production of IL-11 in cells cultured under hypoxic conditions. Silencing of IL11 dramatically abrogated the ability of hypoxia to increase anchorage-independent growth and significantly reduced tumor growth in xenograft models. Notably, these results were phenocopied by partial knockdown of STAT1 in a human prostate cancer cell line (PC3), suggesting that this pathway may play an important role in mediating the effects of IL-11 under hypoxic conditions. In conclusion, these results identify IL11 as an oxygen- and VHL-regulated gene and provide evidence of a pathway “hijacked” by hypoxic cancer cells that may contribute to tumor progression.

Authors

Barbara Onnis, Nicole Fer, Annamaria Rapisarda, Victor S. Perez, Giovanni Melillo

Activation of inflammasome signaling mediates pathology of acute P. aeruginosa pneumonia

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The respiratory tract is exceptionally well defended against infection from inhaled bacteria, with multiple proinflammatory signaling cascades recruiting phagocytes to clear airway pathogens. However, organisms that efficiently activate damaging innate immune responses, such as those mediated by the inflammasome and caspase-1, may cause pulmonary damage and interfere with bacterial clearance. The extracellular, opportunistic pathogen Pseudomonas aeruginosa expresses not only pathogen-associated molecular patterns that activate NF-κB signaling in epithelial and immune cells, but also flagella that activate the NLRC4 inflammasome. We demonstrate that induction of inflammasome signaling, ascribed primarily to the alveolar macrophage, impaired P. aeruginosa clearance and was associated with increased apoptosis/pyroptosis and mortality in a murine model of acute pneumonia. Strategies that limited inflammasome activation, including infection by fliC mutants, depletion of macrophages, deletion of NLRC4, reduction of IL-1β and IL-18 production, inhibition of caspase-1, and inhibition of downstream signaling in IL-1R– or IL-18R–null mice, all resulted in enhanced bacterial clearance and diminished pathology. These results demonstrate that the inflammasome provides a potential target to limit the pathological consequences of acute P. aeruginosa pulmonary infection.

Authors

Taylor S. Cohen, Alice S. Prince

Calcium influx through L-type Ca_V1.2 Ca²⁺ channels regulates mandibular development

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The identification of a gain-of-function mutation in CACNA1C as the cause of Timothy Syndrome (TS), a rare disorder characterized by cardiac arrhythmias and syndactyly, highlighted unexpected roles for the L-type voltage-gated Ca²⁺ channel Ca_V1.2 in nonexcitable cells. How abnormal Ca²⁺ influx through Ca_V1.2 underlies phenotypes such as the accompanying syndactyly or craniofacial abnormalities in the majority of affected individuals is not readily explained by established Ca_V1.2 roles. Here, we show that Ca_V1.2 is expressed in the first and second pharyngeal arches within the subset of cells that give rise to jaw primordia. Gain-of-function and loss-of-function studies in mouse, in concert with knockdown/rescue and pharmacological approaches in zebrafish, demonstrated that Ca²⁺ influx through Ca_V1.2 regulates jaw development. Cranial neural crest migration was unaffected by Ca_V1.2 knockdown, suggesting a role for Ca_V1.2 later in development. Focusing on the mandible, we observed that cellular hypertrophy and hyperplasia depended upon Ca²⁺ signals through Ca_V1.2, including those that activated the calcineurin signaling pathway. Together, these results provide new insights into the role of voltage-gated Ca²⁺ channels in nonexcitable cells during development.

Authors

Kapil V. Ramachandran, Jessica A. Hennessey, Adam S. Barnett, Xinhe Yin, Harriett A. Stadt, Erika Foster, Raj A. Shah, Masayuki Yazawa, Ricardo E. Dolmetsch, Margaret L. Kirby, Geoffrey S. Pitt

WNT signaling determines tumorigenicity and function of ESC-derived retinal progenitors

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Tumor formation constitutes a major obstacle to the clinical application of embryonic stem cell–derived (ESC-derived) cells. In an attempt to find major extracellular signaling and intrinsic factors controlling tumorigenicity and therapeutic functionality of transplanted ESC-derived retinal progenitor cells (ESC-RPCs), we evaluated multiple kinds of ESC-RPCs in a mouse retinal degeneration model and conducted genome-wide gene expression profiling. We identified canonical WNT signaling as a critical determinant for the tumorigenicity and therapeutic function of ESC-RPCs. The function of WNT signaling is primarily mediated by TCF7, which directly induces expression of Sox2 and Nestin. Inhibition of WNT signaling, overexpression of dominant-negative Tcf7, and silencing Tcf7, Sox2, or Nestin all resulted in drastically reduced tumor formation and substantially improved retinal integration and visual preservation in mice. These results demonstrate that the WNT signaling cascade plays a critical role in modulating the tumorigenicity and functionality of ESC-derived progenitors.

