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Therapeutic potential of hen egg white peptides for thetreatment of intestinal inflammation

Maggie Leea, Jennifer Kovacs-Nolana, Tania Archboldb, Ming Z. Fanb, Lekh R. Junejac,Tutomu Okuboc, Yoshinori Minea,*

aDepartment of Food Science, University of Guelph, Guelph, Ontario, CanadabDepartment of Animal and Poultry Science, University of Guelph, Guelph, Ontario, CanadacTaiyo Kagaku, Co. Ltd., Yokkaichi, Mie 510-0844, Japan

A R T I C L E I N F O

Article history:

Received 18 December 2008

Accepted 6 January 2009

Available online 3 March 2009

1756-4646/$ - see front matter � 2009 Elsevidoi:10.1016/j.jff.2009.01.005

* Corresponding author: Tel.: +1 519 824 4120E-mail address: ymine@uoguelph.ca (Y. M

A B S T R A C T

Inflammatory bowel disease (IBD) is a chronic and recurring inflammation of the gastroin-

testinal tract; however, current pharmaceutical treatments are only moderately effective

and have potential long-term toxicity, therefore novel IBD therapies are required. Hen

egg white has been shown to be an abundant source of novel immunomodulating proteins

and peptides. The anti-inflammatory activity of egg white peptides was examined using a

porcine model of dextran sodium sulphate (DSS)-induced colitis. DSS was administered for

five days via intra-gastric catheter to induce experimental colitis, followed by five days of

treatment with egg white peptides (EWP) or saline.

Supplementation with EWP attenuated the DSS-induced clinical symptoms, including

weight loss, mucosal and submucosal inflammation, crypt distortion, and colon muscle

thickening, and restored gut barrier function by decreasing intestinal permeability and

increasing mucin gene expression. Furthermore, treatment with EWP significantly reduced

the local expression of pro-inflammatory cytokines TNF-a, IL-6, IL-1b, IFN-c, IL-8, and IL-17,

suggesting that EWP is a promising novel therapeutic for the treatment of IBD.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Inflammatory bowel disease (IBD) is a chronic inflammation

of the gastrointestinal tract, and exists in two forms: ulcera-

tive colitis (UC) which typically affects the large intestine,

and Crohn’s disease (CD) which can occur in any part of

the gastrointestinal tract (Podolsky, 2002; Xavier & Podolsky,

2007; Torres & Rios, 2008). While the causes of IBD are still

relatively unknown, there are indications that it may be

caused by the inability of the gastrointestinal immune sys-

tem to differentiate between intestinal microflora and harm-

ful pathogens (Podolsky, 2002; Xavier & Podolsky, 2007;

Torres & Rios, 2008). This abnormal recognition of antigens

er Ltd. All rights reserved

x52901; fax: +1 519 824 6ine).

by cells of the mucosal innate immune system leads to a

deregulated immune reaction, characterized by an overpro-

duction of inflammatory cytokines and trafficking of effector

leukocytes into the intestine, resulting in uncontrolled

inflammation and tissue injury (Scaldaferri & Fiocchi, 2007;

Etchevers et al., 2008).

The prevalence of IBD in developed countries continues

to increase (Saro & Sicilia, 2008), and it represents a signifi-

cant economic burden on the healthcare system as well as

on the quality of life of IBD patients (Etchevers et al., 2008;

Kappelman et al., 2008). Current treatments, which include

corticosteroids and immunosuppressive agents such as aza-

thioprine, have shown limited therapeutic efficacy and have

.

631.

Table 1 – Size distribution of peptides in EWPpreparation.

M.W. (kDa) 12.5–6.5 6.5–1.3 1.3–0.25 0.25–0.07 <0.07% 6.48 6.65 46.81 35.2 4.86

162 J O U R N A L O F F U N C T I O N A L F O O D S 1 ( 2 0 0 9 ) 1 6 1 – 1 6 9

been associated with severe side effects and long-term tox-

icity (Atreya & Neurath, 2008), and despite treatment the

need for surgical intervention has remained unchanged

(Etchevers et al., 2008). Therefore, there is a pressing need

for novel safe and effective therapeutic agents for the treat-

ment of IBD.

