INFECTION AND IMMUNITY, Feb. 2008, p. 796811 Vol. 76, No.

2
0019-9567/08/$08.000 doi:10.1128/IAI.00093-07
Copyright 2008, American Society for Microbiology. All Rights Reserved.

Modulation of Intestinal Goblet Cell Function during Infection by an

Attaching and Effacing Bacterial Pathogen
Kirk S. B. Bergstrom,1 Julian A. Guttman,2 Mohammad Rumi,1 Caixia Ma,1 Saied Bouzari,1
Mohammed A. Khan,1 Deanna L. Gibson,1 A. Wayne Vogl,3 and Bruce A. Vallance1*
Division of Gastroenterology, BCs Childrens Hospital,1 Michael Smith Laboratories,2 and Department of
Cellular and Physiological Sciences, Division of Anatomy and Cell Biology,3 University of
British Columbia, Vancouver, British Columbia, Canada
Received 17 January 2007/Returned for modification 8 March 2007/Accepted 27 October 2007

The attaching and effacing (A/E) bacterial pathogens enteropathogenic Escherichia coli and enterohemor-
rhagic E. coli and the related mouse pathogen Citrobacter rodentium colonize their hosts intestines by infecting
the apical surfaces of enterocytes, subverting their function, and they ultimately cause diarrhea. Surprisingly,
little is known about the interactions of these organisms with goblet cells, which are specialized epithelial cells
that secrete the protective molecules Muc2 and trefoil factor 3 (Tff3) into the intestinal lumen. C. rodentium
infection leads to dramatic goblet cell depletion within the infected colon, yet it is not clear whether C.
rodentium infects goblet cells or if this pathology is pathogen or host mediated. As determined by immuno-
staining and PCR, both the number of goblet cells and the expression of genes encoding Muc2 and Tff3 were
significantly reduced by day 10 postinfection. While electron microscopy and immunostaining revealed that C.
rodentium directly infected a fraction of colonic goblet cells, C. rodentium localization did not correlate with
goblet cell depletion. To assess the role of the host immune system in these changes, Rag1 knockout (KO) (T-
and B-cell-deficient) mice were infected with C. rodentium. Rag1 KO mice did not exhibit the reduction in the
number of goblet cells or in mediator (Muc2 and Tff3) expression observed in infected immunocompetent mice.
However, reconstitution of Rag1 KO mice with T and B lymphocytes from C57BL/6 mice restored the goblet cell
depletion phenotype during C. rodentium infection. In conclusion, these studies demonstrated that while colonic
goblet cells can be subject to direct infection and potential subversion by A/E pathogens in vivo, it is the host
immune system that primarily modulates the function of these cells during infection.

Enteropathogenic Escherichia coli (EPEC) and enterohem-
orrhagic E. coli (EHEC) are food- and waterborne pathogens
that are attaching and effacing (A/E) bacteria, a class of non-
invasive enteric bacterial pathogens (49). These bacteria infect
the apical surfaces of intestinal epithelial cells, causing a sig-
nature histopathology characterized by intimate attachment to
the host plasma membrane and localized effacement of the
brush border immediately surrounding the attached bacteria
(16, 74). As a result of this infection and the resulting host
inflammatory response, EPEC and EHEC cause severe diar-
rhea and other complications, leading to the death of hundreds
of thousands of children worldwide each year (7, 28, 49). Al-
though these pathogens are human specific and thus difficult to
model in vivo, Citrobacter rodentium is a natural A/E bacterial
pathogen of mice that is closely related to EPEC and EHEC
(11). C. rodentium infection causes colonic epithelial cell pro-
liferation and crypt elongation, as well as inflammation and
diarrhea (20, 38). Because C. rodentium produces A/E lesions
that are virtually indistinguishable from those produced by
EPEC and EHEC (38), it has been widely used as a model to
study A/E bacterial pathogenesis in vivo (11, 12, 21, 25).
During infection by several enteric bacterial pathogens, in-
* Corresponding author. Mailing address: ACB, Rm. K4-188, 4480
Oak Street, BCs Childrens Hospital, Vancouver, British Columbia,
Canada V6H 3V4. Phone: (604) 875-2345, ext. 5118. Fax: (604) 875-
3244. E-mail: bvallance@cw.bc.ca.
K.S.B.B. and J.A.G. contributed equally to this work.
Published ahead of print on 5 November 2007.
cluding A/E pathogens, intestinal epithelial cells can be subject
to direct modulation by the pathogens (4, 22, 50, 67). A/E
pathogens utilize a type III secretion system (T3SS) to secrete
various bacterial effectors encoded in their genomes (e.g., the
transmembrane intimin receptor Tir) (18, 31) directly into host
cells to cause disease. These virulence factors act in an orches-
trated manner to subvert intracellular signaling pathways
within host cells, altering various cellular processes, including
cytoskeletal (61), organelle (39, 47), and barrier (10, 19, 39)
functions. This strategy allows the bacteria not only to inti-
mately attach to and form A/E lesions on epithelial surfaces (6,
32) but also to suppress inflammatory responses and host de-
fenses (44, 65). Through the release of various cytokines, host
immune cells can also modulate intestinal epithelial function
by altering epithelial cell proliferation (5, 54), migration (8),
and permeability (24, 75). Such immunomodulation of epithe-
lial function is thought to represent a critical effector mecha-
nism by which the host is able to mediate clearance of invading
enteric pathogens, as demonstrated with diverse classes of
pathogens, such as viruses (5) and helminths (8, 35, 48). How-
ever, while this mechanism has been characterized best for
parasitic infections, the role of immunomodulation of intesti-
nal epithelial cells during enteric bacterial infections, including
A/E pathogen infections, remains largely undefined.
Infection by several enteric pathogens, including C. roden-
tium, leads to a dramatic reduction in the number of pheno-
typically distinct goblet cells, which is termed goblet cell de-
pletion (38). Intestinal goblet cells are highly polarized
secretory cells that are present throughout the intestinal tract

