Gastroenterol Hepatol (N Y). Sep 2012; 8(9): 582–588.

PMCID: PMC3594957

Emerging Therapeutic Options for Celiac Disease

Potential Alternatives to a Gluten-Free Diet Anita Bakshi, MD, Sindu Stephen, MD, Marie L. Borum, MD, and David B. Doman, MD

Author information ► Copyright and License information ►

Abstract

Celiac disease (CD), also known as gluten sensitivity enteropathy or celiac sprue, is an immune-

mediated enteropathy that is triggered in genetically susceptible individuals by the ingestion of

gluten—the major storage protein of wheat, barley, and rye.1 Globally, CD is one of the most

common autoimmune disorders. The clinical presentations of the disease vary, with either typical

intestinal symptoms or a spectrum of atypical extraintestinal symptoms. Clinically silent forms

can also occur, which are often difficult to diagnose. Given the wide spectrum of presentations,

the diagnosis is often missed.2 CD can occur at any age, is more prevalent than was previously

thought, and can affect a variety of organ systems.3 Early recognition and treatment of CD are

important to prevent complications such as malnutrition, osteoporosis, infertility, and

gastrointestinal malignancies. The only currently approved treatment for CD is dietary exclusion

of foods containing gluten; unfortunately, a majority of patients have difficulty complying with

this diet, and the response to therapy is poor in up to 30% of patients, resulting in persistent or

recurrent symptoms, inadequate cure, and/or refractory disease.4

CD is more common than was previously thought, and recent studies have shown a much higher

global prevalence rate.3 In the past, CD was considered to be a rare disorder in North America,

mostly affecting individuals of northern European descent, and the disease was usually

diagnosed in childhood. At that time, diagnosis was made in patients with typical gastrointestinal

symptoms and classic symptoms of malabsorption, with confirmation by small intestinal

biopsy.5 The discovery of highly sensitive and specific serologic markers—including antigliadin,

antiendomysium, and antitransglutaminase antibodies— has allowed clinicians to evaluate the

true prevalence of CD and identify patients with clinically mild, atypical, or even silent forms of

CD.6

A recently published, large, international, multicenter study investigated a wide population

sample and found that the overall prevalence of CD is 1%, on average, with large variations

among countries.7 The prevalence of CD in the general population of the United States is

approximately 1:133 (0.75%). Most cases of CD remain undiagnosed until later in life, with an

average age at diagnosis of 45 years. The average time to diagnosis is 10—12 years, as many

patients do not present with classic symptoms of diarrhea, weight loss, and abdominal pain.8The

National Institutes of Health estimates that about 3 million people in the United States have CD

and that more than 95% of people with the condition remain undiagnosed. Physicians should be

aware that CD has a wide spectrum of presentations and that this condition may occur at any age,

in both sexes, and in a wide variety of clinical circumstances.9,10

CD is a chronic, inflammatory, small intestinal disorder that can lead to severe villous atrophy,

malab-sorption, and malignancy. Susceptibility to CD, its activation, and the ensuing

inflammatory cascade involve a combination of environmental and genetic factors that trigger

immunologic mechanisms.11 CD is the only autoimmune disease with a known trigger, which is

the ingestion of the gluten proteins found in wheat, barley, and rye. All patients must express the

antigen-presenting molecules human leukocyte antigen (HLA)-DQ2 and/or HLA-DQ8, the

presence of which is the single most important predisposing genetic factor for CD.12 HLA-DQ2

and HLA-DQ8 then bind gluten peptides that have undergone deamidation by transglutamin-ase

2 (TG2), an enzyme tissue transglutaminase; this deamidation increases the gluten peptides’

affinity for HLA-DQ2 and HLA-DQ8 and results in a more destructive intestinal CD4+ T-cell

response.13 Once activated, gluten-reactive CD4+ T cells produce cytokines and induce an

inflammatory cascade that results in intestinal inflammation—characterized by villous atrophy,

crypt hyperplasia, and infiltration of inflammatory cells—which leads to malabsorption.12 Based

on this mechanism of action, the current standard treatment of choice is strict, lifelong adherence

to a gluten-free diet that eliminates wheat, rye, and barley.

A gluten-free diet is presently the therapy of choice for CD, as it improves gastrointestinal

symptoms within a few weeks and has been shown to cause a histologic and serologic response

within 1—2 years. If patients strictly adhere to this diet, vitamin deficiencies resolve, and the risk

of concomitant autoimmune disease and CD-associated malignancies is reduced.14 However,

many patients fail to comply with this lifelong restrictive diet, as gluten is a common ingredient

in diets throughout the world, and gluten-free foods are not widely available. Even if patients

make every effort to avoid gluten in their diets, small levels of contamination frequently occur in

food products, and many people inadvertently consume gluten-containing foods. Gluten-free

foods are also more expensive than their gluten-containing counterparts. In addition, health-

related quality of life has been shown to be lower in people with CD while they are on a gluten-

free diet.15 Therefore, maintaining this diet for life is challenging, and poor adherence often

leads to incomplete resolution of symptoms.

Even in fully compliant patients, a gluten-free diet fails to induce clinical or histologic

improvement in 7—30% of patients. After secondary causes of nonresponse have been

investigated—including alternative diagnoses or complications of CD—persistent symptoms are

attributed to refractory disease. Approximately 5% of patients may have refractory CD, in which

symptoms persist despite strict adherence to a gluten-free diet. Refractory CD may be classified

as type 1, in which there is a normal intraepithelial lymphocyte phenotype, or type 2, in which

there is a clonal expansion of an aberrant intraepithelial lymphocyte population.16 Intraepithelial

T lymphocytes are considered to be aberrant when they express cytoplasmic CD3 but lack

surface expression of the T-cell markers CD3, CD4, CD8, and the T-cell receptor.17 The

intraepi-thelial lymphocyte expansion may be driven by overex-pression of interleukin-15 by the

epithelium.18 Type 1 refractory CD usually responds to steroid therapy, but type 2 refractory

disease carries a more dismal prognosis, as it is usually steroid-refractory and is associated with

an increased risk of lymphoma. Current research is focused on nondietary therapies and

treatment of refractory and diet-unresponsive CD.

Recently, studies have found that, in addition to environmental and genetic predispositions,

abnormalities in the structure of the small intestine play a major role in the pathogenesis of CD.

In most people, links known as tight junctions help keep enterocytes connected. In patients with

CD, however, the junctions come apart, allowing a large number of indigestible gluten fragments

to escape into the underlying tissue and incite immune system cells. New research has identified

a protein that is secreted by intestinal epithelial cells, called zonulin, which induces tight junction

disassembly; increased expression of this protein results in increased intestinal

permeability.19 In CD, gliadin (the toxic component of gluten) binds to the intestinal receptor

CXCR3, which then initiates exaggerated zonulin secretion, resulting in abnormally high levels

of zonulin.20 Zonulin makes the intestine more permeable and allows gluten to seep out of the

gut, where it then interacts freely with the genetically sensitized elements of the immune system

that cause intestinal damage. Therefore, CD treatments are being directed toward reducing this

permeability.20,21

Recent advances and insights have improved understanding of the molecular basis for CD; with

further knowledge of the pathophysiology of this disease, many new targets for therapy have

been identified and are currently being developed (Table 1). Alternative therapeutic strategies are

directed at disrupting 1 of the 3 major pathways that cause the disease, including the

environmental trigger (gluten), genetic susceptibility, and unusual gut permeability.21

Table 1

New Therapeutic Agents for Celiac Disease and Their Mechanisms of Action

Go to:

Novel Therapies

Genetically Modified Gluten

Because removing gluten from the diet results in clinical, serologic, and histologic improvement

in most patients, this approach is currently the recommended treatment of choice for CD, and

many patients have been forced to eat bread made with gluten-free flour. Bread is one of the

most commonly consumed foods in the Western diet, and it is typically made from grains that

contain gluten, such as wheat. Wheat gluten is the protein that strengthens and binds dough in

baking; thus, gluten is an important component of bread. However, gluten-free flour is now

available as a substitute; this flour is made from a variety of materials, such as almonds, rice,

sorghum, corn, and legumes. Flourless breads made with gluten-free grains have also been

created; they use amaranth flour, arrowroot flour, brown rice flour, buckwheat flour, chia flour,

chickpea flour, corn flour, cornmeal hemp flour, maize flour, millet flour, potato flour, quinoa

flour, soya flour, tapioca flour, teff flour, and white rice flour. Although proteins found in these

alternatives are a source of complex carbohydrates, they lack B vitamins, and vitamin

deficiencies may occur. These alternatives also lack many of the essential nutrients and flavor of

wheat flour.4

Therefore, genetically modified gluten with reduced immunogenicity is being developed as a

potential future option to decrease the toxicity that occurs in CD. CD is caused by T-cell

responses to peptides derived from the gluten proteins found in wheat, which bind to DQ2 or

DQ8 molecules and cause an inflammatory response mediated by interferon. In particular,

deamidation of certain glutamine residues to glutamate by the transglutaminase enzyme results in

peptides that are more acidic and thus have greater affinity for DQ2 and DQ8, resulting in a

heightened T-cell—mediated inflammatory response.22 Gianfrani and colleagues found that

blocking these glutamine residues with lysine methyl ester (Lys-CH3) strongly inhibited the

immune response to immunotoxic peptides in T cells from patients with CD.23 In their study, a

transamidation reaction attached Lys or Lys-CH3 to a glutamine residue of α-gliadin p56—68,

an immunotoxic derivative of gluten. The altered peptide had a reduced affinity for the DQ2

molecule, compared with deamidated peptides, and consequently reduced interferon-γ release

from T cells of patients with CD. Treating wheat flour with microbial transglutaminase in the

presence of Lys-CH3 neutralized the immunotoxic-ity of the digested products. Therefore,

transamidation of wheat flour with an appropriate amine group donor can be used to block T-

cell—mediated gliadin activity and gliadin immunotoxicity.23

In addition, a recent study found a large genetic variation in wheat products and the amount of T-

cell— stimulatory peptides present in the wheat accessions. The study found that it was possible

to select and breed gluten proteins that lack 1 or more of the known T-cell—stimulatory

sequences. This breeding could allow selection of wheat that contains low amounts of T-cell—

stimulatory sequences and thus is suitable for consumption by CD patients.24The results of this

study suggest that wheat products can be enzymatically engineered to eliminate their

immunotoxic effects on individuals with CD. This finding demonstrates that there is a potential

to reintroduce detoxified wheat into the diets of patients with CD and thus provide well-balanced

meals and better quality of life.

Zonulin Inhibitor

The discovery of Zot, an enterotoxin elaborated by Vibrio cholerae that reversibly opens tight

junctions, has enhanced understanding of the complex mechanisms that regulate the intestinal

epithelial paracellular pathway.25Zot regulates tight junctions in a rapid, reversible, and

reproducible fashion; based on this knowledge, researchers were able to discover zonulin, which

is a similar, endogenous modulator of epithelial tight junctions.26 Gliadin is known to cause

increased secretion of zonulin, which alters intestinal permeability, facilitates the transport of

gluten, and triggers an inflammatory process. This mechanism has been the target of advanced

research that focuses on blocking tight junction modulators.

Alessio Fasano, one of the lead investigators in this area, helped to develop a promising new

zonulin inhibitor known as larazotide acetate (AT-1001, Alba Therapeutics). This drug inhibits

the human protein zonulin, which regulates intestinal permeability. This agent is currently in

phase IIb trials, following positive results in 2 randomized, placebo-controlled, human trials

conducted in 2009. The first of these studies showed that lar-azotide acetate reduced gluten-

induced intestinal barrier dysfunction, production of inflammatory molecules, and

gastrointestinal symptoms in CD patients. The second, larger study showed that CD patients who

received placebo produced antibodies against tissue transglutaminase, but the group treated with

larazotide acetate did not.21

In the most recent phase of testing, 75% of CD patients who were treated with a placebo pill and

exposed to gluten developed classic symptoms, but symptoms occurred in only 14% of those

who were treated with larazotide acetate and exposed to gluten. This drug is designed to be taken

prior to the consumption of a gluten-containing meal, and it effectively blocks the toxic effect of

zonulin. Phase III clinical trials are now being planned to evaluate the safety, efficacy, and

maintenance of the effect of larazotide acetate.

This agent is a potentially effective drug that, if it continues to show promise, should be available

within the next 5 years and could improve quality of life among patients with CD by allowing

them to enjoy gluten-containing foods for the first time. Recently, Alba Therapeutics received

approval from the US Food and Drug Administration (FDA) to expand studies of larazotide

acetate to other autoimmune disorders, including type 1 diabetes and Crohn’s disease, as these

conditions are also associated with high levels of zonulin. If approved, lar-azotide acetate would

stop the autoimmune process by blocking a specific trigger, as opposed to previous therapeutic

options that have been directed at decreasing the body’s overall immune response or producing a

general anti-inflammatory effect.21

Therapeutic Vaccine

A peptide-based therapeutic vaccine that is currently being developed is a promising treatment

for patients with CD. In contrast to other nondietary therapies that have been proposed, a

peptide-based therapeutic vaccine is being developed to specifically modify the pathogenic T-

cell response rather than reduce the amount of gluten pep-tide presented to the T cell. Currently,

the vaccine would be effective only in patients with genotype HLA-DQ2, which is about 90% of

patients with CD.

