- Email: [email protected]
Inhibitory role of oxytocin on TNFα expression assessed in vitro and in vivo
G Model
DIABET-938; No. of Pages 4 Diabetes & Metabolism xxx (2017) xxx–xxx
Available online at
ScienceDirect www.sciencedirect.com
Short Report
Inhibitory role of oxytocin on TNFa expression assessed in vitro and in vivo S. Garrido-Urbani a, N. Deblon b, A.L. Poher b, A. Caillon b, P. Ropraz a, F. Rohner-Jeanrenaud b, J. Altirriba b,* a b
Department of Pathology and Immunology, Medical Faculty, University Medical Center, University of Geneva, Geneva, Switzerland Laboratory of Metabolism, Department of Internal Medicine Specialties, Faculty of Medicine, University of Geneva, Geneva, Switzerland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 July 2017 Received in revised form 6 September 2017 Accepted 7 October 2017 Available online xxx
Aim. – Oxytocin administration to diet-induced obese (DIO) rodents, monkeys and humans decreases body weight and fat mass with concomitant improvements in glucose metabolism. Moreover, several studies show an immunomodulatory role of oxytocin in a number of settings (such as atherosclerosis, injury, sepsis). This study aims to shed some light on the effects of oxytocin on macrophage polarization and cytokine production, as well as its possible impact on these parameters in adipose tissue in DIO mice with impaired glucose metabolism. Methods. – Mouse bone marrow cells were differentiated into macrophages and treated with oxytocin. Macrophage proliferation, cytokine secretion and macrophage populations were determined. For experiments in vivo, DIO mice were treated with oxytocin for 2 weeks. Body weight and composition and glucose tolerance were subsequently followed. At the end of treatment, adipose tissue macrophage populations, plasma cytokine levels and cytokine expression in adipose tissue were determined. Results. – In bone marrow-derived macrophages, oxytocin induced an anti-inflammatory phenotype (decreased M1/M2 ratio). In M1-derived macrophages, oxytocin decreased TNFa secretion, with no effects on the other cytokines tested nor any effect on cytokine secretion by M2-derived macrophages. Oxytocin treatment in DIO mice in vivo led to decreased body weight accompanied by an improvement in glucose tolerance, with no changes in plasma cytokine levels. In adipose tissue, oxytocin decreased Tnfa expression without modifying the M1/M2 macrophage ratio. Conclusion. – Oxytocin treatment decreases TNFa production both in vitro (in bone marrow-derived macrophages) and in vivo (in epididymal adipose tissue) in DIO mice. This effect may also be contributory to the observed improvement in glucose metabolism. C 2017 Elsevier Masson SAS. All rights reserved.
Keywords: Adipose tissue Inflammation Oxytocin TNFa
Introduction Oxytocin is well known for its therapeutic effects during delivery and lactation, whereas its use in the treatment of pathologies such as psychiatric disorders and obesity is still under investigation. In the context of obesity and diabetes, oxytocin has been shown to efficiently decrease body weight in rodents, monkeys and humans, with concomitant improvements in glucose metabolism [1–5]. The oxytocin receptor is widely expressed, including in monocytes and macrophages [6], although the direct oxytocin
* Corresponding author at: Laboratoire du Me´tabolisme, Centre Me´dical Universitaire, Dpt PHYME, 5e`me e´tage, Lab C05.2132.a, 1, rue Michel Servet, CH1211 Gene`ve 4, Switzerland. E-mail address: [email protected] (J. Altirriba).
effects on these cells is unclear. While the studies of Szeto et al. [6,7] have shown effects on THP-1 and RAW 264.7 monocyte cell lines, peritoneal murine macrophages and human monocytederived macrophages, including decreasing interleukin (IL)-6 expression and secretion after lipopolysaccharide (LPS) stimulation, the studies of Clodi et al. [8] and Ross et al. [9] in human monocytes and macrophages were able to neither replicate these results nor observe any oxytocin effects on secretion of various cytokines. Regarding its effects in vivo, oxytocin was shown to decrease the inflammation present in several pathological conditions (including atherosclerosis, burn/acid injury, sepsis and myocardial infarction) [9–12]. In the present study, the effects of oxytocin in vitro and in vivo on bone marrow-derived macrophages and epididymal adipose tissue in obese mice were examined to assess its effects on cytokine production and secretion as well as on macrophage polarization.