Authors

Lu Cui, Yuan Guan, Zepeng Qu, Jingfa Zhang, Bing Liao, Bo Ma, Jiang Qian, Dangsheng Li, Weiye Li, Guo-Tong Xu, Ying Jin

Hepatic glucose sensing is required to preserve β cell glucose competence

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Abstract

Liver glucose metabolism plays a central role in glucose homeostasis and may also regulate feeding and energy expenditure. Here we assessed the impact of glucose transporter 2 (Glut2) gene inactivation in adult mouse liver (LG2KO mice). Loss of Glut2 suppressed hepatic glucose uptake but not glucose output. In the fasted state, expression of carbohydrate-responsive element-binding protein (ChREBP) and its glycolytic and lipogenic target genes was abnormally elevated. Feeding, energy expenditure, and insulin sensitivity were identical in LG2KO and control mice. Glucose tolerance was initially normal after Glut2 inactivation, but LG2KO mice exhibited progressive impairment of glucose-stimulated insulin secretion even though β cell mass and insulin content remained normal. Liver transcript profiling revealed a coordinated downregulation of cholesterol biosynthesis genes in LG2KO mice that was associated with reduced hepatic cholesterol in fasted mice and reduced bile acids (BAs) in feces, with a similar trend in plasma. We showed that chronic BAs or farnesoid X receptor (FXR) agonist treatment of primary islets increases glucose-stimulated insulin secretion, an effect not seen in islets from Fxr^–/– mice. Collectively, our data show that glucose sensing by the liver controls β cell glucose competence and suggest BAs as a potential mechanistic link.

Authors

Pascal Seyer, David Vallois, Carole Poitry-Yamate, Frédéric Schütz, Salima Metref, David Tarussio, Pierre Maechler, Bart Staels, Bernard Lanz, Rolf Grueter, Julie Decaris, Scott Turner, Anabela da Costa, Frédéric Preitner, Kaori Minehira, Marc Foretz, Bernard Thorens

Rapamycin-treated human endothelial cells preferentially activate allogeneic regulatory T cells

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Abstract

Human graft endothelial cells (ECs) can act as antigen-presenting cells to initiate allograft rejection by host memory T cells. Rapamycin, an mTOR inhibitor used clinically to suppress T cell responses, also acts on DCs, rendering them tolerogenic. Here, we report the effects of rapamycin on EC alloimmunogenicity. Compared with mock-treated cells, rapamycin-pretreated human ECs (rapa-ECs) stimulated less proliferation and cytokine secretion from allogeneic CD4⁺ memory cells, an effect mimicked by shRNA knockdown of mTOR or raptor in ECs. The effects of rapamycin persisted for several days and were linked to upregulation of the inhibitory molecules PD-L1 and PD-L2 on rapa-ECs. Additionally, rapa-ECs produced lower levels of the inflammatory cytokine IL-6. CD4⁺ memory cells activated by allogeneic rapa-ECs became hyporesponsive to restimulation in an alloantigen-specific manner and contained higher percentages of suppressive CD4⁺CD25^hiCD127^loFoxP3⁺ cells that did not produce effector cytokines. In a human-mouse chimeric model of allograft rejection, rapamycin pretreatment of human arterial allografts increased graft EC expression of PD-L1 and PD-L2 and reduced subsequent infiltration of allogeneic effector T cells into the artery intima and intimal expansion. Preoperative conditioning of allograft ECs with rapamycin could potentially reduce immune-mediated rejection.

