Diabetes-Associated Myelopoiesis Drives Stem CellMobilopathy Through an OSM-p66Shc Signaling PathwayMattia 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 hematopoieticstem/progenitor cells (HSPCs) from the bone marrow(BM), which can worsen the outcomes of HSPC trans-plantation and of diabetic complications. In this study,we examined the oncostatin M (OSM)–p66Shc pathwayas a mechanistic link between HSPC mobilopathy andexcessive myelopoiesis. We found that streptozotocin-induced diabetes in mice skewed hematopoiesis towardthe myeloid lineage via hematopoietic-intrinsic p66Shc.The overexpression of Osm resulting from myelopoiesisprevented HSPC mobilization after granulocyte colony-stimulating factor (G-CSF) stimulation. The intimatelink between myelopoiesis and impaired HSPC mobili-zation after G-CSF stimulation was confirmed in humandiabetes. Using cross-transplantation experiments, wefound that deletion of p66Shc in the hematopoieticor nonhematopoietic system partially rescued defectiveHSPC mobilization in diabetes. Additionally, p66Shc me-diated the diabetes-inducedBMmicrovasculature remod-eling. Ubiquitous or hematopoietic restrictedOsmdeletionphenocopied p66Shc deletion in preventing diabetes-associated myelopoiesis and mobilopathy. Mechanisti-cally, we discovered that OSM couples myelopoiesis tomobilopathy by inducing Cxcl12 in BM stromal cells vianonmitochondrial p66Shc. Altogether, these data indicatethat cell-autonomous activation of the OSM-p66Shc path-way leads to diabetes-associated myelopoiesis, whereasits transcellular hematostromal activation links myelopoi-esis to mobilopathy. Targeting the OSM-p66Shc pathwayis a novel strategy to disconnect mobilopathy from mye-lopoiesis and restore normal HSPC mobilization.

Diabetes is associated with low-grade inflammation, whichcontributes to chronic complications (1,2). A skewed dif-ferentiation of common myeloid progenitors (CMPs) trans-lates hyperglycemia into production of proinflammatorycells (3). Such enhanced myelopoiesis propagates inflam-mation from the bone marrow (BM) to the adipose and thevasculature, leading to insulin resistance and atherosclerosis(3,4). In parallel, mobilization of hematopoietic stem/progenitor cells (HSPCs) from the BM to peripheral blood(PB) after stimulation with granulocyte colony-stimulatingfactor (G-CSF) is impaired in murine (5,6) and humandiabetes (7,8), a condition termed mobilopathy (9).

We herein hypothesize that myelopoiesis and mobilopa-thy, described as two distinct pathological features of thediabetic BM, are instead mechanistically linked. Disentan-gling the processes linking myelopoiesis to mobilopathy hasrelevant clinical implications. First, pharmacologic mobili-zation of HSPCs is the gold standard for HSPC transplan-tation (10), and failure to collect robust numbers of HSPCscan delay engraftment, thereby worsening the outcome ofpatients with diabetes undergoing transplantation (5). Sec-ond, reduction of circulating HSPCs in patients with di-abetes predicts the future development of micro- andmacrovascular complications (11,12). Glucose control effec-tively prevents myelopoiesis and partially rescues HSPCmobilization (3,13), but many patients fail to achievenecessary glucose targets. Therefore, disconnectingmobilopathy from myelopoiesis can provide a direct ther-apeutic strategy to restore normal HSPC mobilization.

1Veneto Institute of Molecular Medicine (VIMM), Padova, Italy2Department of Medicine–DIMED, University of Padova, Padova, Italy3Department of Surgery, Oncology and Gastroenterology, University of Padova,Padova, Italy4Istituto Oncologico Veneto (IOV)-IRCCS, Padova, Italy5European Institute of Oncology (IEO), Milan, Italy6Department of Biomedical Sciences, University of Padova, Padova, Italy

Corresponding author: Gian Paolo Fadini, gianpaolofadini@hotmail.com or gianpaolo.fadini@unipd.it

Received 24 January 2019 and accepted 15 March 2019

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db19-0080/-/DC1.

M.A. and S.C. contributed equally to this study.

© 2019 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

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COMPLIC

ATIO

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Recent studies highlight that murine and human di-abetes cause BM microvascular remodeling (14) and au-tonomic neuropathy (6,15), both of which can affect HSPCtraffic (16,17). We previously found that BM denervationin diabetic mice accounts for impaired response to G-CSFand is mediated by p66Shc (6). Unlike p46 and p52, p66Shcfunctions both as an adaptor protein for membranereceptors and a redox enzyme. Upon phosphorylation atSer36, p66Shc translocates to the mitochondrial intermem-brane space where it catalyzes the production of hydrogenperoxide (18), contributing to processes linked to oxidativestress, including diabetic complications (19,20).

Besides sympathetic nervous system activation, deple-tion of BM macrophages is a key event in the mobilizationcascade induced by G-CSF, because macrophage paracrineactivity sustains CXCL12 production (21). We have iden-tified oncostatin M (OSM) as the macrophage-derivedsoluble factor that induces Cxcl12 expression in stromalcells, thereby antagonizing HSPC mobilization (22). OSMis a cytokine of the interleukin 6 family, which signals viamitogen-activated protein kinase (MAPK) and the Januskinase (JAK)/STAT pathways, leading to pleiotropic func-tions, including modulation of inflammation and boneformation (23,24). In murine diabetes, excess BM macro-phages result in persistent OSM signaling, inability toswitch off CXCL12 levels after G-CSF, and impairedHSPCmobilization (22). Thus, OSM represents a candidatelink between myelopoiesis and mobilopathy. In view of thesimilar benefits of p66Shc deletion and OSM inhibition onthe diabetic stem cell mobilopathy (6,22), we have hypoth-esized that the two pathways are mechanistically con-nected. In the current study, we therefore examined theinterplay between OSM and p66Shc in determining thelink between myelopoiesis and mobilopathy observed inexperimental and human diabetes.

