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Abstract
Evidence has mounted that insulin can be synthesized in various brain regions including the hypothalamus. However, the distribution and functions of insulin-expressing cells in the hypothalamus remain elusive. Herein, we show that in the mouse hypothalamus, the perikarya of insulin-positive neurons are located in the paraventricular nucleus (PVN) and their axons project to the median eminence; these findings define parvocellular neurosecretory PVN insulin neurons. Contrary to corticotrophin-releasing hormone expression, insulin expression in the PVN was inhibited by restraint stress (RS) in both adult and young mice. Acute RS–induced inhibition of PVN insulin expression in adult mice decreased both pituitary growth hormone (GH) mRNA level and serum GH concentration, which were attenuated by overexpression of PVN insulin. Notably, PVN insulin knockdown or chronic RS in young mice hindered normal growth via the down-regulation of GH gene expression and secretion, whereas PVN insulin overexpression in young mice prevented chronic RS–induced growth retardation by elevating GH production. Our results suggest that in both normal and stressful conditions, insulin synthesized in the parvocellular PVN neurons plays an important role in the regulation of pituitary GH production and body length, unveiling a physiological function of brain-derived insulin.
Authors
Jaemeun Lee, Kyungchan Kim, Jae Hyun Cho, Jin Young Bae, Timothy P. O’Leary, James D. Johnson, Yong Chul Bae, Eun-Kyoung Kim
Abstract
Acute rejection (AR) in renal transplantation is an established risk factor for reduced allograft survival. The incidence of AR is 10-20% despite standard of care immunosuppression, suggesting molecular pathways exist which are inadequately suppressed by current therapy. Molecules with regulatory control among these could serve as important targets for therapeutic manipulation to prevent rejection. Here, an integrative network-based computational strategy incorporating gene expression and genotype data of human renal allograft biopsy tissue was applied, to identify the master regulators- the key driver genes (KDGs)- within dyregulated AR pathways. A 982 meta-gene signature with differential expression in AR versus non-AR, was identified from a meta-analysis of microarray data from 735 human kidney allograft biopsy samples across seven data sets. Among the upregulated genes, enriched biologic processes include the immune response, leucocyte activation and antigen processing and presentation; where monocytes, macrophages and dendritic cells were identified as the major immune cell populations. Genomic key driver analysis of this signature predicted 14 KDGs. Expression of the KDGs provided risk stratification for subsequent graft loss with high prediction accuracy at 2 years post transplant (AUC=0.913) and 2 years post biopsy (AUC=0.889) in separate clinical cohorts. Interrogation of two drug-repositioning resources identified compounds with predicted efficacy against individual KDGs or a key driver-based gene set respectively, and therefore be repositioned for AR prevention. Minocycline, an FDA-approved tetracycline antibiotic, was chosen for experimental validation in a murine cardiac allograft model of AR. Minocycline alone attenuated the inflammatory profile of AR compared with controls, and when co-administered with immunosuppression prolonged graft survival. This study demonstrates the proof-of-concept that a network-based strategy, using gene expression and genotype data assists target prioritization for therapeutics in renal allograft rejection.
Authors
Zhengzi Yi, Karen L. Keung, Li Li, Min Hu, Bo Lu, Leigh Nicholson, Elvira Jimenez-Vera, Madhav C. Menon, Chengguo Wei, Stephen I. Alexander, Barbara Murphy, Philip J. O’Connell, Weijia Zhang
Abstract
Critical illness is accompanied by the release of large amounts of the anaphylotoxin, C5a. C5a suppresses antimicrobial functions of neutrophils which is associated with adverse outcomes. The signalling pathways that mediate C5a-induced neutrophil dysfunction are incompletely understood. Healthy donor neutrophils exposed to purified C5a demonstrated a prolonged defect (7 hours) in phagocytosis of Staphylococcus aureus. Phosphoproteomic profiling of 2712 phosphoproteins identified persistent C5a signalling and selective impairment of phagosomal protein phosphorylation on exposure to S. aureus. Notable proteins included early endosomal marker ZFYVE16 and V-ATPase proton channel component ATPV1G1. A novel assay of phagosomal acidification demonstrated C5a-induced impairment of phagosomal acidification which was recapitulated in neutrophils from critically ill patients. Examination of the C5a-impaired protein phosphorylation indicated a role for the phosphatidylinositol 3-kinase VPS34 in phagosomal maturation. Inhibition of VPS34 impaired neutrophil phagosomal acidification and killing of S. aureus. This study provides a phosphoproteomic assessment of human neutrophil signalling in response to S. aureus and its disruption by C5a, identifying a defect in phagosomal maturation and new mechanisms of immune failure in critical illness.
