Impact of Vitamin D Supplementation on Influenza Vaccine Response and Immune Functions in Deficient Elderly Persons: A Randomized Placebo-Controlled Trial

Background: Immunosenescence contributes to reduced vaccine response in elderly persons, and is worsened by deficiencies in nutrients such as Vitamin (Vit-D). The immune system is a well-known target of Vit-D, which can both potentiate the innate immune response and inhibit the adaptive system, and so modulate vaccination response.Objective: This randomized placebo-controlled double-blind trial investigated whether Vit-D supplementation in deficient elderly persons could improve influenza seroprotection and immune response.Design: Deficient volunteers (Vit-D serum <30 ng/mL) were assigned (V1) to receive either 100,000 IU/15 days of cholecalciferol (D, n = 19), or a placebo (P, n = 19), over a 3 month period. Influenza vaccination was performed at the end of this period (V2), and the vaccine response was evaluated 28 days later (V3). At each visit, serum cathelicidin, immune response to vaccination, plasma cytokines, lymphocyte phenotyping, and phagocyte ROS production were assessed.Results: Levels of serum 25-(OH)D increased after supplementation (D group, V1 vs. V2: 20.7 ± 5.7 vs. 44.3 ± 8.6 ng/mL, p < 0.001). No difference was observed for serum cathelicidin levels, antibody titers, and ROS production in D vs. P groups at V3. Lower plasma levels of TNFα (p = 0.040) and IL-6 (p = 0.046), and higher ones for TFGβ (p = 0.0028) were observed at V3. The Th1/Th2 ratio was lower in the D group at V2 (D: 0.12 ± 0.05 vs. P: 0.18 ± 0.05, p = 0.039).Conclusions: Vit-D supplementation promotes a higher TGFβ plasma level in response to influenza vaccination without improving antibody production. This supplementation seems to direct the lymphocyte polarization toward a tolerogenic immune response. A deeper characterization of metabolic and molecular pathways of these observations will aid in the understanding of Vit-D's effects on cell-mediated immunity in aging. This clinical trial was registered at clinicaltrials.gov as NCT01893385

Despite a regular spontaneous physical activity, a long-term high-fat diet promotes mammary cancer development in mice.

