Blueberry supplementation attenuates oxidative stress within monocytes and modulates immune cell levels in adults with metabolic syndrome: a randomized, double-blind, placebo-controlled trial

Anand R. Nair a, Nithya Mariappan ad, April J. Stull bc and Joseph Francis *a
aComparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA. E-mail: jfrancis@lsu.edu; Tel: +1 225-578-9414
bDepartment of Human Ecology, University of Maryland Eastern Shore, Princess Anne, MD 21853, USA
cPennington Biomedical Research Center, Louisiana State University System, Baton Rouge, LA 70808, USA
dDepartment of Anesthesiology and Perioperative Medicine, Division of Molecular and Translational Biomedicine, School of Medicine, The University of Alabama at Birmingham (UAB), Alabama, 35294, USA

Received 5th June 2017 , Accepted 27th September 2017

First published on 28th September 2017


Background: Blueberries (BB) have been shown to improve insulin sensitivity and endothelial function in obese and pre-diabetic humans, and decrease oxidative stress and inflammation, and ameliorate cardio-renal damage in rodents. This indicates that blueberries have a systemic effect and are not limited to a particular organ system. In order for blueberries to exert beneficial effects on the whole body, the mechanism would logically have to operate through modulation of cellular humoral factors. Objective: This study investigated the role of blueberries in modulating immune cell levels and attenuating circulatory and monocyte inflammation and oxidative stress in metabolic syndrome (MetS) subjects. Design: A double-blind, randomized and placebo-controlled study was conducted in adults with MetS, in which they received a blueberry (22.5 g freeze-dried) or placebo smoothie twice daily for six weeks. Free radical production in the whole blood and monocytes, dendritic cell (DC) levels, expression of cytokines in monocytes and serum inflammatory markers were assessed pre- and post-intervention. Results: Baseline free radical levels in MetS subjects’ samples were not different between groups. Treatment with blueberries markedly decreased superoxide and total reactive oxygen species (ROS) in whole blood and monocytes compared to the placebo (p ≤ 0.05). The baseline DC numbers in MetS subjects’ samples in both groups were not different, however treatment with blueberries significantly increased myeloid DC (p ≤ 0.05) and had no effect on plasmacytoid cells. Blueberry treatment decreased monocyte gene expression of TNFα, IL-6, TLR4 and reduced serum GMCSF in MetS subjects when compared to the placebo treatment (p ≤ 0.05). Conclusions: The findings of the current study demonstrate that blueberries exert immunomodulatory effects and attenuate oxidative stress and inflammation in adults with MetS.


Introduction

Metabolic syndrome (MetS) comprises a cluster of risk factors that indicate a higher risk for cardiovascular diseases. Cardiovascular disease is the leading cause of death in the United States and produces immense economic and health burdens.1 The salient features of MetS include obesity, hypertension, insulin resistance and dyslipidemia, which are well-known contributing factors to the development of cardiovascular diseases.2 Studies have reported that MetS develops as a result of an imbalance among dietary intake, regulation of cardiac function, glucose metabolism and sedentary lifestyle.3 This imbalance contributes to an augmented inflammatory response and elevated oxidative stress in the body.4

The goal to reduce the incidence of MetS warrants the use of preventative strategies to reduce inflammation, reactive oxygen species (ROS) and oxidative stress. Although a wide range of pharmacological therapeutics is available to combat the factors that lead to the development of MetS, most have adverse side effects. In this context, the need for novel non-pharmacological approaches to delay the progression of MetS is growing. Many fruits, especially blueberries, have been shown to be excellent sources of antioxidant compounds, such as anthocyanins and phenolics.5 Antioxidants serve to counterbalance the effect of oxidants (i.e., ROS) and protect the body from cellular damage that leads to many negative health outcomes.6 The importance of blueberries as a functional food in alleviating the MetS has been highlighted by recent research findings. In preclinical studies, a blueberry-enriched diet has been demonstrated to improve mitochondrial integrity, and reduce inflammation and oxidative stress.7–10 Blueberry polyphenols were found to improve insulin sensitivity and endothelial function in humans.11–13 In addition, we demonstrated that blueberry supplementation improved insulin sensitivity, decreased oxidative stress and inflammation, and protected against cardiovascular and renal damage in normal animals14 and an animal model of MetS.15 These findings indicate that blueberry consumption has effects on the whole body and are not limited to a particular organ system. In order for blueberries to exert beneficial effects on the whole body, the mechanism would logically have to operate through modulation of cellular humoral factors, such as the blood leukocytes, specifically the monocytes.16

