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Abstract
Recombinant adeno-associated virus (rAAV)-mediated gene delivery can efficiently target muscle tissues to serve as “biofactories” for secreted proteins in prophylactic and therapeutic scenarios. Nevertheless, efficient rAAV-mediated gene delivery is often limited by host immune responses against the transgene product. The development of strategies to prevent anti-transgene immunity is therefore crucial. The employment of endogenous microRNA (miRNA)-mediated regulation to detarget transgene expression from antigen presenting cells (APCs) has shown promise for reducing immunogenicity. However, the mechanisms underlying miRNA-mediated modulation of anti-transgene immunity by APC detargeting are not fully understood. Using the highly immunogenic ovalbumin (OVA) protein as a proxy for foreign antigens, we show that rAAV vectors containing miR142 binding sites efficiently repress co-stimulatory signals in dendritic cells, significantly blunt the cytotoxic T cell response, allow for sustained transgene expression in skeletal myoblasts, and attenuate clearance of transduced muscle cells in mice. Furthermore, the blunting of humoral immunity against circulating OVA correlates with detargeting of OVA expression from APCs. This demonstrates that incorporating APC-specific miRNA binding sites into rAAV vectors provides an effective strategy for reducing transgene-specific immune response. This approach holds promise for clinical applications where the safe and efficient delivery of a prophylactic or therapeutic protein is desired.
Authors
Yuanyuan Xiao, Manish Muhuri, Shaoyong Li, Wanru Qin, Guangchao Xu, Li Luo, Jia Li, Alexander J. Letizia, Sean K. Wang, Ying Kai Chan, Chunmei Wang, Sebastian P. Fuchs, Dan Wang, Qin Su, M. Abu Nahid, George M. Church, Michael Farzan, Li Yang, Yuquan Wei, Ronald C. Desrosiers, Christian Mueller, Phillip W.L. Tai, Guangping Gao
Abstract
The mechanisms regulating translation and splicing are not well understood. We provide insight into a new regulator of translation, OGFOD1 (2-oxoglutarate and iron dependent oxygenase domain-containing protein 1), which is a prolyl-hydroxylase that catalyzes the posttranslational hydroxylation of Pro-62 in the small ribosomal protein S23. We show that deletion of OGFOD1 in an in vitro model of human cardiomyocytes decreases translation of specific proteins (e.g., RNA-binding proteins) and alters splicing. RNA sequencing showed poor correlation between changes in mRNA and protein synthesis, suggesting that posttranscriptional regulation was the primary cause for the observed differences. We found that loss of OGFOD1 and the resultant alterations in protein translation modulates the cardiac proteome, shifting it towards higher protein amounts of sarcomeric proteins such as cardiac troponins, titin and cardiac myosin binding protein C. Furthermore, we found a decrease of OGFOD1 during cardiomyocyte differentiation. These results suggest that loss of OGFOD1 modulates protein translation and splicing, thereby leading to alterations in the cardiac proteome and highlight the role of altered translation and splicing in regulating the proteome..
Authors
Andrea Stoehr, Leslie Kennedy, Yanqin Yang, Sajni Patel, Yongshun Lin, Kaari L. Linask, Maria M. Fergusson, Jun Zhu, Marjan Gucek, Jizhong Zou, Elizabeth Murphy
Abstract
Susceptibility to chronic beryllium (Be) disease is linked to HLA-DP molecules possessing a glutamic acid at the 69th position of the β-chain (βGlu69), with the most prevalent βGlu69-containing molecule being HLA-DP2. We have previously shown that HLA-DP2 transgenic (Tg) mice exposed to Be oxide (BeO) develop mononuclear infiltrates in a peribronchovascular distribution and a beryllium-specific, HLA-DP2-restricted CD4+ T cell response. In addition to T cells, B cells constituted a major portion of infiltrated leukocytes in the lung of BeO-exposed HLA-DP2 Tg mice and sequester BeO particles within ectopic lymphoid aggregates and granulomas. B cell depletion was associated with a loss of lymphoid aggregates and granulomas as well as a significant increase in lung injury in BeO-exposed mice. The protective role of B cells was innate in origin, and BeO-induced B cell recruitment to the lung was dependent on MyD88 signaling. Similar to BeO-exposed HLA-DP2 mice, B cells also accumulate in the lungs of CBD subjects, located at the periphery and surrounding the granuloma. Overall, our data suggest a novel modulatory role for B cells in the protection of the lung against sterile particulate exposure, with B cell recruitment to the inflamed lung occurring in an antigen-independent and MyD88-dependent manner.