It is well documented that hen eggs contain numerous

proteins and peptides that exert beneficial bioactive effects

(Kovacs-Nolan et al., 2005; Mine & D’Silva, 2008). Egg white

proteins, in particular have been shown to possess a number

of novel biological functions including antimicrobial, anti-vir-

al, anti-cancer, and protease inhibiting activities (Li-Chan

et al., 1995; Kovacs-Nolan et al., 2005; Mine & D’Silva, 2008).

Several egg white proteins, including lysozyme, ovomucin,

ovalbumin and ovotransferrin, which collectively make up

around 73% of total egg white composition, have also demon-

strated potent immune-modulating activity (reviewed in Kov-

acs-Nolan et al., 2005; Mine & D’Silva, 2008), indicating that

egg white may serve as a valuable source of immunomodulat-

ing proteins. Moreover, the activity of these egg white pro-

teins has been shown to be enhanced following proteolytic

digestion (Tezuka & Yoshikawa, 1995; Tanizaki et al., 1997;

Pellegrini et al., 2000).

Dextran sodium sulphate (DSS) is a polysaccharide that

has been widely used in rodents to model IBD, and induces

intestinal inflammation which is characterized by weight

loss, bloody diarrhea, epithelial cell damage, mucosal ulcers,

and neutrophil infiltration, as well as an increased production

of inflammatory cytokines including tumor necrosis factor

(TNF)-a, interleukin (IL)-6, IL-12, interferon (IFN)-a and IL-1b

(Wirtz & Neurath, 2000; Melgar et al., 2005; Wirtz et al.,

2007). Recently, DSS has also been used successfully to mimic

IBD in pigs (Mackenzie et al., 2003; Bassaganya-Riera &

Hontecillas, 2006). Pigs share a similar gastrointestinal mor-

phology and physiology with humans (Miller & Ullrey, 1987),

and therefore may be a more suitable model for the evalua-

tion of IBD therapeutics.

In this study, we report, for the first time, the anti-inflam-

matory and immunomodulatory effects of EWP using a por-

cine model of DSS-induced colitis. EWP supplementation

attenuated clinical symptoms and weight loss, and restored

intestinal epithelial barrier integrity. Furthermore, EWP mod-

ulated local gene expression, reducing inflammation and

restoring the cytokine balance, indicating its therapeutic po-

tential for the treatment of IBD.

2. Materials and methods

2.1. Materials

All reagents were purchased from Sigma-Aldrich (St. Louis,

MO, USA) unless otherwise stated.

2.2. Egg white peptides

Egg white hydrolysate, containing egg white peptides (EWP)

was provided by Taiko Kagaku Ltd R&D (Mie, Japan) and was

prepared as follows: briefly, liquid egg white was subjected

to enzymatic hydrolysis using food-grade aminopeptidase

(EC3.4.11.1; Novozymes, Bagsvaerd, Denmark) of Aspergillus

sp. origin. The hydrolysis reaction was carried out at 55–

60 �C for 24 h. The reaction was stopped by heating at 90 �Cfor 30 min, and the soluble fraction was then filtered through

a 10 kDa molecular weight cut-off (MWCO) membrane and

spray-dried. The size distribution of the peptides, as

determined by gel permeation chromatography, is shown in

Table 1.

2.3. Animals and experimental design

Yorkshire piglets aged 3–4 days and weighing 3–4 kg were ob-

tained from the Arkell Swine Research Station (Guelph, ON,

Canada). During the study piglets were housed individually

in steel cages equipped with heating lamps, and fed three

times a day with a commercial milk replacement formula

(Soweena Litter Life; Merrich’s Inc., Middleton, WI). The ani-

mals were surgically fitted with an intra-gastric catheter (Mi-

cro-Renathane, O.D. 0.8 mm, Braintree Scientific Inc.,

Braintree, MA) for administration of DSS and EWP treatment.

