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Previous Article | Next Article 
Infection and Immunity, November 2006, p. 6075-6084, Vol. 74, No. 11
0019-9567/06/$08.00+0 doi:10.1128/IAI.00721-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Evidence that Tight Junctions Are Disrupted Due to Intimate Bacterial Contact and Not Inflammation during Attaching and Effacing Pathogen Infection In Vivo
Julian A. Guttman,1
Fereshte N. Samji,1
Yuling Li,1
A. Wayne Vogl,2 and
B. Brett Finlay1*
The University of British Columbia, Michael Smith Laboratories, 301-2185 East Mall, Vancouver, BC, Canada V6T
1Z4,1
The University of British Columbia, Department of Cellular and
Physiological Sciences, Division of Anatomy and Cell
Biology, Life Sciences Centre, 3.401-2350 Health
Sciences Mall, Vancouver, BC, Canada V6T 1Z32
Received 4 May 2006/
Returned for modification 10 August 2006/
Accepted 18 August 2006
 |
ABSTRACT
|
|---|
It
is widely accepted that tight junctions are altered during infections
by attaching and effacing (A/E) pathogens. These disruptions have been
demonstrated both in vitro and more recently in vivo. For in vivo
experiments, the murine model of A/E infection with Citrobacter
rodentium is the animal model of choice. In addition to effects on
tight junctions, these bacteria also colonize the colon at high levels,
efface colonocyte microvilli, and cause hyperplasia and inflammation.
Although we have recently demonstrated that tight junctions are
disrupted by C. rodentium, the issue of direct
effects of bacteria on epithelial cell junctions versus the indirect
effects of inflammation still remains to be clarified. Here, we
demonstrate that during the C. rodentium infections,
inflammation plays no discernible role in the alteration of tight
junctions. The distribution of the tight junction proteins, claudin-1,
-3, and -5, are unaffected in inflamed colon, and junctions appear
morphologically unaltered when viewed by electron microscopy.
Additionally, tracer molecules are not capable of penetrating the
inflamed colonic epithelium of infected mice that have cleared the
bacteria. Finally, infected colonocytes from mice exposed to
C. rodentium for 14 days, which have high levels of
bacterial attachment to colonocytes as well as inflammation, have
characteristic, altered claudin localization whereas cells adjacent to
infected colonocytes retain their normal claudin distribution. We
conclude that inflammation plays no discernible role in tight junction
alteration during A/E pathogenesis and that tight junction disruption
in vivo appears dependent only on the direct intimate attachment of the
pathogenic bacteria to the
cells.
|
INTRODUCTION
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|---|
Humans and animals infected with the attaching and effacing (A/E)
pathogens enterohemorrhagic Escherichia coli, enteropathogenic
E. coli (EPEC), and Citrobacter rodentium suffer from
serious diarrhea, or diarrhea-like conditions, that can result in
death. These pathogens attach to the surface epithelial cells of the
intestine and inject effector proteins through a type III secretion
system (TTSS) into host cells
(18,
32). These pathogenic
proteins commandeer various host cell functions and structures that
contribute to the disease process. Some of the host structures targeted
by A/E pathogens include the host cell's microvilli
(7,
24), mitochondria
(19,
27,
31), and tight junctions
(3,
6,
23,
26).
In intestinal
epithelial cells, tight junctions are the most luminal cell-cell
junctions of the apical junction complex
(10). These junctions
have two functions. One is to act as permeability barriers, separating
the luminal environment from the adluminal region of the epithelium;
the other is to segregate apically located membrane proteins from those
at the basolateral membrane. Tight junctions are often viewed as
membrane fusions by transmission electron microscopy because the
membranes of adjacent cells are held in extremely close opposition by
the transmembrane protein occludin and members of the claudin family
that form the junctions
(11,
13,
45). Both of these
protein groups contain four transmembrane-spanning regions but do not
share any sequence homology (reviewed in reference
15). They attach
intracellularly to the adaptor molecules zonula occludens 1 (ZO-1),
ZO-2, and ZO-3. ZO-1 links the transmembrane proteins to the actin
cytoskeleton (9,
45). Work on these two
groups of proteins has yielded striking differences as to their
importance in maintaining the barrier function of the tight junctions.