Authors

Chen Wang, Tai Yi, Lingfeng Qin, Roberto A. Maldonado, Ulrich H. von Andrian, Sanjay Kulkarni, George Tellides, Jordan S. Pober

Formylpeptide receptor-2 contributes to colonic epithelial homeostasis, inflammation, and tumorigenesis

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Abstract

Commensal bacteria and their products provide beneficial effects to the mammalian gut by stimulating epithelial cell turnover and enhancing wound healing, without activating overt inflammation. We hypothesized that N-formylpeptide receptors, which bind bacterial N-formylpeptides and are expressed by intestinal epithelial cells, may contribute to these processes. Here we report that formylpeptide receptor-2 (FPR2), which we show is expressed on the apical and lateral membranes of colonic crypt epithelial cells, mediates N-formylpeptide–dependent epithelial cell proliferation and renewal. Colonic epithelial cells in FPR2-deficient mice displayed defects in commensal bacterium–dependent homeostasis as shown by the absence of responses to N-formylpeptide stimulation, shortened colonic crypts, reduced acute inflammatory responses to dextran sulfate sodium (DSS) challenge, delayed mucosal restoration after injury, and increased azoxymethane-induced tumorigenesis. These results indicate that FPR2 is critical in mediating homeostasis, inflammation, and epithelial repair processes in the colon.

Authors

Keqiang Chen, Mingyong Liu, Ying Liu, Teizo Yoshimura, Wei Shen, Yingying Le, Scott Durum, Wanghua Gong, Chunyan Wang, Ji-Liang Gao, Philip M. Murphy, Ji Ming Wang

GSK3β regulates physiological migration of stem/progenitor cells via cytoskeletal rearrangement

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Abstract

Regulation of hematopoietic stem and progenitor cell (HSPC) steady-state egress from the bone marrow (BM) to the circulation is poorly understood. While glycogen synthase kinase-3β (GSK3β) is known to participate in HSPC proliferation, we revealed an unexpected role in the preferential regulation of CXCL12-induced migration and steady-state egress of murine HSPCs, including long-term repopulating HSCs, over mature leukocytes. HSPC egress, regulated by circadian rhythms of CXCL12 and CXCR4 levels, correlated with dynamic expression of GSK3β in the BM. Nevertheless, GSK3β signaling was CXCL12/CXCR4 independent, suggesting that synchronization of both pathways is required for HSPC motility. Chemotaxis of HSPCs expressing higher levels of GSK3β compared with mature cells was selectively enhanced by stem cell factor–induced activation of GSK3β. Moreover, HSPC motility was regulated by norepinephrine and insulin-like growth factor-1 (IGF-1), which increased or reduced, respectively, GSK3β expression in BM HSPCs and their subsequent egress. Mechanistically, GSK3β signaling promoted preferential HSPC migration by regulating actin rearrangement and microtubuli turnover, including CXCL12-induced actin polarization and polymerization. Our study identifies a previously unknown role for GSK3β in physiological HSPC motility, dictating an active, rather than a passive, nature for homeostatic egress from the BM reservoir to the blood circulation.

Authors

Kfir Lapid, Tomer Itkin, Gabriele D’Uva, Yossi Ovadya, Aya Ludin, Giulia Caglio, Alexander Kalinkovich, Karin Golan, Ziv Porat, Massimo Zollo, Tsvee Lapidot

NO triggers RGS4 degradation to coordinate angiogenesis and cardiomyocyte growth

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Abstract

Myocardial hypertrophy is an adaptation to increased hemodynamic demands. An increase in heart tissue must be matched by a corresponding expansion of the coronary vasculature to maintain and adequate supply of oxygen and nutrients for the heart. The physiological mechanisms that underlie the coordination of angiogenesis and cardiomyocyte growth are unknown. We report that induction of myocardial angiogenesis promotes cardiomyocyte growth and cardiac hypertrophy through a novel NO-dependent mechanism. We used transgenic, conditional overexpression of placental growth factor (PlGF) in murine cardiac tissues to stimulate myocardial angiogenesis and increase endothelial-derived NO release. NO production, in turn, induced myocardial hypertrophy by promoting proteasomal degradation of regulator of G protein signaling type 4 (RGS4), thus relieving the repression of the Gβγ/PI3Kγ/AKT/mTORC1 pathway that stimulates cardiomyocyte growth. This hypertrophic response was prevented by concomitant transgenic expression of RGS4 in cardiomyocytes. NOS inhibitor L-NAME also significantly attenuated RGS4 degradation, and reduced activation of AKT/mTORC1 signaling and induction of myocardial hypertrophy in PlGF transgenic mice, while conditional cardiac-specific PlGF expression in eNOS knockout mice did not induce myocardial hypertrophy. These findings describe a novel NO/RGS4/Gβγ/PI3Kγ/AKT mechanism that couples cardiac vessel growth with myocyte growth and heart size.