After a three day recovery period, the animals were randomly

assigned into one of three groups: positive control (Pos; n = 8)

which received DSS followed by saline; EWP group (n = 6),

which received DSS followed by treatment with EWP; and neg-

ative control (Neg; n = 6), which received only saline through-

out the experiment. All procedures were carried out in

accordance with the Canadian Council of Animal Care’s Guide

to the Care and Use of Experimental Animals, and were ap-

proved by the University of Guelph Animal Care Committee.

2.4. Induction of colitis and EWP treatment

To induce colonic inflammation, animals were intra-gastrical-

ly infused with DSS (1.25 g/kg BW) (MP Biochemicals, Solon,

OH, USA) dissolved in saline for five days. Negative control

animals received only saline during this period. Following five

days of DSS administration, animals in the EWP group were

infused with 150 mg/kg BW of EWP for five days, while ani-

mals in the Neg and Pos groups received sterile saline. Ani-

mals were then euthanized and colon tissues were collected

and rinsed with protease inhibitor phenylmethylsulphonyl

fluoride (PMSF) in saline. Colon tissues were stored in 10% for-

malin for histological analysis, or flash frozen in liquid nitro-

gen for enzyme-linked immunosorbent assay (ELISA) and

real-time RT-PCR analyses.

2.5. Physical assessment of inflammation

Animals were monitored daily and body weight (BW), food in-

take, and stool consistencies were recorded.

2.6. In vivo intestinal permeability analyses

D-Mannitol, was used to assess in vivo intestinal permeability

as previously described by Thymann et al. (2006), with modi-

J O U R N A L O F F U N C T I O N A L F O O D S 1 ( 2 0 0 9 ) 1 6 1 – 1 6 9 163

fications. Briefly, on the last day of treatment, animals were

infused with D-mannitol, at a concentration of 0.6 g/kg BW,

via intra-gastric catheter. Blood was collected at 0, 35 and

70 min post-infusion into heparinized tubes, centrifuged at

800g for 5 min to obtain plasma, and stored at �20 �C until

further analysis.

The measurement of plasma D-mannitol concentrations

was adapted from previous studies (Lunn et al., 1989; Graefe

et al., 2003). Plasma was boiled for 5 min and centrifuged at

21,000g for 60 min. The supernatant was collected and incu-

bated at pH 8.6 and 40 �C with 0.1 U/mL mannitol dehydroge-

nase (Megazyme International Ireland Ltd., Co. Wicklow,

Ireland) for 150 min, and nicotinamide adenine dinucleotide

(NADH) production was measured spectrophotometrically at

340 nm. D-Mannitol concentrations were determined from a

D-mannitol standard curve.

2.7. Histological analysis

Immediately following sacrifice, colon tissues were placed

into 10% formalin for 24 h and transferred to 70% ethanol.

Approximately 5–6 tissue cross-sections of 2–3 mm thickness

were cut and placed into histology cassettes in 70% ethanol.

Tissues were fixed onto slides and stained with hematoxylin

and eosin (H&E). Slides were examined using a Leica DMR

microscope (Leica Microsystems GmbH, Wetzlar, Germany),

and muscle thickness was analyzed using Openlab 4.0.4 soft-

ware (Improvision, Coventry, UK).

2.8. Measurement of pro-inflammatory cytokines byELISA

Colon tissues were homogenized in HBSS containing 2 lg/mL

N-tosyl-L-phenylalanine chloromethyl-ketone, 2 lg/mL N-a-

p-tosyl-L-lysine ketone, 2 lg/mL leupeptin, 2 lg/mL hemisul-

phate, 2 lg/mL aprotinin, 2 lg/mL pepstatin A, and 100 mM

PMSF, using a PowerGen 700D homogenizer (Thermo Fisher

Scientific, Waltham, MA, USA). The homogenized tissues

were centrifuged at 12,000g for 15 min and the supernatant

was collected and analyzed by ELISA. IL-6 and tumor necrosis

factor (TNF)-a concentrations were measured using Porcine

IL-6 and TNF-a Quantikine� ELISA Kits according to the man-

ufacturer’s instructions (R&D Systems, Inc. Minneapolis, MN,

USA).