In order to create an effective peptide-based therapy for CD, an important step is the

identification of the gluten peptides that trigger intestinal T-cell responses when patients with

CD consume wheat, rye, or barley. Recently, researchers from the Australian company Nexpep

analyzed the gluten protein and broke it down into about 2,700 distinct fragments. These

fragments were then added to the blood of 200 CD patients, and the immune responses to the

fragments were compared to the responses seen after the same patients consumed wheat bread,

rye muffins, and boiled barley. Three peptides—gliadin, hordein, and secalin—were found to

trigger a heightened immune response.27

The company then designed Nexvax2, which combines the 3 key, gluten-derived peptides into a

vaccine. This vaccine is administered in weekly injections in order to desensitize CD patients to

the toxic effects of gluten. The vaccine recently underwent a phase I clinical study. The

randomized, placebo-controlled, double-blind, phase I study compared weekly intradermal

injections of Nexvax2 versus placebo in CD patients with the HLA-DQ2 genotype who were on

a strict gluten-free diet. In the 3-week study, the safety and tolerability profile of Nexvax2 was

similar to that of placebo. Gastrointestinal symptoms were more common in patients given the

highest dose of the vaccine, confirming the selection of the toxic peptides that can eventually

induce tolerance.28 The symptoms and mobilization of gluten-specific T cells observed after

administration of Nexvax2 were similar to those triggered by acute, oral gluten exposure in

HLA-DQ2 patients on a gluten-free diet. Similar to traditional desensitization therapy for

allergies, the peptide-based vaccine is designed to be given through injections in multiple small

doses over a period of time in order to create immune tolerance to the selected gluten fragments

and to lower the toxicity of related gluten molecules.29 This approach will ultimately prevent the

T cells from initiating the immune cascade that damages the small bowel. The vaccine is

expected to enter phase IIa clinical trials to evaluate its efficacy.

Probiotics with Enzymes

CD is a T-cell—driven intolerance to wheat gluten. The gluten-derived T-cell epitopes are

proline-rich and highly resistant to proteolytic degradation within the gastrointestinal tract. The

abundance and location of proline residues contribute to the resistance of the 33-mer gliadin

peptide to breakdown by endogenous proteases in the gastrointestinal tract. An additional

advance in the alternative treatment of CD includes oral supplementation with prolyl

oligopeptidases to help degrade toxic gliadin peptides before they reach the mucosa. Shan and

coauthors identified a unique 33—amino acid peptide (from the 266—amino acid α2-gliadin)

that is resistant to degradation in the gastrointestinal tract; this 33—amino acid peptide contains

several T-cell stimulatory epitopes that initiate the inflammatory response to gluten in CD

patients.30 This peptide was able to be degraded and lost its antigenicity in both in vitro and in

vivo assays when it was exposed to a bacterial prolyl endopeptidase (PEP) derived

fromFlavobacterium meningosepticum, suggesting an oral bacterial peptidase could be used to

detoxify the immunodominant gliadin epitopes.31 Researchers in Ireland tested the combination

of bacterial-derived and barley-derived proteases—PEP and endoprotease B—isoform 2,

respectively—and demonstrated that, when given orally to CD patients, they can break down

gluten to nontoxic fragments; therefore, these proteases may be a beneficial treatment and might

allow CD patients to include modest amounts of gluten in their diets.32

In addition, bacterial enzyme preparations and intact probiotic preparations have also been

shown to directly alter the function of intestinal cells. Supplementation with a variety of bacterial

strains can help inhibit gluten/gliadin-induced damage in the small intestine.

Researchers in Finland added probiotic bacteria to cultures of intestinal epithelial cells to

determine the bacteria’s effect on gliadin-induced cellular damage. Two probiotic bacterial

species were evaluated: Lactobacillus fermentum and Bifidobacterium lactis. In this study, B.

lactis was able to inhibit permeability caused by gliadin. In addition, both B. lactis and L.

fermentum were able to protect against cell ruffling and alterations in tight junctions. The

bacteria alone, without gliadin, did not cause any significant changes to the intestinal epithelial

cells. Inclusion of probiotics appears to be able to reduce the damage caused by eating gluten-

contaminated foods and may even accelerate mucosal healing after the initiation of a gluten-free

diet.33 Therefore, the addition of probiotics with enzymes, which cause detoxification of gliadin

and promotion of intestinal healing, could be a potentially useful treatment for CD patients.

Transglutaminase Inhibitors

Human TG2, the enzyme that is involved in the patho-genesis of CD, catalyzes the

transamidation and deamida-tion of glutamine residues in peptides. TG2 has a critical role in the

pathogenesis of CD in that it deamidates glutamine residues from gluten peptides and converts

them into glutamic acids, thus increasing their binding affinity to HLA-DQ2 and HLA-DQ8

receptors, which in turn mediates the patient’s T-cell response.34 Given that TG2 increases the

pathologic effect of gluten peptides, TG2 inhibitors are potential therapeutic agents for the

treatment of CD.

There are 3 classes of TG2 inhibitors that differ based on their mechanisms of action:

competitive amine inhibitors, reversible inhibitors, and irreversible inhibitors. Competitive amine

inhibitors are the most common glutaminase inhibitors; they inhibit TG2 activity by competing

with natural amine substrates, such as protein-bound lysine residues, in the transamidation

process. Therefore, TG2 is still enzymatically active, and transamidation continues to occur in

the presence of competitive amine inhibitors. However, the resulting iso-peptide crosslink is

mainly formed between the natural glutamine substrate and the competitive amine inhibitor

rather than between the natural glutamine substrate and natural amine substrate.35 Reversible

TG2 inhibitors prevent enzyme activity by blocking substrate access to the active site without

covalently modifying the enzyme. TG2 cofactors, such as guanosine triphosphate and guanosine

diphosphate, are examples of allosteric, reversible inhibitors of the enzyme.36 Finally,

irreversible TG2 inhibitors prevent enzyme activity by covalently modifying the enzyme, thereby

preventing substrate binding.

Molberg and coworkers showed that culturing small intestinal biopsies from CD patients with

either TG2-treated (deamidated) or non-TG2-treated (non-deamidated) gluten digests resulted in

the generation of patient T-cell lines that preferentially recognized deamidated gluten peptides

rather than nondeamidated gluten peptides.37 Also, by using cystamine to block the activity of

endogenous TG2 in the CD patient biopsies, the authors demonstrated that more than half of the

resultant T cells had reduced proliferative responses to deamidated gluten digests compared to

non-cysta-mine-treated controls; they also showed that these cell lines still did not respond well

to the nondeamidated digests.37 In another study, Maiuri and coauthors showed that the 2-[(2-

oxopropyl)thio]imidazolium inhibitor L682777 was able to prevent the in situ crosslinking of

gluten peptides to endogenous proteins in tissue sections taken from both CD patients and

controls.38 Also, the authors showed that incubation of intact CD small intestinal biopsies with

L682777 prevented T-cell activation induced by the nondeamidated form of an immunodominant

gluten peptide.38 These studies suggest that treatment of CD biopsies with TG2 inhibitors

decreases the induced response of gluten-reactive T cells; these studies also suggest that

irreversible inhibition of endogenous TG2 in CD patient biopsies can prevent gluten peptide

deamidation and ultimately reduce T-cell activation.

Conclusion

CD, a genetically driven, aberrant immune response to dietary gluten, is more common than was

previously thought. CD can present with typical intestinal manifestations or atypical

extraintestinal manifestations. CD has traditionally been treated using a gluten-free diet, which

may be problematic from a standpoint of patient compliance. New insights into CD

pathophysiology have led to research into novel therapeutic opportunities. Research approaches

include engineering gluten-free grains, decreasing intestinal permeability by blockage of the

epithelial zonulin receptor, inducing oral tolerance to gluten with a therapeutic vaccine, and

degrading immunodominant gliadin peptides using probiotics with endopeptidases or

transglutaminase inhibitors. These treatment options have shown encouraging preliminary results

in phase II and phase III clinical trials. These non-diet-based therapies hold promise for

enhanced, lifelong CD management with improved patient compliance. If successful, these novel

approaches raise the possibility of reintroduction of gluten, in amounts to be determined, into the

diets of CD patients. Nonetheless, a gluten-free diet is the mainstay of CD therapy for the

immediate foreseeable future, pending FDA approval of any of these treatment options.

Go to:

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Nutrients. Mar 2013; 5(3): 771–787.

Published online Mar 12, 2013. doi: 10.3390/nu5030771

PMCID: PMC3705319

The Dietary Intake of Wheat and other Cereal Grains and Their Role in Inflammation

Karin de Punder1 and Leo Pruimboom1,2,*

Author information ► Article notes ► Copyright and License information ►

Abstract

Go to:

1. Introduction

Inflammation is the response of the innate immune system triggered by noxious stimuli,

microbial pathogens and injury. When a trigger remains, or when immune cells are continuously

activated, an inflammatory response may become self-sustainable and chronic. Chronic

inflammation has been associated with many medical and psychiatric disorders, including

cardiovascular disease, metabolic syndrome, cancer, autoimmune diseases, schizophrenia and

depression [1,2,3]. Furthermore, it is usually associated with elevated levels of pro-inflammatory

cytokines and acute phase proteins, such as interferons (IFNs), interleukin (Il)-1, Il-6, tumor

necrosis factor-α (TNF-α), and C-reactive protein (CRP). While clear peripheral sources for this

chronic inflammation are apparent in some conditions (i.e., fat production of cytokines in the

metabolic syndrome), in other disorders, such as major depression, the inflammatory source is

not completely understood. Genetic vulnerability, psychological stress and poor dietary patterns

have all been repeatedly implicated as being of significant importance in the development of an

inflammatory phenotype [3,4,5]. Dietary factors associated with inflammation include a shift

towards a higher n-6:n-3 fatty acid ratio [5] and a high intake of simple sugars [6]. Other

substances in our daily food, like those found in wheat and other cereal grains, are also capable

of activating pro-inflammatory pathways.

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2. Wheat Grain, Gluten and Disease

2.1. Wheat Allergy and Intolerance

The ingestion of wheat products has been reported to be responsible for IgE-mediated allergic

reactions. Wheat-dependent exercise-induced anaphylaxis is a syndrome in which the ingestion

of a product containing wheat followed by physical exercise can result in an anaphylactic

response. Several proteins present in wheat, most notably gluten proteins have been shown to

react with IgE in patients [7]. Other allergic responses that appear to be related to a range of

wheat proteins include baker’s asthma, rhinitis and contact urticaria [7,8].

More common than wheat allergies are conditions involving wheat intolerance, including celiac

disease (CD), which is estimated to affect 1% of the population of Western Europe, and

dermatitis herpetiformis, which has an incidence between about 2-fold and 5-fold lower than CD

[9]. The close association between type 1 diabetes and CD [10] and the observation that

autoimmune diseases seem to be more prevalent in celiac patients and their relatives [11] thus

links the intake of wheat with several other conditions.

2.2. Wheat Grain and Gluten

Gluten is the main structural protein complex of wheat consisting of glutenins and gliadins.

When wheat flour is mixed with water to form dough, the gluten proteins form a continuous

network which provides the cohesiveness and viscoelasticity that allows dough to be processed

into bread, noodles and other foods. The protein contents of wheat varies between 7% and 22%

with gluten constituting about 80% of the total protein of the seed [9]. Glutenins are the fraction

of wheat proteins that are soluble in dilute acids and are polymers of individual proteins.

Prolamins are the alcohol-soluble proteins of cereal grains and are specifically named gliadins in

wheat. Gliadins are monomeric proteins and are classified into three groups: α/β-gliadins, γ-

gliadins, and ω-gliadins [7].

2.3. Gluten, Gliadin and CD

Gliadin epitopes from wheat gluten and related prolamins from other gluten-containing cereal

grains, including rye and barley, can trigger CD in genetically susceptible people. The symptoms

of this disease are mucosal inflammation, small intestine villous atrophy, increased intestinal

permeability and malabsorption of macro- and micronutrients. CD, a chronic inflammatory

disorder mediated by T-cells, is preceded by changes in intestinal permeability and pro-

inflammatory activity of the innate immune system. Gliadin immunomodulatory peptides can be

recognized by specific T-cells, a process that can be enhanced by the deamidation of gliadin

epitopes by tissue transglutaminases that convert particular glutamine residues into glutamic acid

resulting in a higher affinity for HLA-DQ2 or DQ8 expressed on antigen-presenting cells (APC)

[10]. Serum antibodies, among which are antibodies against tissue transglutaminases, are also

found in CD. The HLA-DQ2 or HLA-DQ8 is expressed in 99.4% of the patients suffering from

CD [10], however, interestingly enough, there is a group of HLA-DQ2/DQ8-negative patients

suffering from gastrointestinal symptoms that respond well to a gluten-free diet. This group of

“gluten-sensitive” patients does not have the CD serology and histopathology, but does present

the same symptoms and shows improvements when following a gluten-free diet [12,13].