Blood monocytes are leukocytes consisting of at least two functionally distinct subsets, namely inflammatory and stationary monocytes. The leukocytes in the blood are a highly important population of cells that protect against infection or injury. Recent findings indicate that activated monocytes differentiate into dendritic cells, which then polarize naïve T-cells into Th1 or Th2 cells regulating inflammatory balance.17,18 It has been shown that the dendritic cell number is decreased in cardiovascular19 and diabetic patients.20 In addition, it is evident that the propagation of many diseases occurs via NADPH oxidase (NOX), a family of enzymes that combines NADPH and oxygen to actively generate superoxide.21 This enzyme is present in macrophages, monocytes and neutrophils to form ROS within the phagosomal membrane to clear pathogens and damaged cells (4). It is possible that the health benefits of blueberries may be exerted by decreasing the neutrophil NADPH oxidase activity.11 Thus, protection by phagocytes is crucial, but not without side effects, as these ROS leak out of the phagocytes and can produce detrimental effects on bystander cells.22 In an environment rich in oxidative stress, like in MetS or diabetes, these bystanders might drive increased activation of immune system, cell damage and disease progression. Thus, it is evident that monocytes are vital in regulating the immune system and oxidative stress, and the consumption of blueberries rich in polyphenols may have a significant impact on the inflammatory status and oxidative stress levels associated with the MetS.

This ancillary study used pre- and post-intervention blood samples collected from a published parent study conducted by Stull et al.12 Stull et al. and colleagues investigated the impact of blueberry consumption on modifying blood pressure and endothelial function. They found that blueberries did not improve blood pressure, but improved (i.e., increased) endothelial function over six weeks in subjects with MetS. This ancillary study sought to provide further evidence of the benefits of blueberries in a population at risk for developing type 2 diabetes and cardiovascular disease. Therefore the objective of this study was to determine whether blueberry supplementation modulates immune cell levels and attenuates circulatory and monocyte inflammation and oxidative stress in subjects with MetS. We hypothesized that the beneficial effects of blueberries in subjects with MetS were due to an increase in immune cell levels, and decrease in inflammation and oxidative stress in the circulation and also within the monocytes.

Methods

Subjects

Participants in the parent study were recruited from the Greater Baton Rouge, LA, USA area. All participants gave written informed consent before participating in the study. We included participants who were at least 20 years old and met the definition of MetS as defined by the World Health Organization (WHO).2 We excluded from the study all the subjects that smoked and with known diseases, such as diabetes, liver, heart or kidney disease. Further, the participants abstained from using nonprescription drugs, vitamins, dietary and herbal supplements two weeks prior to the start of the study and throughout the duration of the study.

Study design

The study design was previously reported (clinicaltrials.gov; NCT01399138).12 Briefly, a six-week randomized, double-blind, placebo-controlled and parallel arm clinical intervention trial was conducted at Pennington Biomedical Research Center, Baton Rouge, LA, USA. Recruitment began in July 2010 and continued until June 2012. The study was approved by Pennington Biomedical Research Center Institutional Review Board for human subjects (approval number 10014). The participants who met all the inclusion criteria were randomly assigned to either the blueberry (MetS + BB, n = 15) or placebo (MetS + P, n = 12) group. Block randomization was used with random functions in SAS 9.3 to ensure well-balanced placebo and blueberry groups. The randomization table was created by the biostatistician and the subjects were randomized to the groups by the kitchen staff. The blueberry and placebo smoothies, and participants were identified by code numbers. The study participants and investigators were blinded until after the study and data analysis were completed. The participants were required to visit the clinic weekly over the six-week period.

The participants consumed twice daily a 12-oz (∼356 ml) yogurt and skim-milk based smoothie with 22.5 g of freeze-dried blueberry powder added (total 45 g day−1) or an identical smoothie without the blueberry powder (i.e., placebo). The smoothies were identical in physical appearance, macronutrient content, fiber content, consistency and taste as described previously.12 The participants were instructed by a registered dietitian to consume the first smoothie during breakfast time and the second one during dinner time. The 45 g of freeze-dried blueberry powder equated to approximately 2 cups of fresh blueberries and was supplied by the United States Highbush Blueberry Council, CA, USA. The blueberry powder was made from a 50/50 blend of two varieties of blueberries, Tifblue (Vaccinium ashei) and Rubel (Vaccinium corymbosum). The nutritional composition of the blueberry powder was analyzed by Medallion Labs, Minneapolis, MN, USA, as described previously.12

