Journal of Functional Foods 57 (2019) 112–120

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Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Procyanidin B2 rich cocoa extracts inhibit inflammation in Caco-2 cell T

model of in vitro celiac disease by down-regulating interferon-gamma- or
gliadin peptide 31-43-induced transglutaminase-2 and interleukin-15
Kristen Kramer1, Millicent Yeboah-Awudzi, Nicholas Magazine, Joan M. King, Zhimin Xu,
⁎
Jack N. Losso
School of Nutrition and Food Sciences, Louisiana State University, Baton Rouge, LA 70803, United States

A R T I C LE I N FO A B S T R A C T

Keywords: The objective of this research was to investigate the efficacy of procyanidin B2 rich cocoa extracts on interferon-
Celiac disease gamma (IFN-γ) or gliadin peptide p31-43-induced transglutaminase-2 and interleukin-15 (IL-15) in Caco-2 cell
Caco-2 model of in vitro celiac disease. Cysteamine was used as a positive control inhibitor of TG2. Procyanidin B2-rich
Cocoa extract reduced IFN-γ- and gliadin p31-43-induced TG2 activity by 77% and 45%, respectively. Cocoa extract
procyanidin B2
containing 4.9–289 µg/mL of procyanidin B2 dose-dependently reduced IFN-γ-induced IL-15 secretion to the
Caffeine
Theobromine
level of IL-15 in control cells. Similarly, cocoa extract containing 4.9–289 µg/mL of procyanidin B2 dose-de-
Transglutaminase-2 pendently reduced gliadin p31-43-induced IL-15 to the level in control cells. Procyanidin B2-rich cocoa extracts
Interleukin-15, -1β, -6, -8 (IL-15, IL-6, IL-8, IL- reduced the activities of other inflammatory biomarkers including COX-2, IL-1β, IL-6, and IL-8 in both IFN-γ and
1β) p31-43-treated Caco-2 cells. Caffeine or theobromine, at the concentration found in the cocoa extracts, did not
contribute to the activity of procyanidin B2 against TG2 or IL-15.

1. Introduction
Gliadin, a storage protein found in cereal grains including wheat,
rye, and barley, is the major environmental factor associated with the
development of celiac disease (CD). Gliadin is enriched in glutamine
and proline residues and incompletely digested by gastric, pancreatic,
and brush border enzymes because these enzymes lack post-proline
cleaving activity. The incomplete hydrolysis of gliadin generates glu-
tamine-rich peptides including peptide 31-43 (p31-43) and 57-68 (p57-
68). At the small intestine, these peptides are absorbed via transcellular
or paracellular routes where, in susceptible individuals, the peptides are
deamidated by the enzyme tissue transglutaminase-2 (TG2). TG2 is
constitutively expressed in the lamina propria and released upon tissue
damage. TG2 deamidates gliadin peptides and forms immunotoxic
peptides that have a high affinity for the human leukocyte antigen
(HLA)-DQ2 or HLA-DQ8 molecules on antigen presenting cells. The
quantity of cell surface HLA-DQ2 gliadin peptide complex co-determine
the likelihood of disease development (Stepniak & Koning, 2006). The
deamidated gliadin peptides including p31-43 and to some extent p57-
68 promote a strong activation and expansion of gliadin-specific in-
terferon-gamma (IFN-γ) producing CD4+ T cells (Bayardo, Punzi,
Bondar, Chopita, & Chirdo, 2012). IFN-γ, a major cytokine in the small
intestinal mucosa of active CD patients is the most potent inducer of
TG2 in small intestinal mucosa and in cell cultures such as Caco-2 and
HT-29 (Bayardo et al., 2012). The interaction of deamidated gliadin
peptides and CD4+ T cells induce the production of antibodies against
TG2 and gliadin peptides. The antibodies specific for TG2 have been
associated with the manifestation of celiac disease and required for
effective CD diagnosis.
Other inducers of TG2 include interleukin (IL)-1 β, IL-15, IL-6, and
tumor necrosis factor (TNF)-α. IL-15 is chronically upregulated in the
epithelium and the intestinal lamina propria and has been identified as
the hallmark of CD and correlated very well with the degree of tissue
mucosal damage and villous atrophy (Abadie & Jabri, 2014; Di
Sabatino et al., 2006) In CD patients on a gluten-free diet, IL-15 is
highly expressed in the epithelium only, but, does not induce villous
atrophy (Abadie & Jabri, 2014). IL-15 contributes to the weakening of
tight junctions which increases intestinal permeability and allows more
incompletely digested gliadin peptides into the lamina propria.
Blocking IL-15 signaling is a putative therapy in CD.
Nuclear Factor κβ (NF-κβ) is also upregulated in CD after activation
by IFN-γ and TNF- α. Other inflammatory biomarkers that have been

⁎
Corresponding author.
E-mail address: jlosso@lsu.edu (J.N. Losso).
1
Present address: Abbott Nutrition, Columbus, OH, United States.