2.4. Gliadin and Immunity

There are at least 50 gliadin epitopes that exert immunomodulatory, cytotoxic and gut-

permeating activities that can be partially traced back to different domains of α-gliadin. Where

some immunomodulatory gliadin peptides activate specific T-cells, others are able to induce a

pro-inflammatory innate immune response [10]. Stimulation of immune cells by gliadin is not

only restricted to CD patients; the incubation of peripheral blood mononuclear cells (PBMC)

from healthy HLA-DQ2-positive controls and CD patients with gliadin peptides stimulated the

production of IL-23, IL-1β and TNF-α in all donors tested. Nevertheless, the production of

cytokines was significantly higher in PBMC derived from CD patients [14]. Similar results were

obtained by Lammers et al. [15], who demonstrated that gliadin induced an inflammatory

immune response in both CD patients and healthy controls, though IL-6, Il-13 and IFN-γ were

expressed at significantly higher levels in CD patients. IL-8 production was only expressed in a

subset of healthy and CD individuals after stimulation with a specific gliadin peptide and

appeared to dependent on the CXCR3 chemokine receptor only in CD patients. Sapone et

al. [16] showed that in a subset of CD patients, but not in gluten-sensitive patients (with 36% of

the studied individuals in this group being HLA-DQ2/DQ8-positive), there is an increased IL-17

mRNA expression in the small-intestinal mucosa compared to healthy controls. The same group

showed that in a subset of gluten-sensitive patients (with about 50% of the studied individuals

being HLA-DQ2/DQ8-positive) there is a prevailing stimulation of the innate immune system,

while in CD, both the innate and adaptive immune system are involved [13].

2.5. Gliadin and Intestinal Permeability

In order for gliadin to interact with cells of the immune system, it has to overcome the intestinal

barrier. Gliadin peptides cross the epithelial layer by transcytosis or paracellular transport.

Paracellular transport occurs when intestinal permeability is increased, a feature that is

characteristic for CD [17]. It is indicated by several studies that increased intestinal permeability

precedes the onset of CD and is not just a consequence of chronic intestinal inflammation

[18,19]. Gliadin has been demonstrated to increase permeability in human Caco-2 intestinal

epithelial cells by reorganizing actin filaments and altering expression of junctional complex

proteins [20]. Several studies by Fasano et al. show that the binding of gliadin to the chemokine

receptor CXCR3 on epithelial IEC-6 and Caco2 cells releases zonulin, a protein that directly

compromises the integrity of the junctional complex [21,22]. Although zonulin levels were more

up-regulated in CD patients, zonulin was activated by gliadin in intestinal biopsies from both CD

and non-CD patients [21,22], suggesting that gliadin can increase intestinal permeability also in

non-CD patients, yet increased intestinal permeability was not observed in a group of gluten-

sensitive patients [13].

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3. Increased Intestinal Permeability

3.1. Increased Intestinal Permeability is Associated with Disease

Chronically increased intestinal permeability (or leaky gut syndrome) allows for the increased

translocation of both microbial and dietary antigens to the periphery which can then interact with

cells of the immune system. Shared amino acid motifs among exogenous peptides (HLA-derived

peptides and self-tissue) may produce cross-reactivity through immunological mimicry, thereby

disturbing immune tolerance in genetically susceptible individuals [23]. Not surprisingly,

increased intestinal permeability has been associated with autoimmune diseases, such as type 1

diabetes [24], rheumatoid arthritis, multiple sclerosis [18], but also with diseases related to

chronic inflammation like inflammatory bowel disease [18,25], asthma [26], chronic fatigue

syndrome and depression. The latter two conditions see patients with significantly greater values

of serum IgA and IgM to LPS of gram-negative enterobacteria compared to controls, implying

intestinal permeability is increased in these patients [27,28,29].

3.2. Intestinal Barrier Function and Inflammation

The intestinal barrier allows the uptake of nutrients and protects from damage of harmful

substances from the gut lumen. Macromolecules that can be immunogenic like proteins, large

peptides, but also bacteria and lectins, can be endocytosed or phagocytosed by enterocytes

forming the epithelial layer of the gut. Absorbed proteins will generally enter the lysosomal route

and will be degraded to small peptides. Normally, only small amounts of antigen pass the barrier

by transcytosis and interact with the innate and adaptive immune system situated in the lamina

propria. Highly specialized epithelial microfold (M) cells function as active transporters of

dietary and microbial antigens from the gut lumen to the immune system, where either a pro-

inflammatory or tolerogenic immune response can be generated. The paracellular route is

regulated by the junctional complex that allows the passage of water, solutes and ions, but under

normal conditions provides a barrier to larger peptides and protein-sized molecules. When the

barrier function is disrupted, there is an increased passage of dietary and microbial antigens

interacting with cells of the immune system [25,30] (Figure 1).

Figure 1

Increased intestinal permeability allows for the passage of microbial and dietary antigens across

the epithelial layer into the lamina propria, where these antigens can be taken up by APC and

presented to T-cells. JC, junctional complex.

3.3. The Role of Zonulin Signaling on Intestinal Permeability

Intestinal permeability is a measure of the barrier function of the gut which relates to the

paracellular space surrounding the brush border surface of the enterocytes and the junctional

complexes consisting of tight junctions, adherent junctions, desmosomes and gap junctions [31].

The junctional complexes are regulated in response to physiological and immunological stimuli,

like stress, cytokines, dietary antigens and microbial products [31]. As mentioned before,

zonulin, a protein identified as prehaptoglobulin-2 (the precursor of haptoglobin-2) is also a

regulator of intestinal permeability. Haptoglobin-2, together with haptoglobin-1, is one of the

two gene variants of the multifunctional protein haptoglobin and is associated with an increased

risk for CD (homozygotes and heterozygotes) and severe malabsorption (homozygotes) [32,33].

The haptoglobulin-2/zonulin allele has a frequency of about 0.6 in Europe and the U.S.A, but

varies throughout the world depending on racial origin [34].

3.4. High Zonulin Levels are Observed in Auto-Immune and Inflammatory Diseases

Zonulin signaling is proposed to cause rearrangements of actin filaments and induces the

displacement of proteins from the junctional complex, thereby increasing permeability

[18,32,35]. Gliadin peptides initiate intestinal permeability through the release of zonulin,

thereby enabling paracellular translocation of gliadin and other dietary and microbial antigens,

which by interacting with the immune system give rise to inflammation. In this manner, a vicious

cycle is created in which, as a consequence of the persistent presence of pro-inflammatory

mediators, intestinal permeability will increase even further. High zonulin levels (together with

increased intestinal permeability) have been observed in autoimmune and inflammatory diseases

like CD, multiple sclerosis, asthma and inflammatory bowel disease and the haptoglobin

polymorphism is associated with rheumatoid arthritis, ankylosing spondylitis, schizophrenia and

certain types of cancer [32].

The zonulin inhibitor Larozotide acetate was tested in an inpatient, double-blind randomized

placebo-controlled trial. The group of CD patients in the placebo group that were exposed to

gluten showed a 70% increase in intestinal permeability, while no changes were seen in the

group exposed to Larazotide acetate. Also gastrointestinal symptoms were significantly more

frequent in the placebo group [32]. These results suggest that in CD patients, when intestinal

barrier function is restored, autoimmunity will disappear while the trigger (gluten) is still there.

Besides gliadin from wheat gluten, the lectin wheat germ agglutinin (WGA) has also been shown

to stimulate cells of the immune system and increase intestinal permeability, as we will now

discuss further.

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4. Wheat Germ Agglutinin (WGA)

4.1. Dietary WGA

Lectins are present in a variety of plants, especially in seeds, where they serve as defense

mechanisms against other plants and fungi. Because of their ability to bind to virtually all cell

types and cause damage to several organs, lectins are widely recognized as anti-nutrients within

food [36]. Most lectins are resistant to heat and the effects of digestive enzymes, and are able to

bind to several tissues and organs in vitro and in vivo (reviewed by Freed 1991 [37]). The

administration of the lectin WGA to experimental animals caused hyperplastic and hypertrophic

growth of the small intestine, hypertrophic growth of the pancreas and thymus atrophy [36].

Lectin activity has been demonstrated in wheat, rye, barley, oats, corn and rice, however the best

studied of the cereal grain lectins is WGA [38].

The highest WGA concentrations are found in wheat germ (up to 0.5 g/kg [39]). Although

unprocessed wheat germ, like muesli, contains far higher amounts of active WGA than do

processed wheat germ products, WGA activity is still apparent in several processed breakfast

cereals as assessed by hemagglutination and bacterial agglutination assays [40,41]. A summary

of the amount of active WGA in commonly consumed wheat derived products is listed in Table

1.

Table 1

Amount of active WGA in wheat-derived products.

4.2. WGA Binds to Cell Surface Glycoconjugates

WGA binds to N-glycolylneuraminic acid (Neu5Ac), the sialic acid predominantly found in

humans [44], allowing it to adhere to cell surfaces like the epithelial layer of the gut. The surface

of many prokaryotic and eukaryotic cells are covered with a dense coating of glycoconjugates,

also named glycocalyx. Sialic acids are a wide family of nine-carbon sugars that are typically

found at the terminal positions of many surface-exposed glycoconjugates and function for self

recognition in the vertebrate immune system, but they can also be used as a binding target for

pathogenic extrinsic receptors and molecular toxins [45,46,47]. WGA binding to Neu5Ac of the

glycocalyx of human cells (and pathogens expressing Neu5Ac) allows for cell entry and could

disturb immune tolerance by evoking a pro-inflammatory immune response (discussed below).

4.3. WGA and Immunity

WGA induces inflammatory responses by immune cells. For example, WGA has been shown to

trigger histamine secretion and granule extrusion from non-stimulated rat peritoneal mast cells

[48], induce NADP-oxidase activity in human neutrophils [49] and stimulate the release of the

cytokines IL-4 and IL-13 from human basophils [50]. In human PBMC, WGA induced the

production of IL-2, while simultaneously inhibiting the proliferation of activated lymphocytes

[51]. WGA stimulated the secretion of IL-12, in a T- and B-cell-independent manner in murine

spleen cells. IL-12, in turn, activated the secretion of IFN-γ by T or natural killer cells [52]. In

murine peritoneal macrophages WGA induced the production of the pro-inflammatory cytokines

TNF-α, IL-1β, IL-12 and IFN-γ [53]. Similar results have been observed in isolated human

PBMC, given that nanomolar concentrations of WGA stimulated the release of several pro-

inflammatory cytokines. In the same study a significant increase in the intracellular accumulation

of IL-1β was measured in monocytes after WGA exposure [54]. These results indicate that, when

delivered in vitro, WGA is capable of directly stimulating monocytes and macrophages, cells

that have the ability to initiate and maintain inflammatory responses. Monocytic cells have been

shown to engulf WGA via receptor-mediated endocytosis or by binding to non-receptor

glycoproteins [55].

Human data showing the influence of WGA intake on inflammatory markers are lacking,

however, antibodies to WGA have been detected in the serum of healthy individuals [56].

Significantly higher antibody levels to WGA were measured in patients with CD compared to

patients with other intestinal disorders. These antibodies did not cross-react with gluten antigens

and could therefore play an important role in the pathogenesis of this disease [57].

4.4. WGA and Intestinal Permeability

After ingestion, WGA is capable of crossing the intestinal barrier. In animal models, WGA has

been shown to reach the basolateral membrane and walls of the small blood vessels in the

subepithelium of the small intestine [36]. WGA can be phagocytosed by binding to membrane

non-receptor glycoproteins, a process that has been observed in Caco-2 cells [58]. WGA can also

be endocytosed by antigen sampling M-cells [59,60] or by enterocytes via binding to epidermal

growth factor receptors [61]. Another possible route for lectin entry into the periphery is by

paracellular transport, a process that can be further aggravated by the binding of gliadin to the

chemokine receptor CXR3 on enterocytes.

WGA itself has been found to affect enterocyte permeability. Investigations by Dalla

Pellegrina et al. [54] showed,in vitro, that exposure to micromolar concentrations of WGA

impairs the integrity of the intestinal epithelial layer, allowing passage of small molecules, like

lectins. At the basolateral side of the epithelium, WGA concentrations in the nanomolar range

induced the secretion of pro-inflammatory cytokines by immune cells [54]. This may further

affect the integrity of the epithelial layer, heightening the potential for a positive feedback loop

between WGA, epithelial cells and immune cells. When combined, these mechanisms are likely

able to significantly increase the percentage of consumed WGA that can cross the epithelial layer

compared to the low percentage of WGA crossing by means of transcytosis (0.1%) alone [54].

This suggests that, together with gliadin, WGA can increase intestinal permeability, resulting in

an increase of translocating microbial and dietary antigens interacting with cells of the immune

system.

