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• W2510871661 abstract "•Uncoupling protein 2 (UCP2)-deficient bone marrow cells underwent a stronger adenosine triphosphate decline following H2O2 treatment.•UCP2-deficient bone marrow had a larger amount of lin−Sca-1+c-kit+ (LSK) cells in young mice.•UCP2-deficient bone marrow revealed a skewing towards myeloid cell lines throughout aging.•UCP2-deficient bone marrow correlated with diminished erythroid cells in young and old animals.•UCP2 deficiency was followed by significantly higher red blood cell distribution width in young and old animals. Progress of age-related hematopoietic diseases such as myelodysplastic syndrome has previously been linked to enhanced levels of reactive oxygen species (ROS). Uncoupling protein 2 (UCP2) was found to reduce mitochondrial ROS production through uncoupling of the respiratory chain. The impact of UCP2 loss and elevated ROS on hematopoiesis during aging has not yet been investigated. In this study, UCP2 knockout mice were analyzed at aging stages of 3, 12, and 24 months with respect to oxidative and energy status of bone marrow cells. Further, the cellular bone marrow subpopulation composition was characterized, as were the differential blood counts at all time points. UCP2 knockout mice revealed enhanced levels of mitochondrial superoxide in elderly animals. Following oxidative stress, adenosine triphosphate (ATP) levels decreased more in the knockout mice than in the wild type. Investigation of bone marrow and blood counts of the knockout mice revealed an enhanced amount of monocytes and neutrophils, as well as a decreased amount of B cells and impaired erythropoiesis throughout aging. In summary, UCP2 induces protective effects on ROS and ATP levels during aging. Additionally, the results suggest an imbalance in hematopoiesis because of the lack of UCP2. Progress of age-related hematopoietic diseases such as myelodysplastic syndrome has previously been linked to enhanced levels of reactive oxygen species (ROS). Uncoupling protein 2 (UCP2) was found to reduce mitochondrial ROS production through uncoupling of the respiratory chain. The impact of UCP2 loss and elevated ROS on hematopoiesis during aging has not yet been investigated. In this study, UCP2 knockout mice were analyzed at aging stages of 3, 12, and 24 months with respect to oxidative and energy status of bone marrow cells. Further, the cellular bone marrow subpopulation composition was characterized, as were the differential blood counts at all time points. UCP2 knockout mice revealed enhanced levels of mitochondrial superoxide in elderly animals. Following oxidative stress, adenosine triphosphate (ATP) levels decreased more in the knockout mice than in the wild type. Investigation of bone marrow and blood counts of the knockout mice revealed an enhanced amount of monocytes and neutrophils, as well as a decreased amount of B cells and impaired erythropoiesis throughout aging. In summary, UCP2 induces protective effects on ROS and ATP levels during aging. Additionally, the results suggest an imbalance in hematopoiesis because of the lack of UCP2. An age-related impairment of the hematopoietic system leads to a higher susceptibility to infections, as well as a higher incidence of myelodysplastic syndrome (MDS), in elderly individuals [1Geiger H. Denkinger M. Schirmbeck R. Hematopoietic stem cell aging.Curr Opin Immunol. 