796
VOL. 76, 2008
but are most abundant in the distal colon and rectum (59),
where they make up 16% of the total epithelial cell population
in mice (29). These specialized epithelial cells are thought to
play an important protective role in the intestine by synthesiz-
ing and secreting several mediators, including the mucin
MUC2 (46) and the small peptide trefoil factor 3 (TFF3) (63).
MUC2 (in mice, Muc2) is a high-molecular-weight glycopro-
tein that is stored within granules in the apical compartment of
the cell. Under basal conditions or under the influence of host
or bacterial stimuli (14), goblet cells release MUC2-containing
granules into the lumen, where they hydrate and form the
structural basis for the mucus gel layer overlying the intestinal
epithelium (69). This mucus layer plays important physiologi-
cal roles in the gut; it simultaneously lubricates the intestinal
surface, limits passage of luminal molecules into the mucosa,
functions as a dynamic defensive barrier against enteric patho-
gens (14, 59), and acts as a substrate and niche which the
commensal flora can colonize and from which this flora can
derive nutrients (57). TFF3 (in mice, Tff3) is another goblet
cell-derived molecule belonging to a family of small cysteine-
rich secretory peptides that are expressed in a region-specific
manner throughout the gastrointestinal tract (63). A potent
inducer of cell migration and an inhibitor of apoptosis (64),
TFF3 plays a critical role in wound healing by promoting ep-
ithelial restitution following mucosal injury (45). In addition,
TFF3 is thought to synergize with colonic mucins to enhance
the protective barrier properties of the mucus layer against
bacterial toxins (36). Evidence of the importance of goblet cells
in maintaining overall health has come from studies of mice
lacking either Muc2 or Tff3. These mice are highly susceptible
to experimental colitis (45) or have profound defects in intes-
tinal homeostasis under basal conditions (70).
Considering the critical role that goblet cells appear to play
in host defense against enteric pathogens, the observation that
these cells are depleted during C. rodentium infection may
have important implications regarding the pathogenesis of this
infection, as well as infection by clinically important pathogens,
including Shigella (53, 60) and Campylobacter (37), where the
goblet cell depletion phenotype is also observed. At present,
whether the goblet cell depletion seen during C. rodentium
infection reflects the death or functional alteration of goblet
cells is not clear, nor has the expression of goblet cell media-
tors been assessed in this model. Similarly, it is not clear if this
pathology reflects direct infection and subversion of goblet cell
function by C. rodentium, perhaps in an attempt to bypass
mucosal defenses, or alternatively, if the goblet cell depletion
is mediated by the host as a currently cryptic form of host
defense. We hypothesized that goblet cell function during C.
rodentium infection is subject to modulation by both the patho-
gen and components of the hosts immune system. With this
hypothesis in mind, the current study addressed the mecha-
nisms underlying the intestinal goblet cell depletion that occurs
during C. rodentium infection.
MATERIALS AND METHODS
Mice. Six- to 8-week-old female C57BL/6 mice and Rag1 knockout (KO) mice
(with a C57BL/6 background) were purchased from Jackson Laboratories (Bar
Harbor, ME). Mice were kept in sterilized, filter-topped cages, handled in tissue
culture hoods, and fed autoclaved food and water under specific-pathogen-free
conditions. Sentinel animals were routinely tested for common pathogens. The
GOBLET CELLS AND C. RODENTIUM INFECTION 797
protocols employed were approved by the University of British Columbias
Animal Care Committee and were in direct accordance with guidelines drafted
the Canadian Council on the Use of Laboratory Animals.
Bacterial strains and infection of mice. Mice were infected by oral gavage with
0.1 ml of an overnight culture in Luria broth containing approximately 2.5 108
CFU of wild-type C. rodentium (formerly Citrobacter freundii biotype 4280 strain
DBS100) (33). For transmission electron microscope (TEM) studies, mice were
also infected with a mutant escN C. rodentium strain lacking a functional T3SS
(12).
Tissue collection. Uninfected control mice or mice at days 6 and 10 postin-
fection (p.i.) were anesthetized with Halothane and killed by cervical dislocation,
and colons resected for further analysis; the colons were divided in half to
separate the proximal and distal portions. Tissues were immediately placed in
10% neutral buffered formalin (Fisher) for histological studies, or they were
placed in RNA later (Qiagen) and stored at 86C for subsequent RNA extrac-
tion or in 4% paraformaldehyde for subsequent freezing and cryosectioning. The
paraformaldehyde-fixed tissues were washed in phosphate-buffered saline (PBS),
incubated in 20% sucrose in PBS overnight at 4C, and then embedded in
Shandon Cryomatrix embedding medium (Thermoelectron Corporation), and
the mold was frozen by partial immersion in liquid N2-precooled 2-methylbutane
and stored at 20C until it was used.
Bacterial counting. Whole mouse colons, including stools, were washed thor-
oughly in PBS (pH 7.4), placed in 1.5 ml of PBS, and homogenized at 19,000 rpm
for 45 s using a Polytron homogenizer (Kinematica). Tissue homogenates were
serially diluted in PBS, plated onto Luria broth agar plates, and incubated
overnight at 37C, and bacterial colonies were counted the following day.
RNA extraction and quantitative RT-PCR. Colon tissues stored in RNAlater
(Qiagen) at 86C were thawed on ice and weighed, and the total RNA was
extracted using a Qiagen RNeasy kit by following the manufacturers instruc-
tions. Tissues were homogenized in 1 ml of buffer RLT (supplied with the Qiagen
RNeasy kit) using a Polytron homogenizer for 1 min at 26,000 rpm. Total RNA
was quantified using a Bio-Rad SmartSpec (Bio-Rad), and 1 to 2 g of RNA was
reverse transcribed using a Qiagen Omniscript reverse transcription (RT) kit
(Qiagen) according to the manufacturers instructions. cDNA was diluted 1:25 in
RNase- and DNase-free H2O, and 5 l was added to a 15-l PCR mixture.
Conventional semiquantitative PCR was carried out with an Eppendorf Master-
cycler, using the primers for murine Muc2, Tff3, or -actin as a housekeeping
control. The sequences of all primer sets used, the PCR conditions, and the cycle
numbers are shown in Table 1. Agarose gels were stained with SYBR Safe DNA
gel stain (Molecular Probes) and visualized with a Chemi Doc XRS system
(Bio-Rad). Densitometric analysis was carried out using ImageJ software 1.38x
(downloaded from the National Institutes of Health website [http://rsb.info.nih
.gov/ij/download.html]). For quantitative PCR, Bio-Rad Supermix was used at a
1:2 dilution, and real-time PCR was carried out using a Bio-Rad MJ Mini-
Opticon. Quantitation was carried out using GeneEx Macro OM 3.0 software,
which employs the 2Ct method for real-time quantification of gene expression.
Melting point analysis confirmed the specificity for each of the PCRs, and the
PCR efficiency for each of primer set was incorporated into the final calculations.
Histological staining. Briefly, 5-m paraffin sections were deparaffinized by
heating them at 55 to 65C for 10 min, cleared with xylene, and rehydrated
through an ethanol gradient to water. For periodic acid-Schiff (PAS) staining,
standard histological techniques were used. For immunostaining, either rabbit
polyclonal antisera that recognized the murine colonic mucin Muc2 (1:50; a gift
from Jan Dekker), rabbit polyclonal antisera generated against rat Tff3 (1:200; a
gift from D. Podolsky), or rat antisera against C. rodentium Tir (1:500; a gift from
W. Deng) were used as the primary antibody. The primary antibodies were
diluted in PBS containing 1% bovine serum albumin. For immunoperoxidase
staining, antigen retrieval was performed by incubating deparaffinized, rehy-
drated slides in 10 mM citric acid (pH 6.0) at 90 to 100C for 20 min, followed
by cooling to room temperature. Horseradish peroxidase-linked goat anti-rabbit
immunoglobulin G (IgG) (1:200; Genetex) was used as the secondary antibody.
All antibody incubations were carried out in the presence of 0.2% Triton X-100
(Sigma) to facilitate cell permeabilization. The immunoreaction was developed
using SigmaFast DAB substrate (Sigma). Stained sections were washed in water,
counterstained with Gills hematoxylin, dehydrated using ethanol, cleared using
xylene, and mounted using Entellan (EM Biosciences). For double-immunofluo-
rescence studies with anti-Muc2 and anti-Tir, no permeabilization or antigen
retrieval methods were used to minimize staining for untranslocated Tir still
present within the bacteria. To obtain frozen sections, 6-m sections were cut,
placed onto Superfrost/Plus slides (Fisher), and subsequently stained with rat
anti-mouse CD3 (clone 17A2; 1:300; BioLegend) overnight at 4C or with rat
anti-mouse CD45R/B220 (clone RA3-6B2; 1:100; Becton Dickinson) for 1 h at
room temperature. Immunofluorescent labeling for all stains was carried out
798 BERGSTROM ET AL.
TABLE 1. Primer sets and PCR conditions used in this study
Primera
Target mRNA
Direction Sequence
Muc2 Forward 5-CTGACCAAGAGCGAACACAA-3
Reverse 5-CATGACTGGAAGCAACTGGA-3
Tff3c Forward 5-CAGATTACGTTGGCCTGTCTCC-3
Reverse 5-ATGCTTGCTACCCTTGGACCAC-3
TNF-d Forward 5-ATGAGCACAGAAAGCATGATC-3
Reverse 5-TACAGGCTTGTCACTCGAATT-3
IL-17A (IL-17)f Forward 5-GCTCCAGAAGGCCCTCAGA-3
Reverse 5-CTTTCCCTCCGCATTGACA-3
-Actin Forward 5-CAGCTTCTTTGCAGCTCCTT-3
Reverse 5-CTTCTCCATGTCGTCCCAGT-3
a
The same primer sets were used for end point and quantitative PCR experiments described in text.
b
In all PCR experiments there was an initial denaturation step of 95C for 5 min before PCR cycling, and in end point PCR experiments there was an extension
step of 72C for 10 min after the final cycle.
c
Tff3 primer sequences were obtained from reference 27.
d
TNF- primer sequences were obtained from reference 62.
e
NA, not applicable.
f
IL-17 primer sequences were obtained from reference 23.
with the appropriate secondary antibody using AlexaFluor 488-conjugated goat
anti-rat IgG, AlexaFluor 568-conjugated goat anti-rabbit IgG, or AlexaFluor
568-conjugated goat anti-rat IgG (Invitrogen). Tissues were mounted using Pro-
Long Gold Antifade (Invitrogen) that contained 4,6-diamidino-2-phenylindole
(DAPI) for DNA staining. Sections were viewed at 350, 488, and 594 nm with a
Zeiss AxioImager microscope. Images were obtained using a Zeiss AxioImager
microscope equipped with an AxioCam HRm camera operating through Axio-
Vision software (version 4.4).
Quantitative histological studies. (i) Goblet cell enumeration. PAS- and he-
matoxylin-stained sections prepared at various time points were photographed,
and the total numbers of epithelial cells and PAS-positive cells were determined
for 20 to 30 longitudinally sectioned crypts per section. The number of goblet
cells was expressed as the total number of PAS-positive cells per 100 epithelial
cells. Phenotypically mature goblet cells were assessed based on the intensity of
staining, the size of the apical region, the location on the crypt base-to-surface
axis, and morphology, similar to the method described by Katz and coworkers
(30).
(ii) Quantification of infected crypts. For crypt infection studies involving
double immunostaining for Muc2 and Tir, crypts exhibiting goblet cell depletion