Studies using occludin null mice have demonstrated that occludin is
dispensable with regard to tight junction barrier function
(36,
37). These mice, devoid
of occludin, have tight junctions that function normally
(36). Conversely, mice
deficient in all claudins targeted thus far have catastrophic tight
junction deficiencies
(12,
16,
30). In the colon,
claudin-1 (51), -2, -3,
-4, and -5 (35) are
present in colonocytes.
Until recently, most
research on tight junction disruption induced by A/E pathogens has
focused on EPEC infections in cell culture (in vitro)
(6,
23,
43,
46,
48,
50). Generally it is
accepted that in vitro, EPEC causes a redistribution of the tight
junction proteins occludin, ZO-1, and claudin-1 as well as a decrease
in transepithelial resistance (TER)
(26). These alterations
are thought to be due to the effector proteins EspF and Map
(mitochondria-associated protein)
(6,
23,
48). We previously have
used a naturally occurring A/E murine infection model to test whether
tight junctions are disrupted in vivo
(17). This model uses the
A/E pathogen C. rodentium to infect mice. This pathogen
colonizes the colon, collapses microvilli, and produces a diarrhea-like
phenotype in infected mice
(22). We demonstrated
that C. rodentium disrupts tight junctions in mice and that
this disruption is dependent on the bacterial type III effector protein
EspF but not Map
(17).
Although the
evidence is controversial, it has been hypothesized that inflammatory
effects participate in tight junction alteration during infection
(28). Studies
have shown that the addition of tumor necrosis factor-
to
cultured colonic cells (HT-29/B6) decreases tight junction strands
(14) and that the
addition of both tumor necrosis factor- and gamma interferon
reduces the expression of claudin-2 but not the expression of claudin-3
and -4 in T84 cells (34).
However, the addition of interleukin-13 increases the expression of
claudin-2, while the levels of claudin-3 and -4 are unchanged in T84
cells (34). This result
occurs with an increase in dextran permeability and a decrease in TER
(34). Western blots of
claudin-1, -2, -3, and -5 from mice deficient in interleukin-2, which
have colonic inflammation, show a dramatic increase in protein levels
in membrane preparations from the colon
(1). In humans with
inflammatory bowel disease a report by Gassler and coworkers
(13a) suggests that
ZO-1, occludin, claudin-1, and claudin-2 are all reduced. These results
are contrary to those in a recent report by Prasad and coworkers
(34) in which they
demonstrate that claudin-2 expression increases and claudin-3 and -4
expression decreases in patients with Crohn's disease and ulcerative
colitis/inflammatory bowel disease.
There is some suggestion that
EPEC causes the release of inflammatory mediators in vitro (in cell
culture) (4,
38,
41). To date, only one
study has attempted to link inflammatory effects with tight junction
alteration caused by A/E pathogen infection
(39). In that study
EPEC-infected cells were pretreated with drugs that inhibit
mitogen-activated protein (MAP) or extracellular signal-regulated
kinase (ERK) kinase (39).
Although these inhibitors have numerous effects on cells in general,
they apparently had no effect on the decrease in TER during EPEC
infection in vitro
(39).
To determine
what role A/E pathogen-induced inflammation has on tight
junctions in vivo, we infected C57BL/6 mice with C. rodentium
and then evaluated the status of colonic tight junctions at a time
point (21 days) when bacteria have been cleared from the colon but
inflammation persists. We confirmed that, at this time point, bacteria
were not attached to colonocytes and that inflammatory cell infiltrate
was evident in the lamina propria. Immunolocalization of claudin-1, -3,
and -5 on infected tissue sections labeled the lateral boundaries of
colonocytes and appeared unaltered compared to uninfected or
escN C. rodentium-infected controls.