https://doi.org/10.1016/j.jff.2019.03.039
Received 13 August 2018; Received in revised form 21 March 2019; Accepted 22 March 2019
1756-4646/ © 2019 Elsevier Ltd. All rights reserved.
K. Kramer, et al.
identified and are upregulated in the sera of CD patients include cy-
clooxygenase-2 (COX-2), cytokines IL-6, IL-1β, and IL-8 (Cinova et al.,
2007; Fernandez-Jimenez et al., 2013; Vincentini, Maialetti, Gonnelli,
& Silano, 2015). The increased production of IL-15, COX-2 and TG2 in
enterocytes occurs within three hours of contact and is primarily re-
sponsible for the destruction of the intestinal mucosa, causing the major
symptoms of CD.
Genetics plays a major role in the development of CD. HLA-DQ2
locus has been identified in 95% of individuals with CD (Petersen et al.,
2014). The remaining 5% of patients with CD are HLA-DQ8+ HLA-
DQ2− (Karell et al., 2003). Another genetic factor that contributes to
the risk of CD development is the “leaky gut” that allows more in-
completely digested gliadin peptides absorption through the intestinal
epithelium and enhances the deamidation of the peptides by TG2.
The prevalence of CD is 0.5–1% of the population and increasing
(Lohi et al., 2007). CD is associated with an increased risk of small
intestinal adenocarcinoma and non-Hodgkin’s lymphoma. One effective
treatment for CD is a strict, lifelong adherence to a gluten-free diet and
drugs that includes total avoidance of all types of wheat products. It has
been shown that even 50 mg of gliadin a day can trigger the in-
flammatory process in CD patients (Abadie & Jabri, 2014). However,
the low availability and high costs of a gluten-free diet may affect CD
patient compliance, which stresses the need for additional therapeutic
or non-therapeutic options. In addition, the consumer-perceived health
advantages of gluten-free foods pose a challenge to the wheat-based
products industry.
Therapies for CD include (1) Oral enzyme therapy that uses prolyl
endopeptidases, derived from bacteria, fungi or cereals, to break the
bond between proline and glutamine and convert gliadin peptides into
non-toxic peptides that can reach the intestine and not trigger the in-
flammatory response typically seen in CD. However, some enzymes
have shown limited benefits to celiac patients (Freeman, 2015). Others
which are acid sensitive need to be encapsulated to avoid degradation
in the stomach (Makharia, 2014); (2) Tight junction (TJ) enhancers that
reduce the transport of gliadin peptides through the intestine. Larazo-
tide acetate is a TJ regulator peptide that inhibits disassembly and
dysfunction of TJ epithelial cells. Larazotide has been investigated with
success in clinical trials, showing no difference in serology between CD
on a gluten challenge with larazotide acetate and the control (Kelly
et al., 2013; Leffler et al., 2012); (3) TG2 inhibition because TG2 plays a
crucial role in the pathogenesis of CD, therefore preventing TG2 acti-
vation may reduce the effects of gliadin-induced inflammation. TG2
inhibitors have not yet reached clinical trials and are still in the dis-
covery phase, though proof-of-concept studies have shown promising
results in vitro and ex vivo.
Since diet can induce CD in susceptible individuals, diet can also
treat CD. Food products or beverages capable of inhibiting im-
munotoxic gluten peptide-induced inflammation in small intestinal
epithelium could mitigate gluten toxicity. Consumption of polyphenol-
rich cocoa products reduced IL-1β, IL-6, and IL-8 in moderately hy-
percholesterolaemic individuals (Sarria et al., 2014). Cocoa that has not
gone through the Dutch process contains procyanidins, caffeine and
theobromine, which all are anti-inflammatory. Cocoa that has under-
gone the alkalinization process has reduced levels of procyanidins
(Stanley, Smithson, Neilson, Anantheswaran, & Lambert, 2015).
The objective of this research was to investigate the efficacy of
procyanidin B2 rich cocoa extracts on IFN-γ or p31-43 -induced TG2
and IL-15 in Caco-2 cell model of in vitro celiac disease.
2. Materials and methods
2.1. Materials
Natural process cocoa powder was generously donated by Mars
Chocolate North America, Inc. (Hackettstown, NJ). Procyanidin B2
standard (Fig. 1) was purchased from ChromaDex (Irvine, CA).
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Journal of Functional Foods 57 (2019) 112–120
Theobromine (Fig. 1), caffeine (Fig. 1), and cysteamine were obtained
from Sigma-Aldrich (Bellefonte, PA). α-gliadin peptide 31-43 (Gln or
p31-43) was obtained from Anaspec (Fremont, CA). IFN-γ was pur-
chased from Thermo Fisher Scientific (Waltham, MA). LDS sample
buffer, 4–12% Bis-Tris SDS polyacrylamide gel were from Invitrogen
(Carlsbad, CA). Primary antibodies against β-actin, COX-2, and TG2
were purchased from Cell Signaling Technology (Danver, MA). Con-
jugated secondary antibodies (anti-rabbit and anti-mouse) were pur-
chased from Santa Cruz Biotechnology (Santa Cruz, CA). Human IL-8,
IL-6 and IL-1β kits were from Peprotech (Rocky Hill, NJ). Human IL-15
ELISA kit was purchased from Affymetrix eBioscience (San Diego, CA).
