The Journal of Nutrition

Nutritional Immunology

A Carbohydrate Beverage Reduces Monocytes

Expressing TLR4 in Children with Overweight
or Obesity
Grace M Niemiro,1,2 Nathan A Chiarlitti,3,4 Naiman A Khan,1 and Michael De Lisio1,3,4

1
Department of Kinesiology and Community Health, University of Illinois at Urbana-Champaign, Urbana, IL, USA; 2 Department of
Pediatrics, University of Arizona College of Medicine, Tucson, AZ, USA; 3 School of Human Kinetics, Brain and Mind Institute, Centre on
Neuromuscular Disease, University of Ottawa, Ottawa, ON, Canada; and 4 Regenerative Medicine Program, Ottawa Hospital Research
Institute, Ottawa, ON, Canada

Downloaded from https://academic.oup.com/jn/article/150/3/616/5673204 by guest on 06 April 2021

ABSTRACT
Background: Childhood obesity is increasing, with about one-third of children overweight or obese. Obesity is
characterized by a state of chronic low-grade inflammation that is related to cardiometabolic comorbidities. Inflammatory
monocytes, which are classified into 3 different groups—classical, intermediate, and nonclassical monocytes, with Toll-
like receptor 4 (TLR4+ ) expression indicating a proinflammatory state—underlie several obesity-associated morbidities.
Objectives: This study aimed to assess the responses of monocyte populations to beverages of differing macronutrient
composition in children with healthy weight (HW) or overweight/obesity (OW/OB).
Methods: Ten HW children (5th to 84.9th percentile; mean age 12.29 ± 2.5 y) and 7 children with OW/OB (85th to
99.99th percentile; mean age 11.96 ± 3.8 y) completed the study. Adiposity was determined via DXA. Using a double-
blinded, randomized, crossover design, participants consumed either a high-carbohydrate (CHO; 210 kcal; 0 g fat/56 g
carbohydrates/0 g protein) or a whole-egg–based high-protein/fat (EGG; 210 kcal; 15 g fat/0 g carbohydrates/18 g protein)
beverage. Venous blood was collected at baseline and 2 h postprandially for evaluation of metabolic and inflammatory
responses. Repeated measures ANOVA and Pearson correlations were conducted.
Results: Consuming the CHO beverage significantly reduced the primary outcome: TLR4+ expression on classical
monocytes in children with OW/OB only (25.60% decrease from baseline in OW/OB compared with 1.61% increase in
HW). Children with OW/OB had significantly less percentages of TLR4+ nonclassical monocytes than HW (47.66% lower
after CHO). Insulin and glucose (secondary outcomes), were significantly higher after the CHO condition compared with
baseline (230.61% and 9.93% increase, respectively). Changes in glucose were significantly and negatively related to
changes in monocyte populations in the CHO condition.
Conclusions: These data suggest that high-carbohydrate beverages alter monocyte populations in the blood in children
with OW/OB, which is related to glucose metabolism. These findings have implications for nutritional recommendations
in children with overweight/obesity. National Clinical Trial registry trial number: NCT03597542. J Nutr 2020;150:616–622.

Keywords: inflammation, innate immune system, inflammatory cytokines, diet, adolescents

Introduction 2) nonclassical monocytes, which are involved in inflammation,

antiviral functions, and tissue repair (7, 8); and 3) intermediate
Childhood obesity in the Western and developing world is
monocytes, which are a minor subset thought to be in a
increasing at an alarming rate, with about one-third of children
transition state toward the nonclassical type (8). Toll-like
overweight or obese (1). Obesity is connected with chronic low-
receptor 4 (TLR4) is a cell surface receptor on monocytes that
grade inflammation that is thought to be partly responsible
upon activation, results in subsequent inflammatory cytokine
for its cardiometabolic comorbidities (2–4). Inflammatory
production, creating an immediate immune response (9).
monocytes circulate in the peripheral blood and infiltrate
Previous data have shown that both adults and children with
peripheral tissue where they mature into macrophages (5).
obesity have higher concentrations of monocytes expressing
Their concentration in peripheral blood is routinely used in
TLR4 (10, 11), possibly due to chronically elevated levels of
clinical settings as an indicator for chronic inflammation, stress
inflammation induced by obesity.
responses, immune-mediated disease, necrosis, and in severe
Recent evidence has suggested that macronutrient compo-
cases, sepsis (6). There are 3 subtypes of monocytes in humans:
sition of a meal can induce an inflammatory response (12).
1) classical monocytes, which mature into macrophages (6, 7);

Copyright  C The Author(s) 2019.