Based on their food-intake and eating pattern, the participants were counseled by a registered dietitian on the importance of maintaining their body weight by eliminating approximately 500 kcal day−1 from their daily intake to compensate for the energy consumed in the smoothies. The participants were cautioned about gaining weight during the duration of the study and their body weight was monitored on a weekly basis to verify that there was no significant weight gain. To eliminate consumption of other anthocyanin-rich foods and drinks, the subjects were asked to avoid consumption of berries, grapes and juices containing these fruits and wine, during the study. Further, to monitor compliance, the participants were instructed to return unconsumed blueberry or placebo smoothies when they picked up their next week of smoothies, and this information was recorded.

Whole blood samples and isolation of mononuclear cells

Blood samples were obtained from the subjects at the beginning of this study and after six weeks of smoothie consumption. The blood samples were analyzed at Louisiana State University in Comparative Biomedical Sciences, School of Veterinary Medicine. The samples were collected around the same time of the day to minimize variations, and monocytes were isolated immediately. Human peripheral blood mononuclear cells were isolated using a density-gradient method using Histopaque (Cat. #1077, Sigma Aldrich, US). Briefly, 5–6 ml of histopaque at room temperature was aliquoted into a 15 ml conical tube. An equal amount of whole blood was slowly overlaid on top of the histopaque in the conical tube, centrifuged at 1000 rpm for 20 min at 4 °C. The peripheral blood mononuclear cells that float over histopaque, at the end of centrifugation, were carefully aspirated. These isolated mononuclear cells were washed twice using phosphate-buffered saline (PBS) at 1000 rpm for 10 min at 4 °C.

To further ensure that we used a specific population of monocytes, we used a magnetic bead separation method to positively isolate cells that were CD14+ by microbead technology. Briefly, cells were incubated with CD14 and CD16 microbeads obtained from MACS Miltenyl Biotech Inc., CA, USA and CD14highCD16 and CD16+ monocytes were separated using autoMACS Pro separator, according to the manufacturer's protocol. The monocytes were eluted in 1 ml of elution buffer and had a cell count of 3 × 106 cells per μl, which was used for further molecular analyses. The peripheral blood obtained from MetS subjects from the blueberry and placebo groups were analyzed by flow cytometry.

Myeloid and plasmacytoid dentritic cells

We measured both myeloid and plasmacytoid dentritic cells using four-color flow cytometry. Briefly, 100 μl blood was incubated with CD123-PE- (phycoerythrin), anti-HLA-DR-PerCP- (peridinin chlorophyll protein), lineage 1 cocktail (CD3, CD12, CD16, CD20, CD56)-FITC (fluorescent isothiocyanate)- and CD11c-APC (allophycocyanin)-conjugated mAbs for 20 min at room temperature. The erythrocytes were then lysed by FACS lysing solution (Beckton Dickinson). The cells were washed gently with PBS and the stained cells were analyzed with a FACScaliber flow cytometer and the FlowJo software. The peripheral blood samples were stained with anti-HLA-DR mAb and the lineage cocktail to gate lin-1 negative and HLA-DR positive cells. The gated cells were then further observed for the expression of CD11c and CD123 to determine the two distinct dendritic cell lineages, myeloid (LinHLA-DR+CD11c+) and plasmacytoid (LinHLA-DR+CD123+) dentritic cells, respectively. The total number of white blood cells was determined using an automated cell counter (Nexcelom Cellometer Auto 2000). The percentage of myeloid and plasmacytoid dentritic cells were derived from the total number of dentritic cells as determined by flow cytometric analysis. Also, the number of myeloid and plasmacytoid dendritic cells were adjusted for the total white blood cells.

Samples were acquired on a FACScan flow cytometer (BD Biosciences, San Jose, CA) utilizing a 15 mW 488 nm argon-ion laser. A total of 10[thin space (1/6-em)]000 cells per sample were acquired on a Macintosh G5 workstation (Apple Computer, Cupertino, CA) running Cellquest Pro software (BD Biosciences, San Jose, CA). Cell debris was eliminated by gating on intact cells based on dot plots of forward scatter versus side scatter. Fluorescence analyses in the form of histograms were illustrated using Cellquest Pro software (BD Biosciences, San Jose, CA).