2014; 29: 86-92Crossref PubMed Scopus (45) Google Scholar]. Elevated levels of reactive oxygen species (ROS) were suggested to promote leukemia development in an MDS mouse model [2Chung Y.J. Robert C. Gough S.M. Rassool F.V. Aplan P.D. Oxidative stress leads to increased mutation frequency in a murine model of myelodysplastic syndrome.Leuk Res. 2014; 38: 95-102Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar]. Uncoupling protein 2 (UCP2) was reported to attenuate production of ROS, especially superoxide, through mild uncoupling of the respiratory chain [3Lambert A.J. Brand M.D. Superoxide production by NADH:ubiquinone oxidoreductase (complex I) depends on the pH gradient across the mitochondrial inner membrane.Biochem J. 2004; 382: 511-517Crossref PubMed Scopus (386) Google Scholar]. Uncoupling protein 2 is closely related to UCP1, which is expressed predominantly in brown adipose tissue and is responsible for thermogenesis [4Cannon B. Nedergaard J. Brown adipose tissue: Function and physiological significance.Physiol Rev. 2004; 84: 277-359Crossref PubMed Scopus (4525) Google Scholar]. However, it was found that UCP2 is not involved in thermogenesis but is expressed in various other tissues, such as kidney, spleen, muscle, brain, liver, and erythroid cells [5Andrews Z.B. Horvath T.L. Uncoupling protein-2 regulates lifespan in mice.Am J Physiol Endocrinol Metab. 2009; 296: E621-E627Crossref PubMed Scopus (88) Google Scholar, 6Flachs P. Sponarova J. Kopecky P. et al.Mitochondrial uncoupling protein 2 gene transcript levels are elevated in maturating erythroid cells.FEBS Lett. 2007; 581: 1093-1097Crossref PubMed Scopus (11) Google Scholar]. UCP2 does not influence the basal proton conductance level of the inner mitochondrial membrane. It is activated by reactive alkenals, which in turn are formed by superoxide peroxidation of membrane phospholipids. Thus, superoxide is thought to regulate its own production by a feedback loop involving reactive alkenals and the subsequent activation of UCPs (reviewed by Brand and Esteves [7Brand M.D. Esteves T.C. Physiological functions of the mitochondrial uncoupling proteins UCP2 and UCP3.Cell Metab. 2005; 2: 85-93Abstract Full Text Full Text PDF PubMed Scopus (635) Google Scholar]). The function of UCP2 was investigated in numerous studies of different tissues and the whole organism using a knockout mouse model (UCP2−/−). UCP2−/− mice were found to exhibit higher levels of ROS in liver [8Horimoto M. Fülöp P. Derdák Z. Wands J.R. Baffy G. Uncoupling protein-2 deficiency promotes oxidant stress and delays liver regeneration in mice.Hepatology. 2004; 39: 386-392Crossref PubMed Scopus (95) Google Scholar] and pancreatic islets [9Krauss S. Zhang C.Y. Scorrano L. et al.Superoxide-mediated activation of uncoupling protein 2 causes pancreatic beta cell dysfunction.J Clin Invest. 2003; 112: 1831-1842Crossref PubMed Scopus (333) Google Scholar]. Interestingly, erythroid cells of 3- to 4-month-old UCP2−/− mice were reported to express higher levels of mitochondrial ROS but lower levels of total intracellular ROS in isolated erythroid cells when compared with wild-type animals [10Elorza A. Hyde B. Mikkola H.K. Collins S. Shirihai O.S. UCP2 modulates cell proliferation through the MAPK/ERK pathway during erythropoiesis and has no effect on heme biosynthesis.J Biol Chem. 2008; 283: 30461-30470Crossref PubMed Scopus (28) Google Scholar]. In the same study, knockout animals displayed attenuated reticulocyte regeneration after induction of hemolytic anemia [10Elorza A. Hyde B. Mikkola H.K. Collins S. Shirihai O.S. UCP2 modulates cell proliferation through the MAPK/ERK pathway during erythropoiesis and has no effect on heme biosynthesis.J Biol Chem. 2008; 283: 30461-30470Crossref PubMed Scopus (28) Google Scholar]. UCP2−/− mice were also found to have a decreased life span [5Andrews Z.B. Horvath T.L. Uncoupling protein-2 regulates lifespan in mice.Am J Physiol Endocrinol Metab. 