Manuscript received June 3, 2019. Initial review completed September 25, 2019. Revision accepted November 8, 2019.
616 First published online December 11, 2019; doi: https://doi.org/10.1093/jn/nxz294.
In adults, a high-carbohydrate meal increased the levels of
inflammatory cytokines and CD11a-expressing inflammatory
immune cells, which peaked 2 h after ingestion (12). Conversely,
no increases in these inflammatory markers were observed
following a meal high in unsaturated fats (12). This is important
because it suggests that the macronutrient composition of
a meal can influence the acute inflammatory response. Ac-
cumulated inflammatory responses to individual meals could
then contribute to chronic inflammation. The effects of meals
with different macronutrient composition on blood monocyte
responses in children of different clinically defined body
weight status have never been previously examined. This is an
important gap in the literature because immune/inflammatory
responses in adults might not predict responses in children (13).
Furthermore, if high-carbohydrate meals are proinflammatory,
then children are particularly susceptible because they are the
targets of many marketing campaigns for high-carbohydrate
foods (14), particularly breakfast cereals (15).
Thus, the purpose of this study was to investigate the
response of inflammatory monocytes, particularly those ex-
pressing the proinflammatory marker, TLR4, and cytokines,
after beverages of different macronutrient composition in
healthy weight (HW) children compared with children with
overweight/obesity (OW/OB). We hypothesized that increased
quantities of inflammatory monocytes and cytokines would be
present in children with OW/OB. Furthermore, we hypothesized
that inflammatory monocytes and cytokines would increase
in response to consuming a beverage rich in carbohydrates
compared with a beverage low in carbohydrates, and that
this response would be exacerbated in children with OW/OB
compared with HW children.
Methods
Participants
This project conformed to the guidelines of the Declaration of Helsinki
and was approved by the Institutional Review Board of the University
of Illinois at Urbana-Champaign. Through databases and flyers,
36 children volunteered to participate in this study from November
2016 to February 2018 (Figure 1). Participants volunteered for the study
provided they were: 1) aged 7–17 y; 2) were free of any cardiometabolic
disease, cancer, or diabetes; 3) were HW or above on the children’s BMI
percentile scale; 4) were not allergic to eggs or artificial sweeteners; 5)
were not taking any anti-inflammatory medication; and 6) provided
written informed assent with parental/guardian written informed
consent. Because only 2 attempts were allowed to draw blood from
participants, difficulty in blood collection resulted in 25 participants
having usable data at baseline. After baseline data were collected,
8 participants had issues ingesting the beverages (3 had problems
ingesting the sugar drink, whereas 5 participants had problems ingesting
Funding for this work was provided by the Egg Nutrition Center (to GMN) and
the National Sciences and Engineering Research Council of Canada (to MDL).
The Egg Nutrition Council provided the freeze-dried egg products free of charge.
Author disclosures: The Egg Nutrition Center provided funding (to GMN) and
freeze-dried egg products to conduct the experiments. The funders were not
involved in data analysis or interpretation and were not involved in publishing
the final manuscript.
Supplemental Table 1 is available from the “Supplementary data” link in the
online posting of the article and from the same link in the online table of contents
at https://academic.oup.com/jn.
GMN and NAC contributed equally to this work.
Address correspondence to MDL (e-mail: mdelisio@uottawa.ca).
Abbreviations used: CHO, carbohydrate beverage condition; EGG, egg beverage
condition; ES, effect size; HW, healthy weight; OW/OB, overweight/obese; PE,
phycoerythrin; TLR4, Toll-like receptor 4.
the egg drink), resulting in 17 participants completing the full protocol.
Participants did not complete the full protocol for the following