Authors
Shaikh M. Atif, Douglas G. Mack, Amy S. McKee, Javier Rangel-Moreno, Allison K. Martin, Andrew Getahun, Lisa A. Maier, John C. Cambier, Rubin Tuder, Andrew P. Fontenot
Abstract
Benign prostatic hyperplasia (BPH) is the most common cause of lower urinary tract symptoms in men. Current treatments target prostate physiology rather than BPH pathophysiology and are only partially effective. Here, we applied next-generation sequencing to gain new insight into BPH. By RNAseq, we uncovered transcriptional heterogeneity among BPH cases, where a 65-gene BPH stromal signature correlated with symptom severity. Stromal signaling molecules BMP5 and CXCL13 were enriched in BPH while estrogen regulated pathways were depleted. Notably, BMP5 addition to cultured prostatic myofibroblasts altered their expression profile towards a BPH profile that included the BPH stromal signature. RNAseq also suggested an altered cellular milieu in BPH, which we verified by immunohistochemistry and single-cell RNAseq. In particular, BPH tissues exhibited enrichment of myofibroblast subsets, whilst depletion of neuroendocrine cells and an estrogen receptor (ESR1)-positive fibroblast cell type residing near epithelium. By whole-exome sequencing, we uncovered somatic single-nucleotide variants (SNVs) in BPH, of uncertain pathogenic significance but indicative of clonal cell expansions. Thus, genomic characterization of BPH has identified a clinically-relevant stromal signature and new candidate disease pathways (including a likely role for BMP5 signaling), and reveals BPH to be not merely a hyperplasia, but rather a fundamental re-landscaping of cell types.
Authors
Lance W. Middleton, Zhewei Shen, Sushama Varma, Anna S. Pollack, Xue Gong, Shirley Zhu, Chunfang Zhu, Joseph W. Foley, Sujay Vennam, Robert T. Sweeney, Karen Tu, Jewison Biscocho, Okyaz Eminaga, Rosalie Nolley, Robert Tibshirani, James D. Brooks, Robert B. West, Jonathan R. Pollack
Abstract
Pulmonary fibrosis is a devastating disease characterized by accumulation of activated fibroblasts and scarring in the lung. While fibroblast activation in physiological wound repair reverses spontaneously, fibroblast activation in fibrosis is aberrantly sustained. Here we identified histone 3 lysine 9 methylation (H3K9me) as a critical epigenetic modification that sustains fibroblast activation by repressing the transcription of genes essential to returning lung fibroblasts to an inactive state. We show that the histone methyltransferase G9a (EHMT2) and chromobox homolog 5 (CBX5, also known as HP1α), which deposit H3K9me marks and assemble an associated repressor complex respectively, are essential to initiation and maintenance of fibroblast activation specifically through epigenetic repression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha gene (PPARGC1A, encoding PGC1α). Both TGFβ and increased matrix stiffness potently inhibit PGC1α expression in lung fibroblasts through engagement of the CBX5/G9a pathway. Inhibition of CBX5/G9a pathway in fibroblasts elevates PGC1α, attenuates TGFβ- and matrix stiffness-promoted H3K9 methylation, and reduces collagen accumulation in the lungs following bleomycin injury. Our results demonstrate that epigenetic silencing mediated by H3K9 methylation is essential for both biochemical and biomechanical fibroblast activation, and that targeting this epigenetic pathway may provide therapeutic benefit by returning lung fibroblasts to quiescence.
