Defensins play a crucial role in protecting mice against oral

Shigella exneri infection

Doo-Hee Shim
a,b
, Sangryeol Ryu
b
, Mi-Na Kweon
a,
a
Mucosal Immunology Section, International Vaccine Institute, Seoul 151-818, Republic of Korea
b
Department of Food and Animal Biotechnology, School of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul
151-742, Republic of Korea
a r t i c l e i n f o
Article history:
Received 17 September 2010
Available online 1 October 2010
Keywords:
Shigella
Inammation
Mucosa
Neonate
Defensin
a b s t r a c t
An earlier study revealed that 4-day-old mice, but not older mice, were infected with invasive Shigella
strains. Here we attempted to determine the underlying mechanism that induces inammation in the
intestines of neonate mice after oral Shigella infection. Wild-type BALB/c mice of different ages (7, 14,
and 35 days old) were orally administered GFP-expressing Shigella exneri 5a M90T strain (5 10
9
CFU) and analyzed for colonization 6 h following infection. We found that Shigella localized in the epithe-
lium, lamina propria, and crypt regions of the small intestines of 7-day-old BALB/c mice. Microarray anal-
ysis revealed that expression levels of cryptdin and various types of cryptdin-related mRNA (e.g., cryptrs-
2, -5, -7, -12 and lysozyme) in the small intestines were signicantly lower in 7-day-old than in older
mice regardless of Shigella infection status. Interestingly, matrix metalloprotease-7 (matrilysin)-decient
(MAT
/
) mice of B6 background had more colonies and more severe symptoms of inammation in the
intestines than did wild-type B6 mice after oral Shigella challenge. This suggests that cryptdin-related
antimicrobial molecules are indispensable for efcient protection against oral Shigella infection.
2010 Elsevier Inc. All rights reserved.
1. Introduction
Shigella species, Gram-negative enteroinvasive bacteria, cause
bacillary dysentery by invading the large intestinal epithelium
and promoting a severe intestinal inammatory response accom-
panied by fever, abdominal cramps, and tenesmus in human and
non-human primates [14]. Shigellosis is a huge global health
problem and in developing countries is a common cause of infant
mortality [36]. A murine shigellosis model would facilitate under-
standing the pathogenesis and characteristics of the disease and
enable screening of vaccine candidates. A previous study demon-
strated that 4-day-old mice were susceptible to oral invasive Shi-
gella challenge but not older mice [7]. Histologically, 4-day-old
mice challenged with an invasive Shigella strain showed bacteria
and inammation in the intestinal region that mimicked human
shigellosis [7]. To date, the underlying mechanism that enables
older mice to acquire resistance against virulent Shigella infection
is not clear.
We know there are key innate and adaptive immune system
differences between human neonates/young infants and adults,
which may explain why infants are unduly susceptible to infection
and tend to respond less rapidly and vigorously to immunization
than older children or adults [8]. Kollmann et al. [9] showed that
in neonatal cells the TLR-mediated innate immunity produces few-
er multiple cytokines simultaneously than found in adult cells.
T-cell-dependent and -independent antibody responses, although
inducible by the third trimester of pregnancy, develop slowly.
Thus, disease may take hold before effective immunity is induced
[10]. In addition, T-cell-independent antibody responses to poly-
saccharide antigens in neonates are absent and therefore cannot
be used for intervention [11,12]. It seems likely that antigen-pre-
senting cells (e.g., macrophages and dendritic cells [DC]) in the gas-
trointestinal mucosa of neonatal mice lack the ability to recognize
bacteria as dangerous [13].
Innate immunity is the rst line of defense against virulent bac-
teria and is important for normal microbiota in intestinal environ-
ments and crucial at early stages of life [14]. A recent study showed
that Paneth cells, major producers of multiple peptides and pro-
teins with antimicrobial activity in the small intestine, develop
after age 7 days [15,16]. Antimicrobial peptides (AMPs) efciently
control microbial growth in the gastrointestinal tract without
0006-291X/$ - see front matter 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2010.09.100
Abbreviations: AMPs, antimicrobial peptides; CFU, colony-forming units; Cyptrs,
cryptdin-related mRNA sequence; DC, dendritic cells; FAE, follicle-associated
epithelium; LP, lamina propria; MAT, matrilysin; PLA(2)-IIA, phospholipase A2
group IIA; PP, Peyers patch; SIgA, secretory IgA; TSB, trypticase soybean-casein
digest broth; Zo-1, zonula occludens protein 1.

Corresponding author. Address: Mucosal Immunology Section, International

Vaccine Institute, Seoul National University Research Park, Nakseongdae-dong
Kwanak-Gu, Seoul 151-818, Republic of Korea. Fax: +82 2 881 1211.