RESEARCH DESIGN AND METHODS

MiceC57BL/6J wild-type (Wt) mice were purchased from TheJackson Laboratory and established as a colony since 2001.p66Shc2/2 mice were originally obtained from Pelicci’slaboratory (European Institute of Oncology, Milan, Italy),a colony was established at our facility in 2010, and micehave been backcrossed on the C57BL/6J backgroundfor .10 generations. Osm2/2 mice on the C57BL/6Jbackground were obtained from GlaxoSmithKline (Steven-age, U.K.), and a colony was established in 2015. For all theexperiments, we used sex- and age-matched animals. As-signment of mice to treatments or experimental groupswas based on a computer-generated random sequence. Allanimal studies were approved by the Venetian Institute ofMolecular Medicine Animal Care and Use Committee andby the Italian Health Ministry.

HumansIndividuals with and without diabetes were recruited at theUniversity Hospital of Padova Division of Metabolic Diseases.

The protocols were approved by the local ethical committeeand conducted in accordance with the Declaration of Hel-sinki as revised in 2000. Cross-sectional data on the asso-ciation between myeloid bias and circulating HSPCs werederived from two previous studies that had been approvedby the local ethics committee (6,25). Total and differentialwhite blood cell (WBC) counts were determined in the samelaboratory for both studies, and CD34+ HSPC levels werequantified by flow cytometry relative to the WBC count.Details are given in the previous publications (6,25). Thestudy for G-CSF–induced mobilization was approved by thelocal ethics committee and is registered in ClinicalTrials.gov (NCT01102699). This was a prospective, parallel-groupstudy of direct BM stimulation with G-CSF in subjects withandwithout diabetes. Specificmethods for quantifying bloodcells and HSPCs were given in the previous publication (7).Informed consent was obtained from all participants.

Animal ModelsDiabetes was induced in 2-month-old mice by a single in-traperitoneal injection of 175 mg/kg streptozotocin (STZ).Blood glucose was measured using a FreeStyle glucometer(Abbott, Abbott Park, IL). HSPCmobilizationwas induced bysubcutaneous injection of 200 mg/kg/day G-CSF daily for 4days. Three-month-old mice were treated with vehicle orcarrier free recombinant mouse OSM (495-MO/CF; R&DSystems, Minneapolis, MN) at 0.5 mg per injection every 6 hfor 48 h before analysis was performed. Total WBC countwas performed using the CELL-DYN Emerald hematologyanalyzer (Abbott) on fresh EDTA-treated mouse blood.

Mouse Embryonic Fibroblast TransductionMouse embryonic fibroblasts (MEFs) were isolated fromE13.5 p66Shc2/2 mice after digestion with trypsin (Corn-ing) and cultured with DMEM and 10% FBS. PINCOretroviral particles were produced from the amphotropicpackaging cell line Phoenix. Cells were infected with anempty vector, a vector encoding mouse full-length p66Shc,a vector encoding the mutants p66ShcS36A (S→A sub-stitution at position 36) and p66ShcQQ (EE→QQ sub-stitutions at positions 132–133). P3 MEFs were infectedwith three rounds of infection with Polybrene Infection/Transfection Reagent (Sigma-Aldrich), followed by 96 h ofselection with 2 mg/mL puromycin. Experiments wereperformed with p4 or p5 cells.

BM TransplantationRecipientmice (3months old) were treatedwith amyeloablativedose of total body irradiation of 10 Gy, split in two doses of5 Gy 3 h apart and followed by an intravenous injection ofBM cells from donor mice (4 3 107/each) isolated byflushing femurs and tibias with sterile ice-cold PBS.

CFU AssayBM cells (3 3 104) were plated in 35-mm Petri dishescontaining 1 mL methylcellulose-based medium Metho-Cult supplemented with 1% penicillin/streptomycin. After

1304 OSM-p66Shc Regulates Mobilopathy/Myelopoiesis Diabetes Volume 68, June 2019

red blood cell lysis, 25 mL/well PB was plated in 24-wellplates containing 0.5 mL MethoCult supplemented with1% penicillin/streptomycin. Colony formation was scoredafter 10 days of culture. When required, murine recombi-nant S100A8/9 heterodimer (BioLegend) was mixed withthe MethoCult medium.

Flow CytometryFlow cytometry was performed on BM cells or EDTA-treated PB. BM cells were isolated by flushing femursand tibias with ice-cold MACS Separation Buffer (MiltenyiBiotec GmbH, Gladbach, Germany) through a 40-mm cellstrainer. Then 100 mL PB or BM cells was incubated withantibodies for 15 min in the dark at room temperature.After red blood cell lysis, samples were resuspended in200mL PBS, and data were acquired with a FACSCanto (BDBiosciences) cytometer, followed by analysis using FlowJosoftware (Tree Star).

BM-Derived Mesenchymal Stem CellsMurine BM-derived mesenchymal stem cells (BM-MSCs)were isolated by flushing the BM of 3-month-old mice andcultured in minimum essential medium-a containing 10%FBS, glutamine (2 mmol/L), and penicillin-streptomycin.Passage 3–6 was used in all experiments. For gene expres-sion analysis, cells were treated with murine recombinantOSM (R&D Systems) for 48 h in serum-free media.

Tissue ProcessingFemur bones were fixed in 4% paraformaldehyde anddecalcified. Bones were then washed with PBS, embeddedin Killik cryostat medium (Bio-Optica, Milan, Italy), andfrozen in liquid nitrogen–cooled 2-methylbutane (Sigma-Aldrich). Longitudinal 10-mm-thick femur sections wereobtained with a Leica CM 1950 cryostat (Leica BiosystemsS.r.l., Milan, Italy), placed on Superfrost Plus slides(J1800AMNZ; Gerhard Menzel GmbH, Braunschweig,Germany), and stored at 280°C.