Authors
Alexander J.T. Wood, Arlette M. Vassallo, Marie-Helene Ruchaud-Sparagano, Jonathan Scott, Carmelo Zinnato, Carmen Gonzalez-Tejedo, Kamal Kishore, Clive S. D’Santos, A. John Simpson, David K. Menon, Charlotte Summers, Edwin R. Chilvers, Klaus Okkenhaug, Andrew Conway Morris
Abstract
Background: A treatment option for ADPKD has highlighted the need to identify rapidly progressive patients. Kidney size/age and genotype have predictive power for renal outcomes, but their relative and additive value, plus associated trajectories of disease progression, are not well defined. Methods: The value of genotypic and/or kidney imaging data (Mayo Imaging Class) to predict the time to functional (end stage kidney disease; ESKD, or decline in estimated glomerular filtration rate; eGFR) or structural (increase in height adjusted total kidney volume; htTKV) outcomes were evaluated in a Mayo Clinic PKD1/PKD2 population; and eGFR and htTKV trajectories from 20-65 years of age modeled and independently validated in similarly defined CRISP and HALT PKD patients. Results: Both genotypic and imaging groups strongly predicted ESKD and eGFR endpoints, with genotype improving the imaging predictions, and vice versa; a multivariate model had strong discriminatory power (C statistic = 0.845). However, imaging but not genotypic groups predicted htTKV growth, although more severe genotypic and imaging groups had larger kidneys at a young age. The trajectory of eGFR decline was linear from baseline in the most severe genotypic and imaging groups, but curvilinear in milder groups. Imaging class trajectories differentiated htTKV growth rates; severe classes had rapid early growth and large kidneys but growth later slowed. Conclusions: The value of imaging, genotypic, and combined data to identify rapidly progressive patients was demonstrated, and reference values for clinical trials provided. Our data indicates that differences in kidney growth rates before adulthood significantly define patients with severe disease. Funding: NIDDK grants: Mayo DK058816, DK090728; CRISP DK056943, DK056956, DK056957, DK056961; HALT PKD DK062410, DK062408, DK062402, DK082230, DK062411, DK062401.
Authors
Sravanthi Lavu, Lisa E. Vaughan, Sarah R. Senum, Timothy L. Kline, Arlene B. Chapman, Ronald D. Perrone, Michal Mrug, William E. Braun, Theodore I. Steinman, Frederic F. Rahbari-Oskoui, Godela M. Brosnahan, Kyongtae T. Bae, Douglas Landsittel, Fouad T. Chebib, Alan S. L. Yu, Vicente E. Torres, Peter C. Harris
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is characterized by a relative paucity of cancer cells that are surrounded by an abundance of non-tumor cells and extracellular matrix, known as stroma. The interaction between stroma and cancer cells contributes to poor outcome, but how proteins from these individual compartments drive aggressive tumor behavior is not known. Here, we report the proteomic analysis of laser-capture microdissected (LCM) PDAC samples. We isolated stroma, tumor, and bulk samples from a cohort with long- and short-term survivors. Compartment-specific proteins were measured by mass spectrometry, yielding the largest PDAC proteome landscape to date. These analyses revealed that in bulk analysis, tumor-derived proteins were typically masked and that LCM was required to reveal biology and novel prognostic markers. We validated tumor CALB2 and stromal COL11A1 expression as compartment-specific prognostic markers. We identified and functionally addressed the contributions of the tumor cell receptor EPHA2 to tumor cell viability and motility, underscoring the value of compartment-specific protein analysis in PDAC.