reasons: inability to obtain sufficient blood volume for analysis, lost
to follow-up, and illnesses between visits. Participants provided written
informed assent with parental/legal guardian written informed consent
to participate in the study, and were compensated for their time. Despite
a large age range for recruitment, there was no difference in mean age
between groups.
Study design
This study was a double-blinded, randomized, crossover design.
Participants arrived at the laboratory on 3 different days ≥4 h fasted and
≥1 wk apart. On the first day, the study was described to participants
and their legal guardians prior to obtaining written informed assent
and consent. Then, participants completed anthropometric measures
including body composition, height, and weight. After completion of
anthropometric variables, participants submitted to a venous blood
draw from an antecubital vein. On visits 2 and 3, participants consumed
either a carbohydrate-free (EGG; egg-based) or high-carbohydrate
Downloaded from https://academic.oup.com/jn/article/150/3/616/5673204 by guest on 06 April 2021
(CHO; maltodextrin-based) beverage. All beverages were consumed
within 15 min. Beverages were provided in a random fashion with
randomization conducted via open software (www.researchrandomizer.
org). Participants then sat quietly for 2 h after ingestion. After 2 h,
participants submitted to a venous blood draw for the post time point.
On visit 3, the participants completed the same protocol as visit 2,
but with the beverage that was not previously ingested. Beverage order
for each participant was randomized, and a research team member
not associated with data analyses administered the beverages. Both
the participant and the researcher analyzing the data were blinded to
condition.
Beverage composition
The EGG beverage was composed of whole spray-dried egg powder
(Oskaloosa Food Products) whereas the CHO beverage was composed
of maltodextrin (NOW Foods). The amount of spray-dried whole-egg
powder was 36 g, equivalent to 3 whole eggs (210 kcal; 15 g fat,
0 g carbohydrates, 18 g protein) given in each beverage. The amount
of maltodextrin was 56 g (210 kcal; 0 g fat, 56 g carbohydrates,
and 0 g protein) given in each beverage. Each powder was dissolved
in 500 mL of water. To make each drink more palatable, 14.88 g
(5 commercially available 3.0-g packets) of artificial sweetener (stevia;
Truvia) was added. The amount of calories provided was selected based
on previous research indicating 210 kcal is the average intake for
children for breakfast (16).
Anthropometric measures, adiposity, and fitness
Participants were separated into HW and OW/OB based on BMI
percentile; with those having a 5th to 84.9th BMI percentile considered
HW, and those having an 85th to 99.9th BMI percentile of their age-
matched peers considered OW/OB (1). Whole body and regional soft
tissue composition were measured by DXA by using either a Discovery
A bone densitometer (software version 12.7.3; Hologic) or a Horizon
W bone densitometer (APEX software version 5.6.0.5; Hologic).
DXA measures of interest included fat percentage body weight, and
visceral fat mass, calculated from an algorithm provided on the APEX
software.
Whole blood analysis
Venous blood draws were conducted after ≥4-h fasting on the first study
day. On study days 2 and 3, participants arrived after ≥4-h fasting,
and blood draws were collected 2 h after beverage consumption. The
2-h time point was based on previous literature showing inflammatory
leukocytes peaked at 2 h after a high-carbohydrate meal in adults (12).
Postbeverage blood draws were collected at approximately the same
time of day (within 3 h) as baseline for each participant to avoid
circadian influences on blood cell populations. Blood was collected into
EDTA-anticoagulant tubes following previously defined protocols with
minor modifications (11). Monocyte quantification was conducted from
200 μL of whole blood using a lyse, no-wash technique as previously
Monocyte response to carbohydrate beverages in children 617
FIGURE 1 CONSORT diagram of participants enrolled in this study. Through databases and flyers, 36 children volunteered to participate in