2.2. Preparation of procyanidin B2 rich cocoa extract
Procyanidin B2-rich cocoa extracts were obtained by dissolving the
raw cocoa powder in deionized water in a 1:4 ratio and shaking for
60 min at room temperature. The extracts were centrifuged and the
supernatants were collected and lyophilized. The extracts were con-
centrated four times. Sugars and organic acids in cocoa powder were
removed using acetone:water:acetic acid (70:28:2) (Counet, Ouwerx,
Rosoux, & Collin, 2004).
2.3. UHPLC analysis of cocoa extracts
Ultra High Performance Liquid Chromatography (UHPLC) was
performed with a Thermo Scientific Ultimate 3000 (Waltham, MA)
equipped with a binary gradient pump, sample injector, a column oven
and a photodiode array detector. The equipment was run under the
following conditions: an HSS C18 Column (100 Å, 1.8 µm,
2.1 mm × 50 mm) with an HSS C18 SB VanGuard Pre-column (100 Å,
1.8 µm, 2.1 mm X 5 mm) (Waters Corporation, Milford, MA) was used
with a flow rate of 0.3 mL/min. Procyanidin extracts were diluted 1:20
with water. Injections of 25 µL were used and the oven was held at a
temperature of 25 °C.
The protocol was adapted from Cooper et al. (2007) that involved
UPLC analysis of major cocoa polyphenols in chocolate. The binary
system phases were (A) water/THF/TFA (98:2:0.1 v/v/v) and (B)
acetonitrile with 0.1% TFA. The two minute gradient was as follows:
0.0–0.2 min, 90–87% A; 0.2–0.75 min, 87–85% A; 0.75–0.775 min,
85–0% A; 0.775–1.25 min, 0% A linear; 1.25–1.275 min, 0–90% A;
1.275–2 min, 90% A re-equilibration time. The standards and cocoa
extracts were analyzed at 280 nm wavelength. The concentrations of
procyanidin B2, caffeine and theobromine in the cocoa extracts were
identified by comparing the retention times of their peak with that of
standards (1–1000 µg/ml).
2.4. Cell cultures
Caco-2 cell line was used as a model of human intestinal epithelial
cells. Caco-2 cells (ATCC, Manassas, VA) were grown in 75 cm2
Dulbecco's Modified Eagle's Medium (DMEM) (GIBCO, Grand Island,
NY), 10% fetal calf serum (GIBCO), 100 units/ml penicillin strepto-
mycin (GIBCO), and 2 mM L-glutamine (GIBCO) at 37 °C in a humidified
atmosphere containing 5% CO2. Upon reaching 80% confluence, cells in
the logarithmic phase were subcultured weekly at a split ratio of 1:3 by
trypsinization.
2.4.1. Effects of procyanidin B2-rich cocoa extract on Caco-2 cell viability
Caco-2 cells (1 × 103 cells per well) were seeded in 96-well plates
for 24 h. Cocoa extract containing between 5.78 ng/mL and 289 µg/mL
procyanidin B-2 in culture medium were added to the cells. Similarly,
cells were incubated without procyanidin B2-rich cocoa extract or with
extracts containing 1.15 µg/mL, 11.6 µg/mL or 28.9 µg/mL of procya-
nidin B2 for 2, 6 or 24 h at 37 °C in an incubator with 5% CO2. After
incubation, cells were washed and incubated for 4 h with a solution of
phenazine methosulfate (PMS) and a tetrazolium compound (MTS)
K. Kramer, et al.
A.
Procyanidin B2
B.
Fig. 1. Structure of procyanidin B2, caffeine, and theobromine.
following the CellTiter 96 Aqueous One solution manufacturer’s in-
structions (Promega, Madison, WI). Cells bioreduced MTS into a for-
mazan product and the absorbance was read at 490 nm using a BioRad
Model 680 microplate reader (Hercules, CA). The quantity of formazan
product was directly proportional to the number of living cells in cul-
ture and was used to compare cell viability versus concentrations of
procyanidin B2. The cell viability assay was performed in triplicate and
results are presented as a percentage of control. The highest con-
centrations of nontoxic procyanidin B2 rich cocoa extract were used for
subsequent experiments.