Authors
Tessa Y.S. Le Large, G. Mantini, Laura L. Meijer, T.V. Pham, N. Funel, Nicole C.T. van Grieken, B. Kok, Jaco C. Knol, H.W.M. van Laarhoven, S.R. Piersma, C.R. Jimenez, G. Kazemier, E. Giovannetti, M.F. Bijlsma
Abstract
Triple-negative breast cancers (TNBCs) are highly heterogeneous and aggressive, with high mortality rates. Although TNBC is typically more responsive to chemotherapy than other breast cancer subtypes, many patients develop chemo-resistance. The molecular processes contributing to chemo-resistance, and the roles of tumor cell-stromal crosstalk in establishing chemo-resistance are complex and largely unclear. Here we report molecular studies of paired TNBC patient-derived xenografts (PDX) established from patient biopsies before and after the development of chemo-resistance. Interestingly, the chemo-resistant model acquired a distinct KRASQ61R mutation that activates K-Ras. The chemo-resistant KRAS-mutant model showed gene expression and proteomic changes indicative of altered tumor cell metabolism. Specifically, KRAS-mutant PDXs exhibit increased redox ratios and decreased activation of AMPK, a protein involved in responding to metabolic homeostasis. Additionally, the chemo-resistant model exhibited increased immunosuppression including expression of CXCL1 and CXCL2, cytokines responsible for recruiting immunosuppressive leukocytes to tumors. Notably, chemo-resistant KRAS-mutant tumors harbored increased numbers of granulocytic myeloid-derived suppressor cells (gMDSCs). Interestingly, previously established gene expression signatures of Ras/MAPK activity correlated with myeloid/neutrophil-recruiting CXCL1/2 expression and negatively with T-cell recruiting chemokines (CXCL9/10/11) across TNBC patients, even in the absence of KRAS mutations. Importantly, MEK inhibition induced tumor suppression in mice while simultaneously reversing metabolic and immunosuppressive phenotypes including chemokine production and gMDSC tumor recruitment in the chemo-resistant KRAS mutant tumors. These results suggest that Ras/MAPK pathway inhibitors may be effective in some breast cancer patients to reverse Ras/MAPK-driven tumor metabolism and immunosuppression, particularly in the setting of chemo-resistance.
Authors
Derek A. Franklin, Joe T. Sharick, Paula I. Gonzalez-Ericsson, Violeta Sanchez, Phillip Dean, Susan R. Opalenik, Stefano Cairo, Jean-Gabriel Judde, Michael T. Lewis, Jenny C. Chang, Melinda E. Sanders, Rebecca S. Cook, Melissa C. Skala, Jennifer Bordeaux, Jehovana Orozco Bender, Christine A. Vaupel, Gary Geiss, Douglas Hinerfeld, Justin M. Balko
Abstract
Arrestin domain containing 3 (ARRDC3) represents a newly discovered α-arrestin involved in obesity, inflammation and cancer. Here we demonstrated a pro-inflammation role of ARRDC3 in H. pylori-associated gastritis. Increased ARRDC3 was detected in gastric mucosa of patients and mice infected with H. pylori. ARRDC3 in gastric epithelial cells (GECs) was induced by H. pylori, regulated by ERK and PI3K-AKT pathways in a cagA-dependent manner. Human gastric ARRDC3 correlated with the severity of gastritis, and mouse ARRDC3 from non-BM-derived cells promoted gastric inflammation. This inflammation was characterized by the CXCR2-dependent influx of CD45+CD11b+Ly6C-Ly6G+ neutrophils, whose migration was induced via the ARRDC3-dependent production of CXCL2 by GECs. Importantly, gastric inflammation was attenuated in ARRDC3-/- mice but increased in protease-activated receptor 1 (PAR1)-/- mice. Mechanistically, ARRDC3 in GECs directly interacted with PAR1 and negatively regulated PAR1 via ARRDC3-mediated lysosomal degradation, which abrogated the suppression of CXCL2 production and following neutrophil chemotaxis by PAR1, thereby contributing to the development of H. pylori-associated gastritis. This study identifies a novel regulatory network involving H. pylori, GECs, ARRDC3, PAR1, and neutrophils, which collectively exert a pro-inflammatory effect within gastric microenvironment. Efforts to inhibit this ARRDC3-dependent pathway may prove valuable strategies in treating of H. pylori-associated gastritis.