Protein Phosphorylation by Flow CytometryConfluent BM-MSCs were treated with SCH772984(4 nmol/L) (Cayman Chemical) overnight or with Stattic(2.5 mmol/L) (Selleckchem) for 1 h before adding recombi-nant OSM (R&D Systems) for 30 min in serum-free media.Cells were detached by scraping and incubated with PEmouse anti-Stat3 (pY705) or PE mouse anti-extracellularsignal–regulated kinase (ERK)1/2 (pT202/pY204) (bothfrom BD Biosciences) in Perm Buffer III (BD Biosciences)according to the manufacturer’s instructions.

ImmunohistochemistryFemur sections were air dried for 20 min and then in-cubated with blocking solution. Sections were incubatedwith primary antibody: anti-laminin (1:50 for 4 days),anti–tyrosine-hydroxylase (Tyr-OH) (1:200 for 4 days),and anti-CD150 (1:50 for 3 days). Sections were thenwashed with PBS and incubated with secondary antibodies.

Slides were mounted with an antifade aqueous mountingmedium. Images were taken with a Leica DM5000B mi-croscope, equipped with a DFC300 FX CCD camera, or withCytell (GE Healthcare, Milan, Italy). Images were thenprocessed with Fiji/ImageJ 1.50 software (National Insti-tutes of Health, Bethesda, MD) or with Adobe PhotoshopCS2 9.0.2 software (Adobe Systems, San Jose, CA).

Morphometric MeasurementsVessel size and shape were measured using Fiji/ImageJ1.50 software. Briefly, random 500 mm2

fields from theepiphyseal and diaphyseal region (three each, at least) ofthe samples were analyzed. Vessel structure was visualizedby laminin staining, and regions of interest were manuallyoutlined in Fiji/ImageJ. Area and shape parameters, such ascircularity, were recorded. The bivariate distribution of areaand circularity was visualized using the Bivariate KernelDensity Estimation 1.0.9 in R 3.1 software. BM innervationwas determined by Tyr-HO staining. Arterioles were iden-tified in the whole femur section, and diameters of arterioleswere measured with Fiji/ImageJ.

Molecular BiologyRNA was isolated from flushed BM or cells by using QIAzolor with Total RNA Purification Micro Kit (Norgen Biotek)and quantified with a NanoDrop 2000 Spectrophotometer(Thermo Fisher Scientific, Waltham, MA). cDNA was syn-thesized using the SensiFAST cDNA Synthesis Kit (Bioline,London, U.K.). Quantitative PCR was performed using theSensiFAST SYBR Lo-ROX Kit (Bioline) via a QuantStudio5 Real-Time PCR System (Thermo Fisher Scientific). A listof primers can be found in Supplementary Table 1.

Statistical AnalysisContinuous data are expressed as mean 6 SEM, whereascategorical data are presented as the percentage. Normal-ity was checked using the Kolmogorov-Smirnov test, andnonnormal data were log-transformed before analysis.Comparison between two or more groups was performedusing the Student t test and ANOVA for normal variablesor the Mann-Whitney U test and Kruskal-Wallis test fornonnormal variables that could not be log-transformed.Bonferroni adjustment was used to account for multipletesting. Linear correlations were checked using the Pear-son r coefficient. Statistical analysis was accepted at P ,0.05. Statistical analysis was performed using GraphPadPrism 6, Matlab, and SPSS 21 software.

RESULTS

Mobilopathy Associates With Myelopoiesis inExperimental DiabetesWe first evaluated whether myelopoiesis and mobilopathycoexist in murine diabetes. We found that STZ-induceddiabetic mice had an approximately twofold expansion ofPB granulocytes compared with nondiabetic mice (P ,0.001) (Fig. 1A and B and Supplementary Fig. 1), resultingin a strikingly six times higher granulocyte-to-lymphocyte

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(G-to-L) ratio (P , 0.001) (Fig. 1C). The BM of diabeticmice contained higher numbers of granulocyte-monocyteprogenitors (GMPs) at the expense of CMPs (Fig. 1D). Asa consequence, the clonogenic assay of BM cells showed anincreased output of macrophage and granulocyte coloniesfrom diabetic versus nondiabetic mice (2.2 times and 2.8times, respectively) (Fig. 1E). The diabetic BM showedexcess macrophages both in basal unstimulated conditionsand after G-CSF stimulation (Fig. 1F). These cells areknown to produce OSM (22), and Osm gene expressionin the BM of diabetic mice was indeed upregulated 7.7times compared with that in nondiabetic mice (P = 0.006)(Fig. 1G). Altogether, these data are consistent with ex-aggerated myelopoiesis and myeloid bias in diabetic mice.

As the resulting overproduction of OSM can hampermobilization (22), we evaluated whether mobilopathyoccurred in the same mice. Preliminary to this, we verifiedthat the baseline PB level of HSPCs (Lin2c-Kit+Sca-1+

[LKS] cells) was nonsignificantly different in diabeticversus nondiabetic mice (Supplementary Fig. 2). AfterG-CSF was administered for 4 days, the HSPC level in-creased by 4.38 times in nondiabetic but not in diabeticmice (Fig. 1H). This difference in the fold change of thePB-LKS cell level between diabetic and nondiabetic micewas highly significant (Fig. 1I): 80% of nondiabetic mice vs.10% of diabetic mice achieved a mobilization response ofat least 1.5-fold (Fig. 1J). The colony-forming unit (CFU)assay from PB cells confirmed the absence of functionalHSPC mobilization in diabetes (Supplementary Fig. 3A).

The profound degree of mobilization impairment allowedus to use the G-CSF mobilization assay as a robust readoutfor mobilopathy in subsequent mouse experiments.