2.4.2. Interactions of procyanidin B2 rich cocoa extracts and in vitro model
of celiac disease
To determine the efficacy of procyanidin B2-rich cocoa extracts to
inhibit TG2 produced by Caco-2 cells or the effect of caffeine, and
theobromine in the cocoa extract, Caco-2 cells (2 × 105 per well) were
seeded overnight in 6-well plates. The cells were untreated or stimu-
lated with 10 ng/mL IFN- γ or 20 μg/mL of p31-43 for 24 h followed by
addition of various concentrations of pure procyanidin B2, cocoa ex-
tract containing various concentrations of procyanidin B2, caffeine
(9.7 μg/mL or 97 μg/mL), or theobromine (9 μg/mL or 90 μg/mL) and
incubated for 72 h. Various concentrations of cysteamine, a known
Journal of Functional Foods 57 (2019) 112–120
Caffeine Theobromine
inhibitor of TG2 were also incubated with Caco-2 cells stimulated either
by IFN- γ or p31-43 as described above.
2.4.3. Western blot analysis of TG2 and COX-2
Cells were washed with Phosphate Buffered Saline (PBS) and col-
lected by scraping in 50 μL of hypotonic buffer (10 mmol/L HEPES pH
7.9, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.2 mmol/L PMSF, 0.5 mmol/L
DTT, 5 mmol/L NaF, 1 mmol/L Na3VO4). The mixture was vortexed for
10 s and incubated for 30 min on ice, then centrifuged at 14,000g for
15 min and the supernatant was retained as cytoplasmic extracts. The
pellet was re-suspended in 50 μL of high-salt buffer (20 mmol/L HEPES
pH 7.9, 25% glycerol, 1.5 mmol/L MgCl2, 1.2 mol/L KCl, 0.1 mmol/L
EDTA, 420 mmol/L NaCl, 0.5 mmol/L DTT, and 0.2 mmol/L PMSF) by
pipetting up and down and vortexing 10 s on high setting. The mixture
was incubated for 30 s on ice and centrifuged at 14,000g for 10 min
(pre-cooled at 4 °C). The supernatant was retained as nuclear fraction
and stored at −80 °C until use. Total protein in cytoplasmic and nuclear
fractions was determined by the Bicinchoninic Acid (BCA) protein assay
(ThermoFisher Scientific, Waltham, MA). Equal amounts of protein
(725 μg) from cytoplasmic protein fractions from control and procya-
nidin-B2 rich cocoa extract-, caffeine-, or theobromine- treated cells
were mixed with LDS sample buffer, boiled for 5 min, and vortexed at a
114
K. Kramer, et al.
high setting. Thirty microliters of each sample were added in each lane
of a 4–12% Bis-Tris SDS polyacrylamide gel (Invitrogen, Carlsbad, CA).
Proteins were transferred to a polyvinylidene fluoride (PVDF) mem-
brane (0.4 μm pore size) (ThermoFisher Scientific, Waltham, MA), then
blocked with 5% bovine serum albumin (BSA) in PBST (PBS with 0.1%
Tween-20) for 1 h. The primary antibody was prepared in 5% BSA and
incubated with the membrane overnight at 4 °C on a shaker. The
membrane was washed three times for 10 min using PBST, then in-
cubated for 1 h with secondary antibody and the washes were repeated.
Visualization of the bound antibody was done in a dark room using
West Pico Substrate, an enhanced chemiluminescent HRP-substrate
(ThermoFisher Scientific, Waltham, MA) and a BioRad ChemiDoc MP
System (Hercules, CA). After analyzing band density, membranes were
stripped and reprobed with β-actin to serve as a loading control. Results
are reported as a ratio of the density of each band to its β-actin.
2.4.4. ELISA for inflammatory cytokines
The supernatants from Caco-2 cells control or treated were collected
after 72 h for analysis of inflammatory cytokines including IL-15, IL-1β,
IL-6, and IL-8. Human IL-15 ELISA was performed using total cell ly-
sates. Each sample was analyzed in triplicate, and the results are ex-
pressed as mean ± SD of the concentration of interleukin.
2.5. Statistical analysis
Each experiment was performed at least three times. The data are
expressed as the mean ± SD and analyzed using one-way analysis of
variance (ANOVA) followed with a Tukey’s test for multiple compar-
isons. The differences were considered significant at the P < 0.05
level. Statistical analysis was conducted using the Statistical Analysis
Software (SAS) (version 9.4).
3. Results
3.1. Concentration procyanidin B2, caffeine, and theobromine in cocoa
extracts
Typically procyanidin B2, caffeine, and theobromine are found in
very low levels in cocoa (under 4 mg per g dry cocoa powder). Efforts
were made to concentrate these compounds through extraction and
lyophilization. The results reported in Table 1 were obtained after four
time concentrations/extractions. Theobromine is found in greater
concentration in cocoa than both procyanidin B2 and caffeine (Fig. 1B)
as observed by others (Ortega et al., 2010; Sarria et al., 2015; Smit &
Blackburn, 2005).
3.2. Effect of cocoa extracts on cell viability
Cocoa extracts varying in concentration of procyanidin B2 from
57.8 μg/mL to 289 μg/mL did not have adverse effects on Caco-2 cell
viability (Fig. 2A). Significant difference was observed in cell viability
for treatment containing 0.6 μg/mL to 28.9 μg/mL of procyanidin B2
(Fig. 2B).