Electron microscopy validated these findings by demonstrating
morphologically intact tight junctions. Functional tracer experiments
showed that the tight junctions remained intact as the chemical tracers
were retained in the lumen of the colon without any penetration into
the mucosa. Additionally, we demonstrated that in tissue in which both
bacteria are attached and inflammation persists, tight junctions are
altered only where bacteria are intimately attached. These findings
provide crucial in vivo evidence that the inflammatory response caused
by A/E pathogens does not play a significant role in tight junction
alteration; instead, it is direct bacterial contact that mediates
junctional disruption.
|
MATERIALS AND METHODS
|
|---|
Chemicals and reagents.
Chemicals and reagents used in this
study were obtained from Sigma-Aldrich Canada (Ontario, Canada).
Paraformaldehyde was acquired from Canmeco (Quebec, Canada), and NaCl
was from Fisher Scientific (British Columbia, Canada). Secondary
antibodies conjugated to horseradish peroxidase and control
immunoglobulins (immunoglobulin G [IgG]) were purchased from Jackson
ImmunoResearch Laboratories, Inc. (Pennsylvania). All secondary
antibodies conjugated to Alexa fluorochromes were purchased from
Molecular Probes (Oregon), as was the Alexa 488-conjugated
streptavidin.
Animals, bacterial strains, and mouse infections.
Four- to six-week-old female C57BL/6
mice were acquired from the Jackson Laboratory and Charles River
Laboratories. Upon arrival at the University of British Columbia animal
care center, animals were left undisturbed for at least 4 days prior to
oral gavage infections with 4 x 108 to 5 x
108 escN or wild-type C.
rodentium (8).
Infections persisted for 7, 14, or 21 days, at which point the mice
were euthanized by cervical dislocation. All experiments were repeated
at least three times.
Tissue preparation, immunolocalization, and neutrophil staining.
Tissue immunolocalization was
performed as described previously
(17). Briefly, tissue was
fixed in 3% paraformaldehyde; 5-µm sections were cut by Wax-It
Histology Services, Inc., and treated with 0.2% Triton X-100 in
phosphate-buffered saline (PBS; 150 mM NaCl, 5 mM KCl, 0.8 mM
KH2PO4, 3.2 mM Na2HPO4, pH
7.3), and samples were washed extensively in PBS and blocked with 5%
normal goat serum in PBS containing 0.05% Tween-20 and 0.1% bovine
serum albumin (TPBS-BSA). Primary antibodies consisted of rabbit
anti-claudin-1, anti-claudin-3, and anti-claudin-5 antibodies used at a
concentration of 0.005 mg/ml (Zymed Laboratories, Inc., California) as
well as a previously characterized
(47) rat serum
anti-C. rodentium Tir antibody used at a 1:100 dilution and a
rat anti-mouse F4/80 antibody directly conjugated to biotin and used at
a concentration of 1.315 µg/ml to 3.85 µg/ml (Serotec)
in TPBS-BSA with 1% normal goat serum. These antibodies were incubated
on the tissue sections overnight at 4°C. The material was
washed extensively with the TPBS-BSA and then incubated for 90 min at
37°C with a goat anti-rabbit secondary antibody conjugated to
Alexa 568. The slides were again washed extensively and
stained with DAPI (4',6'-diamidino-2-phenylindole).
Coverslips were mounted using Vectashield (Vector Labs, Ontario,
Canada). The tissue was visualized using a Zeiss Axiophot
microscope.
As controls for claudin-1, -3, and -5
immunolocalization, primary antibodies were replaced with normal rabbit
IgG at identical concentrations to the primary antibodies. Specific
staining was not detected on normal rabbit IgG-stained control sections
(data not shown).
Neutrophils were labeled according to the
manufacturer's instructions using a Naphthol AS-D Chloroacetate
Esterase kit (Sigma). Tissue was counterstained using
hematoxylin.
Electron microscopy and thick sections.