Mobilopathy Associates With Myelopoiesis in HumanDiabetesWe then checked whether myelopoiesis and mobilopathyoccurred simultaneously in human diabetes. We first ana-lyzed cross-sectional data of two studies wherein circulatingWBC types and levels of CD34+ HSPCs were determined inthe same sample (6,25) (Supplementary Table 2). In a pooledcohort of 344 subjects, the patients with diabetes (n = 108;74% type 2) displayed 25% lower levels of HSPCs and a 24%higher neutrophil-to-lymphocyte (N-to-L) ratio than indi-viduals without diabetes (Fig. 2A). Higher N-to-L ratio andlower CD34+ HSPCs remained significantly associated withdiabetes after adjusting for age, sex, BMI, hypertension,lipids, coronary artery disease, and retinopathy (Supplemen-tary Table 3). We also found a significant inverse correlationbetween the N-to-L ratio and the steady-state level of PBHSPCs (r = 20.28) (Fig. 2B). Considering glucose control asa continuous variable in the entire cohort, we found a sig-nificant inverse correlation between HbA1c and HSPC levels(r = 20.23; P , 0.001) (Fig. 2C) and a direct correlationbetween HbA1c and the N-to- L ratio (r = 0.21; P , 0.001)(Fig. 2D), which persisted (both with P, 0.01) after adjust-ing for the above-mentioned confounders. These data sug-gest that myeloid bias is linked to a reduction in HSPC levelsin human diabetes, possibly driven by hyperglycemia.

Figure 1—Mobilopathy and myelopoiesis in experimental diabetes. Panels A–G report the comparison between diabetic (n = 16) andnondiabetic control (n = 12) mice in total WBC counts (A), absolute counts of lymphocyte (Lympho), monocytes (Mono), and granulocytes(Granulo) (B), as well as the G-to-L ratio (C). D: Comparison of FACS-defined CMPs and GMPs. E: Results of the CFU assay from BM cells.GEMM, granulocyte-erythroid-macrophage-MK colonies; GM, granulocyte-macrophage colonies; M, macrophage colonies; G, granulocytecolonies. F: Percentages of BM macrophages over total BM cells in diabetic vs. control mice in the unstimulated and G-CSF–stimulatedconditions. G: Gene expression of Osm in the BM of diabetic vs. control mice. *P , 0.05 for the comparison between diabetic and controlmice. Panels H–J illustrate HSPC mobilization in diabetic vs. nondiabetic mice. HSPCs, defined as LKS, were quantified before and afterG-CSF administration and are reported as fold change from baseline in nondiabetic control mice (n = 20) and in diabetic mice (n = 20).H: Individual lines, indicating single mice, are shown along with the average fold change (95% CI) for each time point and the respectiveP values. I: Comparison of the fold change in LKS cell levels after G-CSF between control and diabetic mice. J: Comparison of thepercentage of mice achieving a mobilization response of at least 1.5-fold in the diabetic vs. nondiabetic control condition. Histograms indicatemean 6 SEM. Box plots indicate median with interquartile range, and whiskers indicate range.

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Second, we evaluated whether myeloid bias was asso-ciated with mobilopathy by analyzing data from a previousprospective study wherein patients with and withoutdiabetes (n = 43) received low-dose G-CSF to test HSPCmobilization (7). The fold change in G-CSF–inducedHSPC levels versus baseline was significantly lower,and the pre–G-CSF N-to-L ratio tended to be higher(P = 0.06) in patients with diabetes versus those withoutdiabetes (Fig. 2E). Remarkably, there was a significantinverse correlation between the N-to-L ratio and HSPCmobilization (r = 20.32; P = 0.03) (Fig. 2F). Theseresults cannot prove causality, because secondaryanalyses of previously collected cohort data can be sub-jected to bias and prone to false-positive signals. None-theless, we confirm that myelopoiesis and mobilopathyare associated in human diabetes as they are in murinediabetes.

Deletion of p66Shc Prevents Diabetes-AssociatedMyelopoiesis and MobilopathyHaving shown that myelopoiesis and mobilopathy concurin murine and human diabetes, we explored the mecha-nisms driving their association. We first focused onp66Shc, which we previously showed to be responsiblefor diabetes-associated BM denervation and mobilopathy(6). BM p66Shc gene expression was more than twofoldhigher in diabetic versus control mice (P = 0.003) (Sup-plementary Fig. 4), consistent with prior data in mice andhumans (26,27). In the nondiabetic condition, we found nodifferences in WBC count and subtypes, G-to-L ratio, BMcolonies, and CMPs/GMPs, as well as BM macrophagesbetween Wt and p66Shc2/2 mice (Fig. 3A–G). However, inp66Shc2/2 mice, diabetes did not increase PB granulocytescounts, and granulocytes were significantly lower than inWt diabetic mice (Fig. 3D and Supplementary Fig. 5), as

Figure 2—Myelopoiesis and mobilopathy in human diabetes. A: Comparison of circulating CD34+ HSPCs and the N-to-L ratio in a pooledcohort of patients without diabetes (n = 236) and with diabetes (n = 108). *P, 0.05.B: Linear correlation between HSPC levels and the N-to-Lratio. Regression coefficients are reported for the entire cohort (alongwithP values) and for the patients without andwith diabetes separately.Linear correlation between HbA1c and HSPC levels (C ) or the N-to-L ratio (D): the regression line with its 95% CI is shown along with theregression coefficients and P values. E: Comparison between patients with and without diabetes in the increase (fold change) in HSPC levelsafter G-CSF and in the baseline N-to-L ratio. *P, 0.05. F: Linear correlation between the baseline N-to-L ratio and the increase (fold change)in HSPC levels after G-CSF stimulation. Regression coefficients are reported for the entire cohort (along with the P value) and for the patientswithout and with diabetes separately. Histograms indicate mean 6 SEM.