2009; 296: E621-E627Crossref PubMed Scopus (88) Google Scholar]. However, the longitudinal influence on the hematopoietic system during aging remains unclear. In our study, we investigated hematopoiesis in the context of aging by analyzing bone marrow cells (BMCs) and peripheral blood in UCP2−/− mice and the wild-type strain C57BL/6J (B6/J). Our findings revealed an impact of the UCP2 knockout on the susceptibility of BMCs to oxidative stress, as well as on the subpopulations of bone marrow (BM) and peripheral blood. UCP2−/− mice were purchased from Charles River Laboratories (Sulzfeld, Germany). C57BL/6J wild-type strain animals were bred in the central animal facility of the University of Rostock. For analyses, male and female animals at the ages of 3, 12, and 24 months were investigated. Performed experiments were in line with the German Animal Protection Act represented by Animal Care Committee of Mecklenburg—Western Pomerania. Animals were sacrificed under narcosis (ketamine 65 mg/kg body weight and xylazine 13 mg/kg body weight) by cervical dislocation. Femurs and tibias were freed of any tissue, cut at both sides, and flushed with phosphate-buffered saline. Cells were collected in Falcon tubes and kept on ice until further procedures. Blood samples were obtained from the retro-orbital plexus using an EDTA-coated capillary and collected in EDTA-coated tubes (Sarstedt, Nürnbrecht, Germany). Samples were diluted fourfold with saline for analysis on an Advia 2120 hematology analyzer (Siemens Healthcare, Erlangen, Germany). Levels of ROS and adenosine triphosphate (ATP) were measured in whole BMCs without any further cell depletion or enrichment. For complete intracellular ROS measurement, BMCs were incubated with 50 μmol/L 2′,7′-dichlorofluorescein diacetate (DCFH-DA, Sigma, Taufkirchen, Germany) for 30 min. Fluorescence of 5 × 104 cells was measured using the Glomax device (Promega, Mannheim, Germany) at a wavelength of 485 nm. Bone marrow cells were incubated with MitoSOX (MS, Invitrogen, Darmstadt, Germany) to evaluate the amount of cells containing mitochondrial superoxide. After staining with 10 nmol/L MS for 10 min at 37°C, cells were incubated in 100 μL annexin binding buffer containing 4 μL annexin V–allophycocyanin (APC) (Becton-Dickinson, Heidelberg, Germany). Cells were analyzed with the FACSCalibur (Becton-Dickinson), and the proportion of nonapoptotic, MS positive cells was counted. Levels of ATP were analyzed by using the ATP Bioluminescence Assay Kit HS II (Roche, Mannheim, Germany). In brief, bioluminescence of cell lysates of 5 × 104 cells mixed with an automatic injection of luciferase reagent was measured in triplicate using the Glomax device. By use of a standard serial dilution of ATP, the concentration in cell lysates could be determined. To investigate the influence of oxidative stress on BMCs, cells were treated with 10 and 100 μmol/L H2O2 for 30 min prior to measurement. To evaluate the proportions of the different subpopulations in the BM, cells were stained with fluorescent antibodies (all obtained from Becton-Dickinson) against different surface antigens: lymphocytes (IgG2 anti-CD3ε phycoerythrin [PE], Catalog No. 553240); IgG2a anti-CD45R fluorescein isothiocyanate (FITC, Catalog No. 553087); erythroid cells (IgG2b anti-Ter-119 APC, Catalog No. 557909); IgG1 anti-CD71 PE (Catalog No. 553267); stem cells (IgG2b anti-c-kit PE, Catalog No. 553355); IgG2a anti-Sca-1 PE-Cy7 (Catalog No. 558162); lineage cocktail APC (Catalog No. 558074); IgG2a anti-CD34 FITC (Catalog No. 560238); monocytes and granulocytes (IgG2b anti-CD45 FITC [Catalog No. 553079], IgG2b anti-CD11b PE [Catalog No. 553311], IgG2b anti-Gr1 APC [Catalog No. 553129]); and corresponding isotype controls (hamster-IgG2 PE [Catalog No. 550085], iso lineage cocktail APC [Catalog No. 558074], rat-IgG2a FITC [Catalog No. 553929], rat-IgG2b FITC [Catalog No. 553988], rat-IgG2a PE-Cy7 [Catalog No. 557855], rat-IgG2b PE [Catalog No. 556925], rat-IgG1 PE [Catalog No. 554685]). Stained cells were analyzed with FACSCalibur (Becton Dickinson). Total nucleated cells (TNCs), were gated and the proportions of the subpopulations were determined. Results within each experiment were described using means + standard deviations. Significance between strains was calculated using the Mann–Whitney U test (SPSS, Version 22). A p value < 0.05 was considered to