Authors
Giovanni Ligresti, Nunzia Caporarello, Jeffrey A. Meridew, Dakota L. Jones, Qi Tan, Kyoung Moo Choi, Andrew J. Haak, Aja Aravamudhan, Anja C. Roden, Y. S. Prakash, Gwen Lomberk, Raul A. Urrutia, Daniel J. Tschumperlin
Abstract
Despite current immunosuppressive strategies, long-term lung transplant outcomes remain poor due to rapid allogenic responses. Using a stringent mouse model of allo-airway transplantation, we identify the CCR4-ligand axis as a central node driving secondary lymphoid tissue homing and activation of the allogeneic T cells that prevent long-term allograft survival. CCR4 deficiency on transplant recipient T cells diminishes allograft injury and when combined with CTLA4-Ig leads to an unprecedented long-term lung allograft accommodation. Thus, we identify CCR4-ligand interactions as a central mechanism driving allogeneic transplant rejection and suggest it as a potential target to enhance long-term lung transplant survival.
Authors
Vyacheslav Palchevskiy, Ying Ying Xue, Rita Kern, Stephen S. Weigt, Aric L. Gregson, Sophie X. Song, Michael C. Fishbein, Cory M. Hogaboam, David M. Sayah, Joseph P. Lynch, III, Michael P. Keane, David G. Brooks, John A. Belperio
Abstract
Impaired insulin secretion in type 2 diabetes (T2D) is linked to reduced insulin granule docking, disorganization of the exocytotic site, and an impaired glucose-dependent facilitation of insulin exocytosis. We show in β-cells from 80 human donors that the glucose-dependent amplification of exocytosis is disrupted in T2D. Spatial analyses of granule fusion, visualized by total internal reflection fluorescence (TIRF) microscopy in 24 of these donors, demonstrate that these are non-random across the surface of β-cells from donors with no diabetes (ND). The compartmentalization of events occurs within regions defined by concurrent or recent membrane-resident secretory granules. This organization, and the number of membrane-associated granules, is glucose-dependent and notably impaired in T2D β-cells. Mechanistically, multi-channel Kv2.1 clusters contribute to maintaining the density of membrane-resident granules and the number of fusion ‘hotspots’, while SUMOylation sites at the channel N- (K145) and C-terminus (K470) determine the relative proportion of fusion events occurring within these regions. Thus, a glucose-dependent compartmentalization of fusion, regulated in part by a structural role for Kv2.1, is disrupted in β-cells from donors with type 2 diabetes.
Authors
Jianyang Fu, John Maringa Githaka, Xiaoqing Dai, Gregory Plummer, Kunimasa Suzuki, Aliya F. Spigelman, Austin Bautista, Ryekjang Kim, Dafna Greitzer-Antes, Jocelyn E. Manning Fox, Herbert Y. Gaisano, Patrick E. MacDonald
Abstract
Background: The lymphocyte-depleting antibody alemtuzumab is a highly effective treatment of relapsing-remitting multiple sclerosis (RRMS); however 50% of patients develop novel autoimmunity post-treatment. Most at risk are individuals who reconstitute their T-cell pool by proliferating residual cells, rather than producing new T-cells in the thymus; raising the possibility that autoimmunity might be prevented by increasing thymopoiesis. Keratinocyte growth factor (palifermin) promotes thymopoiesis in non-human primates. Methods: Following a dose-tolerability sub-study, individuals with RRMS (duration ≤10 years; expanded disability status scale ≤5·0; with ≥2 relapses in the previous 2 years) were randomised to placebo or 180mcg/kg/day palifermin, given for 3 days immediately prior to and after each cycle of alemtuzumab, with repeat doses at M1 and M3. The interim primary endpoint was naïve CD4+ T-cell count at M6. Exploratory endpoints included: number of recent thymic-emigrants (RTEs) and signal-joint T-cell receptor excision circles (sjTRECs)/mL of blood. The trial primary endpoint was incidence of autoimmunity at M30. Findings: At M6, individuals receiving palifermin had fewer naïve CD4+T-cells (2.229x107/L vs. 7.733x107/L; p=0.007), RTEs (16% vs. 34%) and sjTRECs/mL (1100 vs. 3396), leading to protocol-defined termination of recruitment. No difference was observed in the rate of autoimmunity between the two groups Conclusion: In contrast to animal studies, palifermin reduced thymopoiesis in our patients. These results offer a note of caution to those using palifermin to promote thymopoiesis in other settings, particularly in the oncology/haematology setting where alemtuzumab is often used as part of the conditioning regime. Trial Registration: ClinicalTrials.gov NCT01712945 Funding: MRC and Moulton Charitable Foundation