https://doi.org/10.1016/j.diabet.2017.10.004 C 2017 Elsevier Masson SAS. All rights reserved. 1262-3636/
Please cite this article in press as: Garrido-Urbani S, et al. Inhibitory role of oxytocin on TNFa expression assessed in vitro and in vivo. Diabetes Metab (2017), https://doi.org/10.1016/j.diabet.2017.10.004
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Methods Isolation of bone marrow-derived macrophages Femoral and tibia bones were sampled from male C57BL/6JRj mice. Bone marrow cells were expelled by infusion of bone marrow medium and resuspended in conditioned Dulbecco’s modified Eagle’s medium (DMEM; containing macrophage colony-stimulating factor, 10% fetal calf serum, 20% L929, penicillin–streptomycin). The cells were thereafter cultured for 7 days, with medium replacement every 2 days, and then incubated in the same medium, but complemented with different oxytocin concentrations for 48 h to analyze their differentiation profiles. In other experiments, prior to incubation with different oxytocin concentrations, the cells were first differentiated for 72 h into resting (M2) and inflammatory (M1) macrophages with IL-4 and interferon (IFN)-g, respectively. The differentiated macrophages were treated with oxytocin for 48 h, and cell supernatants were collected to quantify the cytokines secreted. Proliferation assay After bone marrow-differentiated macrophages (BMDM) were isolated and differentiated as described above, the BMDM were incubated with different doses of oxytocin for 1 h before incubation with 5-ethynyl-20 -deoxyuridine (EdU). After 24 h of incubation with EdU and oxytocin, EdU incorporation was analyzed by flow cytometry. Mice The principles of laboratory animal care (European and local government guidelines) were followed. Male 8-week-old C57BL/ 6JRj mice were fed either a high-fat (60% of energy from fat) or chow diet for 10 weeks. During their final 2 weeks, they were subcutaneously treated with oxytocin (50 nmol/day) or a solvent (saline) via osmotic mini-pumps, as previously described [4]. At the end of the experiment, trunk blood was collected in EDTA tubes containing aprotinin. Biochemical measurements Plasma and supernatant cytokine levels were measured by multiplex analysis. Isolation of adipose tissue leucocytes Isolated epididymal white adipose tissue (eWAT) was minced, placed in HEPES-buffered DMEM [supplemented with 10 mg/mL of fatty acid-poor bovine serum albumin (FAP-BSA)] and centrifuged at 1000 g for 10 min at room temperature (RT). The tissue suspension was digested for 1 h at 37 8C (containing Liberase DH and DNAse I) under agitation, then filtered through a 70-mm cell strainer and centrifuged at 1000 g for 10 min. Pelleted cells were collected as the stromal vascular fraction and resuspended in erythrocyte lysis buffer at RT for 10 min. The erythrocyte-depleted stromal fraction was centrifuged at 500 g for 5 min, and the pellet resuspended in 5-mM phosphate-buffered saline (PBS)EDTA and 0.2% FAP-BSA [fluorescence-activated cell sorting (FACS) buffer]. RNA isolation and real-time PCR
PCR System, using SYBR Green. Expression levels were normalized against ribosomal protein S29 (Rps29) and represented as a percentage of the saline-treated group (set at 100%). Primer sequences (Table S1; see supplementary materials associated with this article online) were designed by Primer Express Software. FACS analysis Cell population and replication were analyzed by flow cytometry. The cell markers used were CD45 Alexa Fluor 488, APC F4/80 antibody, CD11c PerCP-Cy7 and CD206 Biotinylated Antibody, followed by Streptavidin-PerCP. Acquisition was performed with a Gallios FACS analyzer and the analysis by Kaluza software (Beckman Coulter Inc., Brea, CA, USA). Statistical analysis Quantitative data are expressed as means SEM. Outlier analysis was performed by ROUT test. Statistical significance was determined by Student’s t test or by analysis of variance (ANOVA), with Dunnett’s or Sidak’s post hoc test. Correlations were analyzed using a Spearman test (GraphPad Prism). Reagents/software Details of these can be found in Table S2 (see supplementary materials associated with this article online). Results In vitro To determine whether oxytocin has a direct effect on macrophages, experiments were performed in vitro, wherein bone marrow-derived M1 and M2 macrophages were treated with different oxytocin concentrations, and their effects on differentiation, polarization and proliferation were examined. Oxytocin had no impact on macrophage proliferation (Fig. S1; see supplementary materials associated with this article online), whereas it decreased M1 differentiation with no effect on the M2 population, thereby leading to an anti-inflammatory phenotype (decreased M1/M2 ratio; Fig. 1a–d). Moreover, following measurement of a panel of 23 cytokines in supernatants (Figs. S2 and S3; see supplementary materials associated with this article online), it was observed that oxytocin dose-dependently decreased