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was the G-to-L ratio (Fig. 3E). Furthermore, the diabetes-induced increase in myeloid CFUs (Fig. 3F), CMP/GMPimbalance (Fig. 3G), and excess BM macrophages (Fig. 3H)

were completely prevented in p66Shc2/2 mice. As a result,the surge in BM expression ofOsm observed in diabetic Wtmice, which derives from BM macrophages (22), was

Figure 3—p66Shc deletion protects from diabetes-induced myelopoiesis and mobilopathy. Myelopoiesis was evaluated in nondiabetic anddiabeticWt and p66Shc2/2mice (n. 10/group, unless specified) by comparing PBWBC count (A), WBC types (B–D), the G-to-L ratio (E), theBM cell CFU assay (F ), FACS-defined BM CMPs and GMPs (G), and the percentage of BM macrophages (n = 5/group) (H). GEMM,granulocyte-erythroid-macrophage-MK colonies; GM, granulocyte-macrophage colonies; M, macrophage colonies; G, granulocyte colo-nies. *P, 0.05 diabetic vs. control (Ctrl); †P, 0.05 p66Shc2/2 vs. Wt. I and J: CFU assay performed using BM cells fromWt (I) or p66Shc2/2 (J)mice, which were stimulated ex vivo with 2 mg/mL S100A8/9. *P , 0.05 for the comparison with untreated control cells. K: Percentage ofFACS-defined GMP in the BM of nondiabetic Wt and p66Shc2/2 mice treated with vehicle or S100A8/9 (20 mg/kg twice a day for 3 days). L:Mobilization of HSPCs was evaluated in p66Shc2/2 diabetic and nondiabetic mice (n = 5/group) by enumerating circulating LKS cells. *P ,0.05 in post–G-CSF vs. baseline.M: Schematic representation of the generation of hematopoietic and nonhematopoietic p66Shc2/2mice.N:The fold change with 95% CI of LKS cells (calculated as post–G-CSF divided by pre–G-CSF levels) in Wt diabetic mice, p66Shc2/2 diabeticmice, and diabeticmice with crossed BMT (n = 4–5/group).*Significantly different from 1.0, denoted by the dashed line indicating no effect.O:The change in the G-to-L ratio induced by diabetes is plotted for Wt mice, p66Shc2/2 mice, and mice with crossed BMT. The annotationunder panelsN andO indicates the genotype of the host (receiver mice) or the BMdonormice. KO, knockout. *Significantly different from 1.0,denoted by the dashed line.

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absent in p66Shc2/2 mice (Supplementary Fig. 6). The linkbetween hyperglycemia and myelopoiesis has been attrib-uted to the accumulation of advanced glycation end prod-ucts (AGEs) and the receptor for the AGE (RAGE) ligandS100A8/9 (3). Interestingly, RAGE signaling has beenpreviously linked with downstream p66Shc activation(28). We found that S100A8/9 potentiated myelopoiesisin vitro by BM cells of Wt mice, evidenced by a 1.75-foldand a 2.0-fold increase in macrophage and granulocytecolonies, respectively (Fig. 3I). However, such effect wascompletely abolished in p66Shc2/2 BM cells (Fig. 3J).In vivo treatment of nondiabetic Wt mice with S100A8/9 increased GMP and myeloid cell colonies, but such effectwas not observed in p66Shc2/2 mice (Fig. 3K and Supple-mentary Fig. 7). Together, these data indicate that p66Shcis required for the effects of hyperglycemia on myelopoi-esis, possibly by preventing the activity of S100 proteins.

In agreement with our previous study (6), p66Shc de-letion partially rescued HSPC mobilization in diabeticmice, as indicated by the 1.9 times increase in LKS cellcounts after G-CSF administration (Fig. 3L). The CFU assayshowed that mobilized HSPCs were functionally compe-tent, as G-CSF increased PB hematopoietic colonies both indiabetic and nondiabetic p66Shc2/2 mice (SupplementaryFig. 8). To dissect the hematopoietic-intrinsic and -extrinsicroles of p66Shc in regulating HSPC mobilization, we

performed cross-transplantation experiments as illus-trated in Fig. 3M. We confirmed that BM transplantation(BMT) did not impinge on G-CSF responsiveness, as non-diabetic but not diabetic Wt mice that received BMTs fromWt mice were able to mobilize functional HSPCs (Supple-mentary Fig. 3B and C). Wt mice that received BMTs fromp66Shc2/2mice (p66Shc2/2→Wt BMT) and were rendereddiabetic showed a partial restoration of HSPC increaseafter G-CSF stimulation. An almost identical, but partial,improvement was observed in diabetic p66Shc2/2 micewho received BMTs from Wt mice (Wt→p66Shc2/2 BMT),and in both cases, the fold change in HSPC count was lowerthan that in ubiquitous p66Shc2/2 diabetic mice (Fig. 3N).In contrast, while diabetes increased granulocyte countsand the G-to-L ratio in Wt→p66Shc2/2, p66Shc2/2→WtBMT mice were largely protected from elevation of theG-to-L ratio induced by diabetes (Fig. 3O and Supplemen-tary Fig. 9).