Downloaded from https://academic.oup.com/jn/article/150/3/616/5673204 by guest on 06 April 2021

this. Because only 2 attempts were allowed to draw blood from participants, difficulty in blood collection resulted in 25 participants having usable
data at baseline. After baseline data were collected, 8 participants had issues ingesting the beverages, resulting in 17 participants completing
the full protocol.

described (11). Briefly, peripheral blood samples were incubated with statistically significant for all tests. All data are presented as mean (95%
phycoerythrin (PE)-conjugated CD14 (1:100; Invitrogen), fluorescein CI). Sample size was calculated based on a pilot of previously
isothiocyanate–conjugated CD16 (1:100; Invitrogen), and TLR4 (1:20; collected and published data (11) with an ES of 0.67, α = 0.05, and
PE-Cyanine7, clone: HTA125; eBioscience). Gating was conducted as β = 0.2 (1 − β = 0.8), and it was determined that n = 9 per BMI group
previously described (8, 11) based on unstained and single-stained would be sufficient.
controls. Isotype controls, which matched the heavy chain isotype of
each antibody, were also used to determine levels of nonspecific staining.
Compensation controls were used to accommodate spillover between
fluorophores. Flow cytometric analysis was performed using an Attune Results
Focusing Flow Cytometer (Life Technologies). Blood that was not used
for flow quantification was centrifuged for plasma storage (645 × g; Participant characteristics
15 min; 4◦ C). Plasma was divided into aliquots and stored at −80◦ C A total of 17 participants (HW: n = 10, 5 female; OW/OB:
until analysis. In some instances, sufficient blood was not collected for n = 7, 4 female) completed the 3-d experimental protocol.
all analyses. This is reflected in the different samples sizes in the figure Anthropometric information for these participants is presented
legends. in Table 1. The OW/OB group had a trend for higher BMI
(P = 0.06), had a significantly higher BMI percentile (P ≤ 0.05),
Plasma insulin and glucose and whole-body percentage fat (P < 0.05). No differences in
Plasma samples were used for quantification of insulin and glucose. age, height, and body weight were found between groups.
Glucose was determined using a 2900D Biochemistry Analyzer (YSI
Inc) on thawed plasma samples. The same samples, without being CHO beverage increased glucose and insulin
freeze-thawed after glucose analysis, were used for quantification of Glucose and insulin were significantly increased (P ≤ 0.05)
insulin at each time point (Insulin ELISA; Alpco). The ELISA was
after the CHO condition compared with baseline irrespective of
performed per manufacturer’s instructions with samples analyzed in
duplicate.
weight status. Glucose was significantly higher after the CHO
condition compared with baseline (95.98 mg/dL compared
with 87.31 mg/dL; Figure 2A; P = 0.027; ES = 1.3). Insulin
Statistical analysis was significantly higher after the CHO condition compared
Anthropometric variables, cell quantities, and plasma protein concen- with baseline (50.22 μIU/mL compared with 16.98 μIU/mL;
trations were analyzed for normality via the Shapiro–Wilk test, whereas
Figure 2B; P = 0.037; ES = 2.9). Glucose and insulin were not
equal variance was analyzed via the Levene test. Variables were log
significantly increased in the EGG group (P > 0.05).
transformed prior to analysis if normality was not met. Cell quantities
and plasma protein concentrations were analyzed by a 2 × 3 mixed-
model ANOVA (weight class, 2: HW, OW/OB) × (condition, 3: baseline,
CHO, EGG) in Prism 8.2.0 (GraphPad). Preplanned comparisons TABLE 1 Anthropometric information about participants who
between HW and OW/OB within each condition were further resolved completed the full protocol (n = 17)1
via the Tukey post hoc test. Correlations between anthropometric
Variable HW (n = 10) OW/OB (n = 7)
measures and cell parameters of interest were conducted using a
Pearson correlation and are represented in supplementary material Age, y 12.6 ± 2.48 12.0 ± 3.79
(Supplemental Table 1). Changes in cell populations and cytokines BMI, kg/m2 18.5 ± 1.16 30.2 ± 13.6a
were calculated as nontransformed “post-CHO” or “post-EGG” minus BMI percentile 51.5 ± 20.5 95.6 ± 3.84∗
“pre” values. Pearson correlations were conducted on changes in cell Weight, kg 43.4 ± 11.0 73.4 ± 47.4
populations and changes in glucose. Effect size (ES) for significant and Height, cm 153 ± 16.3 150 ± 16.6
trending outcomes was calculated using the formula: d = (xOW/OB −
Fat, % body weight 24.1 ± 6.06 43.2 ± 12.0∗
xHW )/s with d representing Cohen’s D, xOW/OB representing the mean
of the OW/OB group, xHW representing the mean of the HW group, 1
Values are mean ± SD. ∗ P ≤ 0.05; a P = 0.06. HW, healthy weight; OW/OB,
and s representing the standard deviation. P ≤ 0.050 was considered overweight/obese.