Total ROS and superoxide production rates

Total ROS and superoxide production rates were measured in whole blood and monocytes from the subjects.14 The ‘total ROS’ represents all ROS; however, the major sources trapped by the spin trap used are superoxide, hydrogen peroxide, and hydroxyl radical, with other species as minimal contributors. All Electron paramagnetic resonance (EPR) spectroscopy measurements were performed with a BenchTop EPR spectrophotometer e-scan R (Noxygen Science Transfer and Diagnostics, Elzach, Germany). 1-Hydroxy-3 methoxycarbonyl-2,2,5,5-tetramethylpyrollidine (CMH) was used to measure total ROS and superoxide22,23 in whole blood and monocytes.

For the ROS measurement preparation, whole blood (20 μl in PBS) or monocytes (20 μl in PBS) were added to a tube containing 20 μl of CMH (400 μM) and then mixed gently. For the superoxide measurement preparation, 20 μl whole blood or monocytes were added to a tube containing 20 μl of CMH (400 μM) and then added to 0.64 μl of PEG (50 U ml−1). In both cases, the samples were measured in 50 μl disposable glass capillary tubes (Noxygen Science Transfer and Diagnostics) and measured on the Electron paramagnetic resonance spectroscopy instrument.

Circulatory levels of inflammatory and immune markers

The circulatory levels of inflammatory and immune markers were measured using ELISA. The circulating levels of interferon-α (IFN-α; catalog code ELH-IFNa), interleukin-12 subunit p70 (IL12p70; catalog code ELH-IL12P70), granulocyte macrophage colony-stimulating factor (GMCSF; catalog code ELH-GMCSF), fms-related tyrosine kinase 3 ligand (FLT3L; catalog code ELH-FLT3L) were measured from serum collected at the beginning and the end of the six-week period from the subjects, according to the manufacturer's (RayBiotech Inc., Norcross, GA, USA) instructions.

mRNA levels of proinflammatory cytokines

Real-Time PCR was used to determine the mRNA levels of proinflammatory cytokines, such as tumor necrosis factor alpha (TNFα), interleukin-6 (IL-6) and toll-like receptor 4 (TLR4) in the isolated monocytes by using specific primers, as previously described.22,24 Total RNA was isolated using Trizol Reagent (Invitrogen, CA). The RNA concentration was calculated from the absorbance at 260 nm and RNA quality was assured by the 260/280 ratio. The RNA samples were treated with DNase I (Ambion) to remove any genomic DNA. First strand cDNA were synthesized from 2 μg RNA with iScript cDNA synthesis kit (Bio-rad, Hercules, CA). Real-Time PCR was performed in 384-well PCR plates using iTaq SYBR Green Super mix with ROX (Bio-rad) in triplicate using the ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, CA). The PCR cycling conditions were as follows: 50 °C for 2 min, 95 °C for 3 min, followed for 45 cycles (15 s at 95 °C and 1 min at 60 °C). To confirm the specific PCR product, a dissociation step (15 s at 95 °C, 15 s at 60 °C and 15 s at 95 °C) was added to check the melting temperature. Gene expression was measured by the ΔΔCT method and was normalized to 18 s RNA mRNA levels.

Statistical analyses

This study was powered to detect differences between the MetS + BB and MetS + P groups based on the parent study.12 SAS software version 9.4 was used to conduct a repeated measures analysis of variance with the mixed procedure. Fixed effects in the model included Group, Time Point and the interaction of Group × Time Point. The random effect was subject ID within Group. All comparisons used a p ≤ 0.05 to determine significance.