Knowing that myeloid-biased HSPCs reside in mega-karyocytic (MK) niches (29), we analyzed MK density bystaining BM sections with anti-CD150. As previouslynoted by others (30), MKs were increased by 60% indiabetic compared with nondiabetic Wt mice (P =0.004). However, such an effect was not observed inp66Shc2/2 mice (Supplementary Fig. 10), providing a fur-ther explanation for the protection of ubiquitous and

Figure 4—p66Shc deletion ameliorates BM microvascular remodeling in diabetes. A: BM sections were stained with Hoechst (totalcellularity) and anti-laminin to evaluate the microvasculature: based on specific thresholds, vascular items were scored as arterioles,sinusoids, or small vessels of capillary caliber. B: The four aligned panels report the numbers of any vessel, arterioles, sinusoids, andcapillary-size structures per field in diabetic and nondiabeticWt and p66Shc2/2mice. *P, 0.05 diabetic vs. nondiabetic; †P, 0.05 vs.Wt.C:Kernel density plot of vascular items scored based on size (x-axis) and circularity (y-axis): the area of the plot identified by the dashed boxcontains large irregular items (likely sinusoids), which was reduced by diabetes inWt but not in p66Shc2/2mice.D: BM sections were stainedwith Hoechst, anti-laminin (blood vessels), and anti–Tyr-OH, a marker of sympathetic nerve fibers. A representative example froma nondiabetic Wt mouse is shown to illustrate the pattern of Tyr-OH staining. Number of innervated arterioles/field (E) and the fractionof innervated arterioles (F ) over the total number of arterioles. Histograms indicate mean 6 SEM, with superimposed individual data points(n = 5/group). *P , 0.05 vs. nondiabetic control.

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hematopoietic-restricted p66Shc2/2 mice from diabetes-induced myelopoiesis.

Altogether, these data indicate that hematopoietic-intrinsic and -extrinsic mechanisms are responsible forthe rescue of HSPC mobilization by p66Shc deletion,whereas prevention of myelopoiesis is hematopoietic cellintrinsic.

p66Shc Deletion Improves Diabetes-Induced BMMicrovascular RemodelingThe partial restoration of G-CSF–induced HSPC mobilizationinWt→p66Shc2/2 BMT diabetic mice suggested that deletionof p66Shc exerted protective effects on the BM stromaagainst hyperglycemic damage. We previously reported thatp66Shc2/2 mice were protected from BM sympathectomyinduced by diabetes (6). We herein characterized microvas-cular BM remodeling in p66Shc2/2 versus Wt diabetic andnondiabetic mice. In the peculiar BM microcirculation, thenutrient arterial system drains into sinusoids with capillary-size vessels, and the irregular sinusoid lumen is occasionallycompressed to capillary caliber (31). Using an unbiased auto-instructed procedure to score BM vessels (Fig. 4A), we foundthat the total vascular density and numbers of arterioles andsinusoids were similar, but a significant 2.5-fold reductionin capillary-size structures was noted in diabetic versus non-diabetic Wt mice (P = 0.006) (Fig. 4B). Remarkably, thedensity of BM capillary-size vessels was not reduced inp66Shc2/2 diabetic versus nondiabetic mice and was higherin p66Shc2/2 versus Wt diabetic mice (P = 0.03). Witha more detailed morphometric analysis of BM blood vessel

distribution according to size and circularity, we found thatdiabetes also led to a reduction of larger irregular vessels,likely sinusoids, that was prevented by p66Shc deletion (Fig.4C). We then evaluated sympathetic innervation of the BMand found that Tyr-OH+ sympathetic terminals were almostexclusively located close to arteriolar walls (Fig. 4D). Thepercentage of innervated arterioles was significantly reducedby more than twofold in diabetic versus nondiabetic Wtmice, but not in p66Shc2/2mice (P, 0.001) (Fig. 4E and F).Taken together, these findings indicate that deletion ofp66Shc protects BM from microvascular remodeling, whichcan explain the partial rescue of HSPC mobilization innonhematopoietic p66Shc-deleted mice.

Osm Deletion Phenocopies p66Shc DeletionThe hematopoietic cell–intrinsic mechanism wherebyG-CSF exerts its mobilizing activity relies on suppressionof BM macrophages (21). This pathway is independentfrom the stromal effect of G-CSF through nerve terminals,as mice sympathectomized by 6-OH dopamine showeda normal post–G-CSF suppression of BM macrophagesdespite being unable to mobilize HSPCs (SupplementaryFig. 11). This finding indicates that both hematopoieticand nonhematopoietic effects of G-CSF are required toyield a full HSPC mobilizing response and justifies thepartial recovery of mobilization in Wt↔p66Shc2/2 cross-transplanted animals.

We previously demonstrated that antibody-mediatedOSM neutralization allowed HSPC mobilization in diabeticmice by relieving the brake of CXCL12 produced by stromal

Figure 5—Osm deletion protects from diabetes-induced myelopoiesis and mobilopathy. A: Mobilization of HSPCs, defined as LKS cells,induced by G-CSF in nondiabetic and in diabetic Osm2/2 mice (n = 5). *P, 0.05 vs. baseline. B: Percentage of BM macrophages over totalBM cellularity in diabetic and nondiabetic Wt (same as Fig. 1F) and Osm2/2 mice in unstimulated (Unst.) and G-CSF–stimulated conditions(n = 8–10/group). *P, 0.05 vs. control; †P, 0.05 vs. unstimulated; ‡P, 0.05 vs. Wt. Total WBC count (C) and G-to-L ratio (D) in nondiabeticand STZ diabetic Wt andOsm2/2 mice (n. 10/group). Statistics marks as in panel B. E: Schematic representation of the BMT experiment togenerate hematopoietic-restricted Osm-deleted mice. F: Mobilization of HSPCs in nondiabetic and diabetic hematopoietic-restricted Osm2/2

mice (n = 5). *P, 0.05 vs. baseline. Total WBC count (G) and G-to-L ratio (H) in diabetic and nondiabeticWt mice withOsm2/2 BM. Histogramsindicate mean 6 SEM with superimposed individual data points, where appropriate.