Table 1
Concentration of theobromine, caffeine, and procyanidin B-2 in cocoa extracts.
Compound Concentration (mg/g) % (w/w)
Theobromine 22.61 40.3
Caffeine 11.20 20
Procyanidin-B2 21.39 38.2
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Journal of Functional Foods 57 (2019) 112–120
3.3. Effect of procyanidin B2 rich cocoa extracts, caffeine, or theobromine
on TG2 levels
Exposure of Caco-2 cells to IFN-γ resulted in a significant increase of
TG2 (Fig. 3A). The greatest decreases in TG2 level were seen when cells
were treated with cocoa extracts containing 4.9 μg/mL, 11.6 μg/mL,
57.8 μg/mL, or 289 μg/mL of procyanidin B2, with activity from 44.3%
to 77.2%. Addition of p31-43 also led to an increase in TG2 level
(Fig. 3B). Cocoa extracts containing at least 4.9 μg/mL of procyanidin
B2 was also effective at inhibiting p31-43-induced TG2. Similar effects
were also observed with cocoa extracts containing 11.6–289 μg/mL of
procyanidin B2 (Fig. 3B).
Comparison was made with the effect of cocoa extract containing
4.9 μg/mL or 289 μg/mL procyanidin B2 or 19.3 μg/mL- 38.6 μg/mL
cysteamine on IFN-γ-or p31-43-induced TG2. Cocoa extracts containing
289 μg/mL procyanidin B2 was as effective as 38 μg/mL cysteamine on
IFN-γ-induced TG2 (Fig. 4A). In Fig. 4B, it is shown that 11.6 μg/mL of
procyanidin B2 was effective as 19.3 μg/mL cysteamine on p31-43-in-
duced TG2.
Cysteamine, an irreversible TG2 inhibitor (Jeitner, Delikatny,
Ahlqvist, Capper, & Cooper, 2005; Jeon et al., 2006) significantly re-
duced the activity of TG2 induced by IFN-γ by as much as 75%
(Fig. 4A). Fig. 4C and D show the result of incubating IFN-γ- or p31-43-
treated Caco-2 cells with caffeine or theobromine alone at two different
concentrations. The lower concentration (28.9 μg/mL) of procyanidin
B2 is close to what would be expected with the cocoa extracts stan-
dardized to 4.9–11.6 μg/mL procyanidin B2, and the higher con-
centration (289 μg/mL) of procyanidin B2 is close to what would be
seen with cocoa extracts containing 289 μg/mL procyanidin-B2. At
9.7 μg/mL, caffeine produced a 63% decrease in TG2 in IFN-γ treated
cells and a 65% decrease in p31-43-treated cells. At 97 μg/mL caffeine
was more effective on IFN-γ-induced TG2 than p31-43-induced TG2.
Theobromine produced a 56% decrease in IFN- γ-induced TG2. Theo-
bromine at 9 μg/mL was more effective on p31-43-induced TG2 and
produced a 84% decrease in TG2, respectively. At 90 μg/mL theo-
bromine was more effective on IFN-γ-induced TG2 than p31-43-induced
TG2.
3.4. Effect of procyanidin B-2 rich cocoa extracts on CD inflammatory
biomarkers
IFN- γ-treated Caco-2 cells up-regulated COX-2 (Fig. 5A) and pro-
cyanidin B2 rich cocoa extracts at 11.6–289 μg/mL significantly re-
duced the levels of COX-2 (Fig. 5A). p31-43 also up-regulated the level
of COX-2 in Caco-2 cells but at a much lower level than IFN-γ (Fig. 5B).
p31-43 is probably a much weaker inducer of COX-2 than IFN-γ.
IL-15 was significantly higher in cells treated with IFN-γ alone than
in cells treated with procyanidin B2 (Fig. 6A). Incubating with pro-
cyanidin B2 rich cocoa extracts decreased levels of IL-15 similar to that
seen in the control (medium alone), though the effect was not depen-
dent on procyanidin B2 concentration (Fig. 6A). p31-43 induced IL-15
production in Caco-2 cells. (Fig. 6B). Concentration of at least 28.9 μg/
mL procyanidin B2 was required to achieve inhibition of IL-15. IFN-γ or
p31-43 significantly increased IL-6 secretion in Caco-2 cells (Fig. 6C
and D). Procyanidin B2 rich cocoa extracts also decreased the levels of
IFN- γ- or p31-43-induced IL-8 in Caco-2 cells (Fig. 6E and F). The levels
of IL-1 β in gliadin p31-43-treated Caco-2 cells were reduced by the
cocoa extracts (Fig. 6G and H).
4. Discussion
The major findings of this study indicate that procyanidin B2-rich
cocoa extract inhibited IFN-γ- or gliadin p31-43-induced TG2, IL-15, IL-
1β, COX-2, IL-6, and IL-8 in a Caco-2 cell in vitro model of CD.