Authors
Alasdair J. Coles, Laura Azzopardi, Onajite Kousin-Ezewu, Harpreet Kaur Mullay, Sara A.J. Thompson, Lorna Jarvis, Jessica Davies, Sarah Howlett, Daniel Rainbow, Judith Babar, Timothy J. Sadler, J. William L. Brown, Edward Needham, Karen May, Zoya G. Georgieva, Adam E. Handel, Stefano Maio, Mary Deadman, Ioanna Rota, Georg Holländer, Sarah Dawson, David Jayne, Ruth Seggewiss-Bernhardt, Daniel C. Douek, John D. Isaacs, Joanne L. Jones
Glycolytic inhibitor 2-deoxyglucose prevents cortical hyperexcitability after traumatic brain injury
Abstract
Traumatic brain injury (TBI) causes cortical dysfunction and can lead to post-traumatic epilepsy. Multiple studies demonstrate that GABAergic inhibitory network function is compromised following TBI, which may contribute to hyperexcitability and motor, behavioral, and cognitive deficits. Preserving the function of GABAergic interneurons, therefore, is a rational therapeutic strategy to preserve cortical function after TBI and prevent long-term clinical complications. Here, we explored an approach based on the ketogenic diet, a neuroprotective and anticonvulsant dietary therapy which results in reduced glycolysis and increased ketosis. Utilizing a pharmacologic inhibitor of glycolysis (2-deoxyglucose, or 2-DG), we found that acute in vitro application of 2-DG decreased the excitability of excitatory neurons, but not inhibitory interneurons, in cortical slices from naïve mice. Employing the controlled cortical impact (CCI) model of TBI in mice, we found that in vitro 2-DG treatment rapidly attenuated epileptiform activity seen in acute cortical slices 3 to 5 weeks after TBI. One week of in vivo 2-DG treatment immediately after TBI prevented the development of epileptiform activity, restored excitatory and inhibitory synaptic activity, and attenuated the loss of parvalbumin-expressing inhibitory interneurons. In summary, 2-DG may have therapeutic potential to restore network function following TBI.
Authors
Jenny B. Koenig, David Cantu, Cho S. Low, Mary E. Sommer, Farzad Noubary, Danielle Croker, Michael Whalen, Dong Kong, Chris G. Dulla
Abstract
BACKGROUND. Cutaneous neurofibromas (cNF) are physically disfiguring, painful, and cause extensive psychologic harm in patients with neurofibromatosis type 1 (NF1). There is currently no effective medical treatment and surgical procedures are inaccessible to most NF1 patients globally. OBJECTIVE. While research is underway to find an effective medical treatment for cNF, there is an urgent need to develop surgical approach that is accessible to all NF1 patients in the world with the skill set and equipment found in most general medical office settings. Here, we present a robust surgical approach to remove cNF that does not require sterile surgical field, utilizes accessible clinical equipment, and can be performed by any health care providers including family practitioners, and physician assistants. METHODS. In a prospective case-series, patients with NF1 underwent this surgical procedure which removes multiple cutaneous neurofibromas. The Dermatology Life Quality Index was given to subjects before and after the procedure as surrogate for patient satisfaction. RESULTS. 83 tumors were removed throughout the body from twelve individuals. Examination at follow-up visits revealed well-healed scars without infection or adverse events including aberrant scarring. Patient satisfaction with the procedure was high with significant improvements in symptoms, daily activities, leisure, personal relationships, and treatment experience (P = 0.00062). CONCLUSION. This study demonstrates a robust surgical approach to management cutaneous neurofibromas which can be accessed world-wide to individuals with NF1 and performed by a wide-variety of medical specialists with high clinical efficacy and patient satisfaction.