tumour necrosis factor a (TNFa) secretion by M1 macrophages, but had no effect on other cytokines secreted by either M1 or M2 macrophage populations (Fig. 1e, Figs. S2 and S3). In vivo Diet-induced obese (DIO) and lean mice were treated with oxytocin for 2 weeks. The treatment decreased body weight to a greater extent in obese mice, mainly due to its effect of reducing fat mass in an obese paradigm ([4,13] and data not shown). Moreover, DIO mice exhibited improved glucose tolerance in response to such treatment ([13] and data not shown). Plasma cytokine levels were measured, but no changes were detected (Fig. S4; see supplementary materials associated with this article online). Finally, when assessing local adipose tissue inflammation, no change in M1/M2 ratio was observed (Fig. 1f–i), whereas the decreased Tnfa expression observed in vitro was confirmed (Fig. 1j). Discussion
Total RNA from eWAT was isolated using TRIzol, and reversetranscribed using PrimeScript RT Reagent Kit. Real-time polymerase chain reaction (PCR) was carried out with a StepOne Real-Time
As already mentioned, the immunomodulatory effects of oxytocin have been demonstrated in several models of
Please cite this article in press as: Garrido-Urbani S, et al. Inhibitory role of oxytocin on TNFa expression assessed in vitro and in vivo. Diabetes Metab (2017), https://doi.org/10.1016/j.diabet.2017.10.004
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Fig. 1. Oxytocin effects on macrophages and on adipose tissue inflammation in vitro and in vivo. Bone marrow-derived macrophages were incubated with different oxytocin concentrations, and cell populations were analyzed by flow cytometry [(a) gating strategy] to obtain the (b) inflammatory macrophage population (CD45+, F4/80+, CD11c+, CD206), (c) resident macrophage population (CD45+, F4/80+, CD11c+, CD206+) and (d) ratio of both (n = 14–15). In parallel, another batch was isolated and differentiated into resting and inflammatory macrophages with interleukin (IL)-4 and interferon (IFN)-g, then incubated with different oxytocin concentrations: (e) the supernatant was collected and tumour necrosis factor a (TNFa) was measured (n = 3). *P 0.05, ***P 0.001 vs 0 nM. To assess oxytocin effects on adipose tissue inflammation in vivo, mice were rendered obese and glucose-intolerant by a high-fat diet and treated with either oxytocin (Oxt) or vehicle (Sal). At the end of treatment, macrophages were isolated from epididymal white adipose tissue (eWAT) and analyzed by fluorescence-activated cell sorting (FACS) [(f) gating strategy] to obtain the (g) inflammatory macrophage population (CD45+, F4/80+, CD11c+, CD206), (h) resident macrophage population (CD45+, F4/80+, CD11c+, CD206+) and (i) ratio of both; (j) Tnfa expression was measured in eWAT (n = 8–9). *P 0.05 vs saline-treated mice.
inflammation, wherein oxytocin modulated levels of different cytokines in each condition [10–12,14]. However, it is still unclear whether the oxytocin effects on the immune system are direct or indirect. Thus, while Szeto et al. [6,7] demonstrated that oxytocin incubation of LPS-stimulated cells decreased IL-6 secretion and NADPH-dependent superoxide activity, other authors [8,9] did not observe any changes in these parameters. In parallel, oxytocin has been well demonstrated to be an effective treatment against obesity in several animal models and in humans, with concomitant improvements in glucose tolerance [1–5]. Nevertheless, the molecular mechanisms that drive these effects are, as yet, not completely understood. Taking into account that obesity induces an insulin-resistant state in adipose tissue, in cases where M1 proinflammatory macrophages (producing inflammatory mediators linked to insulin resistance, such as TNFa) [15] prevail over M2 anti-inflammatory macrophages [16], it is possible that oxytocin could modulate adipose tissue inflammation, a process that might partly underlie the observed improved glucose metabolism, as previously proposed (see Olefsky and Glass for a review [16]). To clarify the role of oxytocin in adipose tissue inflammation, bone marrow-derived macrophages were differentiated into M1 and M2 phenotypes and stimulated by oxytocin. While oxytocin had no effect on cell replication (Fig. S1), it did influence macrophage polarization towards an anti-inflammatory phenotype (Fig. 1a–d). When cytokine secretion was screened, it was