Authors
Yu-gang Liu, Yong-sheng Teng, Zhi-guo Shan, Ping Cheng, Chuan-jie Hao, Yi-pin Lv, Fang-yuan Mao, Shi-ming Yang, Weisan Chen, Yong-Liang Zhao, Nan You, Quan-ming Zou, Yuan Zhuang
Abstract
Kidney disease is one of the most devastating complications of diabetes, and tubular atrophy predicts diabetic kidney disease (DKD) progression to end stage renal disease. We have proposed that fatty acids bound to albumin contribute to tubular atrophy by inducing lipotoxicity, following filtration across damaged glomeruli, and subsequent proximal tubule reabsorption by a fatty acid transport protein-2 (FATP2)-dependent mechanism. To address this possibility, genetic (Leprdb/db eNOS-/-) and induced (high fat diet plus low dose streptozotocin) mouse models of obesity and DKD, were bred with global FATP2 gene (Slc27a2)-deleted mice, and then phenotyped. DKD-prone mice with the Slc27a2-/- genotype demonstrated normalization of glomerular filtration rate, reduced albuminuria, improved kidney histopathology, and longer lifespan compared to diabetic Slc27a2+/+ mice. Genetic and induced DKD-prone Slc27a2-/- mice also exhibited markedly reduced fasting plasma glucose, with mean values approaching euglycemia, despite increased obesity and decreased physical activity. Glucose lowering in DKD-prone Slc27a2-/- mice was accompanied by beta-cell hyperplasia and sustained insulin secretion. Together, our data indicate that FATP2 uniquely regulates DKD pathogenesis by a combined lipotoxicity and glucotoxicity (glucolipotoxicity) mechanism.
Authors
Shenaz Khan, Robert J. Gaivin, Caroline Abramovich, Michael Boylan, Jorge Calles, Jeffrey R. Schelling
Abstract
M3 muscarinic acetylcholine receptor (M3R) is one of the autoantigens associated with Sjögren’s syndrome (SS) and is localized in exocrine glands where disease specific inflammation occurs. The inflammatory lesion is characterized by infiltration of CD4+ T cells, including clonally expanded Th17 cells. We undertook this study to identify circulating M3R specific Th17 cells, and to determine functional properties of those cells. Using ELISpot method, we identified M3R reactive Th17 cells in the peripheral blood of patients with primary SS (pSS). Among examined 10 pSS, 10 healthy subjects (HS), and 5 IgG4-related disease (IgG4-RD) patients, M3R reactive IL-17 secreting cells were significantly increased in five pSS patients specifically. The commonest T cell epitope, which was analyzed and confirmed by co-culture of isolated CD4+ T cells with antigen presenting cells plus M3R peptides in vitro, was peptide 83-95 of M3R. Peptide recognition was partly in HLA DR restricted manner, confirmed by blocking assay. M3R reactive Th17 cells positivity correlated with higher titers of anti-M3R antibodies, whose systemic disease activity score tended to be higher. Our studies highlight the role of tissue specific autoantigen derived circulating Th17 cells in pSS, for which further work might lead to antigen specific targeted therapy.
Authors
Saori Abe, Hiroto Tsuboi, Hanae Kudo, Hiromitsu Asashima, Yuko Ono, Fumika Honda, Hiroyuki Takahashi, Mizuki Yagishita, Shinya Hagiwara, Yuya Kondo, Isao Matsumoto, Takayuki Sumida
Abstract
One of the major challenges in using pancreatic cancer patient-derived organoids (PDOs) in precision oncology is the time from biopsy to functional characterization. This is particularly true for biopsy specimen with limited tumor cell yield, typical characteristics of biopsies from endoscopic ultrasound-guided fine needle aspirations (EUS-FNAs).Here, we tested conditioned media of individual PDOs for cell-free tumor DNA (cfDNA) to detect driver mutations already early on during the expansion process in order to accelerate the genetic characterization of PDOs as well as subsequent functional testing. Importantly, genetic alterations detected in the PDO supernatant, collected as early as 72h after biopsy, recapitulate the mutational profile of the primary tumor indicating suitability of this approach to subject PDOs to drug testing in a reduced timeframe. In addition, we demonstrate that this workflow is practicable even in patients of whom the amount of tumor material was not sufficient for molecular characterization by established means.Our findings demonstrate that generating PDOs from very limited biopsy material permits molecular profiling and drug testing. With our approach this can be achieved in a rapid and feasible fashion with broad implications in clinical practice.
