Antimicrobial peptides in gastrointestinal inflammation.

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International Journal of Inflammation
Volume 2010, Article ID 910283, 11 pages
doi:10.4061/2010/910283
Review Article
Antimicrobial Peptides in Gastrointestinal Inflammation
Simon Ja¨ger, Eduard F. Stange, and Jan Wehkamp
Department of Internal Medicine I, Robert Bosch Hospital, Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology,
Auerbachstr. 112, 70376 Stuttgart, Germany
Correspondence should be addressed to Jan Wehkamp, jan.wehkamp@ikp-stuttgart.de
Received 28 July 2010; Accepted 18 August 2010
Academic Editor: G. Rogler
Copyright © 2010 Simon Ja¨ger et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Acute and chronic inflammations of mucosal surfaces are complex events in which the effector mechanisms of innate and adaptive
immune systems interact with pathogenic and commensal bacteria. The role of constitutive and inducible antimicrobial peptides
in intestinal inflammation has been investigated thoroughly over the recent years, and their involvement in various disease states
is expanded ever more. Especially in the intestines, a critical balance between luminal bacteria and the antimicrobial peptides is
essential, and a breakdown in barrier function by impaired production of defensins is already implicated in Crohn’s disease. In this
paper, we focus on the role of antimicrobial peptides in inflammatory processes along the gastrointestinal tract, while considering
the resident and pathogenic flora encountered at the specific sites. The role of antimicrobial peptides in the primary events of
inflammatory bowel diseases receives special attention.
1. Introduction
Although a host of different bacteria colonizes the gut from
the oral cavity to the rectum, translocation of bacterial
agents through the intestinal walls remains limited to highly
pathogenic bacteria or predisposing disease states in which
the natural defense mechanisms are compromised.
In the healthy individual, the physical barrier created
by the thin layer of epithelium forms the basis of the
mucosal defense. In addition, the production of an array
of antimicrobial peptides by secretory epithelial cells limits
the invasion and adherence of pathogenic and commensal
bacteria. Salient examples of antimicrobial peptides are
the defensins and cathelicidin LL37, the two major classes
of AMPs in mammals, yet other molecules like elafin or
secretory leukocyte protease inhibitor (SLPI) complement
the effector mechanisms of innate and adaptive immune
systems. Equally important, in the small and large intestine,
goblet cells are responsible for the production of highly
glycosylated proteins, which form a gel-like layer over
the surface epithelium. The outer portion of this layer is
heavily colonized by bacteria, whereas the inner stratum’s
low bacterial load results from the high local levels of
antimicrobial peptides [1].
Recent years have seen a steadily rising interest in antimi-
crobial peptides, and their implication in the pathogenesis
of intestinal processes like Crohn’s disease [2] or necrotizing
enterocolitis [3] as well as their role in psoriasis and atopic
dermatitis, cystic fibrosis and otitis media has garnered the
attention of an increasing group of scientists.
In this paper, we would like to focus on the role of
antimicrobial peptides in inflammatory processes along the
gastrointestinal tract, while considering the resident and
pathogenic flora encountered at the specific sites. The role of
antimicrobial peptides in the pathogenesis of the idiopathic
inflammatory bowel diseases receives special attention.
2. Antimicrobial Peptides of
the Gastrointestinal mucosa
2.1. Defensins. Defensins serve as endogenous antibiotics
with microbicidal activity against Gram-negative and Gram-
positive bacteria, fungi, viruses, and protozoa [4]. One of
their fundamental characteristics is the presence of three
intramolecular disulfide bonds. The pattern of linkage
between the cystein residues allows the classification into
two major groups, the α-defensins and the β-defensins (the
cyclic octadecapeptide called θ-defensin were not found

2 International Journal of Inflammation
in humans so far). The former are linked by a 1–6, 2–
4, 3–5 pattern, the latter 1–5, 2–4, 3–6 pattern, yet their
three-dimensional structure is similar [5, 6]. The total of
six α-defensins includes human neutrophil peptides 1–4
(HNP1–4) produced by granulocytes and human defensin
5 and 6 (HD5 and HD6) produced by Paneth cells. It
should be noted that the Paneth cell defensins are stored
as propeptides and require cleavage by trypsin, which
is stored in Paneth cell granules as a zymogen as well
[7, 8].
β-defensins are mainly produced by epithelial cells [9],
and four subtypes, designated hBD-1 to hBD-4, have been
identified in the human mucosa so far. hBD-1 is ubiquitously
expressed at all surfaces of the human body including the
skin, the respiratory, urogenital and the gastrointestinal
tract [10–15]. hBD-2 and hBD-3 are inducible antimicrobial
peptides expressed by enterocytes throughout the intestinal
tract on demand.