Comparison was made with cysteamine which at 19.3 μg/mL inhibited
both IFN- γ- and p31-43-induced inflammatory biomarkers associated
K. Kramer, et al.
A B
Procyanidin B2 (ȝg/mL) Procyanidin B2 (ȝg/mL)
Control IFN 0.6 4.9 11.6 28.9 57.8 289 Control p31-43 0.6 4.9 11.6 28.9 57.8 289
TG2 TG2
ȕ-actin ȕ-actin
Control IFN 4.9 11.6 28.9 57.8 289 Control p31-43 4.9 11.6 28.9 57.8 289
Procyanidin B2 (ȝg/mL)
with CD. Although the gold standard for the study of treatment of CD
involves the use of in vitro organ culture of specimens of intestinal
mucosa obtained from celiac patients, the use of Caco-2 cells as in vitro
model of CD has some advantages. (1) It makes it possible to dis-
criminate the contribution of each of the two branches of immunity to
the overall IFN-γ- or gliadin-dependent response, (2) It is inexpensive,
(3) The Caco-2 cells may also be used to study in vitro the preventive
effects of bioavailable dietary bioactive compounds toward celiac in-
flammation, and (4) Caco-2 cells are used more extensively than T84
cells in CD research, notably for testing of novel treatments (Lindfors,
Rauhavirta, Stenman, Maki, & Kaukinen, 2012). The disadvantages of
the Caco-2 cell line include the inability to reproduce all the char-
acteristics of a human intestinal epithelium and the transport of lipo-
philic molecules which may be decreased since Caco-2 cells do not
contain a mucus layer with bile acids as human intestinal cells do (Lea,
2015).
The intestinal epithelial uptake of IFN-γ or p31–43 induces an in-
tracellular pro-oxidative environment that activates TG2 and leads to
the innate immune response in CD. Therefore, CD may be controlled by
inhibiting the activation of the innate immune response. IFN-γ is the
most potent inducer of TG2 and when released by T-cells in CD it ac-
tivates the pathway that causes destruction of intestinal mucosa (Karell
et al., 2003; Petersen et al., 2014).
While not significant, increased viability is seen in Caco-2 cells after
addition of cocoa extracts over time, as seen at 24 h of incubation
(Fig. 2). This effect of cocoa extracts on Caco-2 cells is likely the action
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Journal of Functional Foods 57 (2019) 112–120
Fig. 2. Effects of cocoa extracts on Caco-2 cell
viability. (A) Effects of cocoa extracts containing
various concentrations of procyanidin B2 from 0
to 289 µg/mL on Caco-2 cell viability after 24 h.
(B) Effects of cocoa extracts containing various
concentrations of procyanidin B2 from 57.8 to
289 µg/mL on Caco-2 cell viability after 2, 6, or
24 h incubation. Values followed by different
letters are significantly different at p < 0.05.
Fig. 3. Effects of cocoa extracts containing var-
ious concentrations of procyanidin B2 on IFN-γ-
or p31-43-induced TG2. (A) Effect of cocoa ex-
tract containing various concentrations of pro-
cyanidin B2 on the levels of IFN-γ-induced TG2.
(B) Effect of cocoa extract containing various
concentrations of procyanidin B2 on the levels of
p31-43-induced TG2.
Procyanidin B2 (ȝg/mL)
of procyanidin B2 and other bioactive compounds on inhibiting apop-
totic pathways. Procyanidin-B2 and epicatechin are capable of reducing
oxidative stress that leads to apoptosis, which explains the increase in
viability with higher concentrations of procyanidin B2. Their anti-
oxidant properties have been shown to prevent cytotoxicity and have
anti-apoptopic effects in Caco-2 cells (Rodriguez-Ramiro, Ramos,
Bravo, Goya, & Martin, 2011). As stated in the literature review, TG2
also plays a role in cell death, and inhibition of this enzyme from the
cocoa extracts may also explain the increase in the number of living
cells.
Procyanidin B2 rich extracts inhibited IFN- γ-induced TG2 (Fig. 3A)
with some effects of hormesis appearing between 11.6 μg/mL and
28.9 μg/mL of procyanidin B2. TG2 deamidates gliadin residues which
form new peptides that bind with high affinity to HLA-DQ2 and elicit an
inflammatory response from T-cells. TG2 is a cross-linking enzyme that
is up-regulated in the villus atrophy, the hallmark of CD and several
other autoimmune and inflammatory disorders, making TG2 inhibition
an attractive target for therapy for diseases including CD. Additionally
when high levels of TG2 are present, T-cells increase their reactivity to
gliadin (Caccamo, Curro, & Ientile, 2010). The gliadin peptide p31-43 is
associated with innate immunity in CD (Jabri, Kasarda, & Green, 2005).
p31-43 has a direct toxic effect on intestinal cells and increases calcium
mobilization leading to activation of TG2 (Caputo et al., 2012).