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cells (22). Consistent with the notion that OSM retainsHSPCs in the BM niche, nondiabetic Osm2/2 mice dis-played higher HSPC levels in unstimulated PB than Wtmice, which was not further increased by diabetes (Sup-plementary Fig. 2), and HSPCmobilization in diabetic micewas partially rescued toward normal levels (2.2 times) bygenetic Osm deletion (P = 0.01) (Fig. 5A). In addition, weobserved a marked (;80%) reduction of BM macrophagesin Osm2/2 mice, both in the diabetic and nondia-betic condition, which was further suppressed by G-CSF(Fig. 5B). This result suggests that OSM is not only

a macrophage-derived paracrine factor but it is also re-quired for accumulation of BM macrophages in a para-crine-autocrine loop, as already seen by others in the heart(32). Indeed, diabetic Osm2/2 mice had normal WBCcounts (Fig. 5C) but significantly lower levels of PB gran-ulocytes compared with Wt diabetic mice (SupplementaryFig. 12), and the G-to-L ratio was restored toward thelevels seen in nondiabetic mice (Fig. 5D). To avoid theconfounding factor of the absence of nonhematopoieticOSM in ubiquitousOsm2/2mice, we transplanted BM cellsfrom Osm2/2 mice into Wt mice and induced diabetes

Figure 6—Signaling of OSM requires p66Shc. A: Hypothetical model wherein recruitment of p66Shc to OSMR, instead of migration tomitochondria (M), is required for OSM to regulateCxcl12 expression. N, nucleus.B: Gene expression ofCxcl12 in BM-MSCs isolated fromWtor p66Shc2/2mice and treated with OSM (30 ng/mL) or vehicle (control) (n = 5/condition). *P, 0.05 vs. control.C: Gene expression ofCxcl12in MEFs isolated from p66Shc2/2 mice and transfected with an empty vector or vector encoding for Wt p66Shc (p66WT), serine 36 mutatedp66Shc (p66S36A), or catalytically inactive p66Shc (p66qq) and treated with OSM or vehicle (control) (n = 4/condition). *P, 0.05 vs. control.D:Phosphorylation of ERK1/2 (p-ERK1/2) on threonine 202 or tyrosine 204 and phosphorylation of STAT3 (p-STAT3) on tyrosine 705 wasevaluated by flow cytometry in Wt and p66Shc2/2 MSCs treated with vehicle (Ctrl) or mouse OSM (mOSM) with and without an inhibitor ofERK (SCH772984) or STAT (Stattic), respectively (a representative experiment of three replicates is shown). E: Signaling model whereinp66Shc recruited to the OSMR is required for ERK activation, but not for JAK-STAT activation via gp130/OSMR, although both ERK andSTAT are required for OSM to induce Cxcl12. F: Gene expression of Cxcl12 in the BM of Wt, Osm2/2, and p66Shc2/2 mice treated withvehicle (control) or OSM (0.5mg every 6 h for 48 h).*P, 0.05 vs. control.G: Levels of HSPCs, defined as LKScells, inWt,Osm2/2, and p66Shc2/

2mice treatedwith vehicle (control) or OSM. *P, 0.05 vs. control.H: Fold changeof theG-to-L ratio inWt,Osm2/2, andp66Shc2/2mice treatedwith vehicle (control) or OSM. *P , 0.05 vs. control. Histograms indicate mean 6 SEM with superimposed individual data points for eachexperiment.

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4 weeks later (Fig. 5E). After 4 weeks of diabetes, we testedHSPC mobilization after G-CSF treatment and foundthat hematopoietic-restricted Osm deletion rescued HPSCmobilization in diabetic mice toward normal levels (5.2times; P = 0.03) (Fig. 5F). In addition, hematopoietic-restricted knockout of Osm largely prevented the surgein granulocyte levels (Fig. 5G and Supplementary Fig. 12)and in the G-to-L ratio induced by diabetes (Fig. 5H). Thesedata indicated that Osm deletion prevented hyperglyce-mia-induced myelopoiesis and mobilopathy in a hemato-poietic cell–intrinsic manner.

p66Shc Is Required for the Stem Cell–Retaining Activityof OSMSince Osm2/2 mice phenocopied p66Shc2/2 mice in protect-ing against diabetes-induced myelopoiesis and mobilopathy,we hypothesized that OSM signaling required down-stream p66Shc. OSM signals through heterodimers ofthe OSM receptor (OSMR) and gp130, which elicit intra-cellular events leading to activation of theMAPK and JAK-STAT3/5 pathways (33). Shc proteins cooperate withother adaptor proteins to transduce membrane receptorsignals to MAPK. We previously found that both STAT3and MAPK are required for Cxcl12 induction by OSM inBM stromal cells (22). Here, we hypothesized that theadaptor function of p66Shc is required for OSMR signaltransduction to MAPK to induce Cxcl12 (model shown inFig. 6A). We found that the ability of OSM (30 ng/mL;;1 mmol/L) to stimulate Cxcl12 gene expression inBM-derived stromal cells (3.6 times) was completelyabolished in the absence of p66Shc (Fig. 6B). The

concentration of OSM was chosen based on a previousdose-effect curve (22).

In contrast to p46 and p52, p66Shc acts as both anadaptor protein for signaling cascades and a mitochondrialredox protein (18). To dissect whether mitochondrialfunction of p66Shc was required for OSM signaling andCxcl12 induction, we transfected p66Shc2/2 MEFs with anempty vector or vectors encoding Wt p66, 36Ser→Ala mu-tated p66 (which cannot translocate to mitochondria), ora catalytically inactive p66 (p66qq), and then treated MEFswith OSM: Cxcl12 induction by OSM in p66Shc2/2 MEFswas rescued by expression of Wt, Ser36 mutated, or cat-alytically inactive p66Shc but not empty vector (Fig. 6C),suggesting that the adaptor function, and not the mito-chondrial function, of p66Shc was required for OSMsignaling. In addition, we found that activation of ERKby OSM was abolished in BM-derived stromal cells fromp66Shc2/2mice, while activation of STAT3 was unaffected(Fig. 6D). This set of experiments is in line with the modeldepicted in Fig. 6E, where p66Shc is recruited to OSMRand cooperates to activate the MAPK pathway, which,along with STAT3 activation via gp130, is needed to induceCxcl12.