618 Niemiro et al.


FIGURE 2 Plasma glucose and insulin are increased after CHO
consumption in both HW (n = 10) and OW/OB (n = 7) weight
classes. Metabolic responses to EGG or CHO. (A) Plasma glucose
concentrations and (B) plasma insulin concentrations in response to
either an EGG-based beverage or a CHO-based beverage. Data are
presented as means (error bars = 95% CIs), with “a” indicating
P ≤ 0.05 compared with baseline values. CHO, carbohydrate-based
beverage; EGG, egg-based beverage; HW, healthy weight; OW/OB,
overweight/obese.
Beverage consumption reduced the proportion of
intermediate and nonclassical monocytes
independent of BMI
Classical monocyte concentration and proportion did not differ
by group or condition (Figure 3A, B). Intermediate monocyte
concentration (Figure 3C) and proportion (Figure 3D), and
nonclassical monocyte concentration (Figure 3E) did not
differ by group or condition. The proportion of nonclassical
monocytes did not differ by BMI, but was significantly
lower following beverage consumption compared with baseline
(Figure 3F main effect of condition, P = 0.007; ES = 0.75).
TLR4 expression on monocytes was reduced by CHO,
primarily in OW/OB
The concentration of TLR4+ classical monocytes was not
different by group or beverage (Figure 4A); however, the
level of TLR4+ expression on classical monocytes was
significantly reduced in OW/OB following ingestion of the
CHO beverage (Figure 4B; P ≤ 0.05; ES = 1.46). The
proportion of TLR4+ classical monocytes was not different by
group or beverage (Figure 4C). The concentration of TLR4+
intermediate monocytes was not different between groups or
beverage conditions (Figure 4D); however, TLR4+ expression
on intermediate monocytes trended to be lower in OW/OB after
CHO (Figure 4E, interaction effect; P = 0.062; ES = 1.20).
The proportion of TLR4+ intermediate monocytes trended
to be lower in OW/OB following CHO (Figure 4F, effect of
group; P = 0.069; ES = 1.17). The concentration of TLR4+
nonclassical monocytes was not different by group or beverage
(Figure 4G). TLR4+ expression on nonclassical monocytes was
not different by group or beverage (Figure 4H). The proportion
of TLR4+ nonclassical monocytes was significantly lower in
OW/OB at baseline (Figure 4I; P = 0.009; ES = 1.05), and was
significantly lower in OW/OB after CHO (Figure 4I, interaction
effect; P ≤ 0.05; ES = 1.28).
Changes in monocyte populations were negatively
related to changes in plasma glucose in the CHO
condition
Downloaded from https://academic.oup.com/jn/article/150/3/616/5673204 by guest on 06 April 2021
Pearson correlations were conducted to relate changes in plasma
glucose concentration (post-CHO − baseline) to changes in cell
populations (post-CHO − baseline) after the CHO beverage.
The changes in proportion of nonclassical and intermediate
monocytes were negatively related to changes in glucose (r =
−0.57 and r = −0.58, respectively; both P ≤ 0.05). The change
in TLR4+ expression on intermediate monocytes was also
negatively related to changes in glucose (r = −0.55; P ≤ 0.05).
All other correlations were nonsignificant.
Discussion
This study aimed to investigate the response of inflammatory
monocytes, particularly those expressing the proinflammatory
marker TLR4+ , and inflammatory cytokines to a carbohydrate-
free beverage or high-carbohydrate beverage in HW and
OW/OB children. The main findings of this study were that
inflammatory monocytes, particularly those with the proin-
flammatory marker TLR4+ , were lower 2 h after consuming
a high-carbohydrate beverage, particularly in children with
OW/OB. Further, larger increases in blood glucose following
carbohydrate beverage consumption were associated with
greater decreases in monocyte populations. These results
suggest that the macronutrient composition of a meal alters
inflammatory monocyte populations in circulation in children,
and that this effect is related to blood glucose control. These
findings have implications for the role of the immune system in
acute metabolic responses in children with OW/OB.
Contrary to our initial hypothesis, we observed a significant
reduction in inflammatory-primed (i.e., TLR4+ ) monocyte
populations in children with OW/OB, but not HW, whereas no
differences were observed in the carbohydrate-free condition.
The mechanisms responsible for this decrease remain unknown.
We speculate that high-carbohydrate beverage consumption
resulted in monocytes with high TLR4+ expression being
recruited to peripheral tissues, particularly the intestines, to
participate in local inflammatory responses. In support of this
speculation, carbohydrate ingestion increased adhesion markers
on lymphocytes 2 h after consumption in adults (12). Further,
hyperglycemia has recently been shown to be associated with
increased gut permeability (17). This could have resulted in
an exacerbation of the metabolic endotoxemia that is known
to be associated with obesity (18), and promote recruitment
of TLR4+ monocytes to the gut. Previous reports have shown
that in healthy adults, a meal high in fat and carbohydrates
induced higher plasma LPS concentration than a meal high in
Monocyte response to carbohydrate beverages in children 619
 Downloaded from https://academic.oup.com/jn/article/150/3/616/5673204 by guest on 06 April 2021