were defined as crypts with less than three Muc2-positive phenotypically mature
goblet cells and with little Muc2 in the crypt lumen. Crypts that did not exhibit
goblet cell depletion were defined as crypts with three or more strongly Muc2-
positive phenotypically mature goblet cells and an intense secreted Muc2 signal
in the crypt lumen. Positively infected crypts were defined as crypts that were
positive for Tir staining on the cells of the surface epithelium and on cells in the
upper one-third of colonic crypts.
TEM. Mouse colons were immersed for 3 h in a fixative containing 0.1 M
sodium cacodylate, 1.5% paraformaldehyde, and 1.5% glutaraldehyde (pH 7.3).
Following fixation, the material was postfixed for 1 h on ice in 1% osmium
tetroxide in 0.1 M sodium cacodylate (pH 7.3) and stained with 0.1% uranyl
acetate. The material was dehydrated using an ascending alcohol series, followed
by incubation in propylene oxide. The blocks were then left in a 1:1 solution of
propylene oxide-Polybed overnight. The material was embedded in 100% Poly-
bed, and the resin was polymerized at 60C for 24 h. Sections were viewed and
photographed using a Philips 300 electron microscope operated at 60 kV.
In vivo imaging: bacterial strains and generation of bioluminescent C. roden-
tium. Bioluminescent strains of C. rodentium were constructed by introducing
plasmid pT7 (E. A. Meighen, Department of Biochemistry, McGill University)
carrying the entire lux operon from Photorhabdus luminescens. Bioluminescent
colonies were selected on Luria broth agar plates supplemented with 100 g/ml
of ampicillin and were screened using a model 1420 Victor3V multilabel counter
(PerkinElmer). For in vivo tissue imaging of bioluminescent C. rodentium at 6
and 10 days p.i., mice were anesthetized and then euthanized. The colons and
ceca were removed and opened lengthwise so that the lumen was exposed, and
INFECT. IMMUN.
No. of cycles
PCR cycle conditions
(endpoint
(denaturation/annealing/extension)b
PCR only)
94C for 30 s/55C for 30 s/72C for 45 s 23
94C for 30 s/60C for 30 s/72C for 30 s 30
94C for 30 s/59C for 30 s/72C for 45 s NAe
94C for 30 s/60C for 30 s/72C for 30 s NA
94C for 30 s/55 to 60C for 30 s/72C for 30 s 23 or 30
the tissue was washed with sterile PBS. Tissues were then placed in a light-tight
specimen chamber that is part of an in vivo imaging system (IVIS; Xenogen,
Alameda, CA). The bacterial signals were quantified using the software program
LIVING IMAGE (Xenogen) as an overlay on Igor (Wavemetrics, Seattle, WA).
To determine the anatomical location, a pseudocolor image showing the light
intensity (blue [least intense] to red [most intense]) was generated using LIVING
IMAGE software and was superimposed over the grayscale reference image.
Immune cell reconstitution of Rag1 KO mice. The adaptive immune system
was reconstituted in Rag1 KO mice using splenic and mesenteric lymph node
(MLN) populations of T and B lymphocytes as previously described (66). In
brief, wild-type immunocompetent mice were euthanized, and their spleens and
MLNs were aseptically removed. Spleens and MLNs were placed in RPMI
medium with 10% fetal bovine serum, mashed to a pulp with the rubber end of
the plunger from a 1.0-ml syringe, and then forced through a 70-m-pore-size
filter (BD Biosciences), generating a single-cell suspension. Cells were spun
down and resuspended in red blood cell lysis buffer (155 mM NH4Cl, 1 mM
KHO3, 0.01 mM Na2EDTA-2H2O; pH 7.4) for 5 min to lyse the red blood cells.
Following two washes with RPMI medium, cells were pelleted and then resus-
pended in PBS. Cells were then counted, and viability was analyzed by trypan
blue exclusion. Recipient Rag1 KO mice were then inoculated via the tail vein
with 2 108 viable mononuclear cells. Mice were left for 6 weeks and then tested
for the success of reconstitution by staining colonic tissue sections with isolated
lymphoid follicles for the presence of T lymphocytes using the marker CD3 and
for the presence of B lymphocytes using the marker B220.
Statistical analysis. Statistical significance was calculated by using either a
two-tailed Student t test or the Mann-Whitney t test as indicated, with assistance
from GraphPad Prism software (version 4.00; GraphPad Software, San Diego,
CA) (www.graphpad.com). A P value of 0.05 was considered significant. The
results were expressed as means and standard errors of the means or as means
and standard deviations as indicated below.
RESULTS
Depletion of mucus-containing goblet cells correlates with
peak bacterial colonization during C. rodentium infection. Pre-
vious studies have indicated that C. rodentium infection causes
goblet cell depletion within the colons of infected mice (38);
however, the timing of this pathology and how the changes are
related to C. rodentium colonization have not been addressed.
To begin, we examined the time course and spatial character-
istics of C. rodentium colonization in C57BL/6 mice (Fig. 1).
VOL. 76, 2008
FIG. 1. C. rodentium infection peaks at day 10 p.i. in C57BL/6 mice.
Each symbol indicates the mean for three independent infections, each
with three mice per time point. The error bars indicate standard
deviations.
When total pathogen burdens were assessed, C. rodentium was
found to heavily colonize the colon by day 6 p.i., with the
number of bacteria peaking at day 10 p.i. By day 14 p.i, the
number of C. rodentium cells began to drop, and the infection
essentially cleared by day 21 p.i., with only a few thousand
bacteria remaining (Fig. 1 and data not shown). In addition,
consistent with recent studies of C. rodentium infection dynam-
ics by other groups (76), we found that bioluminescent C.
rodentium cells were predominantly localized in the cecum and
distal half of the colon, and little infection was detected in the
proximal colon at days 6 and 10 p.i. (not shown).
We next examined whether the localization of C. rodentium
influenced colonic pathology and the reported goblet cell de-
pletion by comparing tissue sections taken from the distal and
proximal colons of uninfected and infected mice at days 6 and
10 p.i. Using the PAS staining technique to stain goblet cell
mucins, only minimal changes in PAS staining were observed
in any region of the cecum and colon at day 6 pi (data not
shown), and there was little overt change in PAS staining of
goblet cells in the cecum (data not shown) or in the proximal
colon (Fig. 2A and B) at day 10 p.i. However, a dramatic
reduction in PAS staining was observed in tissue sections of
samples taken from the distal colon at day 10 p.i. (Fig. 2C and
D). While a small number of crypts still contained phenotyp-
ically mature mucin-filled goblet cells, characterized by a swol-
len (PAS-stained) apical region and a tapered basolateral com-
partment containing the nucleus, in most colonic crypts there
were almost none of these cells (Fig. 2E). However, examina-
tion under higher magnification revealed that these tissues
contained many cells that exhibited weak PAS staining in their
apical compartments (Fig. 2D). Compared to the cells that
stained strongly with PAS stain (Fig. 2E), these weakly stained
cells were smaller and exhibited a more columnar morphology
(Fig. 2F), reminiscent of the hypotrophic goblet cell pheno-
type observed in the interleukin-10 (IL-10)-deficient mouse
model of spontaneous colitis (41). When we determined the
number of PAS-positive cells per 100 epithelial cells at day
10 p.i., we observed a modest but statistically significant de-
crease in the size of the total PAS-positive population (P
0.036) compared to uninfected controls. However, when we
determined the number of cells possessing the phenotypically
mature goblet cell morphology that stained strongly with PAS
GOBLET CELLS AND C. RODENTIUM INFECTION 799
stain (Fig. 2F), we found that there was a substantially greater
reduction in the size of this population (P 0.002) (Table 2).
As the infection progressed, the hypotrophic goblet cell phe-
notype was still predominant at day 14 p.i., but the number of
large mucin-filled goblet cells began to rebound again by day
21 p.i. (data not shown). Thus, our data indicate that C. ro-
dentium infection leads to a reduction in PAS staining, poten-
tially due to a decrease in mucin glycoprotein content of goblet
cells, rather than an actual loss of the goblet cell lineage. As a
result, we observed only a modest decrease in the total number
of goblet cells, and the dramatic reduction in the large goblet
cells that appeared to be mature explained the previously de-
scribed goblet cell depletion.
Goblet cell-specific gene expression is reduced during infec-
tion. While infection led to a decrease in overall PAS staining
and a reduction in the number of phenotypically mature goblet
cells, it remained unclear whether these effects solely reflected
a reduction in mucin expression and production, which directly
impacts goblet cell morphology (70), or whether they were also
accompanied by altered production of additional goblet cell-
specific mediators. To assess this, the expression of the major
goblet cell mucin Muc2, as well as Tff3, was assayed by semi-
quantitative RT-PCR. As assessed by densitometry of end
point PCR products shown in Fig. 3A, there were slight but
nonsignificant reductions in expression of Muc2 and Tff3
mRNA by day 6 p.i., but there were notably larger and signif-
icant decreases in expression of Muc2 and especially Tff3
mRNA by day 10 p.i. (Fig. 3A, graph). To determine whether
C. rodentium infection impacted Muc2 and Tff3 protein expres-
sion, we performed immunoperoxidase staining for Muc2 and
Tff3 of formalin-fixed paraffin-embedded sections of distal co-
lons from control and infected mice. In control mice, Muc2
protein expression and Tff3 protein expression were abundant
in goblet cells, although the expression patterns were distinct.
While Muc2-positive cells were observed from the base to the
luminal surface of colonic crypts (Fig. 3B, upper left panel),
Tff3-positive goblet cells were confined predominantly to the
upper half of the crypts (Fig. 3B, lower left panel), in agree-
ment with previous reports (52). However, consistent with our
results showing that infection causes down-regulation of Muc2
and Tff3 mRNA, we observed a dramatic reduction in staining
for both the Muc2 and Tff3 proteins at day 10 p.i. throughout
crypts having the depleted goblet cell phenotype (Fig. 3B,
upper and lower right panels, respectively), although there was
still faint Muc2 staining in scattered crypt cells. Thus, our data
show that infection leads not only to reduced mucin (Muc2)
expression and production, resulting in decreased PAS stain-
ing, but also to reduced production of the goblet cell-specific
mediator Tff3; taken together, the results suggest that goblet
cell function is profoundly altered during infection. Consider-
ing that this pathology was most pronounced in regions of the
colon heavily infected by C. rodentium, we next addressed
whether direct infection might play a role in this modulation of
goblet cell function.
C. rodentium directly interacts with goblet cells in vivo. It
remains unclear whether C. rodentium is able to infect cells
other than enterocytes in vivo, such as goblet cells. To address
this question, we first assessed whether C. rodentium cells in
the infected colons were close to or in contact with goblet cells.
Here, we focused on day 6 p.i., when colonization in the distal
800 BERGSTROM ET AL. INFECT. IMMUN.