Authors
Zahra Dantes, Hsi-Yu Yen, Nicole Pfarr, Christof Winter, Katja Steiger, Alexander Muckenhuber, Alexander Hennig, Sebastian Lange, Thomas Engleitner, Rupert Öllinger, Roman Maresch, Felix Orben, Irina Heid, Georgios A. Kaissis, Kuangyu Shi, Geoffrey J. Topping, Fabian Stögbauer, Matthias Wirth, Katja Peschke, Aristeidis Papargyriou, Massoud Rezaee-Oghazi, Karin Feldmann, Arlett P. G. Schäfer, Raphela Ranjan, Clara Lubeseder-Martellato, Daniel E. Stange, Thilo Welsch, Marc E. Martignoni, Güralp Onur Ceyhan, Helmut Friess, Alexander Herner, Lucia Liotta, Matthias Treiber, Guido von Figura, Mohamed Abdelhafez, Peter Klare, Christoph Schlag, Hana Algül, Jens T. Siveke, Rickmer F. Braren, Gregor Weirich, Wilko Weichert, Dieter Saur, Roland Rad, Roland Schmid, Günter Schneider, Maximilian Reichert
Abstract
The bromodomain and extraterminal (BET) family of epigenetic reader proteins are key regulators of inflammatory and hypertrophic gene expression in the heart. We previously identified the activation of pro-inflammatory gene networks as a key early driver of dilated cardiomyopathy (DCM) in transgenic mice expressing a mutant form of phospholamban (PLNR9C) – a genetic cause of DCM in humans. We hypothesized that BETs coactivate this inflammatory process, representing a critical node in the progression of DCM. To test this hypothesis, PLNR9C or age-matched wild type mice were treated longitudinally with the small molecule BET bromodomain inhibitor JQ1 or vehicle. BET inhibition abrogated adverse cardiac remodeling, reduced cardiac fibrosis, and prolonged survival in PLNR9C mice by inhibiting expression of pro-inflammatory gene networks at all stages of disease. Specifically, JQ1 had profound effects on pro-inflammatory gene network expression in cardiac fibroblasts, while having little effect on gene expression in cardiomyocytes. Cardiac fibroblast proliferation was also substantially reduced by JQ1. Mechanistically, we demonstrated that BRD4 serves as a direct and essential regulator of NFkB-mediated pro-inflammatory gene expression in cardiac fibroblasts. Interdicting pro-inflammatory gene expression via BET bromodomain inhibition could be a novel therapeutic strategy for chronic DCM in humans.
Authors
Andrew Antolic, Hiroko Wakimoto, Zhe Jiao, Joshua M. Gorham, Steven R. DePalma, Madeleine E. Lemieux, David A. Conner, Da Young Lee, Jun Qi, Jonathan G. Seidman, James E. Bradner, Jonathan D. Brown, Saptarsi M. Haldar, Christine E. Seidman, Michael A. Burke
Abstract
Macrolide antibiotics exert anti-inflammatory effects; however, little is known regarding their immunomodulatory mechanisms. In this study, using two distinct mouse models of mucosal inflammatory disease (LPS-induced acute lung injury and ligature-induced periodontitis), we demonstrated that the anti-inflammatory action of erythromycin (ERM) is mediated through upregulation of the secreted homeostatic protein DEL-1. Consistent with the anti-neutrophil recruitment action of endothelial cell-derived DEL-1, ERM inhibited neutrophil infiltration in the lungs and the periodontium in a DEL-1-dependent manner. Whereas ERM (but not other antibiotics such as josamycin and penicillin) protected against lethal pulmonary inflammation and inflammatory periodontal bone loss, these protective effects of ERM were abolished in Del1-deficient mice. By interacting with the growth hormone secretagogue receptor (GHSR) and activating JAK2 in human lung microvascular endothelial cells, ERM induced C/EBPβ-dependent DEL-1 transcription, which was mediated by MAPK p38. Moreover, ERM reversed IL-17-induced inhibition of DEL-1 transcription, in a manner that was not only dependent on JAK2 but also on PI3K/AKT signaling. As DEL-1 levels are severely reduced in inflammatory conditions and with aging, the ability of ERM to upregulate DEL-1 may be a novel approach for the treatment of inflammatory and aging-related diseases.