FIGURE 3 The percentage of nonclassical monocytes is decreased after beverage consumption in both HW (n = 10) and OW/OB (n = 7) weight
classes. Changes in (A) classical monocyte concentrations, (B) classical monocyte proportions, (C) intermediate monocyte concentrations, (D)
intermediate monocyte proportions, (E) nonclassical monocyte concentrations, and (F) nonclassical monocyte proportions in response to EGG
and CHO beverages compared with baseline in children. Data are presented as means (error bars = 95% CIs); a P ≤ 0.05 compared with baseline
values. CHO, carbohydrate-based beverage; EGG, egg-based beverage; ev, events; HW, healthy weight; OW/OB, overweight/obese.

fiber and fruit (19), supported also by animal models of obesity
showing that a high-carbohydrate/high-fat diet increases gut
permeability linked to increased endotoxemia (20). However,
more recent evidence has shown this increased gut permeability
to be directly related to a hyperglycemia-induced environment,
rather than a high-fat environment (17). However, this study
used type 1 diabetic mice, so it remains to be seen if these same
results would hold in instances of obesity.
Glucose and insulin homeostasis have been shown to be
disrupted by obesity, and this could be caused by innate immune
system activation (21). Previous research has indicated that
monocytes express insulin receptors and might play a role
in obesity-driven insulin resistance (22, 23). Here, we show
that changes in monocyte content and proportions after high-
carbohydrate, but not carbohydrate-free, beverage consumption
are negatively related to changes in glucose. These findings

620 Niemiro et al.

 Downloaded from https://academic.oup.com/jn/article/150/3/616/5673204 by guest on 06 April 2021
FIGURE 4 The level of TLR4 expression on classical monocytes and percentage of TLR4+ nonclassical monocytes are decreased after CHO
consumption in children with OW/OB. Changes in (A) TLR4+ classical monocyte concentrations, (B) TLR4-expressing classical monocytes,
(C) TLR4+ relative classical monocyte proportions, (D) TLR4+ intermediate monocyte concentrations, (E) TLR4+ -expressing intermediate
monocytes, (F) TLR4+ relative intermediate monocyte proportions, (G) TLR4+ nonclassical monocyte concentrations, (H) TLR4+ -expressing
nonclassical monocytes, and (I) TLR4+ relative nonclassical monocyte proportions in response to EGG and CHO beverages compared with
baseline in children. Data are presented as means (error bars = 95% CIs). a P < 0.05 interaction effect in OW/OB compared with HW after CHO.
b P < 0.05 effect of weight class in OW/OB (n = 5) compared with HW (n = 8), after CHO. CHO, carbohydrate-based beverage; EGG, egg-based

beverage; ev, events; HW, healthy weight; MFI, median flouorescence intensity; OW/OB, overweight/obese; TLR4, Toll-like receptor 4.

suggest that regulation of blood glucose concentrations could
be related to the regulation of monocyte populations. It
has been shown that meals high in fatty acids can increase
both inflammatory cytokines and monocytes, and the increase
in monocytes has been suggested to be a result of the
increased inflammatory cytokines (24). Additionally, fatty acid
stimulation of primary monocytes ex vivo causes shifts in
the cellular media composition, which in turn impairs insulin
responses on myotubes (24). Considering the limitations of in
vitro experiments, these monocyte/macrophage and myotube
cell culture models have repeatedly shown differences in
murine monocyte/macrophage production of inflammatory
cytokines causing changes in myotube insulin responses (24).
In vivo research has shown that injection of nucleotide binding
oligomerizing domain 1 stimulates insulin resistance through
monocytes (24). Although these promising findings highlight
the relation between monocytes and insulin resistance, more
research is needed to fully delineate the role of meal macronu-
trient composition in OW/OB children on inflammatory
responses.
This study has limitations, which are primarily due to
the childhood population being studied. First, we did not
include children’s habitual diet consumption. It is possible that
adaptations to habitual dietary patterns could override some
of the acute inflammatory responses induced by a single meal.
Participants were fasted for 4 h prior to each experimental
session to account for any short-term dietary influences. Second,
we did not collect physical activity data on our participants;
however, participants were instructed to maintain normal levels
of physical activity throughout the trial. Physical activity has
been shown to increase insulin sensitivity, reduce susceptibility
to viral and bacterial infections, and decrease acute and chronic