Biochemical properties of the human defensin family
include a low molecular mass from 3 to 6 kDa and a cationic
charge, which allows these molecules to bind to negatively
charged phospholipid groups on microbial surfaces. The
exact mechanism by which defensins exert their bactericidal
effect has still not been identified, but it has already become
clear that they do not act with a uniform mechanism. One
model, the “Shai-Matsuzaki-Huangh” model, proposes that
after integration of defensins into the cell membrane, its
outer layer expands and strains the inner leaflet of this
bilayer, leading to disruption or formation of toroidal pores
[16]. On the other hand, hBD-3 has been shown to function
rather by inhibiting steps of the biosynthesis of the bacterial
cell wall [17].
Defensins were also noted for their chemotactic proper-
ties. The chemoattractant effect on immature dendritic cells
and CD4+ T cells has been shown to act through chemokine
receptor CCR6 [18]. Chemoattraction of macrophages and
monocytes has been observed as well, but these cells do
not express CCR6. A recent publication now reported that
hBD-2 and hBD-3 are chemotactic for these cell lines in
a CCR2-dependent manner [19]. Other investigations have
shown that hBD-2 induces the migration of mast cells
by activating G-protein-phospholipase C-coupled receptors
and is a specific chemoattractant for human neutrophils
[20, 21].
In a broader concept, Peyrin-Biroulet and Chaimallard
position defensins at the interface between innate and adap-
tive immunity, proposing that NOD2-mediated microbial
recognition leads to secretion of defensins, which in turn
attract immature dendritic cells, help in their maturation and
promote the subsequent activation of T cells [22].
Also, HD-5 may influence the intestinal inflammatory
response by binding to the cell membrane of intestinal
epithelial cells. A subsequent induction of interleukin-(IL-)
8 was observed in a concentration- and structure-dependent
fashion [23, 24].
2.2. Cathelicidins. The second major group of AMPs in
mammals are the cathelicidins. While a signal peptide called
“cathelin prosequence” can be found at their N-terminus,
the C-terminal part is formed by a more variable cationic
region that has antimicrobial activity once cleaved from
the holoprotein. The only cathelicidin identified in humans
was termed LL-37/h-CAP18. Its constitutive expression is
found in various immune cells, in salivary glands, and in
epithelia of respiratory, digestive and reproductive tracts
while keratinocytes and intestinal cells can be induced to
enhance expression. LL-37’s antimicrobial properties are
supplemented by its chemotactic effect on blood cells,
activation of histamin release from mast cells, or induction
of angiogenesis [25].
2.3. Other Antimicrobial Peptides. Antimicrobial activity has
been noted in a multitude of other small molecules. For
example, the chemokines CCL14 and CCL15 are con-
stitutively expressed at high levels in human intestinal
epithelium and display potent antibacterial effects [24].
CLL20/macrophage-inflammatory-protein-3α and an addi-
tional 17 chemokines function as antimicrobials as well
[26]. Elafin and secretory leukocyte protease inhibitor (SLPI)
also exhibit broad spectrum antimicrobial activity against
Gram-positive and Gram-negative bacteria, selected fungi
and viruses [5], though in their principal role, these antipro-
teases serve to maintain tissue integrity by antagonising
aggressive serine proteases like human neutrophil elastase
(HNE) [27]. Yet another epithelial antimicrobial peptide is
bactericidal/permeability-increasing protein (BPI), which is
involved in lipid-mediated killing and the attenuation of
proinflammatory signalling by bacteria. Its sphere of action
covers mostly Gram-negative bacteria [28, 29]. For a quick
overview, Table 1 lists the abovementioned antimicrobials
along with their properties.
3. Antimicrobials in Gastrointestinal Diseases
3.1. Esophagus. Microbial infections of the esophagus repre-
sent a rather uncommon event in healthy individuals. Nev-
ertheless, the immunocompromised host quite frequently
suffers from infections with C. albicans, CMV or HSV, while
bacterial infections remain rare.
Fittingly, despite a high expression of numerous antimi-
crobial peptides, assays with oesophageal tissue showed a
weakened potency to kill C. albicans [30], a fact which
could help explain the susceptibility of esophageal tissues
to infections with this yeast. Kiehne et al. [31] observed
that Candida colonization induced a high expression of a
subset of antimicrobial peptides, especially hBD-2 (shown
in Figure 1) and hBD-3. In a subsequent mechanistic
study the group showed that polymorphonuclear leukocytes
(PMNs) reinforce the defensin expression in the epithelium.