International audienceBreast cancer is one of the most women common malignancies worldwide (1). Among the multiple risk factors, sedentary lifestyle, obesity and postmenopausal are correlated to estrogen and inflammation exposure during a lifetime (2). Physical activity protects against breast cancer development by affecting hormone levels, immune responses, and oxidative defences (3,4). This study aims was to assess the impact of long-term obesity on the benefits of physical activity in preventing and managing breast cancer during mammary tumorigenesis.Ovariectomized 35-week-old C57BL/6 mice were placed in an enriched environment to induce spontaneous physical activity and fed throughout the experiment with a high-fat diet (HFD). After 42 days (Short-term (ST), n=10) or 88 days (Long-term, LT, n=10) of exposure to HFD, syngenic mammary EO771 cells were implanted into the 4th mammary glands and the tumour growth was followed for 30 days. At sacrifice, the tumour microenvironment (TME) immune infiltrate and metabolic parameters were explored using flux cytometry, transcriptomic, enzyme activities and biochemical approaches. The data reported as mean SD were analysed by a Mann-Whitney test.The median survival was significantly reduced in the LT group compared to the ST group (22 days vs 25.5 days, p=0.0296). Whatever the group, the spontaneous physical activity (before tumor implantation (TI): 0.645±0.082 vs 1.065±0.076 km/mice/j; after TI: 0.0803±0.045 vs 0.965±0.217 km/mice/d) and the individual food intake was similar (before TI: 2.786±0.154 vs 2.835±0.066 g/d; after implantation: 2.264±0.208 vs 2.713±0.081 g/d) were similar. At the sacrifice, the visceral adipose tissue mass was higher in the LT group (1029.88±203.77 vs 1533.40±259.79 mg, p=0.04) while the skeletal muscle mass was reduced (354.20±12.79 vs 334.14±7.83 mg (ST group), p=0.0765). In the TME, among the total lymphocytes, the proportion of NK cells was reduced in the LT group (24.54±1.93 vs 0.24±0.09 %, p=0.05) as well as the TCD8+ one, (7.87±3.07 vs 1.01±0.28 %, p=0.002) while the proportion of T regulators tended to increase (0.02±0.01 vs 0.50±0.06 %Treg/T lymphocytes, p=0.1), leading to a collapse of the T8/Treg ratio (421.61±153.84 vs 2.62±1.31, p=0.03). The significant decrease in the tumour triglyceride content in the LT group (0.07±0.01 vs 0.01±0.01 mmol/g of tissue, p=0.0143) was accompanied with an enhanced activity of the glutathione reductase (165.46±5.81 vs 224.83±26.80 UI/mg of proteins, p=0.0286) and a decrease of the glutathione S transferase activity (295.40±15.10 vs 11.61±1.79 UI/mg of proteins, p=0.0143), markers of glutahione recycling involved in the oxidative stress management.In our experimental conditions, the LT HFD is associated with the tumor growth despite the spontaneous physical activity. LT HFD promotes a tolerogenic TME by increasing lipid consumption and oxidative stress and recruiting anti-tumour immune cells. The exploration of the tumor and skeletal muscles by the analysis of cytokines, myokines and free radicals could help to understand the inter-organ exchanges related to tumor development and muscle mass loss. In perspective, the combination of imposed physical activity with immunotherapy treatment could be envisaged to counteract the long-term effects of a hypercaloric diet.1.Sung H et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA A Cancer J Clin. mai 2021;71(3):209‑49. 2.Garcia-Estevez L, Moreno-Bueno G. Updating the role of obesity and cholesterol in breast cancer. Breast Cancer Res. déc 2019;21(1):35. 3.Swain CTV et al. Linking Physical Activity to Breast Cancer via Sex Hormones, Part 1: The Effect of Physical Activity on Sex Steroid Hormones. Cancer Epidemiology, Biomarkers & Prevention. 1 janv 2022;31(1):16‑27. 4.Le Guennec D et al. Spontaneous Physical Activity in Obese Condition Favours Antitumour Immunity Leading to Decreased Tumour Growth in a Syngeneic Mouse Model of Carcinogenesis. Cancers. 23 déc 2021;14(1):59

Long-term high-fat diet limits exercice benefits on mammary tumor growth in mice

International audienceIn postmenopausal condition, body fatness, inducing metabolic and hormonal stresses, promotes the risk of breast cancer. Physical activity (PA), a central component of weight management, limits it by reducing inflammation, insulin resistance, and oxidative stress. We measure the impact of high-fat diet duration on the PA’s beneficial effects during tumor development.Old ovariectomized C57BL/6 mice were fed by a high-fat diet. After 42 days (short lipid impregnation, SLI) or 88 days (long LI, LLI) of high-fat diet, EO771 syngeneic cells were implanted and the tumor was allowed to grow for 5 weeks. In SLI, one group was placed in a standard environment (SE, spontaneous physical activity) and the second in an enriched environment (EE). Under LLI, both groups were in an EE and one was subjected to a moderate PA (treadmill 12m/mn from 30 to 45mn, 5d/W, EEI). At sacrifice, tissue immune cell infiltrate and metabolic parameters were explored. n=10-12/group, mean SEM, Mann-Whitney test.Under SLI, tumour growth is reduced in the EE (p<0.05 vs SE). Under LLI, tumour growth is similar to that of the SLI-SE group, and the imposed PA does not impact it. Mice under SLI significantly lost weight between implantation and sacrifice (SE or EE, p<0.05) but not under LLI. Total adipose tissue and hind leg muscle mass are respectively higher and lower in mice on LLI compared to those on SLI, without effect of PA. In the LLI vs SLI condition, the tumor Th1 and Treg infiltrated cell ratio are higher whereas TCD8+ and NK are the same. In this model, LLI promotes tumour growth and counteracts the benefits of PA by establishing a tolerogenic environment, regardless of the rupture of a sedentary lifestyle by the EE or its reinforcement by the EEI. Future explorations of skeletal muscle, adipose tissue, their metabolism and inter-organ exchanges will provide insights into the mechanisms involved