Monocyte response to carbohydrate beverages in children 621

inflammation (25, 26). It is possible that our participants’
physical activity levels could have influenced monocyte and/or
insulin and glucose responses to the beverages provided in
the study. Due to inherent challenges in conducting blood
draws in children, we had somewhat smaller samples size than
desired and could only collect 1 postprandial time point. These
factors could have contributed to trends, rather than significant
findings, in some of our analyses.
In conclusion, these results show that carbohydrate ingestion
induces a reduction in TLR4 expression on classical mono-
cytes and in the proportion of TLR4-expressing nonclassical
monocytes, and that these monocyte changes are related
to changes in blood glucose. These findings indicate that
meals of different macronutrient composition might contribute
to underlying inflammatory disease associated with obesity,
exacerbated by small changes in inflammatory monocytes
induced by consumption of high-carbohydrate meals. As such,
substituting high-carbohydrate meals with low-carbohydrate
options could improve inflammatory responses to meals and
reduce the negative systemic health effects of obesity in children.
Acknowledgments
We thank Richard Kim for his help in processing samples.
The authors’ contributions were as follows—GMN and
MDL: designed the research; GMN: conducted research; NAK
and MDL: provided essential materials; GMN, NAC, NAK,
and MDL: analyzed the data; GMN and NAC: wrote the
manuscript, with revisions from all authors; MDL: had primary
responsibility for final content; and all authors: read and
approved the final manuscript.
References
1. Ogden CL, Carroll MD, Fryar CD, Flegal KM. Prevalence of obesity
among adults and youth: United States, 2011–2014. NCHS Data Brief
2015;1–8.
2. Park HS, Park JY, Yu R. Relationship of obesity and visceral adiposity
with serum concentrations of CRP, TNF-α and IL-6. Diabetes Res Clin
Pract 2005;69:29–35.
3. Hotamisligil GS. Inflammation and metabolic disorders. Nature
2006;444:860–7.
4. Richard C, Wadowski M, Goruk S, Cameron L, Sharma AM, Field CJ.
Individuals with obesity and type 2 diabetes have additional immune
dysfunction compared with obese individuals who are metabolically
healthy. BMJ Open Diabetes Res Care 2017;5:e000379.
5. Ziegler-Heitbrock L. The CD14+ CD16+ blood monocytes: their role
in infection and inflammation. J Leukoc Biol 2007;81:584–92.
6. Mukherjee R, Barman PK, Thatoi PK, Tripathy R, Das BK,
Ravindran B. Non-classical monocytes display inflammatory features:
validation in sepsis and systemic lupus erythematous. Sci Rep 2015;5:
srep13886.
7. Anbazhagan K, Duroux-Richard I, Jorgensen C, Apparailly F.
Transcriptomic network support distinct roles of classical and non-
classical monocytes in human. Int Rev Immunol 2014;33:470–89.
8. Ziegler-Heitbrock L, Hofer TP. Toward a refined definition of monocyte
subsets. Front Immunol 2013;4:23.
9. Lu Y-C, Yeh W-C, Ohashi PS. LPS/TLR4 signal transduction pathway.
Cytokine 2008;42:145–51.
622 Niemiro et al.
10. Devêvre EF, Renovato-Martins M, Clément K, Sautès-Fridman C,
Cremer I, Poitou C. Profiling of the three circulating monocyte
subpopulations in human obesity. J Immunol 2015;194:3917–23.
11. Niemiro GM, Raine LB, Khan NA, Emmons R, Little J, Kramer AF,
Hillman CH, De Lisio M. Circulating progenitor cells are positively
associated with cognitive function among overweight/obese children.