FIG. 2. Depletion of mucus-containing goblet cells occurs in the distal colon and is pronounced when the C. rodentium burdens peak. (A to
D) PAS staining of formalin-fixed paraffin-embedded tissue sections obtained from the proximal (A and B) and distal (C and D) colons of both
nave (uninfected) mice (A and C) and mice at day 10 p.i (B and D). There was a reduction in overall PAS staining within many crypts in distal
colons at day 10 p.i., although scattered PAS-positive cells were still evident (arrows in panel D). Bars 100 m. DIS, distal colon; PROX,
proximal colon. (E and F) Representative images of PAS-positive cells at day 10 p.i. exhibiting a large mucin-filled goblet cell morphology (arrows
in panel E) or a mucin-depleted columnar morphology (arrows in panel F). The images are representative of at least three independent infections
with two or three mice per time point.

colon was established but phenotypically distinct goblet cells
were still relatively abundant (Table 2). To identify any poten-
tial C. rodentium-goblet cell interactions, we a performed a
dual immunostaining analysis to look for colocalization of
Muc2-positive goblet cells and the locus of enterocyte efface-
ment-encoded virulence factor Tir, which is expressed on the
apical surface of cells only following direct infection by C.
rodentium (13). As shown in Fig. 4A, Tir staining was found on
the surface epithelium and progressed down some crypts,
where Muc2-positive goblet cells also resided. To confirm the
colocalization of Tir with Muc2-positive cells, we examined
tissues at a higher magnification and found positive Tir staining
on the apical surface of enterocytes, as well as on cells that
were positive for Muc2 (Fig. 4B). Next, we examined whether
C. rodentium cells were adherent to colonic goblet cells using
TEM. TEM analysis showed that C. rodentium cells were ad-
herent to the apical plasma membrane of goblet cells (Fig. 4C
and D). Although goblet cells have a morphology and function
VOL. 76, 2008 GOBLET CELLS AND C. RODENTIUM INFECTION 801