E-mail address: mnkweon@ivi.int (M.-N. Kweon).
Biochemical and Biophysical Research Communications 401 (2010) 554560
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j our nal homepage: www. el sevi er . com/ l ocat e/ ybbr c
any induction of potentially harmful inammatory responses [17].
Among AMPs, a-defensin plays a crucial role in enteric antibacte-
rial defense together with other molecules such as lysozyme, b-
defensin, cryptdin-related peptides, and the phospholipase A2
group IIA (PLA(2)-IIA). These are mainly produced by Paneth and
epithelial cells in the small intestine [18,19]. In children and adults
with diarrhea caused by Shigella spp., synthesis of colonic entero-
cyte b-defensin HBD-1 and the cathelicidin LL37 is markedly de-
pressed but expression recovers as the illness resolves [20]. In
addition, a-defensins, termed cryptdin in mice, are abundant con-
stituents of secretory granules in Paneth cells in the small intestine
[17] and are produced from day 14 after birth [21].
In the present study, we found that Shigella organisms can in-
vade the small intestine of neonatal mice until age 7 days but
not later. Microarray analysis revealed lower expression levels of
various types of cryptdin-related mRNA in the small intestine of
7-day-old mice than in the older mice that were less susceptible
to oral Shigella challenge. These data suggest the importance of
defensin (e.g., cryptdin and cryptdin-related molecules) for host
defense against oral Shigella infection in mice and further imply
that modulation of those molecules in adult mice could be useful
for developing a murine shigellosis model.
2. Materials and methods
2.1. Mice
BALB/c and C57BL/6 mice were purchased from Charles River
Co. (Seoul, Korea). MAT
/
mice of C57BL/6 background were pur-
chased from the Jackson Laboratory (Bar Harbor, ME). The animals
were maintained in the animal care facilities of the International
Vaccine Institute (Seoul, Korea). All experiments described were
approved by the ethical committees for animal experiments of
Seoul National University and the International Vaccine Institute.
2.2. Oral infection and survival
We grew the virulent form of streptomycin-resistant, GFP-
expressing Shigella exneri 5a M90T strain in trypticase soybean-
casein digest broth (TSB; Difco, Sparks, MD) containing 20 lg/ml
streptomycin (SigmaAldrich, St. Louis, MO) [7]. Neonate mice
were inoculated orally with 5 10
9
colony-forming units (CFU)
of virulent Shigella strain without any starvation or antibiotic treat-
ment. After oral infection, mice were separated from their mothers.
Mice were monitored for tenesmus, diarrhea at 6 h, and survival at
24 h after oral Shigella infection.
2.3. Bacteria count
To assess the numbers of bacteria in the ileal region of small
intestine (one-fourth of the small intestine beginning at the ce-
cum) and whole large intestine of non-infected and Shigella-in-
fected mice, dissected samples were placed in PBS with
gentamycin (50 lg/ml). Tissues were homogenized and plated
onto TSB agar plates that included streptomycin, and green-col-
ored colonies were enumerated after overnight culture at 37 C.
2.4. Histology
Randomly selected tissues in the ileal region were washed with
PBS including gentamycin and xed in 4% formaldehyde for 1 h at
4 C. For immunohistochemical study, frozen intestines were cut
into 5-lm sections and stained with PE-conjugated anti-CD11c
(BD Pharmingen, San Diego, CA) or Alexa 647-conjugated
NKM16-2-4 mAb (kindly provided by Prof. Hiroshi Kiyono, Univer-
sity of Tokyo, Japan) [22]. The sections were mounted with Perma-
Fluor medium (Thermo, Houston, TX) and viewed under a confocal
scanning laser microscope (Zeiss, Germany).
2.5. Microarray analysis
At 0 and 6 h following Shigella infection, we obtained about
1 cm of the small intestine ileal region from 7-, 14-, and 35-day-
old BALB/c mice. Specimens were homogenized after washes with
PBS with gentamycin to remove attached bacteria in the cell or tis-
sue surface. RNA was extracted by RNA isolation kit (Qiagen,
Valencia, CA). cDNA microarray was performed by Macrogen Co.
(Seoul, Korea) using an illumina BeadStation 500 X (Illumina,
Inc., San Diego, CA).
2.6. Statistics
Data are expressed as the mean S.D. Statistical comparisons
between experimental groups were performed using the Student
t-test.