Two- to
three-millimeter sections of colon were excised from euthanized mice
and fixed at room temperature for 2 to 3 h by immersion in a
solution consisting of 1.5% paraformaldehyde, 1.5% glutaraldehyde, and
0.1 M sodium cacodylate (pH 7.3). The sections of bowel were cut into
smaller pieces (1 to 1.5 mm), washed three times (10 min each wash) in
0.1 M sodium cacodylate buffer (pH 7.3), and then postfixed on ice for
1 h in 1% OsO4 in 0.1 M sodium cacodylate (pH
7.3). The material was washed three times in distilled water and then
stained en bloc in 1% aqueous uranyl acetate. After 1 h, the
samples were washed with distilled water, dehydrated through an
ascending series of ethyl alcohols, infiltrated through propylene oxide
into resin (EPON 812), and then embedded in resin. Thick sections were
cut at a 1-µm thickness, mounted on glass slides, stained with
toluidine blue, and then photographed on a Zeiss Axiophot microscope.
Thin sections were cut at about 700 Å, mounted on grids,
stained with uranyl acetate and lead citrate, and photographed on a
Philips 300 electron microscope operated at 60
kV.
Tracer experiments.
Tracer experiments have been
described previously
(17). Briefly, EZ-link
Sulfo-NHS-Biotin (Pierce Chemical Co., Illinois) was diluted to 2 mg/ml
in PBS-1 mM CaCl2 and slowly injected into the
distal colon for 3.5 min. Following this, 1 cm of colon, just cranial
to the area contacting the needle, was removed and fixed in 3%
paraformaldehyde in PBS for 3 h. The tissue was then washed
in PBS and sectioned by Wax-It Histology Services, Inc. Tissue sections
were incubated with a 1:500 dilution of streptavidin conjugated to
Alexa 488 for 30 min at room temperature and imaged using a Zeiss
Axiophot microscope. As controls tissue sections that were not
pretreated with biotin were incubated with streptavidin to investigate
endogenous biotin activity. These controls were negative
(data not
shown).
|
RESULTS
|
|---|
Tight junctions remain morphologically and functionally intact in the presence of A/E bacterially induced inflammation in vivo.
To assess what role the inflammatory
response has on tight junction disruption caused by A/E pathogens, we
infected mice with wild-type C. rodentium for 21 days and
compared claudin-1, -3, and -5 localization on tissue sections with
material from uninfected and 21-day escN C.
rodentium-infected mice as well as 7-day wild-type C.
rodentium-infected tissue sections. The 21-day time point was
studied because 21 days postinfection, C. rodentium is cleared
by the animal (25), but
significant inflammation persists. The TTSS mutant
escN C. rodentium is cleared by the animal
by about day 7, does not attach to colonocytes, and, consequently, does
not cause disease; an animal infected with escN
C. rodentium is indistinguishable from an uninfected animal
(17). For these reasons,
we use the escN C. rodentium infection as a
sham control. Following 21-day infection, localization of claudin-1,
-3, and -5 in colonocytes appeared unaltered compared to uninfected or
escN C. rodentium-infected mice, whereas
7-day infections demonstrated extensive claudin redistribution away
from the cell periphery (Fig.
1). The characteristic staining pattern remained along the lateral cell
boundaries (Fig. 1,
arrowheads). Wild-type C. rodentium-infected mice had inflamed
colons, as demonstrated by increased cell infiltrate in the lamina
propria (Fig. 1). The
inflammatory cell infiltrate was confirmed by neutrophil and macrophage
(F4/80) staining (Fig.
2 and
3).
View larger version (82K):
[in this window]
[in a new window]
|
FIG. 1. Claudin-1,
-3, and -5 and DAPI (DNA) localization on 21-day wild-type C.
rodentium-infected mouse sections paired with 21-day
escN C. rodentium-infected tissue or uninfected
tissue. Claudin staining of 7-day wild-type C.
rodentium-infected tissue is also presented. Arrowheads indicate
areas of staining at the lateral boundaries of colonocytes. The
asterisks in the merged images indicate regions with high levels of
cellular infiltrate in the lamina propria of 21-day wild-type C.
rodentium-infected inflamed epithelium. WT, wild type; CR. C.