TABLE 2. Goblet cell enumeration in the distal colon of C57BL/6 that are very distinct from the morphology and function of
mice following C. rodentium infectiona their absorptive enterocytic counterparts, they still contain in-
No. of phenotypically tact, albeit shortened and less dense, microvilli (58). Notably,
Total no. of PAS-positive
mature goblet cells/ however, C. rodentium-associated goblet cells also exhibited
Days p.i. cells/100 epithelial cells
100 epithelial cells
(avg SD) evidence of microvillar effacement, a hallmark of A/E lesion
(avg SD)
formation (Fig. 4C). Interestingly, we also occasionally ob-
Uninfected control 32.9 7.9 17.9 2.8
6 28.1 7.3 14.0 4.6 served bacteria within the apical granule mass of goblet cells,
10 21.6 7.3b 7.7 3.2b suggesting that C. rodentium may be internalized by some gob-
a
C. rodentium infection results in depletion of phenotypically mature goblet
let cells (Fig. 4C). In contrast, when mice were infected with
cells in the distal colon during times corresponding to peak infection. Enumer- escN C. rodentium, which lacks a functional T3SS, no C.
ation was performed using PAS- and hematoxylin-stained tissue sections of distal rodentium cells were found to be adherent to goblet cells, nor
colons by determining the number of distinctly PAS-positive cells per 100 epi-
thelial cells, differentiating the cells that had a mature goblet phenotype. The was any effacement of goblet cell (or enterocyte) microvilli
data are values from three independent infections, with each infection group observed in these mice (Fig. 4E). To determine the frequency
containing three mice per time point.
b
P 0.05, as determined by the Mann-Whitney t test. at which goblet cells were infected, we determined the propor-

FIG. 3. C. rodentium infection results in reduction of Muc2 and Tff3 gene expression. (A) Representative RT-PCR analysis of Muc2 and Tff3
gene expression in distal colonic tissues of nave C57BL/6 mice or mice at days 6 and 10 p.i. -Actin was used as a loading control. Differences
in levels of expression relative to the levels in nave (uninfected) mice after normalization to -actin were determined by densitometric analysis,
as shown in the graph on the right. The bars indicate the means for three independently infected mice per time point. The error bars indicate the
standard errors of the means. Asterisk, P 0.05; ns, not significant (P 0.05), as determined by Students t test. (B) Immunoperoxidase staining
for Muc2 (upper panels) and Tff3 (lower panels) protein (brown) in serial sections of distal colons from either nave mice (left panels) or mice
at day 10 p.i. (right panels). In control mice, Muc2-positive cells are present from the crypt base to the luminal surface, whereas Tff3 expression
is restricted to the top half of the crypts (arrows). The arrowheads indicate cells that are coexpressing the Muc2 and Tff3 proteins. Note the lack
of staining in intact crypts at day 10 p.i. (arrows in left panels). Bar 50 m.
 FIG. 4. C. rodentium directly infects colonic goblet cells in vivo. (A) Immunofluorescence staining for the C. rodentium translocated effector Tir (red),
goblet cell-specific Muc2 (green), and DNA (blue) in distal colonic tissues at day 6 p.i. Tir staining is present in Muc2-positive cells (arrowheads) and
Muc2-negative cells within the surface epithelium, progressing down the length of some crypts (arrow). (B) Magnified image of Tir (red) staining (arrows)
on the apical surface of cells staining strongly for Muc2 (green) in the apical compartment and exhibiting distinct goblet cell morphology. GA, goblet cell
apical compartment; GN, goblet cell nucleus; E, enterocyte; L, lumen. Original magnification, 1,000. (C to E) TEM micrographs of distal colons of
C57BL/6 mice taken at day 7 (C) and day 11 (D) following infection with wild-type C. rodentium and at day 7 following infection with escN C. rodentium
(E). C. rodentium is in direct contact with goblet cells in panels C and D (black arrows). In panel C, effacement of microvilli (black arrows) on a goblet
cell and internalization of bacteria into the apical granule mass of the goblet cell (white arrow) can be seen. In panel E, no infection of goblet cells and
intact microvilli (arrow) can be seen following infection with escN C. rodentium. (F) Graph showing the proportions of Muc2-positive (goblet) cells and
Muc2-negative (nongoblet) cells of the surface epithelia that were positive for Tir staining at day 6 p.i. The bars indicate the averages for three mice from
two independent infections. The error bars indicate the standard errors of the means. Asterisk, P 0.05, as determined by Students t test.