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5. Animal Data on Cereal Grain Intake

There are two rodent models of spontaneous type 1 diabetes: the non-obese-diabetic (NOD)

mouse and the diabetes-prone BioBreeding (BBdp) rat. In these animals, a cereal-based diet

containing wheat induced the development of type 1 diabetes, while animals fed a hypoallergenic

diet (gluten free) or a hypoallergenic diet supplemented with casein showed a decreased

incidence and a delayed onset of this disease. BBdp rats fed a cereal-based diet showed increased

intestinal permeability and a significant increase in the percentage of IFN-γ-producing Th1

lymphocytes in the mesenteric lymph nodes in the gut [30]. Compared to animals fed a

hypoallergenic diet, NOD mice fed a wheat-based diet expressed higher mRNA levels of the pro-

inflammatory cytokines IFN-γ and TNF-α and the inflammatory marker inducible NO synthase

in the small intestine. While these diet-induced changes in gut-wall inflammatory activity did not

translate to increased cytokine mRNA in Peyers patches, structures that contribute to immune

regulation to exogenous antigens, it is possible that the gut-signal may promote systemic

inflammation via other mechanisms, such as activating intraepithelial lymphocytes and

mesenteric lymph node cells [62]. These in vivo results show that, in two rodent models of

spontaneous type 1 diabetes, a cereal-containing diet induces the (early) onset of disease and

increases markers of inflammation. In addition, Chignola et al. [63] have shown in rats that a

WGA-depleted diet was associated with reduced responsiveness of lymphocytes from primary

and secondary lymphoid organs after in vitro stimulation and attenuated spontaneous

proliferation when compared to lymphocytes from rats fed a WGA-containing diet, indicating

the stimulatory effect of WGA on cells of the immune system.

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6. Human Studies on Cereal Grain Intake and Inflammation

6.1. Human Epidemiological Data on Cereal Grain Intake and Inflammation

Observational prospective and cross-sectional studies show that the intake of whole grain

products is associated with reduced risks for developing type 2 diabetes, cardiovascular diseases,

obesity and some types of cancer [64]. Inflammation is associated with these conditions and

some studies have shown that associations between the intake of whole grains and decreased

inflammatory markers (CRP, Il-6) are found [65]. Intervention studies, however, do not

demonstrate a clear effect of the intake of whole grains on inflammation [66,67,68,69,70,71] and

it could therefore be that other components in the diet modulate the immune response.

It has been shown that the intake of whole grains is associated with healthier dietary factors and

a healthier lifestyle in general. In a Scandinavian cross-sectional study, the intake of whole

grains was directly associated with the length of education, the intake of vegetables, fruits, dairy

products, fish, shellfish, coffee, tea and margarine and inversely associated with smoking, BMI

and the intake of red meat, white bread, alcohol, cakes and biscuits [72]. Good quality

epidemiological studies attempt to control these confounding factors, but with the consequence

that associations are attenuated or become insignificant.

6.2. Human Intervention Trials on Cereal Grain Intake and Inflammation

To accurately estimate the causal relationship of cereal grain intake and inflammation,

intervention trials provide us with better evidence. Wolever et al. [71] showed that a diet with a

low glycemic index (containing whole grains) compared to high (containing refined grain

products), resulted in sustained reductions in postprandial glucose and CRP levels on the long-

term in patients with type 2 diabetes treated with diet alone. A refined grain is a whole grain that

has been stripped of its outer shell (fiber) and its germ, leaving only the endosperm, resulting in

lower levels of macro- and micronutrients and a higher dietary glycemic index for refined grains

compared to whole grains. Refined wheat products contain less WGA, but still contain a

substantial amount of gluten. It should be noted that whole grains contain phytochemicals, like

polyphenols, that can exert anti-inflammatory effects which could possibly offset any potentially

pro-inflammatory effects of gluten and lectins [73].

The substitution of whole grain (mainly based on milled wheat) for refined grains products in the

daily diet of healthy moderately overweight adults for six weeks did not affect insulin sensitivity

or markers of lipid peroxidation and inflammation [66]. Consistent with these finding are the

results of Brownlee et al. [67], who showed that infrequent whole-grain consumers, when

increasing whole grain consumption (including whole wheat products), responded with no

improvements of the studied biomarkers of cardiovascular health, including insulin sensitivity,

plasma lipid profile and markers of inflammation. The substitution of refined cereal grains and

white bread with three portions of whole wheat food or one portion of whole wheat food

combined with two servings of oats significantly decreased the systolic blood pressure and pulse

pressure in middle-aged, healthy, overweight men and women, yet none of the interventions

significantly affected systemic markers of inflammation [70]. In obese adults suffering from

metabolic syndrome, there were significantly greater decreases in CRP and the percentage of

body fat in the abdominal region in participants consuming whole grains compared to those

consuming refined grains. It must be noted that both diets were hypocaloric (reduced by 500

kcal/d) [69]. Most of the intervention studies mentioned above attempted to increase whole grain

intake and were using refined grain diets as controls, thereby making it very difficult to draw any

conclusions on the independent role of cereal grains in disease and inflammation.

6.3. Health Effects of the Paleolithic Diet

There are few studies that investigate the influence of a paleolithic type diet comprising lean

meat, fruits, vegetables and nuts, and excluding food types, such as dairy, legumes and cereal

grains, on health. In domestic pigs, the paleolithic diet conferred higher insulin sensitivity, lower

CRP and lower blood pressure when compared to a cereal-based diet [74]. In healthy sedentary

humans, the short-term consumption of a paleolithic type diet improved blood pressure and

glucose tolerance, decreased insulin secretion, increased insulin sensitivity and improved lipid

profiles [75]. Glucose tolerance also improved in patients suffering from a combination of

ischemic heart disease and either glucose intolerance or type 2 diabetes and who had been

advised to follow a paleolithic diet. Control subjects who were advised to follow a

Mediterranean-like diet based on whole grains, low-fat dairy products, fish, fruits and vegetables

did not significantly improve their glucose tolerance despite decreases in weight and waist

circumference [76]. Similar positive results on glycemic control were obtained in diabetic

patients when the paleolithic diet was compared with the diabetes diet. Participants were on each

diet for three months, whereby the paleolithic diet resulted in a lower BMI, weight and waist

circumference, higher mean HDL, lower mean levels of hemoglobin A1c, triacylglycerol and

diastolic blood pressure, though levels of CRP were not significantly different [77]. Although the

paleolithic diet studies are small, these results suggest that, together with other dietary changes,

the withdrawal of cereal grains from the diet has a positive effect on health. Nevertheless,

because these studies are confounded by the presence or absence of other dietary substances and

by differences in energy and macronutrient intake, factors that could all affect markers of

inflammation, it is difficult to make a concise statement on the impact of cereal grains on these

health outcomes.

6.4. Rechallenge Trial of Effects of Dietary Gluten

One human intervention study specifically focused on the effects of dietary gluten on

inflammation. Biesiekierski et al. [12] undertook a double-blind randomized, placebo-controlled

rechallenge trial to investigate the influence of gluten in individuals with irritable bowel

syndrome but without clinical features of CD, who reached satisfactory levels of symptom

control with a gluten-free diet. After screening the participants, about 50% of the individuals in

both the gluten and placebo group were HLA-DQ2 and/or HLA-DQ8 positive. Participants

received either gluten or placebo together with a gluten-free diet for six weeks. Endpoints in the

study were symptom assessments and biomarkers of inflammation and intestinal permeability.

The patients receiving gluten reported significantly more symptoms compared to the placebo

group. The most striking outcome of this study was that for all the endpoints measured, there

were no differences in individuals with or without HLA-DQ2/DQ8, indicating that the intake of

gluten can cause symptoms also in individuals without this specific HLA-profile. No differences

in biomarkers for inflammation and intestinal permeability were found between both groups,

however, inflammatory mediators have been implicated in the development of symptoms in

patients with irritable bowel syndrome [78]. It could therefore be inferred that the markers used

to measure inflammation and intestinal permeability were not sensitive enough to detect subtle

changes on the tissue level.

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7. Conclusion

In the present review, we describe how the daily consumption of wheat products and other

related cereal grains could contribute to the manifestation of chronic inflammation and

autoimmune diseases. Both in vitro and in vivostudies demonstrate that gliadin and WGA can

both increase intestinal permeability and activate the immune system. The effects of gliadin on

intestinal permeability and the immune system have also been confirmed in humans. Other cereal

grains containing related prolamins and lectins have not been so extensively studied and,

therefore, more research investigating their impact on intestinal permeability and inflammation is

required. It would be interesting to further elucidate the role of other prolamins on zonulin

release and intestinal permeability.

In CD and gluten-sensitive individuals, adverse reactions to the intake of wheat, rye and barley

are clinically apparent; however, it is important to gain better insights on the effects of the

consumption of these cereal grains in other groups of patients and in healthy individuals. It

would be of high interest to investigate the effects of the withdrawal of cereal grain products

from the diet on inflammatory markers and intestinal permeability in healthy subjects and

patients suffering from inflammation-related diseases and measure the same parameters in a

rechallenge trial. Ideally, in such an intervention study, the diet must be completely controlled

and combined with the appropriate substitution of foods in the cereal grain-deprived diet so that

small dietary variations and alterations in energy intake can be avoided and cannot potentially

influence inflammatory markers.

Until now, human epidemiological and intervention studies investigating the health effects of

whole grain intake were confounded by other dietary and lifestyle factors and, therefore, well-

designed intervention studies investigating the effects of cereal grains and their individual

components on intestinal permeability and inflammation are warranted.

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Acknowledgments

We would like to thank Charles L. Raison and Alicia Lammerts van Bueren for the editorial

work on the manuscript.

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Conflict of Interest

Conflict of Interest

The authors declare no conflict of interest.

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Gastroenterology. Author manuscript; available in PMC Mar 9, 2009.

Published in final edited form as:

Gastroenterology. Jul 2008; 135(1): 194–204.e3.

Published online Mar 21, 2008. doi: 10.1053/j.gastro.2008.03.023

PMCID: PMC2653457

NIHMSID: NIHMS82443

Gliadin Induces an Increase in Intestinal Permeability and Zonulin Release by Binding to the Chemokine Receptor CXCR3

Karen M. Lammers,*‡ Ruliang Lu,*‡ Julie Brownley,*‡ Bao Lu,§ Craig Gerard,§ Karen Thomas,∥ Prasad Rallabhandi,∥Terez Shea-Donohue,* Amir Tamiz,¶ Sefik Alkan,¶ Sarah Netzel-Arnett,# Toni Antalis,# Stefanie N. Vogel,∥ and Alessio Fasano*‡

Author information ► Copyright and License information ►

The publisher's final edited version of this article is available at Gastroenterology

See other articles in PMC that cite the published article.

Abstract

Celiac disease (CD) is an autoimmune enteropathy triggered by ingestion of gluten-containing

grains (eg, wheat, rye, and barley). The disease persists in the continued presence of gliadin, the

toxic component of gluten.1 Other characteristics of CD include a highly specific autoantibody

response against tissue transglutaminase2 and a strong association with specific major

histocompatibility complex haplotypes. Greater than 90%–95% of CD patients carry the HLA-

DQ2, with the remaining carrying the HLA-DQ8 haplotype; however, non-HLA genes have been

implicated in the disease pathogenesis as well.3

Under physiologic conditions, access of gliadin to gutassociated lymphoid tissue is prevented by

competent intercellular tight junctions (TJ) that limit passage of macromolecules (including

gliadin peptides) across the intestinal epithelial barrier.4 In susceptible individuals, however, the

interplay between the initiating stimulus (eg, gliadin) and intestinal cells triggers TJ disassembly.

It has been hypothesized that this is an early biologic change that precedes the onset of gliadin-

induced immune events that eventually lead to the pathology associated with CD.5

One protein that induces TJ disassembly and therefore is thought to be involved in the early

phase of CD is zonulin.6 Increased and persistent production of this protein as determined by

Western immunoblotting7 and enzyme-linked immunosorbent assay (ELISA)8 were observed in

patients with active CD.6 Furthermore, ex vivo studies, using intestinal biopsy specimens in the

microsnapwell system, showed that intestinal biopsy specimens of CD patients mounted a more

pronounced response to gliadin when compared with nonceliac controls, including an increased

and persistent release of zonulin and a significant increase in intestinal permeability.8 It is

noteworthy that epithelial release of zonulin occurs after apical, but not basolateral, exposure to

gliadin.9 The latter finding implies that gliadin interacts with an intestinal luminal receptor and

prompted us to seek the identity of this moiety.

In this paper, we provide evidence that the chemokine receptor CXCR3 serves as the target

receptor for gliadin. Our data demonstrate that, in the intestinal epithelium, CXCR3 colocalizes

with gliadin and that this interaction coincides with recruitment of the adapter protein, MyD88,

to the receptor. We also demonstrated that binding of gliadin to CXCR3 is crucial for the release

of zonulin and subsequent increase of intestinal permeability because CXCR3-deficient mice

failed to respond to gliadin challenge in terms of zonulin release and TJ disassembly.

Go to:

Materials and Methods

Reagents

Gliadin (crude wheat), pepsin, and trypsin were purchased from Sigma (St Louis, MO). Gliadin

was pepsin/trypsin digested (PT-gliadin) as described previously10 with minor

modifications.11 Recombinant α-gliadin was a gift from Dr D. Kasarda (USDA-ARS, Albany,

CA). Recombinant interleukin (rIL)-1, monokine induced by interferon (IFN) γ (rMig/CXCL9),

IFN-γ-inducible protein 10 (rIP-10/CXCL10), IFN-γ-inducible T-cell α-chemoattractant (rI-

TAC/CXCL11), and tumor necrosis factor-α (rTNF-α) were purchased from R&D (Minneapolis,

MN) and Calbiochem (San Diego, CA), respectively. Pertussis toxin or inactivated pertussis

toxin were kindly provided by Dr N. Carbonetti (University of Maryland, Baltimore, MD).