2.9. RNA isolation and analysis of gene expression byreal-time RT-PCR

Real-time RT-PCR analysis was used to measure the gene

expression of various biomarkers in the colon. Total RNA

was extracted from pulverized colon tissue using the AurumTM.

Total RNA Mini Kit (Bio-Rad Laboratories, Inc., Hercules, CA,

USA) and cDNA synthesis was carried out using the iScriptTM

cDNA Synthesis Kit (Bio-Rad Laboratories, Inc.), according to

the manufacturer’s instructions. Real-time PCR was carried

out using iQTM SYBR� Green Supermix (Bio-Rad Laboratories,

Inc.) on a MyiQTM Single Color Real-Time PCR Detection System

(Bio-Rad Laboratories, Inc.) using the following conditions:

denaturation 15 s at 95 �C, annealing 15 s at 56 �C, and exten-

sion 30 s at 72 �C. Porcine primers were designed using Pri-

mer3 v.0.4.0 (Rozen & Skaletsky, 2000) and synthesized by

the University of Guelph Laboratory Services Molecular Biol-

ogy Section (Guelph, ON). Primer pairs used were as follows:

b-actin (Accession no. U07786), 5 0-GGATGCAGAAGGAGAT-

CACG-30 (forward) and 5 0-ATCTGCTGGAAGGTGGACAG-3 0

(reverse); IL-6 (Accession no. M86722) 5 0-AAGGTGATGCCACC-

TCAGAC-3 0 (forward) and 5 0-TCTGCCAGTACCTCCTTGCT-3 0

(reverse); TNF-a (Accession no. X54001) 5 0-ATGGATGGGTG-

GATGAGAAA-30 (forward) and 50-TGGAAACTGTTGGGGAGAAG-30

(reverse); INF-a (Accession no. AY1 88090) 5 0-CCATTCAAAG-

GAGCATGGAT-3 0 (forward) and 5 0-GAGTTCACTGATGGCTTT-

GC-3 0 (reverse); IL-8 (Accession no. M86923) 5 0-TGGCAGTT-

TTCCTGCTTTCT-30 (forward) and 5 0-CAGTGGGGTCCACTC-

TCAAT-3 0 (reverse); IL-1b (Accession no. NM_2 14055)

5 0-CAAAGGCCGCCAAGATATAA-3 0 (forward) and 5 0-GAAAT-

TCAGGCAGCAACAT-3 0 (reverse); IL-17 (Accession no. NM_00

1005729) 5 0-TCATGATCCCACAAAGTCCA-3 0 (forward) and

5 0-AGTCCATGGTGAGGTGAAGC-30 (reverse); MUC1 (Accession

no. XM_001926883) 50-ACCAAGTCCCCTAACCCATC-30 (forward)

and 5 0-TTGGAATTTTCCAGGCAGTC-30 (reverse). Relative

mRNA expression was calculated using the2�DCt formula

(Livak & Schmittgen, 2001), using porcine b-actin as the

housekeeping gene.

2.10. Statistical analysis

Data are presented as means ± SEM of Neg, n = 6, Pos, n = 8,

and EWP, n = 6. Statistical significance was determined using

the GraphPad Prism statistical software (San Diego, CA,

USA). The ANOVA analysis and the Tukey multiple compari-

son test were used to determine the statistical significance.

A P-value of less than 0.05 was defined as significant unless

otherwise stated.

3. Results and discussion

3.1. Effect of EWP on colitis symptoms

DSS-induced intestinal inflammation is characterized by

intestinal epithelial cell damage resulting in bloody diarrhea

and weight loss (Wirtz & Neurath, 2000; Melgar et al., 2005;

Wirtz et al., 2007). As would be expected, piglets treated

with DSS developed mild to severe bloody diarrhea, which

was reduced following treatment with EWP. In humans, co-

lonic inflammation can cause weight loss due to increased

metabolic rate, decreased dietary intake and malabsorbtion

(Klein et al., 1988; Rigaud et al., 1994; Melgar et al., 2005).

