Diabetes Volume 68, June 2019 1303 Diabetes-Associated Myelopoiesis Drives Stem Cell Mobilopathy Through an OSM-p66Shc Signaling Pathway Mattia Albiero,1,2 Stefano Ciciliot,1 Serena Tedesco,1,2 Lisa Menegazzo,1 Marianna D’Anna,1,2 Valentina Scattolini,1,2 Roberta Cappellari,1,2 Gaia Zuccolotto,3,4 Antonio Rosato,3,4 Andrea Cignarella,2 Marco Giorgio,5,6 Angelo Avogaro,2 and Gian Paolo Fadini1,2 Diabetes 2019;68:1303–1314 | https://doi.org/10.2337/db19-0080 Diabetes impairs the mobilization of hematopoietic Diabetes is associated with low-grade inflammation, which stem/progenitor cells (HSPCs) from the bone marrow contributes to chronic complications (1,2). A skewed dif- (BM), which can worsen the outcomes of HSPC trans- ferentiation of common myeloid progenitors (CMPs) trans- plantation and of diabetic complications. In this study, lates hyperglycemia into production of proinflammatory we examined the oncostatin M (OSM)–p66Shc pathway cells (3). Such enhanced myelopoiesis propagates inflam- as a mechanistic link between HSPC mobilopathy and mation from the bone marrow (BM) to the adipose and the excessive myelopoiesis. We found that streptozotocin- vasculature, leading to insulin resistance and atherosclerosis COMPLICATIONS induced diabetes in mice skewed hematopoiesis toward (3,4). In parallel, mobilization of hematopoietic stem/ the myeloid lineage via hematopoietic-intrinsic p66Shc. progenitor cells (HSPCs) from the BM to peripheral blood The overexpression of Osm resulting from myelopoiesis (PB) after stimulation with granulocyte colony-stimulating prevented HSPC mobilization after granulocyte colony- factor (G-CSF) is impaired in murine (5,6) and human stimulating factor (G-CSF) stimulation. The intimate diabetes (7,8), a condition termed mobilopathy (9). link between myelopoiesis and impaired HSPC mobili- zation after G-CSF stimulation was confirmed in human We herein hypothesize that myelopoiesis and mobilopa- diabetes. Using cross-transplantation experiments, we thy, described as two distinct pathological features of the found that deletion of p66Shc in the hematopoietic diabetic BM, are instead mechanistically linked. Disentan- or nonhematopoietic system partially rescued defective gling the processes linking myelopoiesis to mobilopathy has HSPC mobilization in diabetes. Additionally, p66Shc me- relevant clinical implications. First, pharmacologic mobili- diated the diabetes-induced BM microvasculature remod- zation of HSPCs is the gold standard for HSPC transplan- eling. Ubiquitous or hematopoietic restricted Osm deletion tation (10), and failure to collect robust numbers of HSPCs phenocopied p66Shc deletion in preventing diabetes- can delay engraftment, thereby worsening the outcome of associated myelopoiesis and mobilopathy. Mechanisti- patients with diabetes undergoing transplantation (5). Sec- cally, we discovered that OSM couples myelopoiesis to ond, reduction of circulating HSPCs in patients with di- mobilopathy by inducing Cxcl12 in BM stromal cells via nonmitochondrial p66Shc. Altogether, these data indicate abetes predicts the future development of micro- and that cell-autonomous activation of the OSM-p66Shc path- macrovascular complications (11,12). Glucose control effec- way leads to diabetes-associated myelopoiesis, whereas tively prevents myelopoiesis and partially rescues HSPC its transcellular hematostromal activation links myelopoi- mobilization (3,13), but many patients fail to achieve esis to mobilopathy. Targeting the OSM-p66Shc pathway necessary glucose targets. Therefore, disconnecting is a novel strategy to disconnect mobilopathy from mye- mobilopathy from myelopoiesis can provide a direct ther- lopoiesis and restore normal HSPC mobilization. apeutic strategy to restore normal HSPC mobilization. 1Veneto Institute of Molecular Medicine (VIMM), Padova, Italy Received 24 January 2019 and accepted 15 March 2019 2 – Department of Medicine DIMED, University of Padova, Padova, Italy This article contains Supplementary Data online at http://diabetes 3 Department of Surgery, Oncology and Gastroenterology, University of Padova, .diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0080/-/DC1. Padova, Italy M.A. and S.C. contributed equally to this study. 