Authors
Bahir H. Chamseddin, La’Nette Hernandez, Dezehree Solorzano, Juan Vega, Lu Q. Le
Abstract
Chimeric antigen receptor (CAR) T cell therapies have achieved promising outcomes in several cancers, however more challenging oncology indications may necessitate advanced antigen receptor designs and functions. Here we describe a bipartite receptor system comprised of separate antigen targeting and signal transduction polypeptides, each containing an extracellular dimerization domain. We demonstrate that T cell activation remains antigen dependent but can only be achieved in the presence of a dimerizing drug, rapamycin. Studies performed in vitro and in xenograft mouse models illustrate equivalent to superior anti-tumor potency compared to currently used CAR designs, and at rapamycin concentrations well below immunosuppressive levels. We further show that the extracellular positioning of the dimerization domains enables the administration of recombinant re-targeting modules, potentially extending antigen targeting. Overall, this novel regulatable CAR design has exquisite drug sensitivity, provides robust anti-tumor responses, and is uniquely flexible for multiplex antigen targeting or retargeting, which may further assist the development of safe, potent and durable T cell therapeutics.
Authors
Wai-Hang Leung, Joel Gay, Unja Martin, Tracy E. Garrett, Holly M. Horton, Michael T. Certo, Bruce R. Blazar, Richard A. Morgan, Philip D. Gregory, Jordan Jarjour, Alexander Astrakhan
Abstract
Enchondroma and chondrosarcoma are the most common benign and malignant cartilaginous neoplasms. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are present in the majority of these tumors. We performed RNA-seq analysis on chondrocytes from Col2a1Cre;Idh1LSL/+ animals and found that genes implied in cholesterol synthesis pathway were significantly upregulated in the mutant chondrocytes. We examined the phenotypic effect of inhibiting intracellular cholesterol biosynthesis on enchondroma formation by conditionally deleting SCAP (sterol regulatory element-binding protein cleavage-activating protein), a protein activating intracellular cholesterol synthesis, in IDH1 mutant mice. We found fewer enchondromas in animals lacking SCAP. Furthermore, in chondrosarcomas, pharmacological inhibition of intracellular cholesterol synthesis significantly reduced chondrosarcoma cell viability in vitro and suppressed tumor growth in vivo. Taken together, these data suggest that intracellular cholesterol synthesis is a potential therapeutic target for enchondromas and chondrosarcomas.
Authors
Hongyuan Zhang, Qingxia Wei, Hidetoshi Tsushima, Vijitha Puviindran, Yuning J. Tang, Sinthu Pathmanapan, Raymond Poon, Eyal Ramu, Mushriq Al-Jazrawe, Jay S. Wunder, Benjamin A. Alman
Abstract
In the current preclinical study, we demonstrate the therapeutic potential of sarcospan (SSPN) overexpression to alleviate cardiomyopathy associated with Duchenne muscular dystrophy (DMD) utilizing dystrophin-deficient mdx mice with utrophin haploinsufficiency that more accurately represent the severe disease course of human DMD. SSPN interacts with dystrophin, the DMD disease gene product, and its autosomal paralog utrophin, which is upregulated in DMD as a partial compensatory mechanism. SSPN transgenic mice have enhanced abundance of fully glycosylated α-dystroglycan, which may further protect dystrophin-deficient cardiac membranes. Baseline echocardiography reveals SSPN improves systolic function and hypertrophic indices in mdx and mdx:utr-heterozygous mice. Assessment of SSPN transgenic mdx mice by hemodynamic pressure-volume methods highlights enhanced systolic performance compared to mdx controls. SSPN restores cardiac sarcolemma stability, the primary defect in DMD disease, reduces fibrotic response and improves contractile function. We demonstrate that SSPN ameliorates more advanced cardiac disease in the context of diminished sarcolemma expression of utrophin and β1D integrin that mitigate disease severity and partially restores responsiveness to β-adrenergic stimulation. Overall, our current and previous findings suggest SSPN overexpression in DMD mouse models positively impacts skeletal, pulmonary and cardiac performance by addressing the stability of proteins at the sarcolemma that protect the heart from injury, supporting SSPN and membrane stabilization as a therapeutic target for DMD.