Authors
Tomoki Maekawa, Hikaru Tamura, Hisanori Domon, Takumi Hiyoshi, Toshihito Isono, Daisuke Yonezawa, Naoki Hayashi, Naoki Takahashi, Koichi Tabeta, Takeyasu Maeda, Masataka Oda, Athanasios Ziogas, Vasileia Ι. Alexaki, Triantafyllos Chavakis, Yutaka Terao, George Hajishengallis
Abstract
Dendritic cells (DCs) are critical component of immune responses in cancer primarily due to their ability to cross-present tumor associated antigens. Cross-presentation by DCs in cancer is impaired, which may represent one of the obstacles for the success of cancer immunotherapies. Here, we report that polymorphonuclear myeloid derived suppressor cells (PMN-MDSC) blocked cross-presentation by DCs without affecting direct presentation of antigens by these cells. This effect did not require direct cell-cell contact and was associated with transfer of lipids. Neutrophils (PMN) and PMN-MDSC transferred lipid to DCs equally well, however PMN did not affect DC cross-presentation. PMN-MDSC generate oxidatively truncated lipids previously shown to be involved in impaired cross-presentation by DCs. Accumulation of oxidized lipids in PMN-MDSC was dependent on myeloperoxidase (MPO). MPO deficient PMN-MDSC did not affect cross-presentation by DCs. Cross-presentation of tumor associated antigens in vivo by DCs was improved in MDSC depleted or tumor-bearing MPO KO mice. Pharmacological inhibition of MPO in combination with checkpoint blockade reduced tumor progression in different tumor models. These data suggest MPO-driven lipid peroxidation in PMN-MDSC as a possible non-cell autonomous mechanism of inhibition of antigen cross-presentation by DCs and propose MPO as potential therapeutic target to enhance the efficacy of current immunotherapies for cancer patients.
Authors
Alessio Ugolini, Vladimir Tyurin, Yulia Tyurina, Evgenii Tsyganov, Laxminarasimha Donthireddy, Valerian E Kagan, Dmitry I. Gabrilovich, Filippo Veglia
Abstract
Noonan syndrome with multiple lentigines (NSML) is a rare autosomal dominant disorder that presents with cardio-cutaneous-craniofacial defects. Hypertrophic cardiomyopathy (HCM) represents the major life-threatening presentation in NSML. Mutations in the PTPN11 gene that encodes for the protein tyrosine phosphatase (PTP), SHP2, represents the predominant cause of HCM in NSML. NSML-associated PTPN11 mutations renders SHP2 catalytically inactive with an “open” conformation. NSML-associated PTPN11 mutations cause hypertyrosyl phosphorylation of the transmembrane glycoprotein, protein zero-related (PZR) resulting in increased SHP2 binding. Here we show that NSML mice harboring a tyrosyl phosphorylation-defective mutant of PZR (NSML/PZRY242F) that is defective for SHP2 binding fail to develop HCM. Enhanced AKT/S6K signaling in heart lysates of NSML mice was reversed in NSML/PZRY242F mice demonstrating that PZR/SHP2 interactions promote aberrant AKT/S6K activity in NSML. Enhanced PZR tyrosyl phosphorylation in the hearts of NSML mice was found to drive myocardial fibrosis by engaging a Src/NFkB pathway resulting in increased activation of interleukin-6 (IL6). Increased expression of IL6 in the hearts of NSML mice was reversed in NSML/PZRY242F mice and PZRY242F mutant fibroblasts were defective for IL6 secretion and STAT3-mediated fibrogenesis. These results demonstrate that NSML-associated PTPN11 mutations that induce PZR hypertyrosyl phosphorylation trigger pathophysiological signaling that promotes HCM and cardiac fibrosis.
Authors
Jae-Sung Yi, Sravan K. Perla, Liz E. Enyenihi, Anton M. Bennett
Abstract
De novo lipogenesis (DNL) plays a role in the development of hepatic steatosis. In humans with lipodystrophy, reduced adipose tissue causes lower plasma leptin, insulin resistance, dyslipidemia and ectopic triglyceride (TG) accumulation. We hypothesized that recombinant leptin (metreleptin) for 6 months in 11 patients with lipodystrophy would reduce DNL by decreasing insulin resistance and glycemia, thus reducing circulating and hepatic-TG. The percentage of TG-rich lipoprotein particle (TRLP)-TG derived from DNL (%DNL) was measured by deuterium incorporation from body water into palmitate. At baseline, DNL was elevated with levels similar to levels previously shown in obesity-associated nonalcoholic fatty liver disease (NAFLD). After metreleptin, DNL decreased into the normal range. Similarly, absolute DNL (TRLP-TG x % DNL) decreased by 88% to near-normal levels. Metreleptin improved peripheral insulin sensitivity (hyperinsulinemic-euglycemic clamp) and lowered HbA1c and hepatic-TG. Both before and after metreleptin, DNL positively correlated with insulin resistance, insulin doses, and hepatic-TG, supporting the hypothesis that hyperinsulinemia stimulates DNL and that elevated DNL is integral to the pathogenesis of lipodystrophy-associated NAFLD.These data suggest that leptin-mediated improvement in insulin sensitivity increases clearance of blood glucose by peripheral tissues, reduces hepatic carbohydrate flux, and lowers insulinemia, resulting in DNL reductions, and improvements in hepatic steatosis and dyslipidemia.