indicate significance. Animals of the B6/J strain exhibited a significant decrease in basal total intracellular ROS levels from 3 to 24 months (Fig. 1A; Supplementary Table E1, online only, available at www.exphem.org). The UCP2−/− strain exhibited a decline in ROS from 3 to 12 months. Up to 24 months, there was a restoring increase reaching levels similar to those detected in young mice. Middle-aged UCP2−/− mice exhibited significantly lower ROS levels than B6/J mice (1.7 × 103 ± 0.6 vs. 4.3 × 103 ± 3.4 relative fluorescence units [RFU]). To evaluate the response of bone marrow to oxidative stress, cells were treated with H2O2 (10 and 100 μmol/L). Low-dose H2O2 led to an increase in total intracellular ROS with growing response to oxidative stress from 3 to 24 months in B6/J strain mice (2.3- to 4.7-fold). Compared with the B6/J strain, UCP2−/− mice 3 and 12 months old exhibited significantly stronger responses to oxidative stress (3.4- and 6.3-fold) and those 24 months old exhibited a significantly lower response (3.5-fold). The higher dose of H2O2 (100 μmol/L) increased ROS levels further. B6/J mice exhibited similar reaction intensities throughout the aging period (7.3- to 7.6-fold), whereas UCP2−/− exhibited an overall decreasing reaction from 3 to 24 months (7.5- to 5.7-fold). The higher H2O2 concentration (100 μmol/L) did not lead to significantly different reaction intensities between the two strains. Mitochondrial superoxide can be analyzed with MitoSOX (MS), which specifically reacts to superoxide with a red fluorescence signal measured by flow cytometry (Fig. 1B). Here, the B6/J strain exhibited a slight decrease from 3 to 24 months, whereas UCP2−/− mice had an increase and higher levels of superoxide at 24 months compared with B6/J mice (1.7 ± 0.8% vs. 0.6 ± 0.5% MS+ cells). Oxidative stress with low-dose H2O2 was followed by increasing levels of superoxide in both strains. Young and advanced-age mice B6/J mice had similar reactions, whereas UCP2−/− mice exhibited a significant decrease in response to oxidative stress from 3 to 24 months. Here, the reactions of 3- and 24-month-old mice were significantly lower than those of the B6/J strain. High-dose oxidative stress led to a stronger increase in superoxide levels in both strains. Although B6/J mice exhibited a slightly increasing reaction from 3 to 24 months, UCP2−/− animals exhibited decreasing reaction intensities. At 24 months, UCP2−/− mice had lower responses to H2O2 compared with B6/J mice. ATP content was measured by luciferase driven bioluminescence (Fig. 1C). Young and advanced-age animals (3 and 24 months old) of both strains had similar basal ATP levels (0.6–1.0 μmol/L). Low-dose H2O2 did not affect B6/J mice to an age of 12 months, whereas 24-month-old animals reacted with a decrease in ATP to 67.7 % of basal levels. The UCP2−/− strain responded to low-dose H2O2 with a decrease in ATP throughout aging (3 months: 53.0%, 24 months: 49.4%). ATP levels decreased significantly lower in the 3-month-old UCP2−/− animals compared with B6/J mice. High-dose H2O2 decreased ATP levels in both strains at all ages. Here, the UCP2−/− strain also responded more strongly (45.3–29.2%) than the B6/J strain (55.1–34.2%). Isolated BMCs were stained with fluorescence-labeled antibodies against specific surface antigens to analyze distinct subpopulations. The B6/J strain exhibited a relative increase in lin−Sca-1+c-kit+ (LSK) cells from 3 to 24 months (1.1–2.7 % of lin− cells), whereas UCP2−/− mice exhibited slightly decreasing fractions at the same ages (2.4–2.0%). Compared with the B6/J strain, 3-month-old UCP2−/− mice had a significantly higher proportion of LSK cells (Fig. 2A; Supplementary Table E2, online only, available at www.exphem.org). Long-term hematopoietic stem cells (LT-HSCs, CD34dim/−) decreased in the B6/J strain from 3 to 24 months. In contrast, UCP2−/− mice had a relative increase from 3 to 12 