Finally, to gather in vivo evidence that OSM signalingrequires p66Shc, we treated mice with systemic OSMinjections. Gene expression of Cxcl12 in the BM wassignificantly induced in Wt (4.5 times; P = 0.01) and inOsm2/2 mice (7.9 times; P = 0.01) but not in p66Shc2/2

mice (0.8 times; P = 0.77) (Fig. 7F). In parallel, the morethan twofold higher levels of HSPCs observed in thesteady-state basal condition in Osm2/2 and in p66Shc2/2

mice could be significantly suppressed by OSM injection inOsm2/2mice (P = 0.04) but not in p66Shc2/2mice (Fig. 6G).These data support the concept that regulation of HSPCtrafficking by OSM via Cxcl12 requires p66Shc. Further-more, injection of OSM increased circulating granulocytesand reduced lymphocytes in both Wt and Osm2/2 mice,thereby increasing twofold the G-to-L ratio, but this effectwas absent in p66Shc2/2 mice (Fig. 6H), demonstrating thatthe effect of OSM on myelopoiesis is also dependent onp66Shc.

DISCUSSION

Defective HSPC mobilization in response to G-CSF isa consistent finding in experimental and human diabetes(8), but the underlying causes are incompletely under-stood. Our new data indicate that mobilopathy is inti-mately linked with myelopoiesis, an underlying driver ofdiabetes-associated inflammation. We herein show a novelOSM-p66Shc signaling pathway that is overactive in di-abetes against HSPC mobilization via mechanisms that arehematopoietic cell intrinsic and extrinsic (Fig. 7). OSM isproduced by myeloid inflammatory cells that are exceed-ingly present in the diabetic BM as part of the enhancedmyelopoiesis induced by hyperglycemia (22). In turn, OSMsignal transduction is activated in BM stromal cells via

Figure 7—Schematic representation of the link between myelopoi-esis and mobilopathy exerted by the OSM-p66Shc signaling path-way. PMNs, polymorphonuclear cells; MF, macrophages; M,mitochondrion; N, nucleus. Red bullets marked with “+” denotestimulatory effects.

1312 OSM-p66Shc Regulates Mobilopathy/Myelopoiesis Diabetes Volume 68, June 2019

nonmitochondrial p66Shc to induce CXCL12 production,thereby retaining HSPCs in the BM niche (Fig. 6). Notably,p66Shc also mediates microvascular remodeling of the di-abetic BM that can jeopardize HSPC traffic (Fig. 4). G-CSFexerts its mobilizing function by acting on hematopoietic cellsand on the BM stroma (21). Remarkably, both hemato-poietic and nonhematopoietic p66Shc deletion was neededto restoreHSPCmobilization response toG-CSF indiabetes (Fig.3). Hematopoietic-restricted p66Shc deletion partially res-cued mobilization in diabetic mice, along with inhibitionof diabetes-induced MK expansion and myelopoiesis.Hyperglycemia-driven myelopoiesis arises from theskewed hematopoiesis stimulated by RAGE ligands (3), aneffect that we found requires p66Shc. At the same time,hematopoietic Osm deletion prevented diabetes-induced mye-lopoiesis, and the ability of OSM to stimulate myelopoiesis alsorequired p66Shc. The striking similarities between Osm2/2

and p66Shc2/2mice are indeed consistentwith the notion thatOSM couples myelopoiesis with mobilopathy via p66Shc.These data together indicate that activation of the OSM-p66Shc pathway drives diabetes-associated myelopoiesisin a cell-autonomous way, whereas its transcellular hemato-stromal activation links myelopoiesis to mobilopathy.

Understanding HSPC mobilization unresponsiveness toG-CSF has clinical implications for patients undergoingHSPC collection for transplantation purposes (8). Thus,interrupting the OSM-p66Shc pathway provides a thera-peutic strategy in conditions of poor HSPC mobilization,like diabetes. Diabetic stem cell mobilopathy precedesreduction of steady-state PB HSPCs in human diabetes(7), which in turn has been linkedwithworsening of diabeticcomplications (11,12). Mobilopathy preceded the reductionof PBHSPCs also in diabetic mice (Fig. 1 and SupplementaryFig. 2). However, since our new data reveal a causal linkbetween myelopoiesis and mobilopathy, future studiesshould better clarify whether diabetes outcomes are morerelated to alterations in blood inflammatory cells, circulat-ing stem cells, or stem cell mobilization.

In summary, we provide evidence that an overactiveOSM-p66Shc pathway couples diabetes-associated myelo-poiesis with HSPC mobilopathy. In addition to rescuingHSPC mobilization, tackling this pathway in the BM couldprovide a new avenue for the improvements of thediabetes-related inflammation and complication risk.

Funding. The study was supported by the following grants to G.P.F.: EuropeanFoundation for the Study of Diabetes Novartis 2013 grant and Lilly 2016 grant,Ministry of University and Education Progetti di Rilevante Interesse Nazionale(PRIN) grant 2015, Italian Diabetes Society/Lilly grant 2017, and FondazioneCariplo 2016-0922.Duality of Interest.M.A., S.C., and G.P.F. are the inventors of a patent, heldby the University of Padova, on the use of pharmacologic OSM inhibition to allowstem cell mobilization. No other potential conflicts of interest relevant to this articlewere reported.Author Contributions. M.A, S.C., S.T., L.M., V.S., R.C., G.Z., A.R., A.C.,and M.G. performed the research. M.A., S.C., S.T., V.S., and G.P.F. analyzed thedata. M.A., S.C., A.A., and G.P.F. designed the research and wrote the manuscript.

M.A, S.C., S.T., L.M., M.D’A., V.S., R.C., G.Z., A.R., A.C., M.G, A.A., and G.P.F.reviewed and edited the manuscript. G.P.F. is the guarantor of this work and, assuch, had full access to all the data in the study and takes responsibility for theintegrity of the data and the accuracy of the data analysis.

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