Diabetes-Associated Myelopoiesis Drives Stem Cell Mobilopathy Through an OSM-p66Shc Signaling Pathway

Diabetes is associated with low-grade inflammation, which contributes to chronic complications (1,2). A skewed differentiation of common myeloid progenitors (CMPs) translates hyperglycemia into production of proinflammatory cells (3). Such enhanced myelopoiesis propagates inflammation from the bone marrow (BM) to the adipose and the vasculature, 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-stimulating factor (G-CSF) is impaired in murine (5,6) and human diabetes (7,8), a condition termed mobilopathy (9).

We herein hypothesize that myelopoiesis and mobilopathy, described as two distinct pathological features of the diabetic BM, are instead mechanistically linked. Disentangling the processes linking myelopoiesis to mobilopathy has relevant clinical implications. First, pharmacologic mobilization of HSPCs is the gold standard for HSPC transplantation (10), and failure to collect robust numbers of HSPCs can delay engraftment, thereby worsening the outcome of patients with diabetes undergoing transplantation (5). Second, reduction of circulating HSPCs in patients with diabetes predicts the future development of micro- and macrovascular complications (11,12). Glucose control effectively prevents myelopoiesis and partially rescues HSPC mobilization (3,13), but many patients fail to achieve necessary glucose targets. Therefore, disconnecting mobilopathy from myelopoiesis can provide a direct therapeutic strategy to restore normal HSPC mobilization.

Recent studies highlight that murine and human diabetes cause BM microvascular remodeling (14) and autonomic neuropathy (6,15), both of which can affect HSPC traffic (16,17). We previously found that BM denervation in diabetic mice accounts for impaired response to G-CSF and is mediated by p66Shc (6). Unlike p46 and p52, p66Shc functions both as an adaptor protein for membrane receptors and a redox enzyme. Upon phosphorylation at Ser³⁶, p66Shc translocates to the mitochondrial intermembrane space where it catalyzes the production of hydrogen peroxide (18), contributing to processes linked to oxidative stress, including diabetic complications (19,20).

Besides sympathetic nervous system activation, depletion of BM macrophages is a key event in the mobilization cascade induced by G-CSF, because macrophage paracrine activity sustains CXCL12 production (21). We have identified oncostatin M (OSM) as the macrophage-derived soluble factor that induces Cxcl12 expression in stromal cells, thereby antagonizing HSPC mobilization (22). OSM is a cytokine of the interleukin 6 family, which signals via mitogen-activated protein kinase (MAPK) and the Janus kinase (JAK)/STAT pathways, leading to pleiotropic functions, including modulation of inflammation and bone formation (23,24). In murine diabetes, excess BM macrophages result in persistent OSM signaling, inability to switch off CXCL12 levels after G-CSF, and impaired HSPC mobilization (22). Thus, OSM represents a candidate link between myelopoiesis and mobilopathy. In view of the similar benefits of p66Shc deletion and OSM inhibition on the diabetic stem cell mobilopathy (6,22), we have hypothesized that the two pathways are mechanistically connected. In the current study, we therefore examined the interplay between OSM and p66Shc in determining the link between myelopoiesis and mobilopathy observed in experimental and human diabetes.

C57BL/6J wild-type (Wt) mice were purchased from The Jackson Laboratory and established as a colony since 2001. p66Shc^−/− mice were originally obtained from Pelicci’s laboratory (European Institute of Oncology, Milan, Italy), a colony was established at our facility in 2010, and mice have been backcrossed on the C57BL/6J background for >10 generations. Osm^−/− mice on the C57BL/6J background were obtained from GlaxoSmithKline (Stevenage, U.K.), and a colony was established in 2015. For all the experiments, we used sex- and age-matched animals. Assignment of mice to treatments or experimental groups was based on a computer-generated random sequence. All animal studies were approved by the Venetian Institute of Molecular Medicine Animal Care and Use Committee and by the Italian Health Ministry.