Correction to: 25-Hydroxyvitamin D potentializes extracellular cathelicidin release from human PBMC stimulated ex vivo with either bacterial (LPS) or viral (P: IC) mimetics

International audienceBreast cancer is influenced by factors such as diet, a sedentary lifestyle, obesity, and postmenopausal status, which are all linked to prolonged hormonal and inflammatory exposure. Physical activity offers protection against breast cancer by modulating hormones, immune responses, and oxidative defenses. This study aimed to assess how a prolonged high-fat diet (HFD) affects the effectiveness of physical activity in preventing and managing mammary tumorigenesis. Ovariectomised C57BL/6 mice were provided with an enriched environment to induce spontaneous physical activity while being fed HFD. After 44 days (short-term, ST HFD) or 88 days (long-term, LT HFD), syngenic EO771 cells were implanted into mammary glands, and tumour growth was monitored until sacrifice. Despite similar physical activity and food intake, the LT HFD group exhibited higher visceral adipose tissue mass and reduced skeletal muscle mass. In the tumour microenvironment, the LT HFD group showed decreased NK cells and TCD8+ cells, with a trend toward increased T regulatory cells, leading to a collapse of the T8/Treg ratio. Additionally, the LT HFD group displayed decreased tumour triglyceride content and altered enzyme activities indicative of oxidative stress. Prolonged exposure to HFD was associated with tumour growth despite elevated physical activity, promoting a tolerogenic tumour microenvironment. Future studies should explore inter-organ exchanges between tumour and tissues

Mammary tumour microenvironment response to vitamin D and exercise in aged mice fed by high fat diet

International audienceBoth vitamin D (VD) and exercice (Ex) with their anti-carcinogenic and immunomodulatory properties can reduce the incidence of obesity-related breast cancer in post-menopausal situation. This study aimed to investigate the impact of VD supplementation and imposed exercise on the tumour microenvironment (TM) in a mouse breast cancer model.Old ovariectomized C57BL/6 mice under a high-fat diet were randomized into 4 groups (n=10/group) supplemented with VD (1250 vs 125 UI; 450 kcal / 100g) and/or submitted to an exercise (Ex) (treadmill 12m/mn from 30 to 45mn, 5d/W, moderate intensity). Syngenic EO771 cells were implanted at W8 and the protocol ended at W14. TM immune cell infiltrates, cellular expression of VD metabolism actors in cancer cells, liver and kidney, and tissue oxidative stress markers were analysed. Mean SEM, Anova two ways + Tukey test.No effect was observed in the immune cell found in the TM. Ex alone increases the expression of specific immune cells genes such as Cd8a, Eomes, and Nos2. Together, Ex and VD increase the expression of various genes such as Tnfr and Dx5 (p<0.01). In addition, both VD and Ex decreases the Tnfr/Foxp3 ratio, and the Cd8a/Foxp3 ratio (p<0.05). Finally, they modulate VD metabolism enzymes by enhancing the expression of VD receptor, Cyp24a1 involved in the catabolism of VD (p<0.01) while VD alone stimulates the gene expression of CYP27B1 transforming 25-OHD to the active VDR ligand, 1,25-D-OHD, (p=0.02).In our conditions, VD and Ex do not affect the tumour immune infiltrates but they modulated the expression of some immune subpopulation’s genes. Due to the overexpression of Cyp24a1 and high spontaneous activity, expected VD and Ex effects are masked, such as immunomodulatory and direct anti-proliferative effects. Further studies should be carried out to adjust VD supplementation and/or Ex to prevent or slow down tumor growth