Authors
Michelle S. Parvatiyar, Alexandra J. Brownstein, Rosemeire M. Kanashiro-Takeuchi, Judd R. Collado, Karissa M. Dieseldorff Jones, Jay Gopal, Katherine G. Hammond, Jamie L. Marshall, Abel Ferrel, Aaron M. Beedle, Jeffrey S. Chamberlain, Jose Renato Pinto, Rachelle H. Crosbie
Abstract
Monosomy 7 or deletion of 7q (del(7q)) are common clonal cytogenetic abnormalities associated with high grade myelodysplastic syndrome (MDS) arising in inherited and acquired bone marrow failure. Current non-transplant approaches to treat marrow failure may be complicated by stimulation of clonal outgrowth. To study the biological consequences of del(7q) within the context of a failing marrow, we generated induced pluripotent stem cells (iPSCs) derived from patients with Shwachman Diamond Syndrome (SDS), a bone marrow failure disorder with MDS predisposition, and genomically engineered a 7q deletion. The TGFβ pathway was the top differentially regulated pathway in transcriptomic analysis of SDS versus SDSdel(7q) iPSCs. SMAD2 phosphorylation was increased in SDS relative to wild type cells consistent with hyperactivation of the TGFbeta pathway in SDS. Phospho-SMAD2 levels were reduced following 7q deletion in SDS cells and increased upon restoration of 7q diploidy. Inhibition of the TGFbeta pathway rescued hematopoiesis in SDS-iPSCs and in bone marrow hematopoietic cells from SDS patients while it had no impact on the SDSdel(7q) cells. These results identified a potential targetable vulnerability to improve hematopoiesis in an MDS-predisposition syndrome, and highlight the importance of the germline context of somatic alterations to inform precision medicine approaches to therapy.
Authors
Melisa Ruiz-Gutierrez, Özge Vargel Bölükbaşi, Gabriela Alexe, Andriana G. Kotini, Kaitlyn Ballotti, Cailin E. Joyce, David W. Russell, Kimberly Stegmaier, Kasiani Myers, Carl D. Novina, Eirini P. Papapetrou, Akiko Shimamura
Abstract
Atrial fibrillation (AF) is the most common cardiac arrhythmia and accounts for substantial morbidity and mortality. Recently, we created a mouse model with spontaneous and sustained AF caused by a mutation in the NaV1.5 channel (F1759A) that enhances persistent Na+ current, thereby enabling the investigation of molecular mechanisms that cause AF and the identification of novel treatment strategies. The mice have regional heterogeneity of action potential duration of the atria similar to observations in patients with AF. In these mice, we found that the initiation and persistence of the rotational reentrant AF arrhythmias, known as spiral waves or rotors, were dependent upon action potential duration heterogeneity. The centers of the rotors were localized to regions of greatest heterogeneity of the action potential duration. Pharmacologically attenuating the action potential duration heterogeneity reduced both spontaneous and pacing-induced AF. Computer-based simulations also demonstrated that the action potential duration heterogeneity is sufficient to generate rotors that manifest as AF. Taken together, these findings suggest that action potential duration heterogeneity in mice and humans is one mechanism by which AF is initiated and that reducing action potential duration heterogeneity can lessen the burden of AF.