Authors

Irina M. Jaba, Zhen W. Zhuang, Na Li, Yifeng Jiang, Kathleen A. Martin, Albert J. Sinusas, Xenophon Papademetris, Michael Simons, William C. Sessa, Lawrence H. Young, Daniela Tirziu

Tumor VEGF:VEGFR2 autocrine feed-forward loop triggers angiogenesis in lung cancer

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Abstract

The molecular mechanisms that control the balance between antiangiogenic and proangiogenic factors and initiate the angiogenic switch in tumors remain poorly defined. By combining chemical genetics with multimodal imaging, we have identified an autocrine feed-forward loop in tumor cells in which tumor-derived VEGF stimulates VEGF production via VEGFR2-dependent activation of mTOR, substantially amplifying the initial proangiogenic signal. Disruption of this feed-forward loop by chemical perturbation or knockdown of VEGFR2 in tumor cells dramatically inhibited production of VEGF in vitro and in vivo. This disruption was sufficient to prevent tumor growth in vivo. In patients with lung cancer, we found that this VEGF:VEGFR2 feed-forward loop was active, as the level of VEGF/VEGFR2 binding in tumor cells was highly correlated to tumor angiogenesis. We further demonstrated that inhibition of tumor cell VEGFR2 induces feedback activation of the IRS/MAPK signaling cascade. Most strikingly, combined pharmacological inhibition of VEGFR2 (ZD6474) and MEK (PD0325901) in tumor cells resulted in dramatic tumor shrinkage, whereas monotherapy only modestly slowed tumor growth. Thus, a tumor cell-autonomous VEGF:VEGFR2 feed-forward loop provides signal amplification required for the establishment of fully angiogenic tumors in lung cancer. Interrupting this feed-forward loop switches tumor cells from an angiogenic to a proliferative phenotype that sensitizes tumor cells to MAPK inhibition.

Authors

Sampurna Chatterjee, Lukas C. Heukamp, Maike Siobal, Jakob Schöttle, Caroline Wieczorek, Martin Peifer, Davide Frasca, Mirjam Koker, Katharina König, Lydia Meder, Daniel Rauh, Reinhard Buettner, Jürgen Wolf, Rolf A. Brekken, Bernd Neumaier, Gerhard Christofori, Roman K. Thomas, Roland T. Ullrich

Nanogel-based delivery of mycophenolic acid ameliorates systemic lupus erythematosus in mice

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Abstract

The ability to selectively inactivate immune cells with immunosuppressants is a much sought-after modality for the treatment of systemic lupus erythematosus and autoimmunity in general. Here, we designed and tested a novel nanogel drug delivery vehicle for the immunosuppressant mycophenolic acid (MPA). Treatment with MPA-loaded nanogels increased the median survival time (MST) of lupus-prone NZB/W F1 mice by 3 months with prophylactic use (MST was 50 weeks versus 38 weeks without treatment), and by 2 months when administered after the development of severe renal damage (MST after proteinuria onset was 12.5 weeks versus 4 weeks without treatment). Equivalent and greater doses of MPA administered in buffer were not efficacious. Nanogels had enhanced biodistribution to organs and association with immune cells. CD4-targeted nanogels yielded similar therapeutic results compared with nontargeted formulations, with protection from glomerulonephritis and decreases in IFN-γ–positive CD4 T cells. DCs that internalized nanogels helped mediate immunosuppression, as they had reduced production of inflammatory cytokines such as IFN-γ and IL-12. Our results demonstrate efficacy of nanogel-based lupus therapy and implicate a mechanism by which immunosuppression is enhanced, in part, by the targeting of antigen-presenting cells.