Comment prévenir le risque de déficit en vitamine D

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Activation of antioxidant defences of human mammary epithelial cells under leptin depend on neoplastic state

Abstract Background Obesity is associated with oxidative stress, a major factor in carcinogenesis, and with high leptin concentration. The aim of this study was to determine the effects of leptin on the antioxidant response in three human mammary epithelial cells each presenting a different neoplastic status: healthy human mammary epithelial cells (HMEC), oestrogen-receptor positive MCF-7 cells and triple-negative MDA-MB-231 cells. Methods This in vitro kinetic study characterized the cell antioxidant response after 1, 6 and 24 h in the presence of leptin (10 or 100 ng/ml).The antioxidant response was defined in terms of cell glutathione content, gene expression and catalytic activity of antioxidant enzymes (i.e. glutathione peroxidase 1 (Gpx1), glutathione reductase (GR), glutathione S transferase (GST), heme-oxygenase 1 (HO-1) and cyclooxygenase-2 (COX-2)). Oxidative stress occurrence was assessed by lipid hydro peroxide (HPLIP) and isoprostane concentrations in culture media at 24 h. Results At both concentrations used, leptin induced ROS production in all cell models, contributing to various antioxidant responses linked to neoplastic cell status. HMEC developed a highly inducible antioxidant response based on antioxidant enzyme activation and an increase in cell GSH content at 10 ng/ml of leptin. However, at 100 ng/ml of leptin, activation of antioxidant response was lower. Conversely, in tumour cells, MCF-7 and MDA-MB-231, leptin did not induce an efficient antioxidant response, at either concentration, resulting in an increase of lipid peroxidation products. Conclusions Leptin can modulate the oxidative status of mammary epithelial cells differently according to their neoplastic state. These novel results shed light on oxidative status changes in mammary cells in the presence of leptin

Réponse du micro-environnement tumoral mammaire à la vitamine D et/ou à l’exercice