802
VOL. 76, 2008 GOBLET CELLS AND C. RODENTIUM INFECTION 803

FIG. 5. C. rodentium associates with crypts that are strongly positive for Muc2 at day 10 p.i. (A) Dual immunofluorescence staining for C.
rodentium Tir (red) and Muc2 (green) in a distal colon at day 10 p.i. Tir staining (white arrow) is associated with crypts strongly positive for Muc2
(white arrowhead), but little if any Tir staining (yellow arrow) is associated with crypts that are weakly positive for Muc2 (yellow arrowhead). Note
the frequent colocalization of Tir with Muc2-postive cells and luminal Muc2. (B) Proportions of crypts exhibiting depletion of Muc2-positive goblet
cells that are positive for Tir compared to crypts that are not depleted of Muc2-postive goblet cells. The bars indicate the averages for 30 crypts
counted within colonic sections from two individual mice, and the error bars indicate standard deviations.

tions of Muc2-positive (goblet) cells and Muc2-negative (en-
terocyte, nongoblet) cells that were positive for Tir staining on
the surface epithelium, where most C. rodentium cells were
localized. Our results showed that there was a moderate but
significant difference between the proportion of infected
Muc2-positive cells and the proportion of infected Muc2-neg-
ative cells, in that fewer than 50% of Muc2-positive cells were
positive for Tir staining, whereas just over 60% of Muc2-
negative cells were positive for Tir staining (Fig. 4F). These
results indicate that along with colonocytes, goblet cells are
subject to direct infection by A/E pathogens in vivo, although
Muc2-negative cells are more frequently observed to be in-
fected.
C. rodentium predominantly associates with crypts that do
not exhibit goblet cell depletion. Our next goal was to examine
the interactions of C. rodentium with colonic crypts at day
10 p.i. in order to determine whether C. rodentium could di-
rectly mediate the loss of mature goblet cells. If this were the
case, we would expect to find C. rodentium associated with
hypotrophic goblet cells, which we tested by immunofluores-
cently staining for both Tir and Muc2. Strikingly, only minimal
Tir staining was associated with crypts exhibiting hypotrophic
goblet cells, and no C. rodentium cells were identified in prox-
imity to these cells (Fig. 5A). To clarify the relationship be-
tween C. rodentium infection and goblet cell depletion, we
identified individual colonic crypts that were infected by C.
804 BERGSTROM ET AL.
rodentium (i.e., Tir positive) and, as described in Materials and
Methods, differentiated between crypts that exhibited a weak
overall signal for Muc2, indicative of the goblet cell depletion
phenotype, and crypts that displayed a strong signal for Muc2,
indicating that goblet cell depletion did not occur. Interest-
ingly, we found that the proportion of crypts that were infected
and exhibited goblet cell depletion was more than threefold
lower than the proportion of crypts that did not display an
overt loss of goblet cells (Fig. 5B). This trend was even more
dramatic at day 14 p.i., when in virtually all crypts Muc2 ex-
pression was markedly reduced and there was little if any
associated Tir staining (data not shown). These results suggest
that there is a surprising inverse relationship between C. ro-
dentium infection of crypts and goblet cell depletion. It should
be noted that we did observe one or two crypts per colonic
section that were heavily invaded by C. rodentium and were
strongly Tir positive from the surface epithelium to the crypt
base, yet were also depleted of Muc2-positive cells. However,
unlike the typical goblet cell-depleted crypts, these crypts in-
variably looked atrophic and/or necrotic, suggesting that the
loss of Muc2 staining in these crypts was due to destruction of
the crypt epithelium rather than to modulation of goblet cell
function. These data therefore demonstrate that aside from
occasional crypt destruction, the widespread goblet cell deple-
tion seen in colonic crypts during C. rodentium infection is
independent of direct bacterial contact or infection of the
altered goblet cells.
Rag1 KO mice do not suffer goblet cell depletion during C.
rodentium infection. The studies described above suggested
that goblet cell depletion is not a result of direct infection,
since it is frequently observed in uninfected crypts. When al-
ternative mechanisms were considered, the adaptive immune
response was shown to modulate goblet cell function during
infection by intestinal helminths (35, 48). Moreover, as previ-
ous studies have found that a robust adaptive immune re-
sponse to C. rodentium infection occurs by day 10 p.i. (26), we
tested whether the host adaptive immune response mediated
the observed reduction in the number of mature goblet cells
and Muc2 and Tff3 expression. Thus, Rag1 KO mice (lacking
mature T and B lymphocytes) were infected, and their goblet
cell responses were analyzed at day 10 p.i. As observed with
C57BL/6 mice, mucosal thickening associated with epithelial
hyperplasia was observed in Rag1 KO mice, consistent with
previous reports (66). However, compared with infected
C57BL/6 mice (Fig. 6B), no decrease in the number of mature
goblet cells was observed in the Rag1 KO mice at day 10 p.i.,
as assessed by PAS staining (Fig. 6D and 6E); in fact, there was
a modest trend toward increased numbers of PAS-positive cells
at day 10 p.i. compared to uninfected controls (Fig. 6E). More-
over, the majority of goblet cells in the Rag1 KO mice, at both
the base and surface of the crypts, had a highly differentiated
appearance and contained full mucin-filled apical granule
masses that were stained strongly with PAS stain (Fig. 6D).
However, as observed for immunocompetent mice in the stud-
ies mentioned above, we occasionally observed heavily infected
and atrophic crypts that exhibited reduced PAS staining, (Fig.
6D). The maintenance of overall goblet cell function within the
colons of infected Rag1 KO mice was further confirmed by
immunostaining for the Muc2 and Tff3 proteins. High levels of
both proteins were detected in infected Rag1 KO mice at day
INFECT. IMMUN.
10 p.i., and the staining patterns were similar to those observed
in uninfected colons (Fig. 7A). In addition, there was not a
significant decrease in Muc2 or Tff3 gene expression at day
10 p.i. compared with C57BL/6 mice (Fig. 7B). To determine
how the preservation of mature goblet cells is related to the
number of bacteria, we quantified the bacterial burdens at days
6 and 10 p.i. Consistent with previous reports (66), Rag1 KO
mice had greater bacterial burdens than C57BL/6 mice at day
10 p.i. (Fig. 6F), which indicates that the maintenance of goblet
cell number and function in the Rag1 KO mice was not due to
reduced numbers of bacteria in these mice and confirms that
direct C. rodentium infection plays a minor role in the overall
goblet cell depletion observed in C57BL/6 mice. These results
strongly suggested that T and/or B lymphocytes were involved
in the loss of mature goblet cells during C. rodentium infection.
Proinflammatory cytokine expression in C57BL/6 and Rag1
KO mice during C. rodentium infection. Because proinflamma-
tory cytokines, such as tumor necrosis factor alpha (TNF-),
have been implicated in causing goblet cell depletion in the
small intestine in a Salmonella enterica serovar Typhimurium
ligated ileal loop model (1), we compared TNF- gene expres-
sion in C57BL/6 mice and TNF- gene expression in Rag1 KO
mice to begin to address which host molecules are responsible
for the loss of mature goblet cells in the colon during infection.
As shown in Fig. 8, TNF- expression was induced in both
strains at day 10 p.i. but was induced to a greater extent in
C57BL/6 mice; however, the difference did not reach statistical
significance (P 0.0727). These results indicated that cyto-
kines besides TNF- are probably involved in the observed
changes in colonic goblet cells during C. rodentium infection.
We have previously shown that gamma interferon expression is
lower in Rag1 KO mice than in C57BL/6 mice during infection
(66), but recent studies have shown that IL-17A (IL-17), a
proinflammatory T-cell-derived cytokine that has been re-
ported to directly affect intestinal epithelial function (54), is
also upregulated in mice with C. rodentium-induced colitis
(42). Therefore, we compared IL-17 mRNA levels in C57BL/6
and Rag1 KO mice. As expected, we found that in infected
C57BL/6 mice, IL-17 mRNA levels were significantly increased
and were more than 100-fold greater than the levels in unin-
fected mice; in contrast, IL-17 expression in Rag1 KO mice did
not increase to levels greater than those in uninfected C57BL/6
control mice (Fig. 8), despite the greater numbers of bacteria
in the Rag1 KO mice (Fig. 6F). These results, coupled with
those of previous studies (66), show that effector T-cell cyto-
kines are highly expressed at times when goblet cell depletion
is apparent and may play a direct or indirect role in functional
modulation of goblet cells during infection.
Adoptive transfer of T and B lymphocytes rescues the goblet
cell depletion phenotype in Rag1 KO mice. While the results
described above indicated that a functional adaptive immune
system was important for meditating the observed goblet cell
depletion phenotype, we tested this hypothesis directly by re-
constituting Rag1 KO mice with T and B lymphocytes isolated
from spleens and MLNs of C57BL/6 mice (using PBS as a
negative control) and subsequently challenging them with C.
rodentium. At day 10 p.i., PBS-treated (nonreconstituted) mice
or mice reconstituted with T and B lymphocytes were eutha-
nized, and the distal colons were used for histological assess-
ment of goblet cells via PAS staining, as well as the presence of
VOL. 76, 2008 GOBLET CELLS AND C. RODENTIUM INFECTION 805