Authors
Annah P. Baykal, Elizabeth J. Parks, Robert Shamburek, Majid M. Syed-Abdul, Shaji K. Chacko, Elaine Cochran, Megan Startzell, Ahmed M. Gharib, Ronald Ouwerkerk, Khaled Z. Abd-Elmoniem, Peter J. Walter, Mary Walter, Ranganath Muniyappa, Stephanie T. Chung, Rebecca J. Brown
Abstract
Cigarette smoking (CS) and genetic susceptibility determine the risk for development, progression, and severity of chronic obstructive pulmonary diseases (COPD). We posited that an incidental balanced reciprocal chromosomal translocation was linked to a patient’s risk of severe COPD. We determined 46,XX,t(1;4)(p13.1;q34.3) caused a breakpoint in IGSF3 (immunoglobulin superfamily, member 3) gene, with markedly decreased expression. Examination of COPDGene cohort identified 14 IGSF3 SNPs of which, rs1414272 and rs12066192 were directly- and rs6703791 inversely associated with COPD severity, including COPD exacerbations. We confirmed that IGSF3 is a tetraspanin-interacting protein that colocalized with CD9 and integrin B1 in tetraspanin enriched domains. IGSF3-deficient patient-derived lymphoblastoids exhibited multiple alterations in gene expression, especially in the unfolded protein response and ceramide pathways. IGSF3-deficient lymphoblastoids had high ceramide- and sphingosine-1 phosphate-, but low glycosphingolipids- and gangliosides levels; were less apoptotic and more adherent; with marked changes in multiple TNFRSF molecules. Similarly, IGSF3 knockdown increased ceramide in lung structural cells, rendering them more adherent, with impaired wound repair and a weakened barrier function. These findings suggest that, by maintaining sphingolipid and membrane receptor homeostasis, IGSF3 is required for cell mobility-mediated lung injury repair. IGSF3 deficiency may increase susceptibility to CS-induced lung injury in COPD.
Authors
Kelly S. Schweitzer, Natini Jinawath, Raluca Yonescu, Kevin Ni, Natalia Rush, Varodom Charoensawan, Irina Bronova, Evgeny Berdyshev, Sonia M. Leach, Lucas A. Gillenwater, Russell P. Bowler, David B. Pearse, Constance A. Griffin, Irina Petrache
Abstract
Group 2 innate lymphoid cells (ILC2s) in mouse lungs are activated by the epithelium-derived alarmin IL-33. Activated ILC2s proliferate and produce IL-5 and IL-13 that drive allergic responses. In neonatal lungs, IL-33 is spontaneously released resulting in activation of lung ILC2s. Here we report that neonatal lung ILC2 activation by endogenous IL-33 has significant effects on ILC2 functions in adulthood. Most neonatal lung ILC2s incorporated 5-bromo-2’-deoxyuridine (BrdU) and persisted into adulthood. BrdU+ ILC2s in adult lungs responded more intensely to IL-33 treatment than BrdU- ILC2s. In IL-33 deficient (KO) mice, lung ILC2s develop normally but they are not activated in the neonatal period. Lung ILC2s in KO mice responded less intensely to IL-33 in adulthood compared to wild type (WT) ILC2s. While there was no difference in the number of lung ILC2s, there were fewer IL-13+ ILC2s in KO than WT mice. The impaired responsiveness of ILC2s in KO mice was reversed by intranasal administrations of IL-33 in the neonatal period. These results suggest that activation of lung ILC2s by endogenous IL-33 in the neonatal period may “train” ILC2s seeding the lung after birth to become long-lasting resident cells that respond more efficiently to challenges later in life.