4Istituto Oncologico Veneto (IOV)-IRCCS, Padova, Italy 5European Institute of Oncology (IEO), Milan, Italy © 2019 by the American Diabetes Association. Readers may use this article as 6Department of Biomedical Sciences, University of Padova, Padova, Italy long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at http://www.diabetesjournals Corresponding author: Gian Paolo Fadini, [email protected] or gianpaolo .org/content/license. [email protected] 1304 OSM-p66Shc Regulates Mobilopathy/Myelopoiesis Diabetes Volume 68, June 2019 Recent studies highlight that murine and human di- The protocols were approved by the local ethical committee abetes cause BM microvascular remodeling (14) and au- and conducted in accordance with the Declaration of Hel- tonomic neuropathy (6,15), both of which can affect HSPC sinki as revised in 2000. Cross-sectional data on the asso- traffic (16,17). We previously found that BM denervation ciation between myeloid bias and circulating HSPCs were in diabetic mice accounts for impaired response to G-CSF derived from two previous studies that had been approved and is mediated by p66Shc (6). Unlike p46 and p52, p66Shc by the local ethics committee (6,25). Total and differential functions both as an adaptor protein for membrane white blood cell (WBC) counts were determined in the same receptors and a redox enzyme. Upon phosphorylation at laboratory for both studies, and CD34+ HSPC levels were Ser36, p66Shc translocates to the mitochondrial intermem- quantified by flow cytometry relative to the WBC count. brane space where it catalyzes the production of hydrogen Details are given in the previous publications (6,25). The peroxide (18), contributing to processes linked to oxidative study for G-CSF–induced mobilization was approved by the stress, including diabetic complications (19,20). local ethics committee and is registered in ClinicalTrials Besides sympathetic nervous system activation, deple- .gov (NCT01102699). This was a prospective, parallel-group tion of BM macrophages is a key event in the mobilization study of direct BM stimulation with G-CSF in subjects with cascade induced by G-CSF, because macrophage paracrine and without diabetes. Specific methods for quantifying blood activity sustains CXCL12 production (21). We have iden- cells and HSPCs were given in the previous publication (7). tified oncostatin M (OSM) as the macrophage-derived Informed consent was obtained from all participants. soluble factor that induces Cxcl12 expression in stromal cells, thereby antagonizing HSPC mobilization (22). OSM Animal Models is a cytokine of the interleukin 6 family, which signals via Diabetes was induced in 2-month-old mice by a single in- mitogen-activated protein kinase (MAPK) and the Janus traperitoneal injection of 175 mg/kg streptozotocin (STZ). kinase (JAK)/STAT pathways, leading to pleiotropic func- Blood glucose was measured using a FreeStyle glucometer tions, including modulation of inflammation and bone (Abbott, Abbott Park, IL). HSPC mobilization was induced by formation (23,24). In murine diabetes, excess BM macro- subcutaneous injection of 200 mg/kg/day G-CSF daily for 4 phages result in persistent OSM signaling, inability to days. Three-month-old mice were treated with vehicle or switch off CXCL12 levels after G-CSF, and impaired carrier free recombinant mouse OSM (495-MO/CF; R&D HSPC mobilization (22). Thus, OSM represents a candidate Systems, Minneapolis, MN) at 0.5 mg per injection every 6 h link between myelopoiesis and mobilopathy. In view of the for 48 h before analysis was performed. Total WBC count similar benefits of p66Shc deletion and OSM inhibition on was performed using the CELL-DYN Emerald hematology the diabetic stem cell mobilopathy (6,22), we have hypoth- analyzer (Abbott) on fresh EDTA-treated mouse blood. esized that the two pathways are mechanistically con- nected. In the current study, we therefore examined the Mouse Embryonic Fibroblast Transduction interplay between OSM and p66Shc in determining the Mouse embryonic fibroblasts (MEFs) were isolated from 2 2 link between myelopoiesis and mobilopathy observed in E13.5 p66Shc / mice after digestion with trypsin (Corn- experimental and human diabetes. ing) and cultured with DMEM and 10% FBS. PINCO retroviral particles were produced from the amphotropic RESEARCH DESIGN AND METHODS packaging cell line Phoenix. Cells were infected with an Mice empty vector, a vector encoding mouse full-length p66Shc, C57BL/6J wild-type (Wt) mice were purchased from The a vector encoding the mutants p66ShcS36A (S→Asub- Jackson Laboratory and established as a colony since 2001. stitution at position 36) and p66ShcQQ (EE→QQ sub- 2/2 p66Shc mice were originally obtained from Pelicci’s stitutions at positions 132–133). P3 MEFs were infected laboratory (European Institute of Oncology, Milan, Italy), with three rounds of infection with Polybrene Infection/ a colony was established at our facility in 2010, and mice Transfection Reagent (Sigma-Aldrich), followed by 96 h of have been backcrossed on the C57BL/6J background selection with 2 mg/mL puromycin. Experiments were 2/2 for .10 generations. Osm mice on the C57BL/6J performed with p4 or p5 cells. background were obtained from GlaxoSmithKline (Steven-