Authors
Uma Mahesh R. Avula, Jeffrey Abrams, Alexander Katchman, Sergey Zakharov, Sergey Mironov, Joseph Bayne, Daniel Roybal, Anirudh Gorti, Lin Yang, Vivek Iyer, Marc Waase, Deepak Saluja, Edward J. Ciaccio, Hasan Garan, Andrew R. Marks, Steven O. Marx, Elaine Y. Wan
Abstract
Right ventricular dysfunction is highly prevalent across cardiopulmonary diseases and independently predicts death in both heart failure (HF) and pulmonary hypertension (PH). Progression towards right ventricular failure (RVF) can occur in spite of optimal medical treatment of HF or PH, highlighting current insufficient understanding of RVF molecular pathophysiology. To identify molecular mechanisms that may distinctly underlie RVF, we investigated the cardiac ventricular transcriptome of advanced HF patients, with and without RVF. Using an integrated systems genomic and functional biology approach, we identified an RVF-specific gene module, for which WIPI1 served as a hub and HSPB6 and MAP4 as drivers, and confirmed the ventricular specificity of Wipi1, Hspb6, and Map4 transcriptional changes in adult murine models of pressure overload induced RV- versus LV- failure. We uncovered a shift towards non-canonical autophagy in the failing RV that correlated with RV-specific Wipi1 upregulation. In vitro siRNA silencing of Wipi1 in neonatal rat ventricular myocytes limited non-canonical autophagy and blunted aldosterone-induced mitochondrial superoxide levels. Our findings suggest that Wipi1 regulates mitochondrial oxidative signaling and non-canonical autophagy in cardiac myocytes. Together with our human transcriptomic analysis and corroborating studies in an RVF mouse model, these data render Wipi1 a potential target for RV-directed HF therapy.
Authors
Christos Tzimas, Christoph D. Rau, Petra E. Buergisser, Gaston Jean-Louis Jr., Katherine Lee, Jeffrey Chukwuneke, Wen Dun, Yibin Wang, Emily J. Tsai
Abstract
Bi-allelic inactivating mutations in DOCK8 cause a combined immunodeficiency characterised by severe pathogen infections, eczema, allergies, malignancy and impaired humoral responses. These clinical features result from functional defects in most lymphocyte lineages. Thus, DOCK8 plays a key role in immune cell function. Hematopoietic stem cell transplantation (HSCT) is curative for DOCK8 deficiency. While previous reports have described clinical outcomes for DOCK8 deficiency following HSCT, the effect on lymphocyte reconstitution and function has not been investigated. Our study determined whether defects in lymphocyte differentiation and function in DOCK8-deficient patients were restored following HSCT. DOCK8-deficient T and B lymphocytes exhibited aberrant activation and effector function in vivo and in vitro. Frequencies of αβ T and MAIT cells were reduced while γδT cells were increased in DOCK8-deficient patients. HSCT improved, abnormal lymphocyte function in DOCK8-deficient patients. Elevated total and allergen-specific IgE in DOCK8-deficient patients decreased over time following HSCT. Our results document the extensive catalogue of cellular defects in DOCK8-deficient patients, and the efficacy of HSCT to correct these defects, concurrent with improvements in clinical phenotypes. Overall, our findings provide mechanisms at a functional cellular level for improvements in clinical features of DOCK8 deficiency post-HSCT, identify biomarkers that correlate with improved clinical outcomes, and inform the general dynamics of immune reconstitution in patients with monogenic immune disorders following HSCT.