Body weights and food intake were recorded throughout

the trial, and used to determine the weight gain to feed in-

take ratios (Fig. 1), in order to determine the effect of EWP

supplementation on the growth and appetite of the animals.

Animals in the Pos group showed significantly lower gain to

feed ratios when compared to the negative control (Neg)

animals (P < 0.05). In contrast, the animals that were treated

with EWP maintained ratios that were similar to the Neg

group, suggesting that the EWP treatment was able to

reverse the DSS-induced colitis symptoms and weight loss,

and providing evidence of the beneficial properties of EWP

treatment.

Fig. 1 – Body weight gain to feed ratios of Neg (n = 6), Pos

(n = 8) and EWP-treated (n = 6) piglets. Animals were intra-

gastrically infused with saline or DSS for five days, followed

by five days of saline or EWP. Body weights and food intake

were measured daily, and gain: feed ratios (g/mL)

determined. Values shown are means ± SEM. *P < 0.05.

Fig. 2 – Effect of EWP on in vivo gastrointestinal permeability

and colonic mucin gene expression for Neg (n = 6), Pos (n = 8)

and EWP-treated (n = 6) animals. (A) Prior to sacrifice,

animals were intra-gastrically infused with D-mannitol, and

plasma D-mannitol concentrations were determined at 0, 35,

and 70 min post-infusion. (B) Relative gene expression of

the mucin gene MUC1 in the colon tissues was determined

by real-time RT-PCR. Values shown are means ± SEM.*P < 0.05; **P < 0.01.

164 J O U R N A L O F F U N C T I O N A L F O O D S 1 ( 2 0 0 9 ) 1 6 1 – 1 6 9

3.2. Effect of EWP on in vivo gut permeability andintestinal barrier function

The permeability of the intestine is compromised in IBD pa-

tients, and DSS causes damage to the colon which mimics

that of IBD (Kitajima et al., 1999; Clayburgh et al., 2004).

D-Mannitol, a non-metabolizable sugar alcohol, was used to

assess the effect of EWP treatment on gastrointestinal perme-

ability. A linear relationship existed between plasma D-man-

nitol concentrations over time in all groups, and was used

to determine the rate of increase of plasma D-mannitol levels

(Fig. 2A). The rate of increase of plasma D-mannitol in the Pos

group (2.23 ± 0.57 lmol/mL min, r2 = 0.94) was significantly

higher (P < 0.05) than that of animals in the Neg control group

(1.22 ± 0.03 lmol/mL min, r2 = 0.99) and the EWP-treated

group (1.23 ± 0.06 lmol/mL min, r2 = 0.99). There were no dif-

ferences in the rate of plasma D-mannitol increase between

the Neg and the EWP piglets, indicating that EWP supplemen-

tation was able to repair the DSS-induced damage to the

intestinal epithelial barrier.

It has been suggested that the therapeutic restoration of

epithelial barrier function could improve the pathophysiology

and clinical outcomes in IBD, however there is little data

regarding the effects of IBD therapeutics on intestinal barrier

function (McGuckin et al., 2009). Here we demonstrated that

treatment with EWP was able to significantly reduce DSS-in-

duced gut permeability, and improve intestinal epithelial bar-

rier function. The ability of EWP treatment to restore gut

barrier function and reduce permeability may have been

due in part to the capacity of EWP to down-regulate the

expression of cytokines involved in inflammation and tissue

damage. Inflammatory cytokines such as TNF-a, IFN-a and

IL-1b, which were found to be elevated by DSS-treatment, in-

crease intestinal permeability, and contribute to the severity

of pathology and inability to resolve inflammation and repair

wounds (Wang et al., 2005; Al-Sadi et al., 2008). Moreover,

these pro-inflammatory cytokines can trigger the production

of matrix metalloproteinases, which damage intestinal tis-

sues (Mudter & Neurath, 2007). IL-8, expression of which

was also increased here, can also participate in the activation

of these enzymes (Mudter & Neurath, 2007).