Results

A total of 27 participants’ blood samples were analyzed in this ancillary study (MetS + P, n = 12 and MetS + BB, n = 15). The parent study12 had 44 subjects complete the study (n = 23 blueberry; and n = 21 placebo). Because the ancillary study began after the initiation of subject recruitment and data collection for our parent study, we were unable to collect monocytes from 17 subjects. The majority of the MetS + P and MetS + BB groups’ baseline characteristics were comparable except for the percentage of body fat and HDL levels (Table 1).
Table 1 Baseline anthropometrics and serum biochemistry of participants with the metabolic syndrome
  Placebo Blueberry
Variables n = 12 n = 15
Values are Mean ± SEM; blood was drawn from participants after a 10-hour fast; *Blueberry vs. Placebo, p ≤ 0.05. LDL, low-density lipoprotein; HDL, high-density lipoprotein.
Race (African American/Caucasian), n/n 5/7 10/5
Gender (male/female), n/n 2/10 7/8
Age, year 59 ± 3 55 ± 2
Body weight, kg 95.5 ± 4.2 95.6 ± 4.2
BMI, kg m−2 35.2 ± 1.4 34.2 ± 0.8
Body fat, % 43.5 ± 2.3 36.2 ± 1.8*
Fat mass, kg 42.1 ± 3.3 34.9 ± 2.3
Lean mass, kg 54.1 ± 2.9 61.7 ± 3.2
Systolic blood pressure, mm Hg 128.6 ± 4.6 125.9 ± 3.1
Diastolic blood pressure, mm Hg 78.6 ± 3.0 85.3 ± 1.8
Serum biochemistry
Glucose, mmol L−1 102.1 ± 1.8 99.7 ± 1.5
Insulin, pmol L−1 15.2 ± 1.8 17.3 ± 3.2
Triglycerides, mmol L−1 125.5 ± 17.3 161.5 ± 24.0
Cholesterol, mmol L−1 204.2 ± 12.4 216.3 ± 6.4
LDL, mmol L−1 118.2 ± 10.5 134.8 ± 7.6
HDL, mmol L−1 60.9 ± 4.5 49.3 ± 2.8*


Blueberry-supplementation attenuated oxidative stress in the whole blood and monocytes

Baseline (week 0) free radical levels in the whole blood (Fig. 1A and B) and isolated monocytes (Fig. 1C and D) were not different between the MetS + P and MetS + BB groups. At six weeks (week 6), the MetS + BB subjects had a significantly lower total ROS and superoxide radical levels in the whole blood (Fig. 1A and B) and isolated monocytes (Fig. 1C and D) when compared to the MetS + P subjects.
image file: c7fo00815e-f1.tif
Fig. 1 Effect of blueberries on total ROS and superoxide radical in whole blood and monocytes of humans with MetS. Rate of production of (A) total ROS and (B) superoxide radical in whole blood at baseline (week 0) and after 6 weeks (week 6) of placebo or blueberry smoothie consumption; rate of production of (C) total ROS and (D) superoxide radical in monocytes at baseline (week 0) and after 6 weeks (week 6) of placebo or blueberry smoothie consumption; *p ≤ 0.05 MetS + P week 6 vs. MetS + BB week 6.

Blueberry-supplementation modulated the dendritic cells in the blood

We measured both myeloid and plasmacytoid dendritic cells using flow cytometry (Fig. 2A and D). The peripheral blood samples were gated for lin-1 negative and HLA-DR positive cells. The gated cells were then further observed for the expression of markers specific to myeloid and plasmacytoid dendritic cells. At baseline, there were no significant differences in the cell number of plasmacytoid (Fig. 2E) and myeloid (Fig. 2F) dendritic cells between the two groups with MetS subjects. There was no significant difference between the numbers of plasmacytoid dendritic cells between the MetS + P and MetS + BB groups at the end of six weeks (Fig. 2E). However, treatment with blueberries for six weeks significantly elevated the number of myeloid dendritic cells when compared to the placebo supplemented MetS subjects (Fig. 2F).
image file: c7fo00815e-f2.tif
Fig. 2 Effect of blueberries on plasmacytoid and myeloid dendritic cells in humans with MetS. A representative flow cytometric figure of (A) whole blood cells, (B) cells gated for Lin-HLA+, (C) plasmacytoid dendritic cells and (D) myeloid dendritic cells; percentage of (E) plasmacytoid and (F) myeloid dendritic cells at baseline (week 0) and after 6 weeks (week 6) of placebo or blueberry smoothie consumption; DC = dendritic cells; *p ≤ 0.05 MetS + P week 6 vs. MetS + BB week 6.