Authors
Bethany A. Pillay, Danielle T. Avery, Joanne M. Smart, Theresa Cole, Sharon Choo, Damien Chan, Paul E. Gray, Katie Frith, Richard Mitchell, Tri Giang Phan, Melanie Wong, Dianne E. Campbell, Peter Hsu, John B. Ziegler, Jane Peake, Frank Alvaro, Capucine Picard, Jacinta Bustamante, Benedicte Neven, Andrew J. Cant, Gulbu Uzel, Peter D. Arkwright, Jean-Laurent Casanova, Helen C. Su, Alexandra Freeman, Nirali Shah, Dennis D. Hickstein, Stuart G. Tangye, Cindy S. Ma
Abstract
Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent, and potentially morbid, disease that affects one-third of the U.S. population. Normal liver safely accommodates lipid excess during fasting or carbohydrate restriction by increasing their oxidation to acetyl-CoA and ketones, yet lipid excess during NAFLD leads to hyperglycemia and, in some, steatohepatitis. To examine potential mechanisms, flux through pathways of hepatic oxidative metabolism and gluconeogenesis were studied using five simultaneous stable isotope tracers in ketotic (24-hour fast) individuals with a wide range of hepatic triglyceride contents (0-52%). Ketogenesis was progressively impaired as hepatic steatosis and glycemia worsened. Conversely, the alternative pathway for acetyl-CoA metabolism, oxidation in the tricarboxylic (TCA) cycle, was upregulated in NAFLD as ketone production diminished and positively correlated with rates of gluconeogenesis and plasma glucose concentrations. Increased respiration and energy generation that occurred in liver when β-oxidation and TCA cycle activity were coupled may explain these findings, inasmuch as oxygen consumption was higher during fatty liver and highly correlated with gluconeogenesis. These findings demonstrate that increased glucose production and hyperglycemia in NAFLD is not a consequence of acetyl-CoA production per se, but how acetyl-CoA is further metabolized in liver.
Authors
Justin A. Fletcher, Stanislaw Deja, Santhosh Satapati, Xiaorong Fu, Shawn C. Burgess, Jeffrey D. Browning
Abstract
Glucagon, a hormone released from pancreatic alpha-cells, plays a key role in maintaining proper glucose homeostasis and has been implicated in the pathophysiology of diabetes. In vitro studies suggest that intra-islet glucagon can modulate the function of pancreatic beta-cells. However, because of the lack of suitable experimental tools, the in vivo physiological role of this intra-islet cross-talk has remained elusive. To address this issue, we generated a novel mouse model that selectively expressed an inhibitory designer G protein-coupled receptor (Gi DREADD) in α-cells only. Drug-induced activation of this inhibitory designer receptor almost completely shut off glucagon secretion in vivo, resulting in significantly impaired insulin secretion, hyperglycemia, and glucose intolerance. Additional studies with mouse and human islets indicated that intra-islet glucagon stimulates insulin release primarily by activating β-cell GLP-1 receptors. These new findings strongly suggest that intra-islet 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 intra-islet 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
IL-17-producing CD4+ cells (TH17) are pathogenically linked to autoimmunity including to autoimmune kidney disease. Erythropoietin’s (EPO) newly recognized immunoregulatory functions and its predominant intra-renal source suggested that EPO physiologically regulates TH17 differentiation, thereby serving as a barrier to the 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 generation and promotes trans-differentiation of TH17 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 IL-17 and IL-23 receptor genes. In a murine model of TH17-dependent aristolochic acid (ArA)-induced, interstitial kidney disease associated with reduced renal EPO production, we demonstrate that transgenic EPO overexpression or recombinant EPO (rEPO) administration limits TH17 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 induction and clinical/histological expression of pristane-induced glomerulonephritis (associated with decreased intrarenal EPO). rEPO prevents spontaneous glomerulonephritis and TH17 generation in MRL-lpr mice. Together, our findings indicate that EPO physiologically and therapeutically modulate TH17 cells to limit expression of TH17-associated autoimmune kidney disease.
Authors
Chiara Donadei, Andrea Angeletti, Chiara Cantarelli, Vivette D. D'Agati, Gaetano La Manna, Enrico Fiaccadori, Julian Horwitz, Huabao Xiong, Chiara Guglielmo, Susan Hartzell, Joren C. Madsen, Umberto Maggiore, Peter S. Heeger, Paolo Cravedi