α-Gliadin Affinity Column Chromatography

For the preparation of the affinity column, α-gliadin was dissolved in 70% alcohol, mixed with

Affi-Gel 15 Gel, and gently shaken for 4 hours at 4°C. The reaction was terminated by

ethanolamine. Soluble total membrane preparations12 from rabbit small intestine were loaded on

an Affi-gel 15-α-gliadin affinity column, incubated for 90 minutes at 25°C, washed with

phosphate-buffered saline (PBS) containing 0.1% Triton X-100 (Sigma), and eluted with PBS

containing 0.1% Triton X-100 with increasing NaCl concentrations. Fractions were collected and

subjected to SDS-PAGE. The eluted proteins were characterized by MALDI mass spectroscopy

fingerprint analysis (Protein and Nucleic Acid Biotechnology Facility; Stanford University, Palo

Alto, CA).

Transfection Studies

HEK293T cells (2.5 × 106, passages 1–9) were plated in 10-mL culture Petri dishes in complete

culture medium (Dulbecco’s modified Eagle medium [DMEM]; Cellgro, Manassas, VA)

supplemented with 10% heat-inactivated fetal bovine serum (FBS), 50 U/mL penicillin/ 50 g/mL

streptomycin, and 2 mmol/L L-glutamine (Gibco, Carlsbad, CA) and incubated overnight at

37°C in 5% CO2. Cells were transfected with either empty vector (pcDNA3.1; Invitrogen) or

CXCR3 construct generated as previously described13 with minor modifications at a

concentration of 500 ng/well using Superfect transfection reagent (Qiagen, Valencia, CA). After

transfection, fresh complete culture medium was added to the dishes, and cells were incubated

overnight at 37°C in 5% CO2.

α-Gliadin Peptide Library Design and Synthesis

Overlapping (every 10 amino acids), 20mer peptides were designed based on the amino acid

sequence of α-gliadin and synthesized using solid phase synthesis, resulting in a 26 peptide

library (see Supplementary data online at www.gastrojournal.org). Peptide synthesis was carried

out using standard Fmoc chemistry on Rink resin. Peptides were isolated as tri-fluoro acetate

salts at purity levels of greater than 80% by high-performance liquid chromatography.

CXCR3 Binding and Agonist Assays

Both assays were performed at Euroscreen S.A. according to the company protocols

(www.euroscreen.com),14using increasing concentrations of PT-gliadin (for more details,

see Supplementary data online atwww.gastrojournal.org). To establish the binding affinity of the

synthetic gliadin peptides to CXCR3, FITC-labeled CXCR3-binding peptide 4026 was incubated

with CXCR3-transfected HEK293T cells and binding kinetic evaluated by flow cytometry

analysis (for detailed information, see Supplementary data online atwww.gastrojournal.org).

Immunofluorescence Microscopy

HEK293T cells transiently transfected with either pcDNA empty vector or CXCR3 gene-

containing vector were detached by gentle scraping, seeded in Lab-Tek II chamber slides (Nalge

Nunc International, Rochester, NY) at a density of 50,000 cells/well and allowed to attach to the

wells overnight at 37°C in 5% CO2. A separate small aliquot of detached cells was incubated

with 5 µLof allophycocyanin-conjugated anti-human CXCR3 (clone 49801; R&D) or an isotype-

matched control (clone 11711; R&D) mouse monoclonal antibody (mAb) and used for flow

cytometry analysis to verify the expression of CXCR3 on transfectants. Experimental conditions

and staining protocols are described in detail in the Supplementary data online

(see Supplementary data online atwww.gastrojournal.org).

CXCR3 Expression in Intestinal Cell Lines and Tissues

Intestinal epithelial cell lines, IEC6 (rat, passage 36–46) and CaCo-2 (human, passage 30–40),

were grown on Lab-Tek I chamber slides and stained for CXCR3 as described above. To localize

CXCR3 expression in intestinal tissues, 4-µm sections were prepared, and laser capture

microdissection (mouse tissue) or immunohistochemistry (human tissue) were performed as

previously described.15,16 Human intestinal mucosa was obtained from non-CD patients who

underwent a diagnostic upper endoscopy for dyspepsia (no duodenal damage) and CD patients at

the moment of diagnosis (active disease, with a Marsh IIIa–c lesion) during diagnostic

endoscopy. RNA extraction and real-time polymerase chain reaction (PCR) protocols are

described in theSupplementary data online (see Supplementary data online

at www.gastrojournal.org).

Western Blot Analysis and Immunoprecipitation

IEC6 cells were grown in culture flasks and plated in Petri dishes (1 × 106 cells/mL). Confluent

cells were stimulated with PT-gliadin at doses ranging from 100 µg/mL to 1 mg/mL at different

time points (15, 45, and 60 minutes). At the end of stimulation, IEC6 cells were lysed in lysis

buffer containing a cocktail of protease inhibitors. Total protein content was measured using the

Lowry method (Pierce, Rockford, IL). CXCR3 coimmunoprecipitation with the adaptor

molecule MyD88 was performed according to the protocol described in the Supplementary

data online (see Supplementary data online at www.gastrojournal.org).

Microsnapwell System

Intestinal transepithelial electrical resistance (TEER) and changes in TEER in murine small

intestine in response to gliadin exposure were measured using the microsnapwell

system.17 Intestinal segments isolated from either CXCR3−/−18 (backcrossed >10 generations onto

a C57BL/6 background) or C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were

incubated with PT-gliadin (1 mg/mL) added to the mucosal side of the tissue, and TEER was

monitored every 30 minutes for the duration of the experiment. In selected experiments, medium

alone or IP-10/CXCL10 (200 ng/mL), one of the known ligands for CXCR3, was added to the

apical side of the tissue. In selected experiments, tissues were preincubated with IP-10/CXCL10

for 30 minutes, after which IP-10/CXCL10 was removed, and PT-gliadin was added to the

tissue. In a third series of experiments, intestinal segments were preincubated with medium

alone, pertussis toxin (10 ng/mL), or genetically modified (inactivated) pertussis toxin (10

ng/mL) for 30 minutes, followed by addition of PT-gliadin (1 mg/mL). Pertussis toxin or its

inactive genetic mutant were present throughout the stimulation. In a fourth series of

experiments, intestinal tissues were incubated with 4 different peptides (10 µg/mL) from the α-

gliadin synthetic peptide library. TEER data were normalized to the initial value for that specific

data set in each animal.

Zonulin ELISA

Zonulin was measured in the microsnapwell intestinal culture supernatants by ELISA as

previously described.17

Statistical Analysis

Two-tailed Student t tests were used to test differences between 2 groups. Data were paired

where appropriate. Values of P < .05 were regarded as significant.

Go to:

Results

Identification of CXCR3 as the PT-Gliadin Intestinal Binding Protein

To identify the putative gliadin receptor, membrane fractions were prepared from rabbit small

intestine and applied to an Affi-gel α-gliadin affinity column. Three main proteins with estimated

molecular weights of 93, 100, and 107 kilodaltons were eluted from the affinity column and

subjected to MALDI mass spectrometric fingerprint analysis following digestion with trypsin.

The 100-kilodalton band was identified by mass spec/mass spec (MS/MS) as the chemokine

receptor CXCR3, based on sequences derived from 21 peptides, whereas the other bands were

identified as a heat shock protein (93 kilodaltons) and the glutamate receptor (107 kilodaltons)

(data not shown).

CXCR3, a 7-transmembrane G-protein-coupled receptor, 19 is involved in cellular activation and

cell migration (cytoskeleton rearrangement) into inflamed tissues, in particular of γ/δ T

lymphocytes (as observed in CD).13,20 In contrast, the glutamate receptor21 and heat shock

proteins22 are involved in other cellular functions. Based on the observation that CXCR3

activities are potentially relevant to CD pathogenesis, we pursued the possible role of CXCR3 as

a receptor for PT-gliadin.

Characterization of CXCR3 as the PT-Gliadin Intestinal Receptor

To establish whether CXCR3 is the receptor responsible for PT-gliadin-induced mucosal events

leading to increased intestinal permeability, the following series of experiments were performed:

Gliadin Binds to the CXCR3 Receptor

Immunofluorescence microscopy experiments were performed to determine whether PT-gliadin

and CXCR3 colocalize. Transiently transfected, CXCR3-expressing HEK293T cells were

incubated with PT-gliadin and stained for CXCR3 and gliadin. FACS analysis revealed that

>60% of transfected cells expressed CXCR3 (data not shown). After immunofluorescence

staining, colocalization of CXCR3 and gliadin was observed in CXCR3-transfected (Figure 1A–

C) but not in pcDNA3.1-transfected cells (Figure 1D). As additional controls for the specificity

of the observed staining, PT-gliadin-treated CXCR3-transfected cells were stained with isotype

control or secondary Ab alone (Figure 1E). Furthermore, CXCR3-transfected cells were

incubated with the irrelevant protein bovine serum albumin (BSA) (1 mg/mL) and stained with a

specific anti-BSA Ab (Figure 1F). None of the control stainings showed colocalization.

Figure 1

Colocalization of CXCR3 and PT-gliadin in CXCR3-transfected HEK293T cells

To further demonstrate direct and specific gliadin binding to CXCR3, a competitive binding

assay was performed. Our results showed that PT-gliadin caused a concentration-dependent

displacement of the radiolabeled CXCR3 ligand [125]I-TAC from its target receptor on CHO-K1

host cells (Figure 2). However, contrary to the other CXCR3 ligands,14 PT-gliadin binding to

CXCR3 did not activate Ca2+ signaling (see Supplementary data online

at www.gastrojournal.org). To define whether α-gliadin domain(s) are involved in CXCR3

binding, a synthetic peptide library consisting of 26, 10 AA overlapping, 20mer peptides was

subjected to the binding assay. The results show that 2 of these peptides displaced radiolabeled I-

TAC from CXCR3-expressing cells (Table 1). The specificity of this binding was confirmed by

kinetic experiments performed on HEK293T cells transfected with human CXCR3 that showed a

dissociation constant of peptide 4026 of 32 µmol/L (see Supplementary data online

at www.gastrojournal.org).

Figure 2

Dose-response binding curve of PT-gliadin. Concentration-response curve of PT-gliadin

binding to CXCR3

Table 1

Synthetic Peptides

CXCR3 Is Expressed Both in Intestinal Epithelial Cells and Intestinal Immune Cells

To study the receptor expression in intestinal epithelial cells, we measured CXCR3 steady-state

messenger RNA (mRNA) and protein expression in various human and murine intestinal cell

lines and small intestinal tissues. Immunofluorescence analysis of human CaCo-2 cells showed

constitutive CXCR3 expression (Figure 3A and B). Cross-reactivity of the antihuman CXCR3

mAb with rat CXCR3 permitted visualization of CXCR3 expression on the surface of rat IEC6

cells (Figure 3C and D). Real-time reverse-transcription (RT)-PCR analysis of Caco-2 cells

confirmed CXCR3 mRNA expression (Figure 3E).

Figure 3

CXCR3 is expressed on intestinal epithelial cell lines

To confirm intestinal epithelial expression of CXCR3 in vivo, both murine and human intestinal

tissues were analyzed. Murine small intestine was subjected to laser capture microdissection

followed by real-time RT-PCR analysis. Although CXCR3 mRNA expression was more

abundant in the lamina propria (probably because of the large number of CXCR3-positive

immune cells present at this site), measurable expression of the receptor was detected also in

murine intestinal epithelial cells (Figure 4). Immunohistochemical analysis of human small

intestinal tissue stained both for CD3+ cells and CXCR3 confirmed that the receptor is expressed

not only by immune cells but also by enterocytes (Figure 5A–C).

Figure 4

CXCR3 expression in mouse intestinal tissues

Figure 5

Differential mucosal CXCR3 expression in nonceliac and CD patients

CXCR3 Is Up-Regulated During the Active Phase of CD

To investigate whether CXCR3 expression is altered in CD, human small intestinal biopsy

specimens obtained from both non-CD and CD patients were subjected to immunohistochemical

and real-time RT-PCR analysis. CXCR3 staining was detected at higher levels in the lamina

propria and the epithelium of CD patients (Figure 5E) as compared with non-CD controls (Figure

5D). CXCR3 mRNA expression in biopsy specimens revealed a 9.6-fold increase in CXCR3

mRNA expression in CD patients with active disease compared with CXCR3 gene expression in

non-CD patients (P = .004). This disease-associated enhanced mRNA expression returned to

levels seen in non-CD intestinal tissue in CD patients in remission after implementation of a

gluten-free diet (Figure 5G).