observed that oxytocin induced decreased secretion of the proinflammatory cytokine TNFa by M1 macrophages (Fig. 1e). Regarding the range of oxytocin concentrations used in the present study, it was similar to that used in previous reports [8,17], albeit higher than physiological circulating levels (5–250 pM) [7]. Whereas only the 100-nM concentration significantly impacted the M1/ M2 ratio and TNFa secretion, lower doses produced a trend towards similar changes, with significant correlations between oxytocin concentration and M1/M2 ratio (P < 0.01, r = 0.36) and TNFa secretion (P < 0.05, r = 0.64). Therefore, it is conceivable that longer treatments with lower doses closer to the physiological range would produce significant effects. Further future experiments should clarify this point. The secretion of cytokines other than TNFa was unchanged following oxytocin stimulation (Figs. S2 and S3). This is in keeping with the results of other studies [8,9], but in contrast to the data reported by Szeto et al. [6] and Clodi et al. [8], showing that oxytocin decreases IL-6 secretion and has no impact on TNFa release, respectively. The presence of such divergent results could be due to the fact that the present study was performed with M1 and M2 differentiated macrophages treated with IFN-g and IL-4 whereas, in previously reported data, inflammation was induced by LPS stimulation. Although the results obtained in vitro in our study have some limitations (short oxytocin exposure of 24–48 h, use of supraphy-
Please cite this article in press as: Garrido-Urbani S, et al. Inhibitory role of oxytocin on TNFa expression assessed in vitro and in vivo. Diabetes Metab (2017), https://doi.org/10.1016/j.diabet.2017.10.004
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siological concentrations), they have prompted investigations into oxytocin effects in DIO and glucose-intolerant mice in vivo, where oxytocin treatment for 2 weeks induced body weight loss due to a decrease in fat mass, with concomitant improvement in glucose tolerance [13]. In these animals, chronic oxytocin treatment decreased eWAT Tnfa expression (Fig. 1j), but with no changes in levels of other cytokines in eWAT (Fig. S5; see supplementary materials associated with this article online) or in plasma (Fig. S4). However, it is worth noting that plasma inflammatory biomarkers may not always adequately reflect local tissue inflammation. For example, a lack of correlation between adipose tissue Tnfa expression and its plasma levels has been reported [18]. In contrast to our results in vitro, no effect of oxytocin treatment in DIO mice in vivo on macrophage polarization was observed (Fig. 1j). In addition, lean mice treated with oxytocin exhibited no differences in adipose tissue Tnfa expression (Fig. S6; see supplementary materials associated with this article online), which is in agreement with the lack of effect on fat mass previously described in these mice [4]. Finally, it should be mentioned that the present study was focused on eWAT from DIO mice in which macrophages are the main immune cells [19], making this fat depot a good model for our study in vivo. Because various fat depots present different characteristics [19], follow-up studies should now investigate the effect of oxytocin on other fat depots with different immune cells. To summarize, oxytocin exerts a direct effect on macrophages by decreasing TNFa expression and secretion. These effects may be contributing to the observed improvement of glucose metabolism. Funding This study was supported by a European Foundation for the Study of Diabetes (EFSD)/Lilly fellowship award and the Swiss National Science Foundation (Grant 310030 160290/1). Disclosure of interest F.R.J. and N.D. have a patent application (PCT/IB2011/052156) covering novel therapeutic uses of oxytocin. The other authors declare that they have no competing interest.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.diabet.2017. 10.004.
References [1] Blevins JE, Baskin DG. Translational and therapeutic potential of oxytocin as an anti-obesity strategy: insights from rodents, nonhuman primates and humans. Physiol Behav 2015;152:438–49. [2] Deblon N, Veyrat-Durebex C, Bourgoin L, Caillon A, Bussier AL, Petrosino S, et al. Mechanisms of the anti-obesity effects of oxytocin in diet-induced obese rats. PLoS ONE 2011;6:e25565. [3] Zhang H, Wu C, Chen Q, Chen X, Xu Z, Wu J, et al. Treatment of obesity and diabetes using oxytocin or analogs in patients and mouse models. PLoS ONE 2013;8:e61477. [4] Altirriba J, Poher AL, Caillon A, Arsenijevic D, Veyrat-Durebex C, Lyautey J, et al. Divergent effects of oxytocin treatment of obese diabetic mice on adiposity and diabetes. Endocrinology 2014;155:4189–201. [5] Blevins JE, Graham JL, Morton GJ, Bales KL, Schwartz MW, Baskin DG, et al. Chronic oxytocin administration inhibits food intake, increases energy expenditure, and produces weight loss in fructose-fed obese rhesus monkeys. Am J Physiol Regul Integr Comp Physiol 2015;308:R431–8. [6] Szeto A, Sun-Suslow N, Mendez AJ, Hernandez RI, Wagner KV, McCabe PM. Regulation of the macrophage oxytocin receptor in response to inflammation. Am J Physiol Endocrinol Metab 2017;312:E183–9. [7] Szeto A, Nation DA, Mendez AJ, Dominguez-Bendala J, Brooks LG, Schneiderman N, et al. Oxytocin attenuates NADPH-dependent superoxide activity and IL-6 secretion in macrophages and vascular cells. Am J Physiol Endocrinol Metab 2008;295:E1495–501. [8] Clodi M, Vila G, Geyeregger R, Riedl M, Stulnig TM, Struck J, et al. Oxytocin alleviates the