Authors

Michael Look, Eric Stern, Qin A. Wang, Leah D. DiPlacido, Michael Kashgarian, Joe Craft, Tarek M. Fahmy

Chronic activation of a designer G_q-coupled receptor improves β cell function

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Abstract

Type 2 diabetes (T2D) has emerged as a major threat to human health in most parts of the world. Therapeutic strategies aimed at improving pancreatic β cell function are predicted to prove beneficial for the treatment of T2D. In the present study, we demonstrate that drug-mediated, chronic, and selective activation of β cell G_q signaling greatly improve β cell function and glucose homeostasis in mice. These beneficial metabolic effects were accompanied by the enhanced expression of many genes critical for β cell function, maintenance, and differentiation. By employing a combination of in vivo and in vitro approaches, we identified a novel β cell pathway through which receptor-activated G_q leads to the sequential activation of ERK1/2 and IRS2 signaling, thus triggering a series of events that greatly improve β cell function. Importantly, we found that chronic stimulation of a designer G_q-coupled receptor selectively expressed in β cells prevented both streptozotocin-induced diabetes and the metabolic deficits associated with the consumption of a high-fat diet in mice. Since β cells are endowed with numerous receptors that mediate their cellular effects via activation of G_q-type G proteins, our findings provide a rational basis for the development of novel antidiabetic drugs targeting this class of receptors.

Authors

Shalini Jain, Inigo Ruiz de Azua, Huiyan Lu, Morris F. White, Jean-Marc Guettier, Jürgen Wess

Planar cell polarity genes control the connectivity of enteric neurons

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Abstract

A highly complex network of intrinsic enteric neurons is required for the digestive and homeostatic functions of the gut. Nevertheless, the genetic and molecular mechanisms that regulate their assembly into functional neuronal circuits are currently unknown. Here we report that the planar cell polarity (PCP) genes Celsr3 and Fzd3 are required during murine embryogenesis to specifically control the guidance and growth of enteric neuronal projections relative to the longitudinal and radial gut axes. Ablation of these genes disrupts the normal organization of nascent neuronal projections, leading to subtle changes of axonal tract configuration in the mature enteric nervous system (ENS), but profound abnormalities in gastrointestinal motility. Our data argue that PCP-dependent modules of connectivity established at early stages of enteric neurogenesis control gastrointestinal function in adult animals and provide the first evidence that developmental deficits in ENS wiring may contribute to the pathogenesis of idiopathic bowel disorders.

Authors

Valentina Sasselli, Werend Boesmans, Pieter Vanden Berghe, Fadel Tissir, André M. Goffinet, Vassilis Pachnis

Epitope specificity determines pathogenicity and detectability in ANCA-associated vasculitis

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Abstract

Anti-neutrophil cytoplasmic antibody–associated (ANCA-associated) small vessel necrotizing vasculitis is caused by immune-mediated inflammation of the vessel wall and is diagnosed in some cases by the presence of myeloperoxidase-specific antibodies (MPO-ANCA). This multicenter study sought to determine whether differences in ANCA epitope specificity explain why, in some cases, conventional serologic assays do not correlate with disease activity, why naturally occurring anti-MPO autoantibodies can exist in disease-free individuals, and why ANCA are undetected in patients with ANCA-negative disease. Autoantibodies from human and murine samples were epitope mapped using a highly sensitive epitope excision/mass spectrometry approach. Data indicated that MPO autoantibodies from healthy individuals had epitope specificities different from those present in ANCA disease. Importantly, this methodology led to the discovery of MPO-ANCA in ANCA-negative disease that reacted against a sole linear sequence. Autoantibodies against this epitope had pathogenic properties, as demonstrated by their capacity to activate neutrophils in vitro and to induce nephritis in mice. The confounder for serological detection of these autoantibodies was the presence of a fragment of ceruloplasmin in serum, which was eliminated in purified IgG, allowing detection. These findings implicate immunodominant epitopes in the pathology of ANCA-associated vasculitis and suggest that autoantibody diversity may be common to other autoimmune diseases.

Authors

Aleeza J. Roth, Joshua D. Ooi, Jacob J. Hess, Mirjan M. van Timmeren, Elisabeth A. Berg, Caroline E. Poulton, JulieAnne McGregor, Madelyn Burkart, Susan L. Hogan, Yichun Hu, Witold Winnik, Patrick H. Nachman, Coen A. Stegeman, John Niles, Peter Heeringa, A. Richard Kitching, Stephen Holdsworth, J. Charles Jennette, Gloria A. Preston, Ronald J. Falk

Myeloid cell–specific serine palmitoyltransferase subunit 2 haploinsufficiency reduces murine atherosclerosis