months, with a decrease to almost the initial value at 24 months. Although within the strains, B cells did not change significantly during aging, comparison of UCP2−/− with the wild type revealed differences. UCP2−/− mice had significantly lower fractions of B cells compared with B6/J mice at all three ages (7.0–9.0% of TNCs vs. 10.2–12.1% of TNCs). The proportion of T cells increased in both strains from 3 to 24 months. Here, UCP2−/− mice had a significantly higher fraction of T cells at 3 months than B6/J mice. Monocytes and granulocytes fractions increased significantly in B6/J mice during aging, whereas UCP2−/− exhibited constant fractions of both subpopulations. Monocyte proportions were significantly higher in UCP2−/− mice throughout aging (68.3–71.6% of TNCs vs. 54.0–62.4% of TNCs). Additionally, granulocytes were significantly enhanced in UCP2−/− at 3 and 12 months compared with B6/J mice. Complete erythroid cells in bone marrow decreased significantly in B6/J mice from 3 to 24 months, whereas UCP2−/− mice exhibited only a slight overall decrease (Fig. 2B; Supplementary Table E2). The 3- and 24-month-old UCP2−/− mice exhibited significantly lower levels of erythroid cells than the B6/J mice (18.7–14.6% of TNCs vs. 30.3–23.8% of TNCs). Orthochromatic erythroblasts decreased significantly in B6/J mice during aging, whereas UCP2−/− mice had a slight increase. Here, 3- and 12-month-old UCP2−/− animals exhibited significantly fewer orthochromatic erythroblasts than B6/J mice at 3 and 12 months (1.4% of TNCs vs. 2.7–3.1% of TNCs). Blood counts at 3, 12, and 24 months were analyzed to investigate the function of the aging hematopoietic system (Fig. 3; Supplementary Table E3, online only, available at www.exphem.org). Erythrocytes (red blood cells) increased significantly during the aging period in both strains. UCP2−/− mice exhibited a consistently higher percentage of red blood cell distribution width (RDW) compared with B6/J mice, with significantly higher levels in 3-month-old (14.7 ± 0.6% vs. 13.6 ± 0.5%) and 24-month-old (14.7 ± 1.0% vs. 13.3 ± 0.3%) mice. Hematocrit (HCT) increased in both strains, but the increase was more distinct in B6/J mice than in UCP2−/− mice. Leukocytes (white blood cells [WBCs]) exhibited an overall increase in both strains during aging, which was more distinct in the UCP2−/− strain. B6/J mice had a slight increase in lymphocytes during aging, whereas UCP2−/− mice exhibited a more distinct enhancement. The proportion of lymphocytes in WBCs was lower in UCP2−/− mice than in B6/J mice throughout aging, with significantly lower levels at 12 months (78.0 ± 16.0% of WBCs vs. 92.6 ± 1.8% of WBCs). The B6/J strain displayed a minor increase in the level and proportion of neutrophils in WBCs during aging. UCP2−/− exhibited a significant enhancement of the amount of neutrophils, with significantly higher levels at 12 months (0.3 ± 0.1 × 103/μL vs. 0.2 ± 0.1 × 103/μL) and 24 months (0.5 ± 0.1 × 103/μL vs. 0.3 ± 0.1 × 103/μL) compared with B6/J mice. Furthermore, the proportion of neutrophils in WBCs underwent a more distinct increase in UCP2−/− than B6/J mice from 3 to 24 months. UCP2−/− mice are a well-investigated model for examination of the influence of oxidative stress on specific tissues and the whole organism. With this strain, the impact of oxidative stress on life span [5Andrews Z.B. Horvath T.L. Uncoupling protein-2 regulates lifespan in mice.Am J Physiol Endocrinol Metab. 