At 289 μg/mL procyanidin B2 a synergistic effect was seen among
procyanidin B2, caffeine, and theobromine (Fig. 4A and B). Caffeine at
97 μg/mL was able to reduce TG2 activity by 68% in IFN-γ-treated cells
K. Kramer, et al.
and 62% in gliadin-treated cells. Theobromine at 90 μg/mL showed
62% and 47% decrease in TG2 activity, respectively. However, in a
matrix like cocoa that contained both theobromine and caffeine along
with procyanidin B2, there was a 77.2% reduction in relative TG2 ac-
tivity (Fig. 4A and B). This observance explains why cocoa extracts
containing 289 μg/mL procyanidin B2 had greater anti-inflammatory
effects than lower physiological concentrations, and the effect is seen
several times throughout the study. Caffeine was reported to inhibit
TG2 at 10–20 mM (Cho et al., 2012). However, our results show that
caffeine at 9.7 μg/mL or 50 µM did cause a reduction in TG2 level
117
Journal of Functional Foods 57 (2019) 112–120
Fig. 4. Comparison of the effect of cystea-
mine (CS), procyanidin B2, caffeine, or
theobromine on IFN-γ- or p31-43-induced
TG2. (A) Effect of various concentrations of
cysteamine (CS) or procyanidin B2 on IFN-γ
-induced TG2. (B) Effects of various con-
centrations of caffeine or theobromine on
p31-43-induced TG2. (C) Effect of various
concentrations of caffeine (C) or theo-
bromie (T) on IFN-γ -induced TG2. (D)
Effects of various concentrations of caffeine
(C) or theobromine (T) on p31-43-induced
TG2. TG2 level is expressed relative to IFN
or gliadin p31-43 (100%). The figures re-
present mean density ± standard devia-
tion of bands, normalized for corresponding
actin density.
(Fig. 4C and D). Most commercially available cocoa products do not
contain 289 μg/mL of procyanidin B2 which is destroyed during the
Dutch process that the industry uses to improve the solubility of raw
cocoa.
TG2 inhibitors include disulfide compounds such as cystamine and
cysteamine, and α,β unsaturated amides (Marrano et al., 2001; Ozaki
et al., 2011; Pardin, Pelletier, Lubell, & Keillor, 2008). TG2 active site
inhibitors R281 and R283 reduced gliadin-induced inflammation ex
vivo as evidenced by the number of proliferating enterocytes in crypts
and decreased levels of the biomarker IL-15 (Rauhavirta et al., 2013).
Fig. 5. Effect of procyanidin B2 rich cocoa ex-
tract and IFN-γ- or gliadin -31-43-induced COX-
2. A. Effect of cocoa extract containing various
concentrations of procyanidin B2 on the levels of
IFN-γ-induced COX-2. B. Effect of cocoa extract
containing various concentrations of procya-
nidin B-2 on the levels of p31-43-induced COX-2.
K. Kramer, et al. Journal of Functional Foods 57 (2019) 112–120

A B

Fig. 6. Effects of procyanidin B2 rich cocoa extracts on IFN-γ or p31-43-induced interleukin secretions. (a) Effect of cocoa extract containing various concentrations
of procyanidin B2 on the levels of IFN-γ-induced IL-15. (b) Effect of cocoa extract containing various concentrations of procyanidin B2 on the levels of p31-43-
induced IL-15. (c) Effect of cocoa extract containing various concentrations of procyanidin B-2 on the levels of IFN-γ-induced IL-1β. (d) Effect of cocoa extract
containing various concentrations of procyanidin B2 on the levels of p31-43-induced IL-1β. (e) Effect of cocoa extract containing various concentrations of pro-
cyanidin B2 on the levels of IFN-γ-induced IL-6. (f) Effect of cocoa extract containing various concentrations of procyanidin B2 on the levels of p31-43-induced IL-6.
(g) Effect of cocoa extract containing various concentrations of procyanidin B2 on the levels of IFN-γ-induced IL-8. (h) Effect of cocoa extract containing various
concentrations of procyanidin B-2 on the levels of p31-43-induced IL-8.

118
K. Kramer, et al.
Procyanidin dimers including procyanidin B-2 are stable during gastric
transit and reach the small intestine unchanged. In humans, these di-
mers are bioavailable and appear in plasma 2–3 h after digestion (Rios
et al., 2002). Procyanidin B-2 is not toxic, crosses Caco-2 cells by
paracellular transport (Deprez, Mila, Huneau, Tome, & Scalbert, 2001;
Kosinska & Andlauer, 2012) and reaches the intestine unmodified (Sano
et al., 2003). The presence of 10 hydroxyl groups on procyanidin mo-
lecules (Fig. 1a) confers substantial chelating ability to compete with
TG2 for available calcium. Increased intracellular calcium level is a key
event in the inflammatory pathway, not only activating TG2 but also
up-regulating inflammatory cytokines produced by T-cells (Parekh &
Putney, 2005). Concentrations of procyanidin B2 as low as 10 nM
modulate calcium levels and this inhibition of calcium increases in a
concentration-dependent manner (Verstraeten, Mackenzie, Oteiza, &
Fraga, 2008). TG2 has multiple functions in physiological processes
such as blood clotting, wound healing, cell adhesion, cell signaling and
apoptosis. For TG2 inhibition therapy in CD to be approved it would
have to be localized to the small intestine.