Individuals with and without diabetes were recruited at the University Hospital of Padova Division of Metabolic Diseases. The protocols were approved by the local ethical committee and conducted in accordance with the Declaration of Helsinki as revised in 2000. Cross-sectional data on the association between myeloid bias and circulating HSPCs were derived from two previous studies that had been approved by the local ethics committee (6,25). Total and differential white blood cell (WBC) counts were determined in the same laboratory for both studies, and CD34⁺ HSPC levels were quantified by flow cytometry relative to the WBC count. Details are given in the previous publications (6,25). The study for G-CSF–induced mobilization was approved by the local ethics committee and is registered in ClinicalTrials.gov (NCT01102699). This was a prospective, parallel-group study of direct BM stimulation with G-CSF in subjects with and without diabetes. Specific methods for quantifying blood cells and HSPCs were given in the previous publication (7). Informed consent was obtained from all participants.

Diabetes was induced in 2-month-old mice by a single intraperitoneal injection of 175 mg/kg streptozotocin (STZ). Blood glucose was measured using a FreeStyle glucometer (Abbott, Abbott Park, IL). HSPC mobilization was induced by subcutaneous injection of 200 μg/kg/day G-CSF daily for 4 days. Three-month-old mice were treated with vehicle or carrier free recombinant mouse OSM (495-MO/CF; R&D Systems, Minneapolis, MN) at 0.5 μg per injection every 6 h for 48 h before analysis was performed. Total WBC count was performed using the CELL-DYN Emerald hematology analyzer (Abbott) on fresh EDTA-treated mouse blood.

Mouse embryonic fibroblasts (MEFs) were isolated from E13.5 p66Shc^−/− mice after digestion with trypsin (Corning) and cultured with DMEM and 10% FBS. PINCO retroviral particles were produced from the amphotropic packaging cell line Phoenix. Cells were infected with an empty vector, a vector encoding mouse full-length p66Shc, a vector encoding the mutants p66ShcS36A (S→A substitution at position 36) and p66ShcQQ (EE→QQ substitutions at positions 132–133). P3 MEFs were infected with three rounds of infection with Polybrene Infection/Transfection Reagent (Sigma-Aldrich), followed by 96 h of selection with 2 mg/mL puromycin. Experiments were performed with p4 or p5 cells.

Recipient mice (3 months old) were treated with a myeloablative dose of total body irradiation of 10 Gy, split in two doses of 5 Gy 3 h apart and followed by an intravenous injection of BM cells from donor mice (4 × 10⁷/each) isolated by flushing femurs and tibias with sterile ice-cold PBS.

BM cells (3 × 10⁴) were plated in 35-mm Petri dishes containing 1 mL methylcellulose-based medium MethoCult supplemented with 1% penicillin/streptomycin. After red blood cell lysis, 25 µL/well PB was plated in 24-well plates containing 0.5 mL MethoCult supplemented with 1% penicillin/streptomycin. Colony formation was scored after 10 days of culture. When required, murine recombinant S100A8/9 heterodimer (BioLegend) was mixed with the MethoCult medium.

Flow cytometry was performed on BM cells or EDTA-treated PB. BM cells were isolated by flushing femurs and tibias with ice-cold MACS Separation Buffer (Miltenyi Biotec GmbH, Gladbach, Germany) through a 40-μm cell strainer. Then 100 μL PB or BM cells was incubated with antibodies for 15 min in the dark at room temperature. After red blood cell lysis, samples were resuspended in 200 μL PBS, and data were acquired with a FACSCanto (BD Biosciences) cytometer, followed by analysis using FlowJo software (Tree Star).

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

Femur bones were fixed in 4% paraformaldehyde and decalcified. Bones were then washed with PBS, embedded in Killik cryostat medium (Bio-Optica, Milan, Italy), and frozen in liquid nitrogen–cooled 2-methylbutane (Sigma-Aldrich). Longitudinal 10-μm-thick femur sections were obtained with a Leica CM 1950 cryostat (Leica Biosystems S.r.l., Milan, Italy), placed on Superfrost Plus slides (J1800AMNZ; Gerhard Menzel GmbH, Braunschweig, Germany), and stored at −80°C.

Confluent BM-MSCs were treated with SCH772984 (4 nmol/L) (Cayman Chemical) overnight or with Stattic (2.5 μmol/L) (Selleckchem) for 1 h before adding recombinant OSM (R&D Systems) for 30 min in serum-free media. Cells were detached by scraping and incubated with PE mouse anti-Stat3 (pY705) or PE mouse anti-extracellular signal–regulated kinase (ERK)1/2 (pT202/pY204) (both from BD Biosciences) in Perm Buffer III (BD Biosciences) according to the manufacturer’s instructions.