rodentium. Scale bars, 50 µm (21-day micrographs) and 25
µm (7-day
micrographs).
|
|
We
then determined if the unaltered localization of the claudin proteins
correlated with unaltered tight junction morphology. Careful comparison
of colonic epithelial tight junction ultrastructure in 21-day wild-type
C. rodentium-infected animals with that in mice infected with
escN C. rodentium (a sham infection;
indistinguishable from uninfected tissue) indicated to us that there
were no morphological differences with the tight junctions of 21-day
wild-type-infected colonocytes, even though increased cellular
infiltrate was observed in the lamina propria of wild-type-infected
samples (Fig.
4).
Tight junctions maintain barrier function during A/E pathogen-induced inflammation.
Tight junctions in the murine colon
were assessed for functional integrity following 21-day infection with
C. rodentium. The aim of this was to determine if the
unaltered localization of claudin proteins corresponds to functionally
intact tight junctions. We injected a molecular tracer (biotin) into
the distal colon of 21-day wild-type C. rodentium-infected
mice and compared these results to escN C.
rodentium-infected mice. We have used this technique
previously to demonstrate that tight junctions are disrupted in the
presence of wild-type C. rodentium at 7 days postinfection,
when inflammatory cells are not evident in the lamina propria
(17) (Fig.
5).
Following biotin treatment of 21-day wild-type C.
rodentium-infected animals, the biotin tracer lined the luminal
boundary of the colonic epithelium and did not penetrate into the
tissue (Fig.
6). This indicates that the tight junctions during the
inflammatory events caused by A/E pathogen infection are intact and
maintain the epithelial barrier (Fig.
6). No specific tissue
labeling was present in controls (data not
shown).
Tight junctions are altered by C. rodentium intimately attached to colonocytes in vivo.
To investigate whether
or not the intimate attachment of C. rodentium to colonocytes
is a prerequisite for tight junction disruption, murine colon sections
from mice 14 days after C. rodentium infection were labeled
with antibodies to claudin-3. Claudin-3 staining was pursued because of
the superiority of the antibody over claudin-1 or -5 antibodies used in
this study. We previously have shown that the appropriate localization
of claudin-3 at the cell boundaries in colonocytes corresponds to
intact tight junctions whereas the altered localization of claudin-3
corresponded to functional tight junction deficiencies in the colon
(17).
At 14 days
after infection, bacterial colonization and inflammation are at high
levels. Claudin-3 localization was disrupted only in colonocytes that
had intimately attached bacteria (Fig.
7). At areas of the same tissue devoid of bacteria, claudin-3 localization
was unaffected (Fig. 7).
These results further confirm our findings that tight junction
disruption is unaffected by the inflammatory response during A/E
bacterial infection and that any tight junction alteration is due to
the direct contact of the pathogen to the host's cells (Fig.
7). Additionally, this
evidence demonstrates that other host cell mediators likely do not
alter tight junctions during in vivo A/E pathogen
infections.
View larger version (88K):
[in this window]
[in a new window]
|
FIG. 7. (A)
Paired phase, C. rodentium Tir, and DAPI micrographs of
infected tissue. Tir and DAPI colocalization demonstrates that bacteria
labeled by DAPI in infected tissue sections is C. rodentium.
Scale bar, 50 µm. (B) Paired phase, claudin-3, and
DAPI micrographs of uninfected and 14-day wild-type C.
rodentium (WT CR)-infected tissue. Arrows indicate regions of the
epithelium without bacterial attachment that label the lateral
boundaries of colonocytes. Arrowheads and white asterisks identify
regions with bacterial colonization and direct attachment to
colonocytes. All infected regions have altered localization of
claudin-3. The black asterisks in the DAPI and merged images indicate
areas with high levels of cellular infiltrate. Scale bar, 50
µm.
|
|
|
DISCUSSION
|
|---|
We have
previously demonstrated that uninfected and escN
C. rodentium-infected mice display indistinguishable claudin
phenotypes whereas wild-type C. rodentium infections cause
overt tight junction disruption between infected colonocytes of mice
that are exposed for 7 days. At this time point postinoculum,
inflammatory cell infiltrate in the lamina propria of infected mice is
not evident. However, 21 days after wild-type C. rodentium
infection, inflammation persists in the absence of colonized bacteria.