Brain Behav Immun 2016;57:47–52.
12. Torrecilla E, González-Muñoz M, Lahoz C, Mostaza J. Time-
dependent changes in the expression of lymphocyte and monocyte cell
adhesion molecules after meals of different composition. Br J Nutr
2010;104:1650–4.
13. Arif S, Gibson VB, Nguyen V, Bingley PJ, Todd JA, Guy C,
Dunger DB, Dayan CM, Powrie J, Lorenc A, et al. β-cell specific T-
lymphocyte response has a distinct inflammatory phenotype in children
with Type 1 diabetes compared with adults. Diabet Med 2017;34:
419–25.
14. Jensen JD, Ronit K. The EU pledge for responsible marketing of food
and beverages to children: implementation in food companies. Eur J
Clin Nutr 2015;69:896–901.
15. LoDolce ME, Harris JL, Schwartz MB. Sugar as part of a balanced
Downloaded from https://academic.oup.com/jn/article/150/3/616/5673204 by guest on 06 April 2021
breakfast? What cereal advertisements teach children about healthy
eating. J Health Commun 2013;18:1293–309.
16. Kral TVE, Bannon AL, Chittams J, Moore RH. Comparison of the
satiating properties of egg- versus cereal grain-based breakfasts for
appetite and energy intake control in children. Eat Behav 2016;20:
14–20.
17. Thaiss CA, Levy M, Grosheva I, Zheng D, Soffer E, Blacher E,
Braverman S, Tengeler AC, Barak O, Elazar M, et al. Hyperglycemia
drives intestinal barrier dysfunction and risk for enteric infection.
Science 2018;359:1376–83.
18. Frazier TH, DiBaise JK, McClain CJ. Gut microbiota, intestinal
permeability, obesity-induced inflammation, and liver injury. J Parenter
Enter Nutr 2011;35:14S–20S.
19. Ghanim H, Abuaysheh S, Sia CL, Korzeniewski K, Chaudhuri
A, Fernandez-Real JM, Dandona P. Increase in plasma endotoxin
concentrations and the expression of toll-like receptors and suppressor
of cytokine signaling-3 in mononuclear cells after a high-fat, high-
carbohydrate meal: implications for insulin resistance. Diabetes Care
2009;32:2281–7.
20. Serino M, Luche E, Gres S, Baylac A, Bergé M, Cenac C, Waget A, Klopp
P, Iacovoni J, Klopp C, et al. Metabolic adaptation to a high-fat diet is
associated with a change in the gut microbiota. Gut 2012;61:543–53.
21. Bastard J-P, Maachi M, Lagathu C, Kim MJ, Caron M, Vidal H,
Capeau J, Feve B. Recent advances in the relationship between obesity,
inflammation, and insulin resistance. Eur Cytokine Netw 2006;17:4–12.
22. Gerrits AJ, Koekman CA, Yildirim C, Nieuwland R, Akkerman JWN.
Insulin inhibits tissue factor expression in monocytes. J Thromb
Haemost 2009;7:198–205.
23. Mauer J, Chaurasia B, Plum L, Quast T, Hampel B, Blüher M, Kolanus
W, Kahn CR, Brüning JC. Myeloid cell-restricted insulin receptor
deficiency protects against obesity-induced inflammation and systemic
insulin resistance. PLoS Genet 2010;6:e1000938.
24. Chan KL, Boroumand P, Milanski M, Pillon NJ, Bilan PJ, Klip A.
Deconstructing metabolic inflammation using cellular systems. Am J
Physiol Endocrinol Metab 2017;312:E339–47.
25. Walsh NP, Gleeson M, Shephard RJ, Gleeson M, Woods JA, Bishop NC,
Fleshner M, Green C, Pedersen BK, Hoffman-Goetz L, et al. Position
statement. Part one: immune function and exercise. Exerc Immunol Rev
2011;17:6–63.
26. Lee S, Deldin AR, White D, Kim Y, Libman I, Rivera-Vega M, Kuk JL,
Sandoval S, Boesch C, Arslanian S. Aerobic exercise but not resistance
exercise reduces intrahepatic lipid content and visceral fat and improves
insulin sensitivity in obese adolescent girls: a randomized controlled
trial. Am J Physiol Endocrinol Metab 2013;305:E1222–9.