Femur sections were air dried for 20 min and then incubated with blocking solution. Sections were incubated with 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 then washed with PBS and incubated with secondary antibodies. Slides were mounted with an antifade aqueous mounting medium. Images were taken with a Leica DM5000B microscope, equipped with a DFC300 FX CCD camera, or with Cytell (GE Healthcare, Milan, Italy). Images were then processed with Fiji/ImageJ 1.50 software (National Institutes of Health, Bethesda, MD) or with Adobe Photoshop CS2 9.0.2 software (Adobe Systems, San Jose, CA).

Vessel size and shape were measured using Fiji/ImageJ 1.50 software. Briefly, random 500 µm² fields from the epiphyseal and diaphyseal region (three each, at least) of the samples were analyzed. Vessel structure was visualized by laminin staining, and regions of interest were manually outlined in Fiji/ImageJ. Area and shape parameters, such as circularity, were recorded. The bivariate distribution of area and circularity was visualized using the Bivariate Kernel Density Estimation 1.0.9 in R 3.1 software. BM innervation was determined by Tyr-HO staining. Arterioles were identified in the whole femur section, and diameters of arterioles were measured with Fiji/ImageJ.

RNA was isolated from flushed BM or cells by using QIAzol or with Total RNA Purification Micro Kit (Norgen Biotek) and quantified with a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA). cDNA was synthesized using the SensiFAST cDNA Synthesis Kit (Bioline, London, U.K.). Quantitative PCR was performed using the SensiFAST SYBR Lo-ROX Kit (Bioline) via a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific). A list of primers can be found in Supplementary Table 1.

Continuous data are expressed as mean ± SEM, whereas categorical data are presented as the percentage. Normality was checked using the Kolmogorov-Smirnov test, and nonnormal data were log-transformed before analysis. Comparison between two or more groups was performed using the Student t test and ANOVA for normal variables or the Mann-Whitney U test and Kruskal-Wallis test for nonnormal variables that could not be log-transformed. Bonferroni adjustment was used to account for multiple testing. Linear correlations were checked using the Pearson r coefficient. Statistical analysis was accepted at P < 0.05. Statistical analysis was performed using GraphPad Prism 6, Matlab, and SPSS 21 software.

We first evaluated whether myelopoiesis and mobilopathy coexist in murine diabetes. We found that STZ-induced diabetic mice had an approximately twofold expansion of PB granulocytes compared with nondiabetic mice (P < 0.001) (Fig. 1A and B and Supplementary Fig. 1), resulting in a strikingly six times higher granulocyte-to-lymphocyte (G-to-L) ratio (P < 0.001) (Fig. 1C). The BM of diabetic mice contained higher numbers of granulocyte-monocyte progenitors (GMPs) at the expense of CMPs (Fig. 1D). As a consequence, the clonogenic assay of BM cells showed an increased output of macrophage and granulocyte colonies from diabetic versus nondiabetic mice (2.2 times and 2.8 times, respectively) (Fig. 1E). The diabetic BM showed excess macrophages both in basal unstimulated conditions and after G-CSF stimulation (Fig. 1F). These cells are known to produce OSM (22), and Osm gene expression in the BM of diabetic mice was indeed upregulated 7.7 times compared with that in nondiabetic mice (P = 0.006) (Fig. 1G). Altogether, these data are consistent with exaggerated myelopoiesis and myeloid bias in diabetic mice.

As the resulting overproduction of OSM can hamper mobilization (22), we evaluated whether mobilopathy occurred in the same mice. Preliminary to this, we verified that the baseline PB level of HSPCs (Lin⁻c-Kit⁺Sca-1⁺ [LKS] cells) was nonsignificantly different in diabetic versus nondiabetic mice (Supplementary Fig. 2). After G-CSF was administered for 4 days, the HSPC level increased by 4.38 times in nondiabetic but not in diabetic mice (Fig. 1H). This difference in the fold change of the PB-LKS cell level between diabetic and nondiabetic mice was highly significant (Fig. 1I): 80% of nondiabetic mice vs. 10% of diabetic mice achieved a mobilization response of at least 1.5-fold (Fig. 1J). The colony-forming unit (CFU) assay from PB cells confirmed the absence of functional HSPC mobilization in diabetes (Supplementary Fig. 3A). The profound degree of mobilization impairment allowed us to use the G-CSF mobilization assay as a robust readout for mobilopathy in subsequent mouse experiments.