International audienceIntroduction : Divers facteurs vont influencer le cancer mammaire et son développement tels que l’obésité, le statut en vitamine D et l’activité physique. Si la surproduction d’estrogènes associée à l’excès de masse grasse favorise la tumorigenèse en période de post-ménopause, un statut optimal en vitamine D et une activité physique la limite en réduisant l’inflammation, le stress oxydant et en recrutant des cellules de l’immunité cytotoxique tels que les cellules NK. L’objectif de cette étude est d’étudier in vivo l’impact d’une supplémentation en vitamine D associée ou non à un exercice imposé sur la réponse du micro-environnement tumoral.Matériel et méthodes : 44 souris C57BL/6 âgées de 9 mois, ovariectomisées et soumises à un régime riche en lipide, ont été randomisées en 4 groupes supplémentés ou non en vitamine D (HF et HF+D) et/ou soumises à un exercice imposé d’intensité modérée de course sur tapis roulant (HF+Ex et HF+D+Ex). A la semaine 8, des cellules tumorales mammaires syngéniques EO771 ont été implantées dans la 4ème paire de glande mammaire. A l’issue de 6 semaines de croissance tumorale, l’infiltrat immunitaire a été caractérisé par une approche phénotypique en cytométrie en flux. L’expression des gènes caractéristiques des cellules immunitaires, des acteurs du métabolisme de la vitamine D ont été analysés. Le stress oxydant a été étudié via la mesure de l’activité des différentes enzymes associées. Les effets de la supplémentation en vitamine D et/ou de l’exercice ont été exprimés en moyenne ± écart-type et analysés par une ANOVA 2 voies suivie d’un test de Tukey.Résultats : La concentration en vitamine D sérique augmente significativement chez les souris des groupes supplémentés (effet vitamine D : p=0,0032 ; HF : 63,2±26,7 ; HF+D : 106,3±17,3 ; HF+Ex : 63,5±19,7 ; HF+D+Ex : 83,7±27,2 ng/mL) validant ainsi l’expérience. Au niveau intra-tumoral, ni la supplémentation en vitamine D ni l’exercice imposé ne modulent les proportions des sous populations immunitaires. L’exercice entraine l’augmentation de gènes spécifique des différentes populations immunitaires (effet exercice : p < 0,05). Parmi eux on retrouve les gènes Cd8a, spécifique des Lymphocytes TCD8+ (HF : 1±0,14 ; HF+D : 0,81±0,11 ; HF+Ex : 1,22±0,29 ; HF+D+Ex : 1,40±0,22), Eomes, spécifique des cellules NK (HF : 1±0,15 ; HF+D : 1,11±0,19 ; HF+Ex : 1,61±0,26 ; HF+D+Ex : 2,37±0,39) et Nos2, spécifique des cellules M1 (HF : 1±0,37 : HF+D : 0,74±0.36 ; HF+Ex : 1,81±0,34 ; HF+D+Ex : 1,91±0,51). L’exercice et la vitamine D, sans interagir entre eux, augmentent significativement l’expression de divers gènes (effet exercice et effet vitamine D : p < 0,05) tel que Tnfr spécifique des TCD8+ (HF : 1±0,21 ; 1,58±0,57 ; 1,89±0,45 ; HF+D+Ex : 2,62±0,35) et Dx5 spécifique des cellules NK (HF : 1±0,12 ; HF+D : 1,40±0,48 ; HF+Ex : 1,44±0,41 ; HF+D+Ex : 2,16±0,28) mais diminue significativement le ratio Tnfr/Foxp3 (HF : 1±0,45 ; HF+D : 0,91±0,30 : HF+Ex : 0,48±0,21 ; HF+D+Ex : 0,82±0,21). Enfin, l’interaction entre les deux facteurs diminue significativement le ratio Cd8a/Foxp3 (HF : 1±0,65 ; HF+D : 0,46±0,27 ; HF+Ex : 0,31±0,09 ; HF+D+Ex : 0,44±0,09 ; p<0,05). Au niveau du stress oxydant, les activités enzymatiques de la GST, GR et GPx ne sont pas modulées par les 2 facteurs, mais la vitamine D tend à augmenter l’expression du gène Caspase3 (effet vitamine D : p=0,0701 ; HF : 1±0,13 ; HF+D : 1,54±0,53 ; HF+Ex : 0,98±0,16 ; HF+D+Ex : 1,18±0,24). Enfin, les facteurs modulent les acteurs du métabolisme de la vitamine au sein de la tumeur, avec une augmentation significative (p<0,05) de l’expression de Vdr (HF : 1±0,19 ; HF+D : 2,6±0,38 ; HF+Ex : 2,28±0,39 ; HF+D+Ex : 2,25±0,27), Cyp24a1 (HF : 1±0,59 ; HF+D : 8,39±2,58 ; HF+Ex : 10,21±5,24 ; HF+D+Ex : 10,22±3,86) par la vitamine D et l’exercice et une augmentation de l’expression de Cyp27b1 (HF : 1±0,27 ; HF+D : 2,39±0,27 ; HF+Ex : 1,75±0,39 ; HF+D+Ex : 1,99±0,36) par la vitamine D.Conclusion : Dans ce modèle expérimental, la supplémentation en vitamine D et l’exercice n’influent pas sur la proportion des cellules immunitaire du micro-environnement mais modulent l’expression des gènes spécifiques des sous-population immunitaire. Cependant, la surexpression de Cyp24a1, enzyme responsable de l’inactivation de la vitamine D, et une activité physique spontanée élevée masquent certains effets immuno-modulateurs attendus ainsi que son effet antiprolifératif direct. Des études complémentaires doivent être réalisées afin de déterminer plus précisément l’intérêt d’un apport en vitamine D en situation de carcinogenèse tumorale