3. Results
3.1. Virulent Shigella organisms invade small and large intestines of
orally challenged newborn mice
In order to determine whether Shigella organisms invade and
colonize mouse intestines, we adopted 4-, 7-, 14-, and 21-day-
old BALB/c mice and orally administered GFP-expressed S. exneri
5a M90T strain (5 10
9
CFU). At 6 h after oral challenge, we deter-
mined bacteria colonization in the ileal region of the small intes-
tine and in the whole large intestine (Fig. 1A). We conrmed our
ndings by confocal microscopy (Fig. 1B). In contrast to ndings
in an earlier study [7], we found that orally administered S. exneri
5a M90T invaded and colonized both small and large intestines in
4-, 7-, and 14-day-old mice and gradually decreased (Fig. 1A). Or-
ally administered GFP-expressing S. exneri 5a M90T was detected
in both epithelial and crypt regions in the intestines of 7-day-old
mice (Fig. 1B). Next, we used NKM16-4-2 mAb specic for murine
M cells to clarify the invasion patterns of Shigellae in the murine
small intestine [22]. Interestingly, virulent GFP-expressing S. ex-
neri 5a M90T were translocated not only by M cells (Fig. 1C-I)
but also by epithelial cells (Fig. 1C-II) in the follicle-associated epi-
thelium (FAE) of Peyers patches (PP), and lamina propria (LP) of
the small intestine of 7-day-old mice (Fig. 1C). Because a previous
study showed DCs penetrate epithelia to sample bacteria in the gut
lumen [23], we next determined localization of CD11c
+
DCs in the
small intestine following oral challenge with GFP-expressing
S. exneri 5a M90T strain. We found no direct evidence that intes-
tinal DCs are actively involved in sampling Shigella from lumen in
either PP or LP of the small intestine of 7-day-old mice (Fig. 1C).
Thus, when our ndings are considered together, we believe
GFP-expressing virulent S. exneri 5a M90T administered by oral
challenge might invade through both M and epithelial cells and
colonize in the small intestine of 7-day-old of BALB/c mice.
3.2. Lack of cryptdins in the small intestine of 7-day-old mice
In order to clarify which molecules are crucial for bacterial inva-
sion and onset of inammation in 7-day-old neonate mice, total
gene expression levels were compared in the small intestine of
7-, 14- and 35-day-old mice by microarray analysis (Fig. 2). Among
40,000 probes, we found approximately 20- and 60-fold increases
of intelectin 2 expression, which is known as goblet cell lectin
[24], in 14- and 35-day-old mice, respectively. We also found a
D.-H. Shim et al. / Biochemical and Biophysical Research Communications 401 (2010) 554560 555
15-fold increase of clusterin expression, an inammation-associ-
ated protein, in 35-day-old mice [25]. In addition, the intestinal
antimicrobial enzymes [26], lysozyme and PLA (2)-IIA encoding
mRNA expression, were increased by 25- and 50-fold in the small
intestine of 14- and 35-day-old mice when compared with 7-day-
old mice. Further, pIgR expression also increased 4- and 40-fold in
the 14- and 35-day-old mice when compared with 7-day-old mice.
Interestingly, cryptdin and cryptdin-related mRNA (cyptrs) such as
cryptdin-4, and cyptrs-1, -7, -2, -5, -12, -10 (mainly secreted by Pa-
neth cells) were signicantly lacking in the small intestine of 7-
day-old mice when compared with those of 14- and 35-day-old
mice regardless of oral Shigella challenge (Fig. 2). Approximately
20- to 50-fold increases of those cryptdin-related molecules were
found in 14- and 35-day-old mice compared with 7-day-old mice
at steady-state. Expression of cyptrs-7, -10, and -2 were 200-,
160-, and 100-fold higher in the 14-day-old mice and cyptrs-10, -
12, and -7 expression levels were up to 540-, 330-, and 220-fold in-
creased in 35-day-old mice compared with 7-day-old mice after
oral Shigella infection. Other molecules related to T and B cell acti-
vation were not signicantly different in mice aged 7, 14, and
35 days. Among the cell junction and cytoskeleton-related mole-
cules, glypican3, claudin2, and tubulin-b5 expression were 50-, 15-
, and 6-fold higher in the small intestine of 7- and 14-day-old mice
than in 35-day-old mice regardless of infection status (Fig. 2). In
contrast, expression levels of claudin15 and Muc3 were 15- and
20-fold higher in the small intestine of 35-day-old mice than in
7- and 14-day-old mice. These ndings collectively suggest that
lack of cryptdin and cryptdin-related molecules in the small intes-
tine of 7-day-old mice could be a crucial clue for the high suscep-
tibility of young mice to Shigella infection.
Fig. 1. After oral challenge with virulent GFP-expressing S. exneri 5a, the bacteria invade both epithelial cells and crypt regions and colonize the intestines of 7-day-old mice.