The authors speculate that individuals suffering from neu-
tropenia lack this stimulus for the expression of epithe-
lial antimicrobial peptides and thus, a pathophysiologic
explanation for the high incidence of Candida esophagitis
and Candida-related deaths in neutropenic patients can
be proposed [32]. Furthermore, even in esophageal reflux
disease, an induction of β-defensin expression (hBD-2
and hBD-3) could be found, although to a minor degree
[31].

International Journal of Inflammation 3
Table 1: Antimicrobials in the gastrointestinal tract.
Antimicrobial
peptide
Chromosomal
location
Molecular
mass (kDa)
Secretory stimuli
Distribution in
gastrointestinal
tract
Biological function
Changes in
inflammatory bowel
disease
hBD-1 8p23.1 3.5–4.5
Constitutive in
epithelial cells,
IFN-γ and LPS in
monocytes
Ubiquitous in
epithelial cells of
small and large
intestine,
monocytes,
monocyte-derived
dendritic cells
Antimicrobial,
chemotactic
Reduction in colonic
IBD
hBD-2, 3, 4 8p23.1 3.5–4.5
LPS, flagellin
mediated by
NF-κB and AP-1
Epithelial cells,
monocytes
- Antimicrobial,
chemoattractant for
macrophages and
monocytes,
- hBD-2: mast cells
and neutrophils
- Attenuated induction
observed in colonic CD
- Reduced copy
numbers for hBD-2 in
colonic CD
HD-5 and HD-6 8p23.1 3.5–4.5
NOD2 activation
(MDP, LPS) TLR
Granules of ileal
Paneth cells (also
metaplastic Paneth
cells in other areas
of intestinal tract)
Antimicrobial,
induction of IL-8
- Reduction in ileal CD,
more pronounced in
patients with NOD2
mutation
- HD-5 and HD-6
expression due to
metaplastic Paneth cells
in UC and CD colon
Cathelicidin
(“LL-37”)
3p21.3 18
Butyrate, vitamin
D, bile acids, MDP
Epithelial cells,
leukocytes
Antimicrobial,
chemotactic
- Attenuated induction
in colonic CD
- Ileal CD and UC
show regular induction
Elafin 20q13.12 9.8 IL-1, TNF-α
Epithelial cells,
leukocytes
Antiprotease with
antimicrobial and
chemotactic
properties
Attenuated induction
in colonic CD
Secretory
phospholipase A2
16p13.1–p12 14 LPS
Epithelial and
inflammatory cells,
Paneth cell
granules
- Acute phase protein
involved in
eicosanoide
metabolism
- Small intestinal
mucosal defense
?
Lysozyme 12q15 16.5 ?
Gastric, pyloric
and duodenal
glands, small
intestine,
macrophages and
monocytes, not in
colonic tissue
Antimicrobial against
Gram-positive
bacteria, chemotactic
- Small intestine: no
changes observed
- Increased colonic
expression due to
metaplastic Paneth cells
BPI (bactericidal/
permeability-
increasing protein)
20q11.23 50 LPS
Epithelial cells,
neutrophils
Antimicrobial, binds
LPS-compounds
No changes observed,
regular induction in
IBD
3.2. Stomach. The high prevalence and morbidity resulting
from colonization by the Gram-negative bacterium Heli-
cobacter pylori has captured much interest in the role of
antimicrobial peptides in the stomach. Though the mucosa
exhibits a strong inflammatory response against H. pylori
bacteria, clearance of the pathogen is unsuccessful in many
cases.
Helicobacter infection is known to lead to a significant
induction of hBD-2 (see Figure 1), while the defensin gene
expression caused by non-Helicobacter gastritis is much
less pronounced [33], a finding which was confirmed in
a pediatric cohort [34]. In a recent study, it could be
demonstrated that H. pylori induces gastric epithelial cells
to upregulate the endogenous production of hBD-2 [35],

4 International Journal of Inflammation
Stomach
Firmicutes (e.g. enterococcus faecalis (4), left) and bacteroids
(e.g. bacteroides (6), right) those species comprise the majority
of the phyla that make up the human colonic flora. A shift
towards less bacteroidetes and more firmicutes (Bacilli) has
been observed in inflammatory bowel disease
Paneth cell at the crypt base (IHC)
secrete defensins that regulate the
composition of the luminal flora
Interactions of microbes and antimicrobial peptides in the gastrointestinal tract
Small intestine
Liver
Rectum
Colon
Anus
50 µm
Yeast-like cells and pseudohyphae (3)
of C. albicans induce hBD-2 and hBD-3
in the esophagus
Nissle 1917 (5) and other probiotics induce hBD-2.