Incubating Caco-2 cells with IFN-γ or p31-43 also increased the
expression of two markers prominent in innate immunity including
COX-2 (Fig. 5A and B). Dose-dependent effects were observed, though
in each case cocoa extracts containing 289 μg/mL procyanidin B2 sig-
nificantly decreased COX-2. IFN-γ or α-gliadin also increased the se-
cretion of IL-15 (Fig. 6C and D). While TG2 captures the adaptive im-
mune response in CD, IL-15 dominates innate immunity (Vincentini
et al., 2015). IL-15 is present at the cell surface and functions by sti-
mulating intraepithelial lymphocytes (IELs), which are mediators of
cytotoxicity in epithelial cells. IL-15 also induces T-cell proliferation
and levels of IL-15 correlate to the degree of mucosal damage in CD (Di
Sabatino et al., 2006). When IL-15 is blocked, cytotoxicity in Caco-2
cells is reduced, making inhibition of this cytokine an important factor
to prevent CD inflammation.
Significant induction of IL-1β, IL-6, and IL-8 was observed in the
presence of IFN-γ or gliadin added to Caco-2 cells (Fig. 6E–I). Sig-
nificant reduction of these cytokines was observed in the presence of
procyanidin B-2-rich cocoa extracts. IL-1β induces COX-2. Inhibition of
IL-1β correlated with inhibition of COX-2 (Figs. 6G and H and 5A and
B). IL-1β has a larger role in innate immunity, which can explain why
IL-1β showed a greater response to gliadin rather than IFN. Treatment
with cocoa extracts was able to significantly affect the inflammatory
response to p31-43 by decreasing expression of both IL-1β and COX-2,
two biomarkers along with IL-15 that capture innate immunity in CD.
The induction of IL-6 in Caco-2 stimulated by p31-43 (Fig. 6C and
D) has been reported by other studies (Vincentini et al., 2015). The
inflammatory cytokine IL-6 plays a central role in immune response,
and evidence of increased serum IL-6 in untreated CD has been well
documented (O’Keeffe, Mills, Jackson, & Feighery, 1999; Romaldini,
Barbieri, Okay, Raiz, & Cacado, 2002). IFN-γ increases the secretion of
IL-6 (Biondillo, Konicek, & Iwamoto, 1994) (Fig. 6C).
Physiological concentrations of procyanidin dimers in chocolate
products are around 36 μM or 21 μg/g (Cooper et al., 2007). Hormesis is
seen when compounds go above physiological concentrations, and the
increase in pro-inflammation activity seen in cocoa extracts at 28.9 μg/
mL of procyanidin B2 is seen repeatedly in this study and is therefore
indicative of a hormetic effect. However at 289 μg/mL procyanidin B2
in cocoa extracts, there is again significant inhibition of inflammatory
biomarkers including TG2. At concentrations up to 289 μg/mL, pro-
cyanidin B2 rich cocoa extracts had no effect on Caco-2 cell viability.
The antioxidant properties of procyanidin B2 prevent cell cytotoxicity
and procyanidin B2 has anti-apoptopic effects in Caco-2 cells
(Rodriguez-Ramiro et al., 2011). Dias, Perez-Gregorio, Mateus, and De
Freitas (2015) showed that procyanidin trimers and tetramers bind to
gliadin peptides and may be an effective dietary option to prevent CD
development.
While higher concentrations of procyanidin B2 such as 289 μg/mL
exhibited the greatest TG2 inhibition in the IFN-γ treated cells, this
119
Journal of Functional Foods 57 (2019) 112–120
effect was not seen in the gliadin-treated cells, though all concentra-
tions did significantly decrease TG2 activity.
The results of the present study show that procyanidin B2 rich cocoa
extract, can modulate the inflammatory response obtained using an in
vitro model of CD. Turning off the inflammatory response, mediated by
TG2 and IL-15, by cocoa procyanidin B2 may represent a key target in
dietary approach to manage celiac disease.
5. Ethics statement
This research did not include human subjects or animal experi-
ments.
Conflict of interest
None.
Funding
This research was supported by the Louisiana State University
Agricultural Center.
Acknowledgements
We would like to acknowledge M&M Mars North America and
Bloomer for donating the cocoa samples used in this study.
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Further reading
Kaukinen, K., Partanen, J., Maki, M., & Collin, P. (2002). HLA-DQ typing in the diagnosis
of celiac disease. The American Journal of Gastroenterology, 97(3), 695–699.
Leffler, D. A., Kelly, C. P., Green, P. H. R., Fedorak, R. N., DiMarino, A., Perrow, W., ...
Murray, J. A. (2015). Larazotide acetate for persistent symptoms of celiac disease
despite a gluten-free diet: A randomized controlled trial. Gastroenterology, 148(7),
1311–1319.