We then checked whether myelopoiesis and mobilopathy occurred simultaneously in human diabetes. We first analyzed cross-sectional data of two studies wherein circulating WBC types and levels of CD34⁺ HSPCs were determined in the same sample (6,25) (Supplementary Table 2). In a pooled cohort 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 individuals without diabetes (Fig. 2A). Higher N-to-L ratio and lower CD34⁺ HSPCs remained significantly associated with diabetes after adjusting for age, sex, BMI, hypertension, lipids, coronary artery disease, and retinopathy (Supplementary Table 3). We also found a significant inverse correlation between the N-to-L ratio and the steady-state level of PB HSPCs (r = −0.28) (Fig. 2B). Considering glucose control as a continuous variable in the entire cohort, we found a significant inverse correlation between HbA_1c and HSPC levels (r = −0.23; P < 0.001) (Fig. 2C) and a direct correlation between HbA_1c and the N-to- L ratio (r = 0.21; P < 0.001) (Fig. 2D), which persisted (both with P < 0.01) after adjusting for the above-mentioned confounders. These data suggest that myeloid bias is linked to a reduction in HSPC levels in human diabetes, possibly driven by hyperglycemia.

Second, we evaluated whether myeloid bias was associated with mobilopathy by analyzing data from a previous prospective study wherein patients with and without diabetes (n = 43) received low-dose G-CSF to test HSPC mobilization (7). The fold change in G-CSF–induced HSPC 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 without diabetes (Fig. 2E). Remarkably, there was a significant inverse correlation between the N-to-L ratio and HSPC mobilization (r = −0.32; P = 0.03) (Fig. 2F). These results cannot prove causality, because secondary analyses of previously collected cohort data can be subjected to bias and prone to false-positive signals. Nonetheless, we confirm that myelopoiesis and mobilopathy are associated in human diabetes as they are in murine diabetes.

Having shown that myelopoiesis and mobilopathy concur in murine and human diabetes, we explored the mechanisms driving their association. We first focused on p66Shc, which we previously showed to be responsible for diabetes-associated BM denervation and mobilopathy (6). BM p66Shc gene expression was more than twofold higher in diabetic versus control mice (P = 0.003) (Supplementary Fig. 4), consistent with prior data in mice and humans (26,27). In the nondiabetic condition, we found no differences in WBC count and subtypes, G-to-L ratio, BM colonies, and CMPs/GMPs, as well as BM macrophages between Wt and p66Shc^−/− mice (Fig. 3A–G). However, in p66Shc^−/− mice, diabetes did not increase PB granulocytes counts, and granulocytes were significantly lower than in Wt diabetic mice (Fig. 3D and Supplementary Fig. 5), as was the G-to-L ratio (Fig. 3E). Furthermore, the diabetes-induced increase in myeloid CFUs (Fig. 3F), CMP/GMP imbalance (Fig. 3G), and excess BM macrophages (Fig. 3H) were completely prevented in p66Shc^−/− mice. As a result, the surge in BM expression of Osm observed in diabetic Wt mice, which derives from BM macrophages (22), was absent in p66Shc^−/− mice (Supplementary Fig. 6). The link between hyperglycemia and myelopoiesis has been attributed to the accumulation of advanced glycation end products (AGEs) and the receptor for the AGE (RAGE) ligand S100A8/9 (3). Interestingly, RAGE signaling has been previously linked with downstream p66Shc activation (28). We found that S100A8/9 potentiated myelopoiesis in vitro by BM cells of Wt mice, evidenced by a 1.75-fold and a 2.0-fold increase in macrophage and granulocyte colonies, respectively (Fig. 3I). However, such effect was completely abolished in p66Shc^−/− BM cells (Fig. 3J). In vivo treatment of nondiabetic Wt mice with S100A8/9 increased GMP and myeloid cell colonies, but such effect was not observed in p66Shc^−/− mice (Fig. 3K and Supplementary Fig. 7). Together, these data indicate that p66Shc is required for the effects of hyperglycemia on myelopoiesis, possibly by preventing the activity of S100 proteins.