Authors
Catherine A. Steer, Laura Mathä, Hanjoo Shim, Fumio Takei
Abstract
It has been proposed that unmethylated insulin promoter fragments in plasma derive exclusively from β-cells, reflect their recent demise and can be used to assess β-cell damage in type 1 diabetes. Herein we describe an ultrasensitive assay for detection of a β-cell-specific DNA methylation signature, by simultaneous assessment of six DNA methylation markers, that identifies β-cell DNA in mixtures containing as little as 0.03% β-cell DNA (less than one β-cell genome equivalent). With this assay, plasma from non-diabetic individuals (N=218, aged 4-78 years) contained on average only one β-cell genome equivalent/ml. As expected, β-cell cfDNA was significantly elevated in islet transplant recipients shortly after transplantation. We also detected β-cell cfDNA in a patient with KATP congenital hyperinsulinism where substantial β-cell turnover is thought to occur. Strikingly, in contrast to previous reports, we observed no elevation of β-cell-derived cfDNA in autoantibody positive subjects at-risk for type 1 diabetes (N=32), individuals with recent-onset type 1 diabetes (<4 months, N=92), or those with a long-standing disease (>4 months, N=38). We discuss the utility of sensitive beta-cell cfDNA analysis and potential explanations for the lack of a β-cell cfDNA signal in T1D.
Authors
Daniel Neiman, David Gillis, Sheina Piyanzin, Daniel Cohen, Ori Fridlich, Joshua Moss, Aviad Zick, Tal Oron, Frida Sundberg, Gun Forsander, Oskar Skog, Olle Korsgren, Floris Levy-Khademi, Dan Arbell, Saar Hashavya, A.M. James Shapiro, Cate Speake, Carla Greenbaum, Jennifer Hosford, Amanda Posgai, Mark A. Atkinson, Benjamin Glaser, Desmond Schatz, Ruth Shemer, Yuval Dor
Abstract
Rituximab, a B cell-depleting therapy, is indicated for treating a growing number of autoantibody-mediated autoimmune disorders. However, relapses can occur after treatment and autoantibody-producing B cell subsets may be found during relapses. It is not understood if these autoantibody-producing B cell subsets emerge from the failed depletion of pre-existing B cells or are generated de novo. To further define the mechanisms that cause post-rituximab relapse, we studied patients with autoantibody-mediated muscle-specific kinase (MuSK) myasthenia gravis (MG) who relapsed after treatment. We carried out single-cell transcriptional and B cell receptor (BCR) profiling on longitudinal B cell samples. We identified clones present prior to therapy that continued to persist during relapse. Persistent B cell clones included both antibody-secreting cells and memory B cells characterized by gene expression signatures associated with B cell survival. A subset of persistent antibody-secreting cells and memory B cells were specific for the MuSK autoantigen. These results demonstrate that rituximab is not fully effective at eliminating autoantibody-producing B cells and provide a mechanistic understanding of post-rituximab relapse in MuSK MG.
Authors
Ruoyi Jiang, Miriam L. Fichtner, Kenneth B. Hoehn, Minh C. Pham, Panos Stathopoulos, Richard J. Nowak, Steven H. Kleinstein, Kevin C. O'Connor
Abstract
Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy that has no effective treatment. The tumor microenvironment (TME) of PDA employs a multitude of immune derangement strategies to protect PDA from immune elimination. Tumor associated macrophages (TAMs) have been implicated in pathogenesis of immune suppression of PDA-TME, however, its underlying mechanisms remained largely unknown. Using primary patient samples, our studies showed that in comparison with macrophages isolated from normal pancreatic tissues, the phagocytosis activity of PDA-TAM is significantly reduced. We found that the expression of homeobox protein VentX, a master regulator of macrophage plasticity, is significantly decreased in the PDA-TAMs. We demonstrated that VentX is required for phagocytosis and that restoration of VentX expression in PDA-TAMs promotes phagocytosis through regulating the signaling cascades involved in the process. Using an ex-vivo culture model of primary human PDA, we showed that VentX-modulated-TAMs transformed PDA-TME from a pro-tumor milieu to an anti-tumor microenvironment by rectifying differentiation, proliferation and activation of PDA-infiltrating immune cells. Using NSG-PDX models of primary human PDAs, we showed that VentX-modulated-TAMs exert strong inhibition on PDA tumorigenesis in vivo. Taken together, our data revealed a central mechanism underlying immune evasion of PDA and a potential novel venue to improve PDA prognosis.
Authors
Yi Le, Hong Gao, William G. Richards, Lei Zhao, Ronald Bleday, Thomas Clancy, Zhenglun Zhu