neuroendocrine and cytokine response to bacterial endotoxin in healthy men. Am J Physiol Endocrinol Metab 2008;295:E686–91. [9] Ross KM, McDonald-Jones G, Miller GE. Oxytocin does not attenuate the ex vivo production of inflammatory cytokines by lipopolysaccharide-activated monocytes and macrophages from healthy male and female donors. Neuroimmunomodulation 2013;20:285–93. [10] Iseri SO, Gedik IE, Erzik C, Uslu B, Arbak S, Gedik N, et al. Oxytocin ameliorates skin damage and oxidant gastric injury in rats with thermal trauma. Burns 2008;34:361–9. [11] Iseri SO, Sener G, Saglam B, Gedik N, Ercan F, Yegen BC. Oxytocin protects against sepsis-induced multiple organ damage: role of neutrophils. J Surg Res 2005;126:73–81. [12] Jankowski M, Bissonauth V, Gao L, Gangal M, Wang D, Danalache B, et al. Antiinflammatory effect of oxytocin in rat myocardial infarction. Basic Res Cardiol 2010;105:205–18. [13] Maejima Y, Iwasaki Y, Yamahara Y, Kodaira M, Sedbazar U, Yada T. Peripheral oxytocin treatment ameliorates obesity by reducing food intake and visceral fat mass. Aging (Albany NY) 2011;3:1169–77. [14] Szeto A, Rossetti MA, Mendez AJ, Noller CM, Herderick EE, Gonzales JA, et al. Oxytocin administration attenuates atherosclerosis and inflammation in Watanabe Heritable Hyperlipidemic rabbits. Psychoneuroendocrinology 2013;38:685–93. [15] Hotamisligil GS, Shargill NS, Spiegelman BM. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 1993;259:87–91. [16] Olefsky JM, Glass CK. Macrophages, inflammation, and insulin resistance. Annu Rev Physiol 2010;72:219–46. [17] Ndiaye K, Poole DH, Pate JL. Expression and regulation of functional oxytocin receptors in bovine T lymphocytes. Biol Reprod 2008;78:786–93. [18] Kern PA, Ranganathan S, Li C, Wood L, Ranganathan G. Adipose tissue tumor necrosis factor and interleukin-6 expression in human obesity and insulin resistance. Am J Physiol Endocrinol Metab 2001;280:E745–51. [19] van Beek L, van Klinken JB, Pronk AC, van Dam AD, Dirven E, Rensen PC, et al. The limited storage capacity of gonadal adipose tissue directs the development of metabolic disorders in male C57Bl/6J mice. Diabetologia 2015;58:1601–9.
Please cite this article in press as: Garrido-Urbani S, et al. Inhibitory role of oxytocin on TNFa expression assessed in vitro and in vivo. Diabetes Metab (2017), https://doi.org/10.1016/j.diabet.2017.10.004
DIABET-938; No. of Pages 4 Diabetes & Metabolism xxx (2017) xxx–xxx
Available online at
ScienceDirect www.sciencedirect.com
Short Report
Inhibitory role of oxytocin on TNFa expression assessed in vitro and in vivo S. Garrido-Urbani a, N. Deblon b, A.L. Poher b, A. Caillon b, P. Ropraz a, F. Rohner-Jeanrenaud b, J. Altirriba b,* a b
Department of Pathology and Immunology, Medical Faculty, University Medical Center, University of Geneva, Geneva, Switzerland Laboratory of Metabolism, Department of Internal Medicine Specialties, Faculty of Medicine, University of Geneva, Geneva, Switzerland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 July 2017 Received in revised form 6 September 2017 Accepted 7 October 2017 Available online xxx
Aim. – Oxytocin administration to diet-induced obese (DIO) rodents, monkeys and humans decreases body weight and fat mass with concomitant improvements in glucose metabolism. Moreover, several studies show an immunomodulatory role of oxytocin in a number of settings (such as atherosclerosis, injury, sepsis). This study aims to shed some light on the effects of oxytocin on macrophage polarization and cytokine production, as well as its possible impact on these parameters in adipose tissue in DIO mice with impaired glucose metabolism. Methods. – Mouse bone marrow cells were differentiated into macrophages and treated with oxytocin. Macrophage proliferation, cytokine secretion and macrophage populations were determined. For experiments in vivo, DIO mice were treated with oxytocin for 2 weeks. Body weight and composition and glucose tolerance were subsequently followed. At the end of treatment, adipose tissue macrophage populations, plasma cytokine levels and cytokine expression in adipose tissue were determined. Results. – In bone marrow-derived macrophages, oxytocin induced an anti-inflammatory phenotype (decreased M1/M2 ratio). In M1-derived macrophages, oxytocin decreased TNFa secretion, with no effects on the other cytokines tested nor any effect on cytokine secretion by M2-derived macrophages. Oxytocin treatment in DIO mice in vivo led to decreased body weight accompanied by an improvement in glucose tolerance, with no changes in plasma cytokine levels. In adipose tissue, oxytocin decreased Tnfa expression without modifying the M1/M2 macrophage ratio. Conclusion. – Oxytocin treatment decreases TNFa production both in vitro (in bone marrow-derived macrophages) and in vivo (in epididymal adipose tissue) in DIO mice. This effect may also be contributory to the observed improvement in glucose metabolism. C 2017 Elsevier Masson SAS. All rights reserved.