To further examine epithelial barrier integrity, relative

expression of the mucin gene MUC1 was examined. In the co-

lon, the mucus layer, which is composed of mainly mucins,

acts as a physical barrier to protect and maintain epithelium

integrity (Tai et al., 2007). The expression of MUC1 was signif-

icantly decreased (P < 0.01) in Pos animals when compared to

animals in the untreated (Neg) group, but was significantly in-

creased in animals supplemented with EWP (P < 0.05) (Fig. 2B).

Van der Sluis et al. (2006) previously demonstrated that mice

deficient in mucin production spontaneously developed a

colitis-like phenotype, and the decreased expression of mucin

genes has been observed in DSS-induced colitis in mice (Tai

et al., 2007). In the present study, relative mucin gene expres-

sion was indeed reduced as a result of DSS-treatment, but

was restored upon administration of EWP. It has been sug-

gested that while therapeutics that directly stimulate produc-

tion of constituents of the epithelial barrier may not resolve

advanced lesions, they may have promise in maintaining pa-

tients in remission (McGuckin et al., 2009), suggesting a fur-

ther advantage of EWP as a therapeutic in IBD.

J O U R N A L O F F U N C T I O N A L F O O D S 1 ( 2 0 0 9 ) 1 6 1 – 1 6 9 165

3.3. Histopathological observations

The major hallmarks of colonic inflammation include crypt

destruction, mucosal ulceration, and infiltration of lympho-

cytes into the mucosal tissue (Kwon et al., 2005). Representa-

tive histological images of H&E-stained colon sections from

each group are shown in Fig. 3A. In contrast to negative con-

trol animals (a), Pos animals (b) showed severe crypt destruc-

tion, and mucosal and sub-mucosal inflammation and

thickening, which was not evident in the colon of piglets sup-

plemented with EWP (c), indicating that the DSS-induced

ulceration and crypt destruction was reversed by treatment

with EWP.

To further assess the extent of inflammation, colonic

smooth muscle thickness was measured. Smooth muscle

thickness is commonly used to evaluate inflammation along

the gastrointestinal tract (Blennerhassett et al., 1992, 1999).

Previous research has shown that inflammation causes prolif-

eration of the intestinal smooth muscle cells, leasing to in-

creases in muscle mass and muscle cell numbers

(Blennerhassett et al., 1992, 1999), and an accumulation of

collagen leading to a thickening of the intestinal wall (Gra-

ham et al., 1988). Animals in the Pos group showed signifi-

cantly increased muscle thickness (P < 0.01) when compared

to untreated control animals (Fig. 3B). Muscle thickness was

significantly reduced following treatment with EWP

(P < 0.01), but was still greater than that of Neg animals, likely

due to the short duration of treatment. Muscular hypertrophy

and colon thickening are often observed in UC patients, and

the resulting shortening of the colon may be one of the causes

Fig. 3 – Effect of EWP on DSS-induced colitis histology and muscl

50· magnification, from (a) Neg control, (b) Pos control, and (c) E

sections from Neg, Pos, and HEL animals. Values shown are me

of diarrhea (Cho et al., 2007). These results further demon-

strate that EWP supplementation was able to attenuate the

DSS-induced damage to the colon, thereby reducing colitis

symptoms.

3.4. Effect of EWP on local cytokine expression

Pro-inflammatory cytokines play a key role in the inflamma-

tory cascade, and have been implicated in the immunopa-

thology of IBD (Garside, 1999) by being involved in the

initiation and amplification of inflammatory responses that

lead to intestinal injury (Sartor, 1994). Both lymphocytes and

macrophages in inflamed intestinal mucosa synthesize and

secrete large numbers of potent pro-inflammatory mediators

(MacDermott, 1996). Increased levels of inflammatory cyto-

kines such as TNF-a, IL-6, IL-8 and IFN-c have been detected

in the serum and colons of patients with IBD (Daig et al.,

1996; Egger et al., 2000). Likewise, in mice, DSS-induced colo-

nic inflammation is mediated in part by the overexpression of

inflammatory cytokines such as TNF-a, IL-6, IL-1b, and IFN-c

Dieleman et al., 1994; Tomoyose et al., 1998).