PT-Gliadin Activates the Zonulin Innate Immune Pathway Through CXCR3

We next established whether CXCR3 is required for the PT-gliadin-induced increase in zonulin

release and subsequent changes in intestinal permeability previously described.8 Intestinal tissues

of wild-type C57BL/6 and CXCR3−/− mice were mounted in microsnapwell chambers, and PT-

gliadin was added to the mucosal (eg, apical) side of the tissue. No differences in intestinal

mucosal morphology or baseline TEER were noted between C57BL/6 and CXCR3−/− mice (data

not shown). An initial series of experiments was designed to evaluate whether the effect of PT-

gliadin on zonulin release and intestinal permeability is CXCR3 dependent. Intestinal segments

from wild-type mice showed a significant 30% drop in TEER (Figure 6A). These TEER changes

were preceded temporally by the release of zonulin following mucosal PT-gliadin challenge

(Figure 6B). Conversely, CXCR3−/− mice did not exhibit changes in either intestinal TEER or

zonulin release in response to gliadin (Figure 6A and B). To establish whether the zonulin

pathway is operative in CXCR3−/− mice, we repeated the permeability experiments using the

zonulin agonist AT1002. A significant drop in TEER was observed when CXCR3−/− tissues were

challenged with the AT1002 compared with baseline (Figure 6C).

Figure 6

The increased intestinal permeability and zonulin release in response to PT-gliadin

challenge is CXCR3-dependent

In addition, we chose 2 CXCR3-binding peptides and 2 peptides that did not show binding to

CXCR3 from the α-gliadin synthetic peptide library and applied them to the luminal side of wild-

type intestinal segments. Only the 2 CXCR3-binding peptides A (4026) and B (4022) induced a

significant decrease in TEER, whereas the nonbinding peptides C (4018) and D (4030) did not

alter intestinal permeability (Figure 6D).

A second set of experiments was performed on intestinal tissue from wild-type mice to assess

whether the effects after gliadin binding to CXCR3 could be induced by other CXCR3 ligands.

One of 3 previously described CXCR3 ligands, IP-10/CXCL10, was applied to the mucosal side

of the intestinal tissue, and TEER w as measured. IP-10/CXCL10 did not cause significant

changes in either TEER or zonulin release compared with medium alone (data not shown).

From these experiments emerges that IP-10/CXCL10 and PT-gliadin induce different cellular

activation patterns after binding to CXCR3. We next evaluated whether IP-10/CXCL10 binding

to CXCR3 affects PT-gliadin-induced changes in intestinal permeability as a result of the

receptor tachyphylaxis. Pretreatment of wild-type intestinal segments with IP-10/CXCL10 for 30

minutes did not overall prevent the effects of PT-gliadin on TEER. However, the time in which

the TEER started to drop following PT-gliadin exposure was delayed by 30 minutes in tissues

pretreated with IP-10/CXCL10 (Figure 6E). These results suggest that CXCR3 receptors could

be temporally unavailable secondary to IP-10/CXCL10 and needed to shuttle back to the cell

surface before PT-gliadin could bind and exert its effects on TEER.

A third series of experiments was performed to examine whether gliadin binding to CXCR3, a

G-protein-coupled receptor, requires G-protein signaling. For these experiments, intestinal tissue

of wild-type mice was mounted in microsnapwells, and PT-gliadin was added to the mucosal

side after pretreatment with medium alone, pertussis toxin (a G protein-coupled receptor

inhibitor), or an inactive genetic mutant of pertussis toxin. PT-gliadin induced the expected drop

in TEER in tissues preincubated with medium alone, and this was prevented by preincubation

with pertussis toxin but not with its inactive mutant (Figure 6F).

PT-Gliadin Binding to CXCR3 Recruits MyD88

We recently reported that PT-gliadin-induced zonulin release is MyD88-dependent.11 To

investigate whether PT-gliadin binding to CXCR3 induces recruitment of the adapter protein

MyD88, IEC6 intestinal epithelial cells were stimulated with PT-gliadin and subjected to

coimmunoprecipitation assays. These assays revealed an association of CXCR3 and MyD88

after PT-gliadin challenge that was concentration and time dependent (Figure 7). This

association was optimal when PT-gliadin was present at a concentration of 1 mg/mL (Figure 7A)

and reached a plateau after 45 minutes of incubation (Figure 7B).

Figure 7

PT-gliadin binding to CXCR3 induces recruitment of MyD88

Go to:

Discussion

TJs are central to the regulation of intestinal permeability because they maintain the contiguity of

intestinal epithelial cells and are capable of prompt and coordinated responses to the many

physiologic challenges to the intestinal epithelial barrier.4 Increased intestinal permeability

appears to be an early biologic change that precedes the onset of autoimmune diseases, including

CD and type I diabetes.8,23,24 The peculiarity of CD is that it is the only autoimmune disease for

which the triggering environmental factor gliadin is known. This offers a unique opportunity to

study the cellular and molecular basis of the autoimmune process using enzymatically digested

gliadin as a stimulus in experimental assays.

We showed a direct effect of gliadin on intestinal barrier function,9 which was confirmed by

others.25 This effect of gliadin is polarized, eg, gliadin increases intestinal permeability only

when administered on the luminal side of the intestinal tissue.9 These data formed the basis for

the present study because a missing link has been the identification of the luminal structure to

which gliadin binds and through which gliadin induces epithelial zonulin release and TJ

disassembly.

Our MS/MS data identified the chemokine receptor CXCR3 and 2 other proteins, a glutamate

receptor and a heat shock protein, as the proteins that bound to α-gliadin. We chose to investigate

the possible role of CXCR3 as a receptor for gliadin because of its function in recruiting γ/δ

lymphocytes, a marker of early stage in CD pathogenesis.26 In contrast, the glutamate receptor is

an intrinsic transmembrane ion channel that is opened in response to binding of a chemical

messenger but has not been described to be involved in cell activation and rearrangement of the

cytoskeleton.21 Heat shock proteins are cytoplasmic proteins involved in intracellular processes

including protein folding and protein conformation and are found extracellularly only as shed

contents from necrotic cells providing a strong danger signal to the immune system.22

The identification of CXCR3 as a receptor for gliadin is important for several reasons. The

chemokine receptor CXCR3 is involved in various pathophysiologic conditions. Its biologic role

is to provide a mechanism for cells that express this receptor to migrate to its ligands, the

chemokines Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11, which share the receptor, but

exert different and nonredundant effects.27 CXCR3 is associated with leukocyte recruitment to

target organs and subsequent T helper cell 1 immune-mediated tissue damage in viral and

bacterial infections28,29 and autoimmune disease states.30,31 CXCR3 is predominantly expressed on

different T-cell subsets, including activated T helper cell 1 (Th1) cells,32 T lymphocytes,33 a

newly identified E-cadherin-bearing CD8+ T-cell subset that specifically homes to the gut,34 and

natural killer cells,35 but its expression has been reported on other cell types as well.36,37

The phenomenon that ligands other than chemokines can bind to chemokine receptors has been

reported previously; for example, human immunodeficiency virus uses the CCR5 chemokine

receptor for cell entry,38 and PGP, a peptide derived from the extracellular matrix, signals

through the CXCR2 receptor on neutrophils causing neutrophil recruitment into the lungs and

production of superoxide.39

With this paper, we report for the first time CXCR3 expression in intestinal epithelium. CXCR3

expression showed the same qualitative distribution in both CD and non-CD intestinal tissues,

but its expression was higher in CD. These differences were paralleled by higher CXCR3 gene

transcription in CD patients with active disease that returned to baseline levels when the disease

was in remission following the implementation of a gluten-free diet. The enhanced CXCR3

mRNA expression in intestinal tissue from active CD patients reflects the importance of CXCR3

expression on both intestinal epithelial cells and intraepithelial lymphocytes and its distinct

regulation in CD. Our immunohistochemical staining studies show that epithelial CXCR3

expression is predominantly related to enterocytes and not to the large number of intraepithelial

CXCR3-expressing γ/δ-positive T lymphocytes that typically infiltrate the intestinal mucosa

during the acute phase of CD.20

The role of CXCR3 in mediating the PT-gliadin-induced zonulin release and subsequent increase

in intestinal permeability was confirmed using CXCR3−/− mice in which PT-gliadin failed to

release zonulin and, consequently, to reduce TEER. Our observation that pretreatment of

C57BL/6 wild-type intestinal tissue with the G-protein inhibitor pertussis toxin prevented the

effect of PT-gliadin on intestinal permeability is consistent with activation through CXCR3 that

leads to subsequent TJ disassembly.

Interestingly, PT-gliadin, as well as 2 α-gliadin synthetic peptides, bound to CXCR3 but did not

cause Ca2+release (see Supplementary data online at www.gastrojournal.org) as reported for the 3

known natural CXCR3 ligands, Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11.14,27 The fact

that IP-10/CXCL10 failed to cause TEER changes suggests that other intracellular signaling

pathway(s) could be responsible for the PT-gliadin-induced zonulin release and TJ disassembly.

Pretreatment with IP-10/CXCL10 caused a delay of TJ disassembly but was not able to inhibit

the effects of PT-gliadin on intestinal permeability, indicating that both proteins act via binding

to CXCR3 but exert different effects with regard to TJ disassembly and zonulin release. Zonulin

characterization revealed that it belongs to a family of serine proteases with structure similarities

with a series of growth hormones, including epidermal growth factor. One can hypothesize that

the effect of gliadin on epidermal growth factor-related signaling as was recently reported40 could

eventually be mediated by zonulin.

Our data suggest that recruitment of the adapter protein MyD88 to CXCR3 is involved. Until

recently, MyD88 has been described to be associated uniquely with signalin via Toll-like

receptors (TLR) and the interleukin (IL)-1R family. TLRs are a family of pattern recognition

receptors that recognize evolutionary highly conserved structures on microorganisms and give

rise to nuclear factor-κB activation and proinflammatory gene transcription.41 This knowledge

was extended recently with the finding that MyD88 can associate with the IFN-γ receptor,

providing an alternative way by which IFN-γ can enhance proinflammatory gene expression.42 In

our experiments, CXCR3 activation by PT-gliadin failed to activate nuclear factor-κB, IRF-3, or

p38 (data not shown). This observation could indicate that CXCR3 associates with another

receptor that, in turn, leads to recruitment of MyD88 “by proxy.” This concept would exclude

both TLR2 and TLR4 as coreceptors because our previous studies ruled out the involvement of

these 2 TLRs in zonulin signalling and increased permeability.11Alternately, PT-gliadin-

dependent CXCR3 activation signals leading to zonulin release may be mediated by a yet

undefined pathway downstream of the recruitment of MyD88. Support for a direct interaction

was suggested by our Clustal W analysis that identified a TIR-like region within the C-terminus

of CXCR3 (Quan Nhu, unpublished observation).

In conclusion, using biochemical, genetic, and physiologic approaches, we identified the

chemokine receptor CXCR3 as the receptor that binds gliadin. Our data suggest that gliadin

binds to CXCR3 on epithelial cells to initiate an increase in intestinal permeability through an

MyD88-dependent release of zonulin that enables the paracellular passage of gliadin (and

possibly other non-self antigens) from the intestinal lumen to the gut mucosa. In genetically

predisposed individuals, gliadin may attract and stimulate other CXCR3-expressing cells,

including T cells, CD3+CD8+ T cells, and natural killer cells,33,34,43 leading to the early activation

of the innate immune arm of the CD inflammatory response.44

Go to:

Supplementary Material

01

Supplementary Data:

Note: To access the supplementary material accompanying this article, visit the online version

ofGastroenterology at www.gastrojournal.org, and at doi: 10.1053/j.gastro.2008.03.023.

Click here to view.(152K, pdf)

Go to:

Acknowledgments

Supported in part by National Institutes of Health grants DK-48373 (to A.F.) and AI-18797 (to

S.N.V.).

The authors thank Dr Anna Sapone, and Rex Sun for their technical assistance with some of the

snapwell experiments, and Manjusha Thakar for her assistance with the immunoprecipitation

experiments.

Go to:

Abbreviations used in this paper

CD

celiac disease

Mig/CXCL9

monokine induced by interferon γ

IP-10/CXCL10

interferon-γ-inducible protein 10

I-TAC/CXCL11

interferon-inducible T-cell α-chemoattractant

PT-gliadin

pepsin/trypsin-digested gliadin

TEER

transepithelial electrical resistance

TJ

tight junctions

Go to:

Footnotes Conflicts of interest: S.N.V. and A.F. have financial relationship with Alba Therapeutics.

Go to:

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Published online Apr 7, 2014. doi: 10.3748/wjg.v20.i13.3542

PMCID: PMC3974521

Anemia in inflammatory bowel disease: A neglected issue with relevant effects

Danila Guagnozzi and Alfredo J Lucendo

Author information ► Article notes ► Copyright and License information ►

Abstract

Core tip: Anemia represents one of the major causes of both decreased quality of life and

increased hospital admissions among inflammatory bowel disease (IBD) patients. This paper

analyses the complex etiological and pathophysiological mechanisms underlying anemia in IBD,

including iron and micronutrients deficiency, effects of proinflammatory mediators and bone

marrow insufficiency secondary to the disease by itself and IBD therapy. By a comprehensive

review of the current diagnostic and therapeutic evidences on anemia in IBD, an state-of-the-art

approach will be provided to effectively manage this challenging and common condition.