Keywords: Adipose tissue Inflammation Oxytocin TNFa
Introduction Oxytocin is well known for its therapeutic effects during delivery and lactation, whereas its use in the treatment of pathologies such as psychiatric disorders and obesity is still under investigation. In the context of obesity and diabetes, oxytocin has been shown to efficiently decrease body weight in rodents, monkeys and humans, with concomitant improvements in glucose metabolism [1–5]. The oxytocin receptor is widely expressed, including in monocytes and macrophages [6], although the direct oxytocin
* Corresponding author at: Laboratoire du Me´tabolisme, Centre Me´dical Universitaire, Dpt PHYME, 5e`me e´tage, Lab C05.2132.a, 1, rue Michel Servet, CH1211 Gene`ve 4, Switzerland. E-mail address: [email protected] (J. Altirriba).
effects on these cells is unclear. While the studies of Szeto et al. [6,7] have shown effects on THP-1 and RAW 264.7 monocyte cell lines, peritoneal murine macrophages and human monocytederived macrophages, including decreasing interleukin (IL)-6 expression and secretion after lipopolysaccharide (LPS) stimulation, the studies of Clodi et al. [8] and Ross et al. [9] in human monocytes and macrophages were able to neither replicate these results nor observe any oxytocin effects on secretion of various cytokines. Regarding its effects in vivo, oxytocin was shown to decrease the inflammation present in several pathological conditions (including atherosclerosis, burn/acid injury, sepsis and myocardial infarction) [9–12]. In the present study, the effects of oxytocin in vitro and in vivo on bone marrow-derived macrophages and epididymal adipose tissue in obese mice were examined to assess its effects on cytokine production and secretion as well as on macrophage polarization.
https://doi.org/10.1016/j.diabet.2017.10.004 C 2017 Elsevier Masson SAS. All rights reserved. 1262-3636/
Please cite this article in press as: Garrido-Urbani S, et al. Inhibitory role of oxytocin on TNFa expression assessed in vitro and in vivo. Diabetes Metab (2017), https://doi.org/10.1016/j.diabet.2017.10.004
G Model
DIABET-938; No. of Pages 4 2
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Methods Isolation of bone marrow-derived macrophages Femoral and tibia bones were sampled from male C57BL/6JRj mice. Bone marrow cells were expelled by infusion of bone marrow medium and resuspended in conditioned Dulbecco’s modified Eagle’s medium (DMEM; containing macrophage colony-stimulating factor, 10% fetal calf serum, 20% L929, penicillin–streptomycin). The cells were thereafter cultured for 7 days, with medium replacement every 2 days, and then incubated in the same medium, but complemented with different oxytocin concentrations for 48 h to analyze their differentiation profiles. In other experiments, prior to incubation with different oxytocin concentrations, the cells were first differentiated for 72 h into resting (M2) and inflammatory (M1) macrophages with IL-4 and interferon (IFN)-g, respectively. The differentiated macrophages were treated with oxytocin for 48 h, and cell supernatants were collected to quantify the cytokines secreted. Proliferation assay After bone marrow-differentiated macrophages (BMDM) were isolated and differentiated as described above, the BMDM were incubated with different doses of oxytocin for 1 h before incubation with 5-ethynyl-20 -deoxyuridine (EdU). After 24 h of incubation with EdU and oxytocin, EdU incorporation was analyzed by flow cytometry. Mice The principles of laboratory animal care (European and local government guidelines) were followed. Male 8-week-old C57BL/ 6JRj mice were fed either a high-fat (60% of energy from fat) or chow diet for 10 weeks. During their final 2 weeks, they were subcutaneously treated with oxytocin (50 nmol/day) or a solvent (saline) via osmotic mini-pumps, as previously described [4]. At the end of the experiment, trunk blood was collected in EDTA tubes containing aprotinin. Biochemical measurements Plasma and supernatant cytokine levels were measured by multiplex analysis. Isolation of adipose tissue leucocytes Isolated epididymal white adipose tissue (eWAT) was minced, placed in HEPES-buffered DMEM [supplemented with 10 mg/mL of fatty acid-poor bovine serum albumin (FAP-BSA)] and centrifuged at 1000 g for 10 min at room temperature (RT). The tissue suspension was digested for 1 h at 37 8C (containing Liberase DH and DNAse I) under agitation, then filtered through a 70-mm cell strainer and centrifuged at 1000 g for 10 min. Pelleted cells were collected as the stromal vascular fraction and resuspended in erythrocyte lysis buffer at RT for 10 min. The erythrocyte-depleted stromal fraction was centrifuged at 500 g for 5 min, and the pellet resuspended in 5-mM phosphate-buffered saline (PBS)EDTA and 0.2% FAP-BSA [fluorescence-activated cell sorting (FACS) buffer]. RNA isolation and real-time PCR