In agreement with our previous study (6), p66Shc deletion partially rescued HSPC mobilization in diabetic mice, as indicated by the 1.9 times increase in LKS cell counts after G-CSF administration (Fig. 3L). The CFU assay showed that mobilized HSPCs were functionally competent, as G-CSF increased PB hematopoietic colonies both in diabetic and nondiabetic p66Shc^−/− mice (Supplementary Fig. 8). To dissect the hematopoietic-intrinsic and -extrinsic roles of p66Shc in regulating HSPC mobilization, we performed cross-transplantation experiments as illustrated in Fig. 3M. We confirmed that BM transplantation (BMT) did not impinge on G-CSF responsiveness, as nondiabetic but not diabetic Wt mice that received BMTs from Wt mice were able to mobilize functional HSPCs (Supplementary Fig. 3B and C). Wt mice that received BMTs from p66Shc^−/− mice (p66Shc^−/−→Wt BMT) and were rendered diabetic showed a partial restoration of HSPC increase after G-CSF stimulation. An almost identical, but partial, improvement was observed in diabetic p66Shc^−/− mice who received BMTs from Wt mice (Wt→p66Shc^−/− BMT), and in both cases, the fold change in HSPC count was lower than that in ubiquitous p66Shc^−/− diabetic mice (Fig. 3N). In contrast, while diabetes increased granulocyte counts and the G-to-L ratio in Wt→p66Shc^−/−, p66Shc^−/−→Wt BMT mice were largely protected from elevation of the G-to-L ratio induced by diabetes (Fig. 3O and Supplementary Fig. 9).

Knowing that myeloid-biased HSPCs reside in megakaryocytic (MK) niches (29), we analyzed MK density by staining BM sections with anti-CD150. As previously noted by others (30), MKs were increased by 60% in diabetic compared with nondiabetic Wt mice (P = 0.004). However, such an effect was not observed in p66Shc^−/− mice (Supplementary Fig. 10), providing a further explanation for the protection of ubiquitous and hematopoietic-restricted p66Shc^−/− mice from diabetes-induced myelopoiesis.

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

The partial restoration of G-CSF–induced HSPC mobilization in Wt→p66Shc^−/− BMT diabetic mice suggested that deletion of p66Shc exerted protective effects on the BM stroma against hyperglycemic damage. We previously reported that p66Shc^−/− mice were protected from BM sympathectomy induced by diabetes (6). We herein characterized microvascular BM remodeling in p66Shc^−/− versus Wt diabetic and nondiabetic mice. In the peculiar BM microcirculation, the nutrient arterial system drains into sinusoids with capillary-size vessels, and the irregular sinusoid lumen is occasionally compressed to capillary caliber (31). Using an unbiased autoinstructed procedure to score BM vessels (Fig. 4A), we found that the total vascular density and numbers of arterioles and sinusoids were similar, but a significant 2.5-fold reduction in capillary-size structures was noted in diabetic versus nondiabetic Wt mice (P = 0.006) (Fig. 4B). Remarkably, the density of BM capillary-size vessels was not reduced in p66Shc^−/− diabetic versus nondiabetic mice and was higher in p66Shc^−/− versus Wt diabetic mice (P = 0.03). With a more detailed morphometric analysis of BM blood vessel distribution according to size and circularity, we found that diabetes 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 BM and found that Tyr-OH⁺ sympathetic terminals were almost exclusively located close to arteriolar walls (Fig. 4D). The percentage of innervated arterioles was significantly reduced by more than twofold in diabetic versus nondiabetic Wt mice, but not in p66Shc^−/− mice (P < 0.001) (Fig. 4E and F). Taken together, these findings indicate that deletion of p66Shc protects BM from microvascular remodeling, which can explain the partial rescue of HSPC mobilization in nonhematopoietic p66Shc-deleted mice.

The hematopoietic cell–intrinsic mechanism whereby G-CSF exerts its mobilizing activity relies on suppression of BM macrophages (21). This pathway is independent from the stromal effect of G-CSF through nerve terminals, as mice sympathectomized by 6-OH dopamine showed a normal post–G-CSF suppression of BM macrophages despite being unable to mobilize HSPCs (Supplementary Fig. 11). This finding indicates that both hematopoietic and nonhematopoietic effects of G-CSF are required to yield a full HSPC mobilizing response and justifies the partial recovery of mobilization in Wt↔p66Shc^−/− cross-transplanted animals.