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INTRODUCTION

Anemia, a frequent systemic complication in patients with inflammatory bowel disease (IBD),

has a complex and multifactorial pathogenesis (Figure (Figure1).1). It is considered a prototype

of a combination of iron deficiency (IDA) and anemia of chronic disease (ACD), which is caused

by the negative effects of an activated immune system at different levels of erythropoiesis[1,2].

Besides IDA and ACD, metabolic disturbances, vitamin deficiencies, and various drug therapies

commonly used in IBD can aggravate anemia in IBD patients[3]. The study of anemia in these

patients thus requires a specific diagnostic and therapeutic approach.

Figure 1

Pathogenesis of multifactorial anemia associated to inflammatory bowel disease. TNF-α:

Tumour necrosis factor-α; IL: Interleukin.

It is important to highlight that anemia has a significant impact on the disease and is one of the

most frequent comorbid conditions associated with mortality in IBD patients[4]. In addition, it

also has a relevant effect on health related quality of life (QoL) and ability to work[5,6]. The fact

that it is also a common cause of hospitalization and delay of discharge[7] only serves to

underscore the need for prompt diagnosis and management of this condition.

Although the correction of anemia in IBD patients can improve the QoL and the quality of

patient management, the specific diagnosis and treatment of anemia is often a low priority for

gastroenterologists and has thus received little attention. A recent study showed that further

diagnostic tests were undertaken in only one-third of patients with proven anemia and that 54.3%

of patients diagnosed with IDA receive no iron supplements[8,9].

This article reviews current data on the diagnosis and treatment of anemia in IBD patients. A

search was conducted in the PubMed, Cochrane, MEDLINE, and Scopus libraries with the

following individual and combined key words: Crohn’s disease, ulcerative colitis, anemia, iron

deficiency anemia, anemia of chronic disease, vitamin B12 deficiency, folic acid deficiency,

myelodysplastic syndrome, refractory anemia, iron supplementation, intravenous iron therapy,

erythropoietin, and inflammatory bowel disease. References cited in the articles retrieved were

also searched in order to identify other potential sources of information. The results were limited

to human studies available in English.

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PREVALENCE OF ANEMIA IN INFLAMMATORY BOWEL DISEASE

The prevalence of anemia in IBD is markedly variable, ranging from 6% to 74% in two

systematic reviews[10,11]. The more recent review calculated a mean prevalence of 17% (16% in

outpatients and 68% in hospitalized patients), with anemia occurring more frequently in patients

with Crohn’s disease (CD) than in those with ulcerative colitis (UC)[3,12]. This variability in the

prevalence of anemia depends on different factors. First, the definition of anemia is not

homogenous in the various studies reviewed. In fact, because the widely accepted World Health

Organization criterion for the diagnosis of anemia [hemoglobin (Hb) below 13 g/dL in men or 12

g/dL in non-pregnant women][13] has been questioned due to racial differences, environmental

conditions, and eating habits[14,15], its use cannot completely reflect the real prevalence of anemia

in different IBD populations. Furthermore, estimations of the prevalence of anemia often depend

on the specific groups of patients studied (for example, hospitalized patients vs outpatients). In

this sense, a study from a Swedish cohort showed that the prevalence of anemia in hospitalized

UC and CD patients was higher than among outpatient populations (5% vs35% and 9% vs 50%,

respectively)[16]. Furthermore, it is important to note that anemia has been poorly studied in

pediatric IBD patients. One recent epidemiological study showed that while the prevalence of

anemia was 72% at the time of diagnosis, the proportion of severely anemic pediatric patients

decreased from 34% to 9% while the number of patients with mild anemia doubled after 1 year

of follow-up[17]. Finally, because Hb levels form part of a widely used disease activity index, the

presence of anemia correlates directly with disease activity, which means that the prevalence of

anemia may change throughout the natural history of the disease. In fact, the prevalence of mild

and moderate anemia in IBD has decreased over time, reflecting improved treatment and

management of the disease. However, the prevalence of severe anemia in IBD patients over the

last 10 years has not decreased in the same manner[18].

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PATHOGENESIS OF ANEMIA IN INFLAMMATORY BOWEL DISEASE

Iron deficiency anemia

Iron deficiency is the most common cause of anemia in IBD patients, with a reported prevalence

of up to 90%[11]. Iron deficiency may be related to “absolute iron deficiency” due to low dietary

intake and blood loss from ulcerated intestinal mucosa (especially in UC patients) along with

reduced iron absorption (especially in CD localized in the upper GI tract), or it may be related to

“functional iron deficiency.”

Iron is an essential mineral for the function of all body cells and is absorbed at the apical surface

of enterocytes to be transported by ferroportin across the basolateral surface of the enterocyte

into the circulation[19]. In the maintenance of iron homeostasis, the peptide hormone hepcidin is a

master regulator that is produced in response to iron overload or upon induction by pro-

inflammatory stimuli such as lipopolysaccharide or interleukin (IL)-6. In fact, inflammatory

conditions can interfere with iron absorption by causing an increase in hepcidin that inhibits

ferroportin activity[20], leading to its internalization and degradation[21]. The inhibition of

ferroportin activity blocks the transfer of absorbed iron from the enterocyte into the circulation

and causes iron retention in the macrophages and monocyte cells[22]. In addition, during

inflammation other events contribute to the retention of iron in these cells, including the

inhibition of ferroportin transcription by pro and anti-inflammatory cytokine action and a

reduction in the half-life of erythrocytes due to oxidative stress and lipidperoxidation, with iron

recycling through erythrophagocytosis[23]. These mechanisms all lead to “functional iron

deficiency,” which means that despite an abundance of iron in the body, it is not available for

erythropoiesis.

Anemia of chronic disease

The exact prevalence of ACD in IBD patients is unknown[24], with its etiology being ascribed to

altered erythropoiesis at different levels[25]. Firstly, chronic inflammation can decrease

erythropoiesis by direct action of interferon (IFN)-γ, IFN-α, tumor necrosis factor (TNF)-α, and

IL-1 in the bone marrow to exert pro-apoptotic effects on erythroid burst-forming units (BFU-E)

and colony-forming units (CFU-E)[26]. Moreover, IL-1, IL-6, TNF-α, and hepcidin may decrease

erythropoietin (EPO) synthesis and impair its biological activity[1,27]. In fact, EPO levels in ACD

have been found to be inadequate in some chronic disease and IBD patients[11,28,29]. Low EPO

production is due to direct inhibition of the activity of the promoter of the EPO gene by IL-1 and

TNF-α, which in turn inhibits the synthesis of EPO in the kidney and acts indirectly on EPO-

producer cells through cytokine-induced toxic radicals[30]. Impairment of the biological activity

of EPO means that much higher amounts of EPO are needed to restore the formation of CFU-E

in the bone marrow. Cytokines can also interfere with the signaling process mediated by the

interaction of EPO and its receptor and can downregulate EPO receptors on erythroid progenitor

cells[26], thus producing cell resistance to EPO activity. Finally, the limited availability of iron

for heme biosynthesis induced by “functional” or “absolute’’ iron deficiency and the inhibition

of iron uptake into erythroid progenitors due to the blocking of the transferrin receptor by alfa1-

antitrypsin (an acute phase protein) negatively affect the biological functions of EPO along with

cell growth and differentiation[1].

Other types of anemia

Vitamin B12 deficiency has been observed in 48% of CD patients and in 5% of UC patients[31]

while folic acid deficiency has been noted in 67% of CD patients and in 30%-40% of UC

patients[31-33]. These types of deficiencies depend on low dietary intake as well as increased

turnover of epithelial cells due to chronic inflammation in the intestinal mucosa and a reduced

absorption in the intestinal tract[34-36]. In CD, several factors influence these deficiencies,

including the inflammatory involvement of ileal mucosa, the presence of fistulas, secondary

bacterial overgrowth with direct consumption of vitamin B12, and extensive surgical resections in

small bowel segments with impaired absorption[37]. Deficiencies in patients with UC derive from

proctocolectomy and ileo-pouch anastomosis, with the prevalence of vitamin B12 deficiency

being affected more by surgical changes leading to impaired function of ileal receptors, reduced

intestinal transit time, and secondary bacterial overgrowth than on the length of the ileal segment

resected[38].

Autoimmune hemolytic anemia (AIHA) is a rare type of anemia observed in UC patients. It can

be due either to the development of antibodies with cross-reactivity with erythrocytes[39] or to the

hemolytic effect of sulfasalazine in patients with glucose-6-phosphate dehydrogenase

deficiency[40]. This association was first described in 1955 by Lorber et al[41] with the most

recent studies calculating that the prevalence of AIHA in UC patients is between 0.2%-1.7%, as

indicated by a positive Coombs test result in 1.8% of patients studied[40]. AIHA can occur

before, after, or at the moment of diagnosing UC. Even when the potential relationship between

disease activity and the occurrence of AIHA is not clear, a correlation with the extension of the

disease has been demonstrated in several reports, which show a prevalence of AIHA of up to

28% in patients with extensive colitis[40].

Anemia can also represent a late manifestation of myelosuppression in IBD patients due to

several factors. Firstly, myelosuppression can be associated with myelodysplastic syndrome

(MDS), with ineffective erythropoiesis and a risk of progression to acute myeloid leukemia.

Some studies have shown a frequent predominance of MDS in CD with colorectal involvement;

however, it should be noted that the prognosis of IBD with concomitant MSD is determined by

the MSD itself[42]. The prevalence of MDS in IBD patients has been estimated to be 0.17%, with

a higher incidence in IBD patients than in the general population (170/100000 IBD

patients/year vs 20-30/100000 of the general population over the age of 70/year)[43]. This is

probably due to a undetermined common pathogenetic mechanism, the long-term use of

immunosuppressive drugs, or chromosomal abnormalities in bone marrow cells that have been

observed in 67% of patients with concomitant IBD and MSD[44,45]. These can induce the

development of colitogenic monocytes, producing a large number of pro-inflammatory cytokines

resistant to apoptosis upon stimulation with microbial antigens. Indeed, one of the first

hypotheses about this association regarded IBD to be an extra-hematologic manifestation of

MSD with a vasculitic process at the level of the mesenteric arteries[46]. Alternatively,

myelosuppression may represent a complication of severe UC with the development of a

systemic inflammatory response syndrome, or even been a side effect of immunosuppressive

drugs. There is an increasing concern about therapy-induced leukemias and myelodysplastic

syndromes in patients treated with thiopurines, which are extensively used as

immunosuppresants in IBD, particularly for maintenance therapy[47]. Data from a large French

cohort of patients (19486) with inflammatory bowel disease identified a relative risk of

developing lymphoproliferative disorders as 5.2 for patients who were treated with thiopurines

compared to those who were not[48].

Finally, additional gastrointestinal diseases that do not usually cause bleeding should be also

considered in case of iron deficiency anemia in those IBD patients who maintain disease into

remission, including colon or gastric cancer o polyps, peptic ulcer, hiatal hernia with linear

erosions, atrophic or Helicobacter pylori-associated gastritis, and celiac disease[49,50].

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DIAGNOSIS OF ANEMIA IN INFLAMMATORY BOWEL DISEASE

Basic laboratory screening for anemia in IBD should consist of hemoglobin and full blood counts

(including reticulocytes to differentiate between regenerative or hypo-regenerative anemia), with

a determination of erythrocyte mean corpuscular volume (MCV) to distinguish between

microcytic, normocytic, and macrocytic anemia as well as a determination of mean corpuscular

Hb (MCH) and reticulocyte Hb content (CHr), if available. Moreover, assessments of both the

level of inflammation by means of C-reactive protein (CRP) and of iron status are required.

There is no single biomarker to diagnose iron deficiency in IBD; a combination of different

biomarkers is needed. In most cases, total store of body iron with serum-ferritin (or ferritin) and

the iron available in the bone marrow with transferrin saturation (TfS) is sufficient to

differentiate between IDA and ACD[51]. However, many of the laboratory measures of iron

status may be unreliable in IBD patients because the inflammation influences all parameters of

iron metabolism to produce “functional iron deficiency[52,53]”. For this reason, in some cases it is

essential to use other, more specific biomarkers of iron status to allow for the differentiation

between predominantly IDA, predominantly ACD, and ACD combined with iron deficiency in

order to provide appropriate, more effective treatment[54] (Table (Table1).1). Further testing for

causes of anemia in IBD may include tests for vitamin B12, folic acid (especially erythrocyte

levels, which, when available, represent the best stable marker of folic acid deficiency),

haptoglobin, lactate dehydrogenase, indirect bilirubin (with Coombs test if hemolytic anemia is

confirmed), and serum creatinine in order to rule out potential hemolysis or renal failure, which

in itself can cause macrocytic or normocytic anemia[51]. It should be noted that if the origin of

anemia is not obvious, IBD patients should be tested for MDS, especially if normocytic and

hypo-regenerative anemia are both present, carrying out a bone marrow study in selected

patients.

Table 1

Laboratory findings in anemia of inflammatory bowel disease patients

Once a diagnosis of IBD has been established, patients in clinical remission should be screened

for anemia at least every 6 to 12 mo, whereas patients with active disease should be tested every

3 mo or at even shorter intervals, depending upon their iron status[55].