The temporal separation of inflammation from A/E bacterial colonization
at 21 days provides a model to study the effects of bacterial
colonization versus inflammation in vivo. We demonstrate here,
morphologically and by the localization of claudin proteins, that tight
junctions are unaltered in the presence of inflammatory cells during
the inflammatory response caused by C. rodentium infection.
These findings correspond to a functionally intact epithelial
barrier.
Thus far, only one study has explored the role of A/E
pathogen-induced inflammation on tight junctions
(39). In that study, the
MAP or ERK kinase inhibitor PD-98059 was used on T84 cells that were
subsequently infected with EPEC. Although these kinases are capable of
activating inflammatory cascades, they can also have direct effects on
tight junctions (2,
21,
49) and can influence
numerous other events such as cell proliferation, protection against
apoptosis, and cell survival
(5,
20,
29,
33,
44). Results from the
Savkovic (39) study found
equivalent decreases in TER caused by EPEC on cultured T84 monolayers
in the presence or absence of PD-98059. This suggested to the authors
that the relationship of cytokine release to tight junction alteration
was not present because TER was still decreased in cells infected in
the presence PD-98059. Although this study used an inhibitor that can
directly act on tight junctions
(2,
21,
49), the effects of both
MAP and ERK in vivo are an area that should still be pursued in
vivo.
Recently, a second mouse model has emerged for the study of
A/E pathogen infections in vivo
(40). In this model, the
human A/E pathogen EPEC was used to infect mice. During infection,
inflammation is minimal, and bacterial counts have been reported in
various parts of the intestine at about 104 CFU
(40). These counts
correspond to those of C. rodentium lacking a functional type
III secretion system (escN), which does not colonize
the colon, and signify extremely low levels of colonization of EPEC in
mice. A recent study by this group using the EPEC murine model
(42) suggested that tight
junctions can be disrupted in vivo in a TTSS-dependent manner by EspF,
but the presented photographic evidence indicated the absence of any
bacteria intimately attached to the cells. Although this suggested that
potential mediators might be released from infected host cells to
influence tight junctions in noninfected cells, our evidence
demonstrates that intimate contact of the bacteria to cells is
essential for barrier alteration to occur.
Here, we present
evidence that the inflammatory response caused by C. rodentium
has no discernible effect on morphology or the barrier function of
tight junctions. We also demonstrate that the alteration of claudin-3
localization in the infected murine colon is completely dependent on
the intimate attachment of the pathogen to the colonic enterocytes. Our
evidence further accentuates the importance of using relevant in vivo
studies and infection models to assess the role of pathogenic
infections on tissues.
|
ACKNOWLEDGMENTS
|
|---|
The authors
thank Wanyin Deng, Bruce Vallance, and Bryan Coburn for their critical
review of the manuscript and Guntram Grassel for his technical
assistance.
J.A.G. is a CAG/CIHR/AstraZeneca and MSFHR
Postdoctoral Fellow. B.B.F. is a Howard Hughes International Research
Scholar, a CIHR Distinguished Investigator, and the UBC Peter Wall
Distinguished Professor. This study was funded through operating grants
from the CIHR and the
HHMI.
|
FOOTNOTES
|
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* Corresponding author. Mailing address: The University of British Columbia, Michael
Smith Laboratories, 301-2185 East Mall, Vancouver, BC, Canada V6T 1Z4.
Phone: (604) 822-2493. Fax: (604) 822-2114. E-mail: bfinlay{at}interchange.ubc.ca. 
Published ahead of print on 5 September 2006.
Editor: A. D. O'Brien
|
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Infection and Immunity, November 2006, p. 6075-6084, Vol. 74, No. 11
0019-9567/06/$08.00+0 doi:10.1128/IAI.00721-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
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Abstract
Introduction