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Abstract

Serine palmitoyltransferase (SPT) is the first and rate-limiting enzyme of the de novo biosynthetic pathway of sphingomyelin (SM). Both SPT and SM have been implicated in the pathogenesis of atherosclerosis, the development of which is driven by macrophages; however, the role of SPT in macrophage-mediated atherogenesis is unknown. To address this issue, we have analyzed macrophage inflammatory responses and reverse cholesterol transport, 2 key mediators of atherogenesis, in SPT subunit 2–haploinsufficient (Sptlc2^+/–) macrophages. We found that Sptlc2^+/– macrophages have significantly lower SM levels in plasma membrane and lipid rafts. This reduction not only impaired inflammatory responses triggered by TLR4 and its downstream NF-κB and MAPK pathways, but also enhanced reverse cholesterol transport mediated by ABC transporters. LDL receptor–deficient (Ldlr^–/–) mice transplanted with Sptlc2^+/– bone marrow cells exhibited significantly fewer atherosclerotic lesions after high-fat and high-cholesterol diet feeding. Additionally, Ldlr^–/– mice with myeloid cell–specific Sptlc2 haploinsufficiency exhibited significantly less atherosclerosis than controls. These findings suggest that SPT could be a novel therapeutic target in atherosclerosis.

Authors

Mahua Chakraborty, Caixia Lou, Chongmin Huan, Ming-Shang Kuo, Tae-Sik Park, Guoqing Cao, Xian-Cheng Jiang

Sudden unexpected death in a mouse model of Dravet syndrome

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Abstract

Sudden unexpected death in epilepsy (SUDEP) is the most common cause of death in intractable epilepsies, but physiological mechanisms that lead to SUDEP are unknown. Dravet syndrome (DS) is an infantile-onset intractable epilepsy caused by heterozygous loss-of-function mutations in the SCN1A gene, which encodes brain type-I voltage-gated sodium channel Na_V1.1. We studied the mechanism of premature death in Scn1a heterozygous KO mice and conditional brain- and cardiac-specific KOs. Video monitoring demonstrated that SUDEP occurred immediately following generalized tonic-clonic seizures. A history of multiple seizures was a strong risk factor for SUDEP. Combined video-electroencephalography-electrocardiography revealed suppressed interictal resting heart-rate variability and episodes of ictal bradycardia associated with the tonic phases of generalized tonic-clonic seizures. Prolonged atropine-sensitive ictal bradycardia preceded SUDEP. Similar studies in conditional KO mice demonstrated that brain, but not cardiac, KO of Scn1a produced cardiac and SUDEP phenotypes similar to those found in DS mice. Atropine or N-methyl scopolamine treatment reduced the incidence of ictal bradycardia and SUDEP in DS mice. These findings suggest that SUDEP is caused by apparent parasympathetic hyperactivity immediately following tonic-clonic seizures in DS mice, which leads to lethal bradycardia and electrical dysfunction of the ventricle. These results have important implications for prevention of SUDEP in DS patients.

Authors

Franck Kalume, Ruth E. Westenbroek, Christine S. Cheah, Frank H. Yu, John C. Oakley, Todd Scheuer, William A. Catterall

Biochemical correlates of neuropsychiatric illness in maple syrup urine disease

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Abstract

Maple syrup urine disease (MSUD) is an inherited disorder of branched chain amino acid metabolism presenting with neonatal encephalopathy, episodic metabolic decompensation, and chronic amino acid imbalances. Dietary management enables survival and reduces risk of acute crises. Liver transplantation has emerged as an effective way to eliminate acute decompensation risk. Psychiatric illness is a reported MSUD complication, but has not been well characterized and remains poorly understood. We report the prevalence and characteristics of neuropsychiatric problems among 37 classical MSUD patients (ages 5–35 years, 26 on dietary therapy, 11 after liver transplantation) and explore their underlying mechanisms. Compared with 26 age-matched controls, MSUD patients were at higher risk for disorders of cognition, attention, and mood. Using quantitative proton magnetic resonance spectroscopy, we found lower brain glutamate, N-acetylaspartate (NAA), and creatine concentrations in MSUD patients, which correlated with specific neuropsychiatric outcomes. Asymptomatic neonatal course and stringent longitudinal biochemical control proved fundamental to optimizing long-term mental health. Neuropsychiatric morbidity and neurochemistry were similar among transplanted and nontransplanted MSUD patients. In conclusion, amino acid dysregulation results in aberrant neural networks with neurochemical deficiencies that persist after transplant and correlate with neuropsychiatric morbidities. These findings may provide insight into general mechanisms of psychiatric illness.