Iron deficiency anemia

Patients are considered to suffer from IDA when they present with low Hb (men < 13 g/dL, non-

pregnant women < 12 g/dL), TfS < 20%, and ferritin concentrations < 30 ng/mL without any

biochemical or clinical signs of inflammation. A low MCH (< 27 pg) or even better a low CHr (<

28 pg) rather than MCV (< 80 fL) have became the most important red cell markers for detecting

iron deficiency in circulating red blood cells. Although MCV is a reliable and widely available

measurement, it tends to be a relatively late indicator in patients who are not actively

bleeding[56]. A normal Hb level does not rule out iron deficiency and with an MCH in the lower

limit of normal (normal range: 28-35 pg) or an increased red cell distribution width (RDW,

normal range: 11-15), one can suspect the presence of mild iron deficiency without anemia[57].

Although the main laboratory marker for iron deficiency with or without anemia is a low ferritin

level (< 30 ng/mL) in the absence of inflammation, in the presence of inflammation a normal

ferritin level (as an acute phase reactant) does not rule out iron deficiency; therefore, TfS should

also be measured. “Functional iron deficiency” in inflammatory conditions should be defined by

low TfS (< 20%) and normal ferritin concentration (> 100 ng/mL), whereas low TfS (< 20%) and

intermediate ferritin values (30-100 ng/mL) suggest “absolute iron deficiency[57]”. Some authors

suggest a cut-off value of TfS < 16% combined with low iron value for the diagnosis of iron

deficiency[51]. Iron deficiency can also be defined by a ferritin index > 3.2 (> 2 if CRP > 5

mg/L). The ferritin index, which reflects the iron supply for erythropoiesis, is calculated as the

ratio between the soluble transferrin receptor (sTfR) and the log of ferritin[58]. The sTfR is a

truncated fragment of the membrane receptor and its levels increase when the availability of iron

for erythropoiesis is low, as occurs in IDA. CHr, which measures the Hb content of reticulocytes,

reflects the direct measurement of available iron for erythropoiesis and is useful for

differentiating IDA from ACD. In particular, CHr has a high sensitivity and specificity for

diagnosing iron deficiency and is less affected by inflammation than TfS and ferritin, but no data

are available for its use in IBD[58].

Anemia of chronic disease

Patients are considered to suffer predominantly from ACD when they present evidence of

inflammation (with increased levels of serum CRP and clinical signs), an Hb concentration < 13

g/dL for men and < 12 g/dL for non-pregnant women, and a low TfS < 20%, but normal or

increased ferritin concentrations > 100 ng/mL. In the presence of intermediate ferritin

concentrations (30-100 ng/mL), a diagnosis of ACD combined with “absolute iron deficiency” is

confirmed if the ferritin index has a value < 2 with normal CHr[54,58-60]. Still, some cases may

require supplementary testing for the differential diagnosis between IDA and ACD. It has

recently been shown that hepcidin levels may replace the ferritin index for the confirmation of

combined IDA and ACD if the hepcidin levels are > 4 nmol/L with a CHr < 28 pg[61]. In fact,

hepcidin levels have been found to be significantly higher in IBD patients compared with healthy

controls, with a significant correlation with ferritin levels, CRP, and disease activity, whereas

those of prohepcidin were observed to be significantly lower[62]. In addition, although other

hematological indices may help in the diagnosis of iron deficiency in ACD, many of them are

only available in specific hematology analyzers and their precise clinical usefulness has yet to be

determined. In a recent study carried out with a Beckman-Coulter LH 780, high values of RDW

and low values of blood cell Size Factor were the best markers for the diagnosis of IDA, whereas

both Reticulocyte Distribution Width-Coefficient of Variation (RDWR-CV) and Reticulocyte

Distribution Width-Standard Deviation (RDWR-SD) were significantly correlated to disease

activity and CRP levels[63].

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TREATMENT OF ANEMIA IN INFLAMMATORY BOWEL DISEASE

Iron supplementation

Iron supplementation should be considered in every patient presenting iron deficiency with or

without anemia. In patients with mild to moderate anemia (Hb ≥ 10 g/dL), the administration of

oral iron at optimal low doses of 60-120 mg/d is the conventional approach recommended by the

Centers for Disease Control and Prevention[9,64]. Oral iron compounds are mostly available as

inorganic ferrous salts, such as ferrous fumarate, ferrous sulphate, and ferrous gluconate

containing 33%, 20%, and 12% of elemental iron, respectively. A single tablet of most of these

ferrous salt preparations provides a sufficient dose for the treatment of iron deficiency[65,66]. In

fact, there is no evidence to support the administration of high doses of iron in comparative

trials[65-67]; on the contrary, excessive doses may actually decrease tolerance and compliance

while increasing gastrointestinal side effects, with a discontinuation of iron treatment in 20% of

patients with or without IBD[68]. Nevertheless, there are several drawbacks associated with oral

iron therapy that must be taken into account. In addition to the generally low bioavailability of

oral iron, intestinal absorption is further compromised in IBD patients due to an inflammation-

driven blockade caused by increased hepcidin levels. For this reason, in patients with active

inflammation and combined ACD/iron deficiency, intravenous administration of iron may be

preferable to oral iron therapy. Moreover, when achieved, the therapeutic effect of oral iron

supplementation is slow, requiring two to three weeks to obtain increased Hb concentrations and

up to two months to achieve normal values. At least six months are needed to replenish iron

stores completely[69]. Moreover, non-absorbed iron salts can be toxic to the intestinal mucosa

and oral iron has been shown to increase intestinal inflammation and possibly colon

carcinogenesis in animal models through the production of reactive oxygen species that mediate

intestinal damage and the alteration of the intestinal bacterial milieu in rodents[70-75].

Intravenous iron therapy is more effective, has a higher response rate, and is better tolerated by

patients, with a lower discontinuation rate due to adverse events than oral iron supplementations

in IBD patients, as demonstrated in a recent systematic review with meta-analysis[76]. However,

it is important to highlight that of 757 articles identified, only three industry-funded articles met

the inclusion criteria for this systematic review[69,77-83]. Nevertheless, intravenous iron therapy

should be considered in patients with severe anemia (< 10 g/dL), with intolerance or inadequate

response to oral iron, or with concomitant erythropoietin treatment and/or presence of active

IBD. It should be noted that the new intravenous iron formulations (iron carboxymaltose, iron

ferumoxytol, and iron isomaltoside) reduce both the risk of free iron reactions as well as that of

immunogenicity without the need for administering a test dose before starting treatment.

Treatment duration is also reduced because the new formulations are safer at higher doses than

traditional intravenous iron formulations (iron sucrose, ferric gluconate, and low molecular

weight iron dextran). Iron carboxymaltose is the only new intravenous iron formulation approved

for use in Europe that has been studied in IBD patients. Its superiority at higher standardized

doses over individually calculated doses of iron sucrose has been demonstrated along with its

efficacy in reducing anemia recurrence as compared to a placebo[84-86]. After 12 wk of follow-up,

ferric carboxymaltose led to higher response rates (66.1% vs 54.1%), a higher proportion of non-

anemic patients (72.8% vs 61.8%), and better treatment adherence (92.5% vs 79.1%) than iron

sucrose, with no difference in treatment-related adverse events (13.9% vs 11.3%). With regard to

side effects, in the ferric carboxymaltose group there were more skin and subcutaneous tissue

disorders (rash, dermatitis, pruritus) and more cases of hypophosphatemia, but fewer infusion

site reactions than in the iron sucrose group. The superiority of high-dose intravenous iron

supplementation in IBD probably depends on the iron overload produced in the macrophages of

the reticulo-endothelial system. This induces an over-expression of ferroportin, which may, in

turn, by-pass the hepcidin block in ACD[57]. Recently, an alternative dosage scheme to the

traditional Ganzoni formula has been presented for ferric carboxymaltose treatment. In the new

protocol, for a baseline Hb > 10 g/dL, the dose is 1.0 g for patients with a body weight < 70 kg

and 1.5 g for patients > 70 kg; the corresponding total doses for serum Hb ≤ 10 g/dL are 1.5 g

and 2.0 g[84]. Phase 3 clinical trials are currently underway to evaluate the use of ferumoxytol in

patients with iron deficiency anemia, including a subgroup with IBD (ClinicalTrial.org identifier:

NCT01114139, NCT01114217 and NCT01114204). However, ferumoxytol may be problematic

in IBD patients because it can interfere with MRI signals due to the paramagnetic nature of its

iron core[19]. In addition, a Phase 3 clinical trial of iron isomaltoside (NCT01017614) and a

Phase 4 study of iron sucrose (NCT01067547) are currently being carried out on IBD patients

with IDA. All treatments being tested strive to achieve ferritin concentrations > 100 μg/L,

measured as early as 8 wk after intravenous iron treatment to obtain a reliable result[55].

Considering that the recurrence of iron deficiency with or without anemia is frequent in IBD

patients[87], regular controls at 12 wk intervals are advisable so that treatment can be restarted

promptly if needed.

Erythropoietin supplementation

Several studies have shown that recombinant human erythropoietin may be effective for treating

ACD in IBD patients[88-95]. In the anemia treatment algorithm, intravenous iron therapy should be

considered as a first-line therapy in patients with severe anemia whereas erythropoietin treatment

should be considered only in patients unresponsive to intravenous treatment, with low EPO

levels, or who are unresponsive to aggressive management of IBD[29,53,96] since EPO can be used

as an adjunct therapy to control the inflammation[97]. Recently, a prospective study on CD

patients showed that EPO combined with enteral nutrition can improve the Hb levels in CD

patients with a treatment success rate of 63.16% in the EPO group compared to none of the

patients in the non-EPO group[98]. When a decision has been made to administer EPO therapy, it

should always be combined with intravenous iron supplementation to meet the increased demand

caused by the “functional iron deficiency” typical in IBD patients[99].

Other treatments

Intramuscular vitamin B12 continues to be the gold standard therapy for vitamin B12 deficiency,

especially in symptomatic patients[100]. A dose of 1000 μg/wk for 8 wk, then 1000 μg once

monthly for maintenance lifelong is recommended[101]. No therapeutic advantages have been

demonstrated for either cyanocobalamine or hydroxycobalamine in terms or serum levels during

maintenance therapy[102]. Effectiveness data for sublingual[103,104] and intranasal[105] routes for

vitamin B12 administration have revealed as promising non-invasive alternatives.

Specific treatment of IBD has been shown to gradually increase Hb levels over time, which

indicates that the presence of anemia is positively associated with disease activity and disease-

associated gut lesions. Some data suggest that anti-TNF-α treatment improves the anemia in a

sub-group of patients with CD. In fact, patients who responded to treatment showed

improvements in their anemia within 2 wk of the first infusion of Infliximab, with a parallel

improvement in their CD activity index and an increase in endogenous EPO levels over time[12].

Infliximab seems to neutralize the inhibitory effects of TNF-α on EPO production, increasing the

availability of iron for erythropoiesis and reducing anemia[30]. The drug produces these effects

through various mechanisms, including reduced cytokine-induced formation of ferritin and

hepcidin, with improvement of intestinal iron absorption and iron release from

macrophages via ferroportin-mediated iron export[21,106,107]. Moreover, Infliximab improved the

proliferation of cultured BFU-E, blocking the inhibitory effects of cytokines on erythroid

progenitor cells[26]. Finally, it induced mucosal healing, thereby reducing the production of pro-

inflammatory cytokines and the amount of blood loss through mucosal ulcers. Other therapies

with potential for treating IBD associated with anemia include treatment with anti-IL-6, which is

the major inflammation-driven inducer of hepcidin, and other new therapies that neutralize

hepcidin, modify EPO and/or erythropoietin receptor sensitivity, or affect cytokines to

effectively stimulate erythropoiesis.

The multi-factorial origin of anemia in IBD implies that several leading mechanisms can be

simultaneously identified in a single patient, including chronic intestinal blood loss, decreased

absorption capabilities of the small bowel secondary to inflammation or resection, bacterial

overgrowth, and an inability of many IBD patients to tolerate the side effects of oral ferrous

sulfate, among others[108]. Each of these causative factors usually requires a specific therapeutic

approach. Disease inflammatory activity and iron deficiency should be the first aspects to be

restored in every patient[109] since they are the main causes of anemia and easily identified.

Although more uncommon, vitamin B12 or folate deficiency, hemolytic and drug-induced anemia

should also be born in mind. Effective treatment is only possible if the contributing factors in a

particular patient are clearly defined and corrected[110].

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CONCLUSION

Anemia is a common multifactorial complication in IBD that increases disease morbidity.

Awareness on the part of gastroenterologists needs to be increased to improve the specific

diagnosis and management of anemia in these patients. New generation IV iron compounds are

currently available to treat iron deficiency effectively in IBD patients. Further studies are needed

to establish standardized treatments to reduce the development and recurrence of anemia as well

as to improve the clinical course of IBD.

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Footnotes P- Reviewers: Cheifetz A, Dickey W S- Editor: Ma YJ L- Editor: A E- Editor: Liu XM

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