2009; 296: E621-E627Crossref PubMed Scopus (88) Google Scholar] and erythropoiesis [10Elorza A. Hyde B. Mikkola H.K. Collins S. Shirihai O.S. UCP2 modulates cell proliferation through the MAPK/ERK pathway during erythropoiesis and has no effect on heme biosynthesis.J Biol Chem. 2008; 283: 30461-30470Crossref PubMed Scopus (28) Google Scholar] has been revealed. However, longitudinal monitoring of UCP2−/− mice with a focus on the hematopoietic system has not been performed so far. Here, we comparatively analyzed UCP2−/− and wild-type B6/J strains at ages 3, 12, and 24 months. We investigated the oxidative and energy status in BMCs, as well as the cellular constitution of subpopulations in the bone marrow, and analyzed blood counts. An overview of the most important differences in the UCP2−/− strain is provided in Figure 4. Earlier studies revealed an enhancement of ROS in several tissues of UCP2−/− mice such as liver [8Horimoto M. Fülöp P. Derdák Z. Wands J.R. Baffy G. Uncoupling protein-2 deficiency promotes oxidant stress and delays liver regeneration in mice.Hepatology. 2004; 39: 386-392Crossref PubMed Scopus (95) Google Scholar], pancreatic islets [9Krauss S. Zhang C.Y. Scorrano L. et al.Superoxide-mediated activation of uncoupling protein 2 causes pancreatic beta cell dysfunction.J Clin Invest. 2003; 112: 1831-1842Crossref PubMed Scopus (333) Google Scholar], and macrophages [11Arsenijevic D. Onuma H. Pecqueur C. et al.Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production.Nat Genet. 2000; 26: 435-439Crossref PubMed Scopus (940) Google Scholar], compared with the wild-type strain. A further study investigated isolated erythroid BMCs of young UCP2−/− mice and revealed significantly higher levels of mitochondrial ROS and significantly lower levels of total intracellular ROS [10Elorza A. Hyde B. Mikkola H.K. Collins S. Shirihai O.S. UCP2 modulates cell proliferation through the MAPK/ERK pathway during erythropoiesis and has no effect on heme biosynthesis.J Biol Chem. 2008; 283: 30461-30470Crossref PubMed Scopus (28) Google Scholar]. Furthermore, in a mitochondrial DNA mutator mouse model, UCP2 deficiency led to no further increase in oxidative stress in heart muscle cells [12Kukat A. Dogan S.A. Edgar D. et al.Loss of UCP2 attenuates mitochondrial dysfunction without altering ROS production and uncoupling activity.PLoS Genet. 2014; 10: e1004385Crossref PubMed Scopus (57) Google Scholar]. Here, we observed no significant differences in young animals with respect to mitochondrial and total cellular ROS in all BMCs. However, 24-month-old UCP2−/− mice exhibited higher levels of mitochondrial superoxide, revealing a higher oxidative load in old animals in our study. Though basal levels of ATP did not differ significantly between strains during aging, the responding decrease in ATP levels after oxidative stress was stronger in UCP2−/− mice. Thus, apparently the anti-oxidative defense after additional oxidative stress seems to include a more intensive cutoff of ATP synthesis, as in wild-type animals. In our study, the lack of UCP2 significantly affected the distribution of BMC subpopulations. Throughout aging, myelopoietic cells of UCP2−/− mice were enhanced compared with those of wild-type mice, whereas erythroid cells were diminished. Significantly larger proportions of LSK and T cells were observed only in young mice. Furthermore, UCP2−/− had significantly lower fractions of B cells in bone marrow throughout aging. The proportion of orthochromatic erythroblasts, the last precursor prior to blood entry, was significantly lower in young and middle-aged UCP2−/− animals than in B6/J mice. Although in peripheral blood no decreases in the amount of erythrocytes were detected, the RDW was significantly higher in UCP2−/− mice, indicating hampered erythropoiesis after all. The myeloid skewing in BMCs in UCP2−/− mice seen throughout aging was also observable in peripheral blood at 12 and 24 months, with significantly higher fractions of neutrophil granulocytes compared with the wild type. Together these results indicate attenuations in lymphopoiesis and erythropoiesis in the UCP2−/− strain throughout aging. Various studies have revealed that middle-aged but not young UCP2−/− mice tend to higher inflammatory responses observed in autoimmune encephalomyelitis with concomitant elevated ROS in T cells [13Vogler S. Pahnke J. Rousset S. et al.Uncoupling protein 2 has protective function during experimental autoimmune encephalomyelitis.Am J Pathol. 