Authors

Emilie R. Muelly, Gregory J. Moore, Scott C. Bunce, Julie Mack, Don C. Bigler, D. Holmes Morton, Kevin A. Strauss

GSK-3α is a central regulator of age-related pathologies in mice

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Abstract

Aging is regulated by conserved signaling pathways. The glycogen synthase kinase-3 (GSK-3) family of serine/threonine kinases regulates several of these pathways, but the role of GSK-3 in aging is unknown. Herein, we demonstrate premature death and acceleration of age-related pathologies in the Gsk3a global KO mouse. KO mice developed cardiac hypertrophy and contractile dysfunction as well as sarcomere disruption and striking sarcopenia in cardiac and skeletal muscle, a classical finding in aging. We also observed severe vacuolar degeneration of myofibers and large tubular aggregates in skeletal muscle, consistent with impaired clearance of insoluble cellular debris. Other organ systems, including gut, liver, and the skeletal system, also demonstrated age-related pathologies. Mechanistically, we found marked activation of mTORC1 and associated suppression of autophagy markers in KO mice. Loss of GSK-3α, either by pharmacologic inhibition or Gsk3a gene deletion, suppressed autophagy in fibroblasts. mTOR inhibition rescued this effect and reversed the established pathologies in the striated muscle of the KO mouse. Thus, GSK-3α is a critical regulator of mTORC1, autophagy, and aging. In its absence, aging/senescence is accelerated in multiple tissues. Strategies to maintain GSK-3α activity and/or inhibit mTOR in the elderly could retard the appearance of age-related pathologies.

Authors

Jibin Zhou, Theresa A. Freeman, Firdos Ahmad, Xiying Shang, Emily Mangano, Erhe Gao, John Farber, Yajing Wang, Xin-Liang Ma, James Woodgett, Ronald J. Vagnozzi, Hind Lal, Thomas Force

Eliminating malignant contamination from therapeutic human spermatogonial stem cells

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Abstract

Spermatogonial stem cell (SSC) transplantation has been shown to restore fertility in several species and may have application for treating some cases of male infertility (e.g., secondary to gonadotoxic therapy for cancer). To ensure safety of this fertility preservation strategy, methods are needed to isolate and enrich SSCs from human testis cell suspensions and also remove malignant contamination. We used flow cytometry to characterize cell surface antigen expression on human testicular cells and leukemic cells (MOLT-4 and TF-1a). We demonstrated via FACS that EpCAM is expressed by human spermatogonia but not MOLT-4 cells. In contrast, HLA-ABC and CD49e marked >95% of MOLT-4 cells but were not expressed on human spermatogonia. A multiparameter sort of MOLT-4–contaminated human testicular cell suspensions was performed to isolate EpCAM⁺/HLA-ABC^–/CD49e^– (putative spermatogonia) and EpCAM^–/HLA-ABC⁺/CD49e⁺ (putative MOLT-4) cell fractions. The EpCAM⁺/HLA-ABC^–/CD49e^– fraction was enriched for spermatogonial colonizing activity and did not form tumors following human-to–nude mouse xenotransplantation. The EpCAM^–/HLA-ABC⁺/CD49e⁺ fraction produced tumors following xenotransplantation. This approach could be generalized with slight modification to also remove contaminating TF-1a leukemia cells. Thus, FACS provides a method to isolate and enrich human spermatogonia and remove malignant contamination by exploiting differences in cell surface antigen expression.

Authors

Serena L. Dovey, Hanna Valli, Brian P. Hermann, Meena Sukhwani, Julia Donohue, Carlos A. Castro, Tianjiao Chu, Joseph S. Sanfilippo, Kyle E. Orwig

Impaired autophagic flux mediates acinar cell vacuole formation and trypsinogen activation in rodent models of acute pancreatitis

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Abstract

Authors

Olga A. Mareninova, Kip Hermann, Samuel W. French, Mark S. O’Konski, Stephen J. Pandol, Paul Webster, Ann H. Erickson, Nobuhiko Katunuma, Fred S. Gorelick, Ilya Gukovsky, Anna S. Gukovskaya