PCR System, using SYBR Green. Expression levels were normalized against ribosomal protein S29 (Rps29) and represented as a percentage of the saline-treated group (set at 100%). Primer sequences (Table S1; see supplementary materials associated with this article online) were designed by Primer Express Software. FACS analysis Cell population and replication were analyzed by flow cytometry. The cell markers used were CD45 Alexa Fluor 488, APC F4/80 antibody, CD11c PerCP-Cy7 and CD206 Biotinylated Antibody, followed by Streptavidin-PerCP. Acquisition was performed with a Gallios FACS analyzer and the analysis by Kaluza software (Beckman Coulter Inc., Brea, CA, USA). Statistical analysis Quantitative data are expressed as means SEM. Outlier analysis was performed by ROUT test. Statistical significance was determined by Student’s t test or by analysis of variance (ANOVA), with Dunnett’s or Sidak’s post hoc test. Correlations were analyzed using a Spearman test (GraphPad Prism). Reagents/software Details of these can be found in Table S2 (see supplementary materials associated with this article online). Results In vitro To determine whether oxytocin has a direct effect on macrophages, experiments were performed in vitro, wherein bone marrow-derived M1 and M2 macrophages were treated with different oxytocin concentrations, and their effects on differentiation, polarization and proliferation were examined. Oxytocin had no impact on macrophage proliferation (Fig. S1; see supplementary materials associated with this article online), whereas it decreased M1 differentiation with no effect on the M2 population, thereby leading to an anti-inflammatory phenotype (decreased M1/M2 ratio; Fig. 1a–d). Moreover, following measurement of a panel of 23 cytokines in supernatants (Figs. S2 and S3; see supplementary materials associated with this article online), it was observed that oxytocin dose-dependently decreased tumour necrosis factor a (TNFa) secretion by M1 macrophages, but had no effect on other cytokines secreted by either M1 or M2 macrophage populations (Fig. 1e, Figs. S2 and S3). In vivo Diet-induced obese (DIO) and lean mice were treated with oxytocin for 2 weeks. The treatment decreased body weight to a greater extent in obese mice, mainly due to its effect of reducing fat mass in an obese paradigm ([4,13] and data not shown). Moreover, DIO mice exhibited improved glucose tolerance in response to such treatment ([13] and data not shown). Plasma cytokine levels were measured, but no changes were detected (Fig. S4; see supplementary materials associated with this article online). Finally, when assessing local adipose tissue inflammation, no change in M1/M2 ratio was observed (Fig. 1f–i), whereas the decreased Tnfa expression observed in vitro was confirmed (Fig. 1j). Discussion
Total RNA from eWAT was isolated using TRIzol, and reversetranscribed using PrimeScript RT Reagent Kit. Real-time polymerase chain reaction (PCR) was carried out with a StepOne Real-Time
As already mentioned, the immunomodulatory effects of oxytocin have been demonstrated in several models of
Please cite this article in press as: Garrido-Urbani S, et al. Inhibitory role of oxytocin on TNFa expression assessed in vitro and in vivo. Diabetes Metab (2017), https://doi.org/10.1016/j.diabet.2017.10.004
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Fig. 1. Oxytocin effects on macrophages and on adipose tissue inflammation in vitro and in vivo. Bone marrow-derived macrophages were incubated with different oxytocin concentrations, and cell populations were analyzed by flow cytometry [(a) gating strategy] to obtain the (b) inflammatory macrophage population (CD45+, F4/80+, CD11c+, CD206), (c) resident macrophage population (CD45+, F4/80+, CD11c+, CD206+) and (d) ratio of both (n = 14–15). In parallel, another batch was isolated and differentiated into resting and inflammatory macrophages with interleukin (IL)-4 and interferon (IFN)-g, then incubated with different oxytocin concentrations: (e) the supernatant was collected and tumour necrosis factor a (TNFa) was measured (n = 3). *P 0.05, ***P 0.001 vs 0 nM. To assess oxytocin effects on adipose tissue inflammation in vivo, mice were rendered obese and glucose-intolerant by a high-fat diet and treated with either oxytocin (Oxt) or vehicle (Sal). At the end of treatment, macrophages were isolated from epididymal white adipose tissue (eWAT) and analyzed by fluorescence-activated cell sorting (FACS) [(f) gating strategy] to obtain the (g) inflammatory macrophage population (CD45+, F4/80+, CD11c+, CD206), (h) resident macrophage population (CD45+, F4/80+, CD11c+, CD206+) and (i) ratio of both; (j) Tnfa expression was measured in eWAT (n = 8–9). *P 0.05 vs saline-treated mice.