Immunohistochemistry for hBD-2 in ulcerative colitis is shown
to the right
E. coli
(1)downregulates Paneth
cell defensin production
(viatype 3-scretion system)
Salmonella typhimurium
in gastric mucosa (2) induces hBD-2 (IHC shown
above to the right) and cathelicidin LL37, and is also associated
with gastric Paneth cell metaplasia andHD5 expression
H.pylori
Esophagus
Figure 1
furthermore the authors showed that this is mediated by the
cytosolic pattern recognition receptor NOD1 (nucleotide-
binding oligomerization domain 1).
Also, an analysis of single nucleotide polymorphisms
in the DEFB1 gene correlated patients with chronic active
H. pylori-induced gastritis with the SNP G-52A, suggesting
an involvement of the constitutive expressed hBD-1 in
susceptibility to this form of gastritis [36]. In the setting
of chronic H. pylori induced gastritis, intestinal metapla-
sia (replacement of the normal mucosa by a columnar
epithelium with characteristics of intestinal epithelia, e.g.,
goblet cells, Paneth cells), is a frequent event. A high
HD-5 expression has been observed by Shen et al. [37],
suggesting that in intestinal metaplasia, where α-defensin
producing Paneth cells are present, this metaplastic change
may strengthen the antibacterial response via production of
HD-5. Aside from the defensins, H. pylori is reported to
induce Cathelicidin LL-37 in gastric epithelial cells [38].
3.3. Inflammation of the Biliary Tree. 10%–20% of adult
populations in developed countries suffer from cholelithiasis
(gallstones). Though more than 80% of patients remain
asymptomatic, infections of the gallbladder or the biliary
tree are common diseases, which require antibiotic treatment
in many cases. The normal sterility of bile is maintained
by the bactericidal effect of bile salts and immunoglobu-
lin A, and a notable expression of hBD-1 and hBD-2 is
documented in biliary tract epithelium and in the liver
[39]. Similarly to other anatomic sites, hBD-1 expression
is constitutive, while in the large intrahepatic bile ducts,
hBD-2 was induced by biliary obstruction or hepatolithiasis,
where these peptides contribute to the local antimicrobial
defense.
Interestingly, in the epithelium of four of five patients
with primary sclerosing cholangitis and in all controls
with normal histology [39], hBD-2 expression remained
low. Furthermore, in all bile samples which were analysed,
hBD-1 could be found constitutively, while hBD-2 was
confined to those with hepatolithiasis [39]. Patients with
primary sclerosing cholangitis, especially following endo-
scopic manipulation, suffer from frequent bouts of infection.
Although further studies are needed, the observed lack of
induction of hBD-2 and possibly other antibacterial peptides
could be implicated in the disease mechanism.

International Journal of Inflammation 5
D’Aldebert et al. found an intense immunostaining for
cathelicidin in human liver biliary epithelium, and showed
that bile salts (chenodeoxycholic acid and ursodeoxycholic
acid), which also possess intrinsic bactericidal properties,
induce cathelicidin expression through different nuclear
receptors. According to their results, either farnesoid X
receptor or vitamin D receptor is involved and upon
activation, promote cathelicidin expression in the biliary
tract [40].
3.4. Intestine. The microbial colonization of the lumen
increases along the intestine, though the number of bacteria
is still very low from the duodenum to the proximal ileum.
The distal ileum contains up to 108 primarily anaerobic
bacteria per gram of luminal contents [41], whereas up
to 1011–1012 bacteria per gram colonize the colon. The
bacterial microflora is crucial for the maintenance of human
health and the development of the mucosal immune system.
Moreover, its contribution to the pathogenesis of the chronic
idiopathic inflammatory bowel diseases is widely acknowl-
edged. In these entities, a shift in microbial composition
towards less Bacteroidetes and more Firmicutes (Bacilli) has
already been observed (see Figure 1).