Blueberry-supplementation decreased the mRNA levels of inflammatory markers in the monocytes

Since MetS is a chronic inflammatory condition, we measured the expression of inflammatory cytokines in the monocytes from MetS subjects. We observed that the mRNA levels of the proinflammatory cytokines (TNFα, IL-6, TLR4) were significantly elevated in the MetS + P subjects when compared to the MetS + BB subjects (Fig. 3A–C). In contrast, after six weeks of consuming the smoothies, the MetS subjects supplemented with the blueberries had a significantly reduced expression of the inflammatory markers TNFα (Fig. 3A), TLR4 (Fig. 3B), IL-6 (Fig. 3C) in the monocytes when compared to the MetS + P subjects.
image file: c7fo00815e-f3.tif
Fig. 3 Effect of blueberries on mRNA levels of inflammatory markers in monocytes of humans with MetS. The gene expression of inflammatory markers (A) TNFα, (B) TLR4, (C) IL-6 in monocytes of MetS patients at baseline (week 0) and after 6 weeks (week 6) of placebo and blueberry smoothie consumption. *p ≤ 0.05, MetS + BB week 6 vs. MetS + P week 6, #p ≤ 0.05, week 0 vs. week 6 (within group). Tumor necrosis factor alpha (TNFα), interleukin 6 (IL6) and toll-like receptor 4 (TLR4). Relative expression to 18 s RNA.

Blueberry-supplementation reduced circulating levels of inflammatory markers

The baseline circulating levels of the serum inflammatory (IFNα, GMCSF) and immune markers (IFNα, IL12p70, FLT3L) were not different between MetS patients from both groups (Fig. 4A–D). At six weeks, the circulating levels of IFNα, IL12p70, and FLT3L were not significantly different in the MetS + BB subjects when compared to the MetS + P subjects (Fig. 4A, B, D). However, GMCSF (Fig. 4C) levels were significantly reduced in the blueberry supplemented subjects when compared to the placebo supplemented subjects at the end of the study.
image file: c7fo00815e-f4.tif
Fig. 4 Effect of blueberries on circulating levels of inflammatory and immune markers in humans with MetS. Serum levels of (A) IFNα, (B) IL12p70, (C) GMCSF and (D) FLT3L at baseline (week 0) and after 6 weeks (week 6) of placebo and blueberry smoothie consumption. *p ≤ 0.05 MetS + P week 6 vs. MetS + BB week 6. Interferon-α (IFN-α), interleukin-12 subunit p70 (IL12p70), granulocyte macrophage colony-stimulating factor (GMCSF), fms-related tyrosine kinase 3 ligand (FLT3L).

Discussion

To our knowledge, this is the first study to find that blueberry supplementation for six weeks significantly attenuated oxidative stress and expression of inflammatory markers in the monocytes, and also modulated the dendritic cell numbers in MetS subjects. In addition, we reported that a blueberry intervention attenuated oxidative stress in the whole blood and decreased circulatory inflammatory markers. These findings put forward a plausible novel mechanism by which blueberries may exert its whole body effects.

It is possible that blueberries exert its beneficial effects through modulation of leukocytes that are an integral part of innate and adaptive immune systems.25 It is well known that blood leukocytes play a major role in protecting the body against injury and infection.26 A specialized subpopulation of leukocytes, called monocytes, are well characterized and have been shown to consist of at least two functionally distinct subsets, namely inflammatory and stationary monocytes. The inflammatory monocytes, as the name suggests, migrate to the site of inflammation and hence possess the ability to propagate diseases characterized by chronic inflammation.27 Inflammation is a well-known risk factor for MetS15 and other cardiovascular diseases28–30 and since MetS is a condition that involves chronic low or high grade inflammation,15 we investigated the possibility of blueberries modulating the inflammatory profile of these monocytes, thereby protecting against the progression of MetS. In the current study, we observed a decrease in the expression of inflammatory cytokines TNFα, IL6 and TLR4 in the monocytes of MetS subjects that consumed the blueberry smoothie for six weeks when compared to the placebo smoothie group at the end of the study. Also, after six weeks, there was an increase in the expression of the inflammatory cytokines in the monocytes from subjects in the placebo smoothie group. We speculate that this could be a result of a higher monocyte turnover in MetS subjects as a result of elevated oxidative stress and chronic inflammation. Evidence from our study implicates higher levels of whole blood oxidative stress in MetS subjects and this increased oxidative stress is also observed in the monocytes of these patients. It is known that increased oxidative stress leads to increased inflammation.31 This eventually contributes to higher levels of monocyte polarization, which leads to a higher monocyte turnover.32 On the contrary, consuming BB for six weeks, provided significant reduction in systemic (whole blood) as well as monocytic oxidative stress. This improvement in the oxidative stress environment within the monocytes, after BB consumption, attenuated inflammation and generation of proinflammatory cytokines within the monocytes. This attenuation in the inflammatory cascade inhibited the polarization of monocytes, thereby leading to a lower monocyte turnover.