FIG. 6. C. rodentium infection does not result in goblet cell depletion phenotype in Rag1 KO mice. (A to D) Histology of PAS- and
hematoxylin-stained distal colonic sections taken from (A) nave C57BL/6 mice, (B) C57BL/6 mice at day 10 p.i., (C) nave Rag1 KO mice, and
(D) Rag1 KO mice at day 10 p.i. The mature PAS-positive cell population is still large in Rag1 KO mice at day 10 p.i. Heavily infected
mucin-depleted atrophic crypts are indicated by arrowheads. Original magnification, 100. Bar 100 m. (E) Quantitation of goblet cells in distal
colons from nave C57BL/6 and Rag1 KO mice or from infected mice at day 10 p.i. The bars indicate the mean numbers of goblet cells per 100
epithelial cells counted in PAS- and hematoxylin-stained sections of the distal colon. The error bars indicate standard errors of the means for at
least three independent infections with one to three mice per time point. Asterisk, P 0.05. (F) Comparison of numbers of bacteria in whole colons
of C57BL/6 mice (filled bars) and Rag1 KO mice (open bars) at day 10 p.i. The bars indicate the means from three independent infections, each
with two or three mice per time point. The error bars indicate the standard deviations. Asterisk, P 0.05, as determined by the Mann-Whitney
t test.

infiltrating lymphocytes via CD3 (T-cell) and B220 (B-cell)
staining. Consistent with the hypothesis that T and/or B cells
mediate the goblet cell depletion phenotype, we observed re-
duced overall PAS staining within crypts of the reconstituted
mice compared with crypts of nonreconstituted mice (Fig. 9A
and B). Examination of the T- and B-lymphocyte populations
within these tissues revealed that while B220-positive cells
were concentrated mainly in isolated lymphoid follicles found
only in reconstituted mice (data not shown), the goblet cell
depletion seen in the reconstituted mice was associated with a
prominent population of CD3-positive cells in the mucosa and
submucosa of the reconstituted mice (Fig. 9C) but not the
806 BERGSTROM ET AL. INFECT. IMMUN.

FIG. 7. Muc2 and Tff3 are abundantly expressed in Rag1 KO mice following C. rodentium infection. (A) Immunohistochemical staining for
Muc2 (upper panels) and Tff3 (lower panels) in the distal colons of nave mice (right panels) and infected mice (left panels). Note the abundance
of Tff3 and Muc2 in crypts that are evidently undergoing hyperplasia, with staining patterns similar to those of nave mice (arrows). Bars 50 m.
Original magnification, 200. (B) Quantitative RT-PCR analysis of Muc2 and Tff3 gene expression in distal colons from nave and infected
C57BL/6 and Rag1 KO mice. Each bar indicates the mean relative expression for four mice from three independent infections compared to nave
mice of the same strain, which were assigned an arbitrary expression value of 1.00. Genes encoding Muc2 and Tff3 were both expressed at
significantly higher levels in Rag1 KO mice than in C57BL/6 mice at day 10 p.i. All samples were normalized to -actin. The error bars indicate
the standard errors of the means. Asterisk, P 0.05, as determined by the Mann-Whitney t test.