2006; 168: 1570-1575Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar], ischemic brain damage with correlating suppression of antioxidant genes [14Haines B.A. Mehta S.L. Pratt S.M. Warden C.H. Li P.A. Deletion of mitochondrial uncoupling protein-2 increases ischemic brain damage after transient focal ischemia by altering gene expression patterns and enhancing inflammatory cytokines.J Cereb Blood Flow Metab. 2010; 30: 1825-1833Crossref PubMed Scopus (61) Google Scholar], and acute pancreatitis [15Müller S. Kaiser H. Krüger B. et al.Age-dependent effects of UCP2 deficiency on experimental acute pancreatitis in mice.PLoS One. 2014; 9: e94494Crossref PubMed Scopus (15) Google Scholar]. These observations match our findings of higher levels of myeloid cells in BM and peripheral blood, representing a possible explanation for an enhanced inflammatory capacity. Additionally, an impaired humoral immune response was observed in UCP2 deficiency [16Cao T. Dong Y. Tang R. Chen J. Zhang C.Y. Zen K. Mitochondrial uncoupling protein 2 protects splenocytes from oxidative stress-induced apoptosis during pathogen activation.Cell Immunol. 2013; 286: 39-44Crossref PubMed Scopus (13) Google Scholar] correlating with the reduced B cells found in our study. Furthermore, it was suggested that after T-cell activation, UCP2 is involved in the regulation of metabolism and proliferation of T cells and is therefore important for the immune response [17Rupprecht A. Bräuer A.U. Smorodchenko A. et al.Quantification of uncoupling protein 2 reveals its main expression in immune cells and selective up-regulation during T-cell proliferation.PLoS One. 2012; 7: e41406Crossref PubMed Scopus (35) Google Scholar]. Thus, the lack of UCP2 might impair the adapted immune response and lead to a compensatory enhanced activation of the innate immune response, which is represented primarily by myelopoietic cells. Hence, the enhanced amount of myelopoietic cells with reduced levels of lymphocytes in BM and blood in our study might be attributable to these circumstances. In summary, the lack of UCP2 enhanced the oxidative load in old mice as well as increased the susceptibility of ATP levels to oxidative stress throughout aging. Furthermore, the composition of BM shifted toward the myeloid cells at all ages investigated. This enhanced myelopoiesis was also observed in peripheral blood in middle-aged and old animals. Consequently, UCP2 appears to have an important influence on the balance between murine myelopoiesis and lymphopoiesis during aging. The distinct signaling pathways influencing hematopoiesis via UCP2 should be further elucidated. This might be accomplished by comprehensive gene and protein expression analyses. Thus, the impact on age-related development of MDS might be clarified and possible treatment strategies suggested. This work was funded by the Federal Ministry of Education and Research (BMBF) as part of the GerontoSys initiative (ROSAge project), Funding No. 0315892A. There is no conflict of interest to declare. Supplementary Table E1Oxidative state of bone marrow cells during agingParameterB6/JUCP2−/−3 months12 months24 months3 months12 months24 monthsROS Basal (RFU)8.6 ± 5.44.3 ± 3.43.4 ± 1.85.8 ± 1.71.7 ± 0.65.0 ± 0.9 H2O2 (fold)10 μmol/L2.3 ± 1.23.1 ± 0.94.7 ± 1.03.4 ± 1.46.3 ± 1.53.5 ± 0.6100 μmol/L7.3 ± 3.67.5 ± 1.27.6 ± 0.87.5 ± 3.310.8 ± 4.15.7 ± 0.9Superoxide Basal (% of TNCs)0.9 ± 0.61.1 ± 0.40.6 ± 0.50.8 ± 0.50.9 ± 0.61.7 ± 0.8 H2O2 (fold)10 μmol/L10.7 ± 3.74.6 ± 2.010.2 ± 1.36.8 ± 3.69.3 ± 8.54.3 ± 2.3100 μmol/L14.7 ± 7.510.1 ± 2.417.6 ± 2.516.5 ± 10.114.0 ± 4.18.8 ± 2.2ATP Basal (μmol/L)0.6 ± 0.21.2 ± 0.30.8 ± 0.30.7 ± 0.40.8 ± 0.31.0 ± 0.2 H2O2 (%)10 μmol/L102.5 ± 26.498.0 ± 52.367.7 ± 26.853.0 ± 26.367.0 ± 23.049.4 ± 7.7100 μmol/L55.1 ± 21.046.9 ± 17.934.2 ± 18.445.3 ± 27.435.0 ± 14.629.2 ± 8.1ATP = adenosine triphosphate; ROS = reactive oxygen species; RFU = relative fluorescence units.Values are means ± standard deviation. Boldface type indicates significant differences of the UCP2−/− strain compared with the B6/J strain at the same aging stage. Open table in a new tab Supplementary Table E2Distribution of bone marrow subpopulations during agingParameterB6/JUCP2−/−3 months12 months24 months3 months12 months24 monthsLSK (% of lin−)1.1 ± 0.41.7 ± 0.42.7 ± 0.92.4 ± 1.01.6 ± 0.52.0 ± 0.6LT-HSC (% of LSK)75.6 ± 8.780.3 ± 4.864.3 ± 7.966.1 ± 13.188.2 ± 6.970.2 ± 8.4(% of TNC) B cells12.1 ± 4.210.2 ± 1.811.9 ± 1.79.0 ± 4.07.0 ± 0.97.0 ± 2.6 T cells2.0 ± 0.44.2 ± 0.84.3 ± 1.12.6 ± 0.63.1 ± 1.04.6 ± 1.9 Monocytes54.0 ± 7.660.8 ± 5.862.4 ± 5.368.3 ± 10.171.6 ± 5.169.6 ± 3.9 Granulocytes52.3 ± 8.061.4 ± 6.061.7 ± 4.865.7 ± 9.870.2 ± 4.965.5 ± 4.8 Erythroids total30.3 ± 4.626.1 ± 4.223.8 ± 5.618.7 ± 7.418.7 ± 8.114.6 ± 2.5 Basophilic/chromatophilic27.6 ± 4.123.0 ± 4.022.2 ± 5.917.3 ± 7.417.3 ± 8.112.9 ± 2.4 Orthochromatic2.7 ± 1.03.1 ± 0.81.5 ± 0.51.4 ± 0.51.4 ± 0.51.8 ± 0.3LSK = lin-, Sca-1+, c-kit+ cells; LT-HSC = long-term hematopoietic stem cells; TNC = total nucleated cells.Values are means ± standard deviation. Boldface type indicates significant differences of UCP2−/− strain compared with B6/J strain at the same aging stage. Open table in a new tab Supplementary Table E3Blood count parameter during agingParameterB6/JUCP2−/−3 months12 months24 months3 months12 months24 monthsRBC (× 106/μL)8.5 ± 0.59.5 ± 0.610.2 ± 1.08.7 ± 1.88.2 ± 2.09.2 ± 1.7RDW (%)13.6 ± 0.514.2 ± 1.713.3 ± 0.314.7 ± 0.614.7 ± 0.614.7 ± 1.0HCT (%)40.9 ± 4.945.6 ± 2.649.2 ± 5.840.5 ± 7.838.7 ± 9.543.0 ± 5.5WBCs (× 103/μL)4.6 ± 1.34.4 ± 1.65.7 ± 3.53.7 ± 1.24.5 ± 1.05.5 ± 3.6Lymphocytes (× 103/μL)4.1 ± 1.24.1 ± 1.55.0 ± 3.03.1 ± 0.93.6 ± 1.36.6 ± 3.7 (% of WBCs)87.9 ± 5.592.6 ± 1.888.9 ± 2.983.9 ± 6.678.0 ± 16.084.8 ± 8.8Neutrophils (× 103/μL)0.2 ± 0.20.2 ± 0.10.3 ± 0.10.2 ± 0.10.3 ± 0.10.5 ± 0.1 (% of WBCs)5.1 ± 3.33.6 ± 1.35.7 ± 2.16.6 ± 1.77.6 ± 3.09.5 ± 6.7RBC = red blood cell; RDW = red blood cell distribution width; HCT = hematocrit; WBCs = white blood cells.Values are means ± standard deviation. Boldface type indicates significant differences of UCP2−/− strain compared with B6/J strain at the same aging stage. Open table in a new tab Supplementary Table E4Numbers of animals used in this study for analysis of ROS, Mitosox, and ATP levels; immunophenotyping; and blood count analysisParameterStrain3 months12 months24 monthsROS levelC57BL/6J766UCP2−/−1166Mitosox levelC57BL/6J766UCP2−/−666ATP levelC57BL/6J766UCP2−/−1266ImmunophenotypingC57BL/6J966UCP2−/−1266Blood countC57BL/6J666UCP2−/−666ATP = adenosine triphosphate; ROS = reactive oxygen species. Open table in a new tab ATP = adenosine triphosphate; ROS = reactive oxygen species; RFU = relative fluorescence units. Values are means ± standard deviation. Boldface type indicates significant differences of the UCP2−/− strain compared with the B6/J strain at the same aging stage. LSK = lin-, Sca-1+, c-kit+ cells; LT-HSC = long-term hematopoietic stem cells; TNC = total nucleated cells. Values are means ± standard deviation. Boldface type indicates significant differences of UCP2−/− strain compared with B6/J strain at the same aging stage. RBC = red blood cell; RDW = red blood cell distribution width; HCT = hematocrit; WBCs = white blood cells. Values are means ± standard deviation. Boldface type indicates significant differences of UCP2−/− strain compared with B6/J strain at the same aging stage. ATP = adenosine triphosphate; ROS = reactive oxygen species." @default.
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• W2510871661 title "Uncoupling protein 2 deficiency results in higher neutrophil counts and lower B-cell counts during aging in mice" @default.
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