Previous evidence suggests that the level of monocyte turnover is a strong predictor of inflammatory disease progression.33,34 Thus, we would assume that in our MetS subjects that consumed the placebo smoothie, the elevated expression of inflammatory cytokines at week six could be a result of the increased monocyte turnover in the cell,35,36 which is indicative of the progressive condition of MetS. Although the placebo patients did not receive an intervention, the monocytes measured at baseline and 6 weeks later were not the same due to the monocytes having an increased turnover in the cell. These current findings support our previously investigated studies that evaluated the antioxidant and anti-inflammatory properties of blueberries in animal models of spontaneous hypertension,37 acute kidney injury14 and MetS.15 Similarly, other researchers9,10 have found that blueberries reduced gene expression and circulatory levels of inflammatory markers (TNFα, IL-10, IL-6) in animal models of obesity and insulin resistance. The uniqueness of the current study was that the anti-inflammatory effects of blueberries were observed in the monocytes. Although previous animal studies and the current study were performed with different designs and experimental models, all the studies reported that blueberry consumption provided protection against increased inflammation and inflammatory diseases. Therefore, these current findings are supportive of the idea that blueberry supplementation has a whole body effect and the effects are not limited to a particular organ system.

The effect of blueberries on reducing inflammatory marker levels is less pronounced in previous human studies. Stull et al.13 and Basu et al.38 demonstrated whole body effects such as improvement in insulin sensitivity and lowering of blood pressure in individuals with MetS, but no changes were observed with the blood inflammatory markers (C-reactive protein, TNFα, MCP-1, adiponectin, IL-6, sICAM-1, sVCAM-1). However, Basu et al.38 observed a decrease in plasma oxidized LDL and serum malondialdehyde and hydroxynonenal concentrations. In addition, other researchers found that blueberries reduced the levels of endogenous and oxidant-induced DNA damage in healthy male volunteers,39,40 but there were no effects on serum markers of inflammation.40 Oxidatively induced DNA damage is a marker of oxidative stress.41 Johnson et al.42 found that eight weeks of blueberry treatment significantly increased superoxide dismutase (SOD) levels when compared with the baseline levels, but the control group SOD levels were also increased in postmenopausal women with pre- and stage 1-hypertension. The previous studies evaluated only the circulatory levels of the inflammatory markers, which could be one explanation for the difference between the previous and current study findings. In addition, the circulatory levels of GMCSF were reduced after blueberry consumption in the current study and this particular inflammatory marker was not evaluated in the previous human studies. It is interesting to note that despite no changes in inflammatory markers, the previous human studies still found that blueberries had a significant effect on parameters of oxidative stress. Similarly, our study observed that blueberries not only decreased oxidative stress in the whole blood, but also in the monocytes.

In addition to attenuating inflammation, our findings from this study demonstrate that blueberry consumption was able to significantly decrease the levels of free radicals in the whole blood, and mainly within the monocytes itself, thereby suggesting an antioxidant-mediated protective ability of blueberries in MetS subjects. Oxidative stress has been well characterized in MetS43,44 and other cardiovascular disease conditions.14,22,28 Increased generation of ROS and a concomitant reduction in antioxidant defense system has been demonstrated to be an early instigator of MetS.44 Previous findings have implicated superoxide as an important free radical agent that can contribute to the development of endothelial dysfunction and cardiovascular disease by inducing vasoconstriction.45,46 ROS can promote cell death and inflammation via induction of mitochondrial dysfunction and researchers have found that blueberries can improve endothelial function11,12 and mitochondrial integrity,7 thereby explaining a possible mechanism of the antioxidant capacity of blueberries in the whole blood and monocytes. Interestingly, our parent study12 found that blueberries improved endothelial function in the same MetS subjects that were used in the current study. According to the current findings, the previously observed improvement in endothelial function with blueberry intake over 6 weeks may be due to the reduction in oxidative stress and inflammation in the whole blood and monocytes.