We previously demonstrated that antibody-mediated OSM neutralization allowed HSPC mobilization in diabetic mice by relieving the brake of CXCL12 produced by stromal cells (22). Consistent with the notion that OSM retains HSPCs in the BM niche, nondiabetic Osm^−/− mice displayed higher HSPC levels in unstimulated PB than Wt mice, which was not further increased by diabetes (Supplementary Fig. 2), and HSPC mobilization in diabetic mice was partially rescued toward normal levels (2.2 times) by genetic Osm deletion (P = 0.01) (Fig. 5A). In addition, we observed a marked (∼80%) reduction of BM macrophages in Osm^−/− mice, both in the diabetic and nondiabetic 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 required for accumulation of BM macrophages in a paracrine-autocrine loop, as already seen by others in the heart (32). Indeed, diabetic Osm^−/− mice had normal WBC counts (Fig. 5C) but significantly lower levels of PB granulocytes compared with Wt diabetic mice (Supplementary Fig. 12), and the G-to-L ratio was restored toward the levels seen in nondiabetic mice (Fig. 5D). To avoid the confounding factor of the absence of nonhematopoietic OSM in ubiquitous Osm^−/− mice, we transplanted BM cells from Osm^−/− mice into Wt mice and induced diabetes 4 weeks later (Fig. 5E). After 4 weeks of diabetes, we tested HSPC mobilization after G-CSF treatment and found that hematopoietic-restricted Osm deletion rescued HPSC mobilization in diabetic mice toward normal levels (5.2 times; P = 0.03) (Fig. 5F). In addition, hematopoietic-restricted knockout of Osm largely prevented the surge in granulocyte levels (Fig. 5G and Supplementary Fig. 12) and in the G-to-L ratio induced by diabetes (Fig. 5H). These data indicated that Osm deletion prevented hyperglycemia-induced myelopoiesis and mobilopathy in a hematopoietic cell–intrinsic manner.

Since Osm^−/− mice phenocopied p66Shc^−/− mice in protecting against diabetes-induced myelopoiesis and mobilopathy, we hypothesized that OSM signaling required downstream p66Shc. OSM signals through heterodimers of the OSM receptor (OSMR) and gp130, which elicit intracellular events leading to activation of the MAPK and JAK-STAT3/5 pathways (33). Shc proteins cooperate with other adaptor proteins to transduce membrane receptor signals to MAPK. We previously found that both STAT3 and MAPK are required for Cxcl12 induction by OSM in BM stromal cells (22). Here, we hypothesized that the adaptor function of p66Shc is required for OSMR signal transduction to MAPK to induce Cxcl12 (model shown in Fig. 6A). We found that the ability of OSM (30 ng/mL; ∼1 μmol/L) to stimulate Cxcl12 gene expression in BM-derived stromal cells (3.6 times) was completely abolished in the absence of p66Shc (Fig. 6B). The concentration of OSM was chosen based on a previous dose-effect curve (22).

In contrast to p46 and p52, p66Shc acts as both an adaptor protein for signaling cascades and a mitochondrial redox protein (18). To dissect whether mitochondrial function of p66Shc was required for OSM signaling and Cxcl12 induction, we transfected p66Shc^−/− MEFs with an empty vector or vectors encoding Wt p66, 36^Ser→Ala mutated p66 (which cannot translocate to mitochondria), or a catalytically inactive p66 (p66^qq), and then treated MEFs with OSM: Cxcl12 induction by OSM in p66Shc^−/− MEFs was rescued by expression of Wt, Ser³⁶ mutated, or catalytically inactive p66Shc but not empty vector (Fig. 6C), suggesting that the adaptor function, and not the mitochondrial function, of p66Shc was required for OSM signaling. In addition, we found that activation of ERK by OSM was abolished in BM-derived stromal cells from p66Shc^−/− mice, while activation of STAT3 was unaffected (Fig. 6D). This set of experiments is in line with the model depicted in Fig. 6E, where p66Shc is recruited to OSMR and cooperates to activate the MAPK pathway, which, along with STAT3 activation via gp130, is needed to induce Cxcl12.