BALB/c mice of different ages were inoculated intragastrically with 5 10
9
CFU of S. exneri 5a and sacriced 6 h later for count of surviving bacteria in the intestines (A). GFP-
expressed S. exneri 5a was observed by confocal microscopy (B). Shigella was localized in the small and large intestines of 7-day-old mice. Results are expressed as mean
values S.D. for two to four mice per group and are representative of three separate experiments. *p < 0.05; compared to non-infected age-matched mice. n.d., Not detected.
(C) GFP-expressed bacteria in the Peyers patch (PP) and lamina propria (LP) in the small intestine of 7-day-old mice were visualized by confocal microscopy after staining for
M cells (A-I) and CD11c
+
cells (A-II). Bar indicates 20 lm.
556 D.-H. Shim et al. / Biochemical and Biophysical Research Communications 401 (2010) 554560
3.3. Severe intestinal inammation in MAT
/
mice after oral Shigella
infection
To conrm the importance of AMPs against Shigella infection in
7-day-old mice, MAT
/
mice, which cannot secrete active forms of
cryptdin, were adopted [27]. Since BALB/c background MAT
/
mice are not commercially available, we used mice with a B6 back-
ground for the comparison group. First we compared bacterial
invasion patterns between BALB/c and B6 background mice follow-
ing oral challenge with S. exneri 5a M90T. The B6 mice had much
less colonization in their small and large intestines than the BALB/c
mice (Figs. 1A and 3C). Of note, however, the 7-day-old (Fig. 3A)
and 14-day-old (Fig. 3B) MAT
/
mice of B6 background had much
higher CFU counts in their small and large intestines than the age-
matched B6 mice at 6 h after oral challenge with S. exneri 5a
M90T. These results were conrmed by confocal microscopy
(Fig. 3C). We further checked for other inammatory symptoms
such as tenesmus and watery diarrhea using wild-type and
MAT
/
mice of B6 background. As expected, the 7-day-old
MAT
/
mice of B6 background had much more severe tenesmus
and watery diarrhea than age-matched wild-type B6 mice at 6 h
after oral challenge with S. exneri 5a M90T (Fig. 3D). When com-
bined our ndings show that cryptdin and cryptdin-related mole-
cules play a critical role in protecting mice from enteric
pathogenic Shigella infection.
4. Discussion
In the present study, an orally administered S. exneri 5a strain
invaded and colonized the small and large intestines of 4- and 7-
day-old BALB/c mice. The low expression of defense molecules
such as lysozyme and several cryptdins in the intestine of neonate
mice might be an important clue for the cause of the intestinal
inammations. The higher susceptibility of MAT
/
mice of B6
background to oral infection with S. exneri 5a (compared with
wild-type B6 mice) further demonstrates the indispensable role
of cryptdin and cryptdin-related molecules in development of oral
Shigella infection in mice.
The innate immune system is important for maintaining
homeostasis of the intestines, the rst line of defense against
pathogens and microbiota [21]. Many AMPs that have an impor-
6.47 11.09 15.71
Fig. 2. Oral challenge of Shigella induces antimicrobial peptides and innate immunity mediator in 14-day-old but not in 7-day-old mice. Total RNA was prepared from
terminal ileum of the corresponding mice 6 h after S. exneri 5a infection for Illumina microarray analysis. Results show hierarchical clustering analysis for six groups and
gene expression, especially those related to antimicrobial peptides and innate immune response before and after Shigella infection in 7-, 14-, and 35-day-old mice. Green,
probes Pthreefold down-regulated; red, probes Pthreefold up-regulated. Each column represents one individual mouse.
D.-H. Shim et al. / Biochemical and Biophysical Research Communications 401 (2010) 554560 557
tant role in innate immunity employ sophisticated and dynamic
mechanisms of action to carry out their roles in antimicrobial host
defense. AMPs in the neonatal intestine and meconium exhibit
broad-spectrum killing of Gram-positive and Gram-negative bacte-
ria including Bacillus megaterium, Escherichia coli, and group B.
streptococci [28]. Defensin is the most abundant and diverse of
the AMPs and has a pivotal role not only in defense from ingested
and commensal microbes in the intestinal lumen but also in the
number and composition of the colonizing microbiota [15,29].