TNF-a and IL-6, in particular, have become attractive tar-

gets for IBD therapy. TNF-a exerts its effects through a num-

ber of mechanisms, including the increased production of

IL-6 and IL-1b, the increased expression of adhesion mole-

cules, as well as the inhibition of apoptosis (Begue et al.,

2006). IL-6 signaling via the signal transducer and activator

of transcription (STAT)-3 pathway has been shown to be crit-

ical for the development of DSS-induced colitis as well as

inflammation in IBD (Lovato et al., 2003; Musso et al., 2005).

e thickness. (A) Representative H&E-stained colon sections at

WP-supplemented animals. (B) Muscle thickness of colon

ans ± SEM. **P < 0.01.

Fig. 4 – Concentrations of TNF-a and IL-6 in the colon. Colon tissues from Neg (n = 6), Pos (n = 8) and EWP-treated (n = 6)

animals were homogenized in HBSS containing protease inhibitors, and TNF-a and IL-6 concentrations in the supernatants

were determined by ELISA. Results are expressed as pg per g of colon tissue. Values shown are means ± SEM. *P < 0.05.

166 J O U R N A L O F F U N C T I O N A L F O O D S 1 ( 2 0 0 9 ) 1 6 1 – 1 6 9

In the present study, the production of both TNF-a and IL-6 in

the colon was significantly elevated by DSS-treatment. How-

ever, treatment with EWP reduced the levels of colonic TNF-

a and IL-6 by 5- and 4-fold, respectively (Fig. 4; P < 0.05) to ba-

sal levels observed in untreated (Neg) animals. These results

are in line with recent evidence that monoclonal antibodies

directed against TNF-a or the IL-6 receptor could reduce

inflammation both in vitro and in vivo (reviewed in Atreya &

Neurath, 2008), and highlight the therapeutic potential of

EWP supplementation. Furthermore mRNA expression levels

of the pro-inflammatory cytokines IL-1b (P < 0.01) and IFN-c

Fig. 5 – Effect of EWP supplementation on cytokine mRNA levels

(n = 6), Pos (n = 8) and EWP-treated (n = 6) animals, and real-tim

Methods. Values shown are means ± SEM. *P < 0.05; **P < 0.01; ***

(P < 0.05), which were elevated in Pos animals, were signifi-

cantly decreased upon EWP treatment (Fig. 5). Expression of

IL-8, a chemokine which contributes to IBD-mediated pathol-

ogy via the recruitment of neutrophils to the intestinal muco-

sa (Umehara et al., 2006) was also significantly increased in

DSS-treated animals (P < 0.001). IL-1b and TNF-a can stimu-

late the increased production of IL-8 from surrounding im-

mune and non-immune cells (MacDermott, 1996), further

contributing to the DSS-induced inflammation observed here.

Treatment with EWP, however, reduced IL-8 (P < 0.01) to levels

similar to those observed in the Neg group.

in the colon. mRNA was extracted from colon tissues of Neg

e RT-PCR was carried out as described in Materials and

P < 0.001.

J O U R N A L O F F U N C T I O N A L F O O D S 1 ( 2 0 0 9 ) 1 6 1 – 1 6 9 167

It has long been considered that the aberrant immune re-

sponse observed in CD was dominated by a T helper (Th) 1

lymphocyte response, while UC was associated with a modi-

fied Th2 response (Shanahan, 2001). However, it is becoming

increasingly clear that a novel subset of IL-17-producing lym-

phocytes, termed Th17 cells, may be critical in IBD pathogen-

esis (Mizoguchi & Mizoguchi, 2008). In fact Ito et al. (2008)

found that IL-17 knock-out mice developed less severe

inflammation and colitis symptoms than wild type mice, sug-

gesting the pivotal role of IL-17 in the pathogenesis of DSS-in-

duced colitis. Th17 cells produce IL-17, IL-6 and TNF-a, which

in turn act on fibroblasts, macrophages, and endothelial and

epithelial cells to elicit the release of inflammatory mediators.