3.4.1. Small Intestinal Inflammation. On the one hand, the
scarcity of bacteria in the ileum can be attributed to the
hostile environment created by acid, bile, and pancreatic
secretions as well as to the phasic propulsive motility of
this part of the gut [42]. On the other hand, adaptive and
innate branches of the immune system contribute as well
to maintain a low microbial density. Paneth cells, which
are a characteristic epithelial lineage of the small intestine
and localize to the bottom of the intestinal crypts, secrete
α-defensins in response to bacterial antigens including
lipopolysaccharide and muramyl dipeptide [43]. A consti-
tutive expression of exceptionally high levels of α-defensins
HD-5 and HD-6 could be demonstrated in human small
intestines [44]. Interestingly, expression of HD-5 exceeds
expression levels of other AMPs produced by the Paneth cell
(lysozyme and sPLa2) by a factor up to 100 [45].
In studies with knockout animals, intestinal extracts from
mice deficient for the cryptdin-processing enzyme matrilysin
and thus lacking functional mature mouse α-defensins
(the mouse homologs to defensins are called cryptdins),
show decreased antimicrobial activity [46], and the authors
furthermore observed that these mice are more susceptible
to orally administered bacterial pathogens as well as to DSS-
induced colitis. Other findings from a transgenic animal
study revealed that human α-defensin HD-5 transgenic
mice are resistant to infection from orally administered S.
typhimurium [47]. Interesting in this context is the fact that
S. typhimurium can downregulate HD-5 expression via a
type-3 secretion system (see Figure 1).
In addition, the Paneth cell defensins can shape the com-
position of microbial species present in the small intestinal
lumen, while the total number of bacteria remains unaffected
[45, 48]. In a mouse model with transgenic expression of
DEFA5, Salzman et al. demonstrated that the colonization
with segmented filamentous bacteria (termed SFB, from
the genus Clostridia) was dramatically decreased when the
mice produced the human α-defensins HD-5. Interestingly
in this context is the fact that mice colonized with SFB were
shown to be more resistant to infection with Citrobacter
rodentium, a close relative to the well-known Escherichia
coli. Paneth cells also exert control over intestinal barrier
penetration by commensals and pathogenic bacteria [49],
apparently mediated by TLR (Toll-like receptor) recognition
and a subsequent induction of antimicrobial peptides. The
signalling was shown to be dependent on the expression
of the MyD88 adaptor protein inside the Paneth cell. The
release of Paneth cell secretions into the intestinal lumen
thus follows stimulation of pattern recognition receptors
(PRR, e.g., Toll-like receptors, NOD-like receptors, RIG-
I-like receptors) with pathogen-associated molecular pat-
terns, termed PAMPs, which are provided by resident and
pathogenic bacteria. Corroborating the concept of a host
driven composition of the microbial flora, Petnicki-Ocweija
et al. showed that in the mouse model, the bactericidal
activity of crypt secretions of the terminal ileum was
severely compromised by NOD2 deletion, and that NOD2
expression depends on the presence of commensal bacteria
[50].
The human NOD2 protein (nucleotide-binding oli-
gomerization domain/caspase recruitment domain (NOD/
CARD) is a cytoplasmic receptor for bacterial molecules
which is predominately expressed in Paneth cells [51].
NOD2 received great attention after it was identified as
a susceptibility gene for Crohn’s disease in 2001 [52, 53].
Structural changes in the leucine-rich repeat region of NOD2
result from two single nucleotide polymorphisms (SNPs)
and an insertion mutation that leads to a frameshift mutation
at Leu1007 (L1007fsinsC). The authors found that homozy-
gosity or compound heterozygosity increases the relative risk
for Crohn’s disease by as much as 40-fold compared with
individuals without mutation. Approximately one third of
patients affected by ileal Crohn’s disease show mutations in
the NOD2 status [22]. Of note, the three common allelic
variants of the NOD2 gene were correlated with an increased
susceptibility only in Caucasians and studies have shown
remarkable differences in the genetic variability of the NOD2
gene in different ethnical populations. The three common
variations could not be found in Asian populations [54, 55]
and in African Americans mutation frequency as well as
the attributable risk were much lower [56]. These findings
could partially explain variations in the frequency of Crohn’s
disease in different world populations.
A link among Crohn’s disease, NOD2, and α-defensins is
strongly suggested by observations made in NOD2-knockout
mice which exhibit a decrease in Paneth cell defensins (crypt-
dins) alongside an impaired mucosal immune response to
orally delivered but not intraperitoneally administrated L.
monocytogenes [57]. Also, a decreased α-defensin mRNA
expression in biopsy specimens of ileal Crohn’s patients,
which was even more pronounced in patients carrying
NOD2 mutations [58, 59], was observed. The decrease
in α-defensins was independent of inflammation in the
specimens and not observed in ulcerative colitis or pouchitis,
an inflammatory control of non-Crohn’s ileitis. Of note,