Cryptdin is produced by the Paneth cells in the small intestine of
mice while defensin is produced by leukocytes in humans, rats,
guinea pigs, and hamsters [30]. Ayabe et al. demonstrated that
MAT
/
mice [27], which cannot process procryptidin precursors,
have more severe symptoms after Salmonella or Listeria infection
than found in cryptdin-compromised mice [31]. The synthesis of
those peptides is impaired in early life in wild-type mice and re-
sults in sensitivity to pathogenic infection [21]. In our current
study, we found a signicant defect of cryptdin-related molecules
in 7-day-old mice (Fig. 2) and detected large numbers of Shigella
organisms in the small intestine of MAT
/
mice at age 7 days
and even at age 14 days (Fig. 3). Several lines of evidence from
other researchers together with our ndings lead us to speculate
that oral infection with S. exneri 5a provokes severe inammation
in 7-day-old mice, likely because they lack the ability to express
AMPs.
Defensin granules are continuously released into the lumen of
the crypt, a process that may be stimulated by cholinergic agonists
[15]. These interact with negatively charged phospholipid groups
on the target cell outer membrane through electrostatic attraction
and/or hydrophobic interaction. This interaction of the peptide
with the bacteria membrane results in displacement of lipids and
alteration of the membrane structure, which consequently breaks
the cell wall, and in certain cases entry of the peptide into the inte-
rior of the target cells and reduction of their activities [29,32].
It is well known that Shigellae initially translocate from the
intestinal lumen region through the M cells in the FAE region
[33]. After translocation from the intestinal lumen, inltrated poly-
morphonuclear cells contribute to the destabilization of epithelial
Fig. 3. 7- (A) and 14-day-old (B) matrilysin (MAT)
/
mice have more GFP-expressing S. exneri 5a colonies in both the small and large intestines than do wild-type C57BL/6
(B6) mice after oral challenge with S. exneri 5a. *p < 0.05; **p < 0.01; compared to Shigella-infected B6 mice. (C) Bacterial colonization in the small and large intestines was
visualized by confocal microscopy. Bar indicates 20 lm. (D) Tenesmus was evaluated for 30 min at 6 h after infection in MAT
/
and wild-type B6 mice. *p < 0.05; **p < 0.01;
compared to wild-type B6 mice. All data are expressed as mean values S.D. and are representative of two independent experiments using four mice per group.
558 D.-H. Shim et al. / Biochemical and Biophysical Research Communications 401 (2010) 554560
permeability and to further bacterial invasion. In the present study,
however, S. exneri 5a invaded not only by way of M cells but also
through epithelial cells of both PP and LP in the neonate mice
(Fig. 1C). The expression levels of molecules involved in the forma-
tion of epithelial tight junctions such as zonula occludens protein 1
(Zo-1), occludin, and E-carderin did not signicantly change as the
mice aged (Fig. 2). Other cell junction-related molecules, such as
glypican3, claudin2, and tubulin-b5, were expressed at much higher
levels in the 7- and 14-day-old mice than in older mice (Fig. 2). In
contrast, claudin15 expression was much lower in the 7-day-old
mice than in older mice (Fig. 2). These ndings suggest the possi-
bility that shigellae acquired by the oral route might easily perme-
ate the intestine of neonate mice because of immature and/or loose
permeability of the intestinal epithelium. However, we have not
yet veried this hypothesis.
Intestinal epithelial cells have polymeric Ig receptors that trans-
fer IgA to the gut lumen as a secretory form of dimeric IgA (SIgA)
[34]. SIgA is abundantly produced in the gut lumen and shows high
afnity for neutralization of toxins and pathogenic microbes and
low afnity to the commensal microbiota [35]. It is especially
important for protection against viral and bacterial infection as it
prevents pathogen entry at the mucosal site [35]. Segmented la-
mentous bacteria are absent during the suckling period, rst ap-
pear at 2 weeks after birth, and correlate with SIgA production
[36]. In the present study, we found high susceptibility to orally
administered S. exneri 5a in neonate mice that had comparably
low levels of pIgR expression (Fig. 2). We speculate that decreased
SIgA secretion levels could be one explanation for high susceptibil-
ity to Shigella infection in these mice.
Comprehensively, our results support the concept that decien-
cies in cryptdin-related antimicrobial molecules in neonate mice
lead to undue susceptibility to oral challenge with a virulent Shi-
gella strain. It is urgent that a vaccine be developed for use in hu-
man neonates to prevent infection with such intestinal pathogenic
bacteria as Shigellae. We hope our ndings in this study will be a
priming point for development of a Shigella vaccine.
Conict of interest
The authors have no nancial conicts of interest.
Acknowledgments
This work is supported by the governments of the Republic of
Korea, Sweden, and Kuwait and the Korean Ministry of Science
and Technology and Grant No. RT104-01-01 from the Regional
Technology Innovation Program of the Ministry of Commerce,
Industry and Energy (MOCIE).
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