This environment recruits neutrophils and creates a general

state of tissue inflammation and damage (Kaiko et al., 2008).

In humans, IL-1b was found to induce IL-17 production, which

was enhanced in the presence of IL-6. IL-1b also enhances

murine Th17 differentiation in vitro and has been shown to

participate in IL-17-mediated disease in mice (Ivanov et al.,

2007). Indeed, similar to IL-6, TNF-a, and IL-1b expression,

we also observed a concomitant increase in IL-17 gene

expression in DSS-treated Pos animals, which was signifi-

cantly reduced by treatment with EWP (Fig. 5; P < 0.01), sug-

gesting that EWP may also modulate gene expression of

Th17 cells, however further study will be required to further

elucidate their role in the Th17 pathway.

Surprisingly, expression levels of the anti-inflammatory

cytokines IL-10 and IL-4 did not appear to correlate with the

reduction in inflammation and colitis symptoms observed

in piglets supplemented with EWP. IL-10 expression was ele-

vated in the Pos animals, and decreased with EWP treatment

(Fig. 5; P < 0.01). IL-10 is constitutively expressed in normal gut

tissue, and inhibits the production of inflammatory cytokines,

however, it is also produced by activated macrophages (Fio-

rentino et al., 1991). Our results support previous observations

of elevated levels of IL-10 in IBD patients as a response to

chronic inflammation (Kucharzik et al., 1995), and it has been

suggested that despite its up-regulation, the intrinsic intesti-

nal bioavailability of IL-10 might be insufficient to control the

local inflammation (Autschbach et al., 1998). The increased IL-

10 levels observed in the Pos animals may be indicative of a

state of ongoing inflammation, whereas EWP treatment

accelerated healing, reduced inflammation and returned IL-

10 to levels similar to those of Neg animals. Likewise, IL-4 is

found in low levels in the colonic mucosa of IBD patients

(Nielsen et al., 1996), and IL-4 gene expression has been found

to be reduced in rodent models of IBD (Bento et al., 2008).

Accordingly, IL-4 expression was significantly reduced in

DSS-treated Pos animals relative to the Neg group (Fig. 5;

P < 0.01), however IL-4 levels remained at significantly lower

levels following EWP treatment (P < 0.01), suggesting that an

up-regulation of anti-inflammatory cytokines was not in-

volved in the attenuation of colitis symptoms observed in

EWP-treated animals.

4. Conclusions

We have demonstrated for the first time the anti-inflamma-

tory and immunomodulating capabilities of egg white pep-

tides in an experimental model of colitis. Treatment of

piglets with DSS resulted in weight loss, severe mucosal and

submucosal inflammation, colonic crypt distortion, muscle

wall thickening, down-regulation of mucin gene expression,

and increased gastric permeability. EWP supplementation

abrogated DSS-induced symptoms, suggesting that EWP

treatment may accelerate colon healing, and restore gut phys-

iology to that observed in the untreated (Neg) animals, as evi-

denced by the weight gain, normal colon histopathology,

increased mucin gene expression, and decreased intestinal

permeability observed in these animals. The anti-inflamma-

tory activity of EWP was also clearly demonstrated. EWP

treatment resulted in a significant down-regulation of inflam-

matory cytokine production, at both the protein and gene

expression levels. The marked reduction in inflammatory

cytokine production observed in EWP-treated animals corre-

sponds well with the attenuation DSS-induced symptoms.

These results suggest that egg white peptides may play a

multifunctional role in amelioration of experimental colitis,

by modulating local gene expression to both down-regulate

the inflammatory milieu, thereby limiting tissue damage

and allowing for recovery to occur, and up-regulating mucin

gene expression to restore gut barrier integrity and function,

indicating that egg white peptides may be a promising novel

therapeutic in the treatment of IBD.

Acknowledgements

The authors would like to thank Dr. A.M. Hayes, Department

of Pathobiology, University of Guelph, for histopathological

analysis, and Chengbo Yang for his assistance with real-time

RT-PCR. Financial support was provided by the Advanced

Foods and Materials Network (AFMNet), Canada.

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