The role of blueberries on dendritic cells was highlighted in the current study. Recent findings indicate that activated monocytes differentiate into dendritic cells,17,18 which then polarize the naïve T cells into Th1 or Th2 cells regulating inflammatory balance. There are studies that have shown that monocyte-derived dendritic cells play a regulatory role in humans with cardiovascular47 and diabetes-associated48 diseases. Further, Zlotnikov-Klionsky et al. have demonstrated that dendritic cells exhibit an immune-regulatory phenotype in MetS,49 thereby highlighting the importance of these considerably small yet highly important population of immune cells in MetS. These interesting findings led us to evaluate if blueberries could also modulate the dendritic cells in human subjects with MetS. We observed a robust elevation in the number of LinHLA-DR+CD11c+ myeloid dendritic cells in MetS subjects that consumed a blueberry smoothie for six weeks compared to the placebo group. However, there was no change in the number of LinHLA-DR+CD123+ plasmacytoid dendritic cells in the blood samples of these subjects. Dendritic cells, being one of the important classes of antigen presenting cells in peripheral blood, are vital in eliciting immune responses in the human body. Further, there is a growing body of evidence suggesting that myeloid DCs are the more potent DCs in modulating T-cell stimulation,50 although this still remains controversial. However, it is well known that myeloid DCs possess higher levels of MHC (major histocompatibility complex) class II and adhesion molecules,51 when compared to their plasmacytoid counterparts. Again, the immuno-regulatory ability of DCs depend less on the type of DCs and more on the activatory or inhibitory stimuli. And since the types of DC that are regulated in human MetS are poorly understood, we assume from our findings in this study that MetS associated stimuli is more targeted towards modulating myeloid dendritic cells.

Our clinical study also has a few limitations that are worth mentioning. First, the distribution of genders in the placebo group was not equal. The placebo group consisted of more females than males and had a higher percentage of total body fat than the blueberry group. Our previous clinical study12 that used MetS participants demonstrated that the improvement in endothelial function in the blueberry-treated MetS subjects remained significant, even after adjusting for percent body fat and gender. Hence, we assumed that beneficial effects of blueberries observed in this study would also be consistent with the previous report. Second, the smoothies used in the current study used yogurt and milk, which have been shown to have beneficial effects on cardiovascular health.52,53 However, both our groups (blueberry and placebo) consumed smoothies with yogurt and milk which indicates that the beneficial effects observed in the current study can be attributed to the presence of blueberries in the diet and not the dairy products. Third, we did not measure the metabolites of blueberries in these patients. Future studies focusing on assessing the various metabolites of blueberries will enable us to understand the active compounds responsible for the protective effects of blueberries in MetS and other diseases. Finally, inclusion of a control group (without MetS) could have helped in understanding the placebo group changes that were observed in our MetS subjects.

In conclusion, we provide evidence that six weeks of a blueberry intervention can potentially exert protective effects in subjects with MetS. We demonstrated possible novel mechanisms for the beneficial effects of blueberries which includes attenuating inflammation and oxidative stress within the monocytes, and also modulating immune cell levels (i.e., dendritic cells) in humans with MetS. Although further genomic- and proteomic-level validation studies of this novel mechanism are warranted to fully evaluate this observation, we indicated for the first time that blueberry consumption does not merely target a particular organ system, but has a much more intriguing whole body protective effect in MetS. Future research observing the effects of longer blueberry supplementation and exploring the changes in immune signaling after the cessation of blueberry treatment in MetS patients would provide further insights into the mechanism(s) and potential of non-pharmacological interventions in conditions such as MetS.

Author contributions

ARN, NM, AJS, JF designed the research. ARN, NM conducted research. ARN, NM analyzed the data. ARN, AJS, JF wrote the paper. JF has primary responsibility for final content. All authors have read and approved the final manuscript.

Conflicts of interest

ARN, NM, AJS, JF – authors have no conflicts of interest.

Abbreviations

MetS + PMetabolic syndrome + placebo
MetS + BBMetabolic syndrome + blueberry
IFN-αInterferon-α
IL12p70Interleukin-12 subunit p70
GMCSFGranulocyte macrophage colony-stimulating factor
FLT3Lfms-Related tyrosine kinase 3 ligand
TNFαTumor necrosis factor alpha
IL-6Interleukin-6
TLR4Toll-like receptor 4
DCDendritic cells

Acknowledgements

Research reported in this publication was supported by the United States Highbush Blueberry Council (JF) and the National Center for Complementary and Integrative Health of the National Institutes of Health under award numbers K01AT006975 (AJS) and P50AT00277 (Botanical Dietary Supplements Research Center). This project used facilities that are supported in part by a NORC Center Grant # 2 P30 DK072476-11A1 entitled “Pennington/Louisiana NORC”. The content is solely the responsibility of the authors and does not necessarily represent the official views of the United States Highbush Blueberry Council and the National Institutes of Health.

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