inflammation, wherein oxytocin modulated levels of different cytokines in each condition [10–12,14]. However, it is still unclear whether the oxytocin effects on the immune system are direct or indirect. Thus, while Szeto et al. [6,7] demonstrated that oxytocin incubation of LPS-stimulated cells decreased IL-6 secretion and NADPH-dependent superoxide activity, other authors [8,9] did not observe any changes in these parameters. In parallel, oxytocin has been well demonstrated to be an effective treatment against obesity in several animal models and in humans, with concomitant improvements in glucose tolerance [1–5]. Nevertheless, the molecular mechanisms that drive these effects are, as yet, not completely understood. Taking into account that obesity induces an insulin-resistant state in adipose tissue, in cases where M1 proinflammatory macrophages (producing inflammatory mediators linked to insulin resistance, such as TNFa) [15] prevail over M2 anti-inflammatory macrophages [16], it is possible that oxytocin could modulate adipose tissue inflammation, a process that might partly underlie the observed improved glucose metabolism, as previously proposed (see Olefsky and Glass for a review [16]). To clarify the role of oxytocin in adipose tissue inflammation, bone marrow-derived macrophages were differentiated into M1 and M2 phenotypes and stimulated by oxytocin. While oxytocin had no effect on cell replication (Fig. S1), it did influence macrophage polarization towards an anti-inflammatory phenotype (Fig. 1a–d). When cytokine secretion was screened, it was
observed that oxytocin induced decreased secretion of the proinflammatory cytokine TNFa by M1 macrophages (Fig. 1e). Regarding the range of oxytocin concentrations used in the present study, it was similar to that used in previous reports [8,17], albeit higher than physiological circulating levels (5–250 pM) [7]. Whereas only the 100-nM concentration significantly impacted the M1/ M2 ratio and TNFa secretion, lower doses produced a trend towards similar changes, with significant correlations between oxytocin concentration and M1/M2 ratio (P < 0.01, r = 0.36) and TNFa secretion (P < 0.05, r = 0.64). Therefore, it is conceivable that longer treatments with lower doses closer to the physiological range would produce significant effects. Further future experiments should clarify this point. The secretion of cytokines other than TNFa was unchanged following oxytocin stimulation (Figs. S2 and S3). This is in keeping with the results of other studies [8,9], but in contrast to the data reported by Szeto et al. [6] and Clodi et al. [8], showing that oxytocin decreases IL-6 secretion and has no impact on TNFa release, respectively. The presence of such divergent results could be due to the fact that the present study was performed with M1 and M2 differentiated macrophages treated with IFN-g and IL-4 whereas, in previously reported data, inflammation was induced by LPS stimulation. Although the results obtained in vitro in our study have some limitations (short oxytocin exposure of 24–48 h, use of supraphy-
Please cite this article in press as: Garrido-Urbani S, et al. Inhibitory role of oxytocin on TNFa expression assessed in vitro and in vivo. Diabetes Metab (2017), https://doi.org/10.1016/j.diabet.2017.10.004
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siological concentrations), they have prompted investigations into oxytocin effects in DIO and glucose-intolerant mice in vivo, where oxytocin treatment for 2 weeks induced body weight loss due to a decrease in fat mass, with concomitant improvement in glucose tolerance [13]. In these animals, chronic oxytocin treatment decreased eWAT Tnfa expression (Fig. 1j), but with no changes in levels of other cytokines in eWAT (Fig. S5; see supplementary materials associated with this article online) or in plasma (Fig. S4). However, it is worth noting that plasma inflammatory biomarkers may not always adequately reflect local tissue inflammation. For example, a lack of correlation between adipose tissue Tnfa expression and its plasma levels has been reported [18]. In contrast to our results in vitro, no effect of oxytocin treatment in DIO mice in vivo on macrophage polarization was observed (Fig. 1j). In addition, lean mice treated with oxytocin exhibited no differences in adipose tissue Tnfa expression (Fig. S6; see supplementary materials associated with this article online), which is in agreement with the lack of effect on fat mass previously described in these mice [4]. Finally, it should be mentioned that the present study was focused on eWAT from DIO mice in which macrophages are the main immune cells [19], making this fat depot a good model for our study in vivo. Because various fat depots present different characteristics [19], follow-up studies should now investigate the effect of oxytocin on other fat depots with different immune cells. To summarize, oxytocin exerts a direct effect on macrophages by decreasing TNFa expression and secretion. These effects may be contributing to the observed improvement of glucose metabolism. Funding This study was supported by a European Foundation for the Study of Diabetes (EFSD)/Lilly fellowship award and the Swiss National Science Foundation (Grant 310030 160290/1). Disclosure of interest F.R.J. and N.D. have a patent application (PCT/IB2011/052156) covering novel therapeutic uses of oxytocin. The other authors declare that they have no competing interest.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.diabet.2017. 10.004.
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Please cite this article in press as: Garrido-Urbani S, et al. Inhibitory role of oxytocin on TNFa expression assessed in vitro and in vivo. Diabetes Metab (2017), https://doi.org/10.1016/j.diabet.2017.10.004