Finally, to gather in vivo evidence that OSM signaling requires p66Shc, we treated mice with systemic OSM injections. Gene expression of Cxcl12 in the BM was significantly induced in Wt (4.5 times; P = 0.01) and in Osm^−/− mice (7.9 times; P = 0.01) but not in p66Shc^−/− mice (0.8 times; P = 0.77) (Fig. 7F). In parallel, the more than twofold higher levels of HSPCs observed in the steady-state basal condition in Osm^−/− and in p66Shc^−/− mice could be significantly suppressed by OSM injection in Osm^−/− mice (P = 0.04) but not in p66Shc^−/− mice (Fig. 6G). These data support the concept that regulation of HSPC trafficking by OSM via Cxcl12 requires p66Shc. Furthermore, injection of OSM increased circulating granulocytes and reduced lymphocytes in both Wt and Osm^−/− mice, thereby increasing twofold the G-to-L ratio, but this effect was absent in p66Shc^−/− mice (Fig. 6H), demonstrating that the effect of OSM on myelopoiesis is also dependent on p66Shc.

Defective HSPC mobilization in response to G-CSF is a consistent finding in experimental and human diabetes (8), but the underlying causes are incompletely understood. Our new data indicate that mobilopathy is intimately linked with myelopoiesis, an underlying driver of diabetes-associated inflammation. We herein show a novel OSM-p66Shc signaling pathway that is overactive in diabetes against HSPC mobilization via mechanisms that are hematopoietic cell intrinsic and extrinsic (Fig. 7). OSM is produced by myeloid inflammatory cells that are exceedingly present in the diabetic BM as part of the enhanced myelopoiesis induced by hyperglycemia (22). In turn, OSM signal transduction is activated in BM stromal cells via nonmitochondrial p66Shc to induce CXCL12 production, thereby retaining HSPCs in the BM niche (Fig. 6). Notably, p66Shc also mediates microvascular remodeling of the diabetic BM that can jeopardize HSPC traffic (Fig. 4). G-CSF exerts its mobilizing function by acting on hematopoietic cells and on the BM stroma (21). Remarkably, both hematopoietic and nonhematopoietic p66Shc deletion was needed to restore HSPC mobilization response to G-CSF in diabetes (Fig. 3). Hematopoietic-restricted p66Shc deletion partially rescued mobilization in diabetic mice, along with inhibition of diabetes-induced MK expansion and myelopoiesis. Hyperglycemia-driven myelopoiesis arises from the skewed hematopoiesis stimulated by RAGE ligands (3), an effect that we found requires p66Shc. At the same time, hematopoietic Osm deletion prevented diabetes-induced myelopoiesis, and the ability of OSM to stimulate myelopoiesis also required p66Shc. The striking similarities between Osm^−/− and p66Shc^−/− mice are indeed consistent with the notion that OSM couples myelopoiesis with mobilopathy via p66Shc. These data together indicate that activation of the OSM-p66Shc pathway drives diabetes-associated myelopoiesis in a cell-autonomous way, whereas its transcellular hemato-stromal activation links myelopoiesis to mobilopathy.

Understanding HSPC mobilization unresponsiveness to G-CSF has clinical implications for patients undergoing HSPC collection for transplantation purposes (8). Thus, interrupting the OSM-p66Shc pathway provides a therapeutic strategy in conditions of poor HSPC mobilization, like diabetes. Diabetic stem cell mobilopathy precedes reduction of steady-state PB HSPCs in human diabetes (7), which in turn has been linked with worsening of diabetic complications (11,12). Mobilopathy preceded the reduction of PB HSPCs also in diabetic mice (Fig. 1 and Supplementary Fig. 2). However, since our new data reveal a causal link between myelopoiesis and mobilopathy, future studies should better clarify whether diabetes outcomes are more related to alterations in blood inflammatory cells, circulating stem cells, or stem cell mobilization.

In summary, we provide evidence that an overactive OSM-p66Shc pathway couples diabetes-associated myelopoiesis with HSPC mobilopathy. In addition to rescuing HSPC mobilization, tackling this pathway in the BM could provide a new avenue for the improvements of the diabetes-related inflammation and complication risk.

Funding. The study was supported by the following grants to G.P.F.: European Foundation 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 Fondazione Cariplo 2016-0922.

Duality of Interest. M.A., S.C., and G.P.F. are the inventors of a patent, held by the University of Padova, on the use of pharmacologic OSM inhibition to allow stem cell mobilization. No other potential conflicts of interest relevant to this article were 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 the data. 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, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.