nonreconstituted mice (Fig. 9D). Moreover, the reduced PAS significant reduction in Tff3 mRNA expression compared to
staining and the large CD3-positive cell population in distal infected nonreconstituted mice (Fig. 9E). Lastly, similar to the
colons of infected reconstituted mice at day 10 p.i. were also results for infected C57BL/6 mice, we observed significantly
accompanied by a reduction in Muc2 mRNA expression and a greater expression of IL-17 mRNA and a slight, nonsignificant
VOL. 76, 2008
FIG. 8. Cytokine gene expression during C. rodentium infection:
quantitative RT-PCR analysis of TNF- and IL-17 expression in distal
colons taken from C57BL/6 and Rag1 KO at day 10 following C.
rodentium infection. Induction of TNF- was moderately increased in
both strains at day 10 p.i.; the levels in C57BL/6 mice were increased,
but the difference was not significant (P 0727). However, IL-17 gene
expression was significantly increased in infected C57BL/6 mice com-
pared to the minimal IL-17 expression observed in infected Rag1 KO
mice. All samples were normalized to -actin. The bars indicate the
means for eight C57BL/6 mice and four Rag1 KO mice from three
independent infections. The error bars indicate the standard errors of
the means. Asterisk, P 0.05, as determined by the Mann-Whitney t
test.
increase in TNF- expression in reconstituted mice compared
to infected nonreconstituted mice at day 10 p.i. (Fig. 9F).
Together, these results show that the adaptive immune re-
sponse, presumably through the actions of T lymphocytes,
plays a central role in regulating goblet cell gene expression,
with downstream effects on goblet cell function and morphol-
ogy during infection by a noninvasive enteric pathogen.
DISCUSSION
While goblet cell depletion during C. rodentium infection
has been reported previously (38), our studies are the first
studies to directly characterize this pathology and specifically
address the underlying mechanisms. We show here that during
C. rodentium-induced colitis, there is a significant reduction in
the size of the mature goblet cell population in the distal colon
during periods corresponding to heavy pathogen burden, and
this is associated with reduced expression of the goblet cell-
specific genes encoding Muc2 and Tff3 at the mRNA level and,
histologically, at the protein level. We also demonstrate that in
addition to colonocytes, goblet cells represent a target of C.
rodentium infection, as the bacteria directly interact with and
infect goblet cells. However, we show that only a portion of
goblet cells in the murine colon are infected, and it is likely the
host adaptive immune system that mediates the majority of the
goblet cell depletion that occurs during C. rodentium infection.
The relationship between the histological changes in the
goblet cell population and the alterations in goblet cell-specific
gene expression seen in this model was intriguing and provided
insight into how the immune system modulates goblet cells
during infection. While infection did lead to a significant re-
duction in the total number of PAS-stained mucin-containing
cells relative to other non-carbohydrate-producing cells, this in
itself is unlikely to account for the dramatic reduction in Muc2
and Tff3 immunostaining that was observed. Rather, the prev-
alence of hypotrophic PAS-positive cells may reflect an altered
GOBLET CELLS AND C. RODENTIUM INFECTION 807
state of goblet cell function that occurs in the distal colon
during infection. As initially suggested by Makkink et al. for
another model of colitis showing goblet cell depletion (41), the
reduced Muc2 protein expression may have been directly re-
sponsible for the hypotrophic goblet cell phenotype that we
observed, since Muc2 is the main morphological determinant
of goblet cell morphology (41, 70). In fact, mice genetically
deficient in this mucin lack phenotypically distinct goblet cells,
yet they are strongly reactive for other goblet cell markers, like
Tff3 (70). However, in addition to reduced Muc2 levels, we also
found a marked reduction in Tff3 protein levels in C57BL/6
mice but not in Rag1 KO mice. Because Tff3 is thought to be
expressed primarily by mature goblet cells (51), this suggests
that the modulation of goblet cell function may reflect immune
system-mediated impairment of the ability of immature goblet
cells to fully differentiate into mature goblet cells.
The biological consequences of the functional modulation of
goblet cells are currently unclear. In some respects it is para-
doxical that the host immune system reduces expression of
Muc2 and Tff3, considering the protective roles that these
goblet cell-derived proteins play in the intestine. Muc2 was
recently shown to be important in maintaining overall mu-
cosal homeostasis, ultimately suppressing spontaneous tu-
mor growth (70), as well as colitis development (68). In vitro
studies have reported that intestinal mucins prevent EPEC
attachment (40, 56) to epithelial cells, as well as bacterial
translocation (17) across epithelial cell monolayers. Moreover,
in mice lacking Tff3 the ability to heal colonic injury induced by
the cytotoxic agent dextran sodium sulfate is impaired, and the
mice suffer an exaggerated and fatal colitis as a result (45).
Thus, down-regulating both these genes could compromise the
host defense when an animal is challenged by an enteric bac-
terial pathogen.
On the other hand, the impact of the loss of these goblet
cell-derived factors may be minimal or even beneficial to the
host during infection with enteric bacteria. For example, sim-
ilar to commensal species (57), enteropathogenic bacteria such
as Yersinia enterocolitica have been demonstrated to use car-
bohydrate-laden mucins as a food source (43), and S. enterica
serovar Typhimurium is thought to bind to intestinal mucins to
facilitate colonization (73). Thus, reducing mucin production
might be important for reducing energy sources for the patho-
genic bacteria, as well as for reducing potential anchoring sites
required for initial colonization. Indeed, as C. rodentium con-
stitutes approximately 90% of the bacterial flora at the peak of
infection (38), reducing a potential nutrient source, such as
mucins, may inhibit pathogen growth. In this regard, the robust
mature goblet cell population observed in infected Rag1 KO
mice may facilitate the increased bacterial burdens that are
observed within the colons of these mice and perhaps even the
previously described impaired clearance of the pathogen (66).
The mechanisms underlying the immune system-driven loss
of mature Muc2- and Tff3-expressing goblet cells are currently
unclear; however, goblet cell depletion has also been observed
in human colonic tissues in association with hyperproliferation
of colonic crypts, in a manner dependent upon activation of T
cells within the lamina propria (15). While infection-induced
alterations in the turnover of the colonic epithelium may be
involved in the observed goblet cell depletion, we observed
that both C57BL/6 mice and Rag1 KO mice showed evidence
808 BERGSTROM ET AL. INFECT. IMMUN.

FIG. 9. Adaptive transfer of T and B cells into Rag1 KO mice restored the goblet cell depletion phenotype during C. rodentium infection. (A
and B) PAS staining of distal colons removed on day 10 p.i. from Rag1 KO mice that were reconstituted with T and B lymphocytes from nave
C57BL/6 mice (Recon) (A) or with PBS (nonreconstituted control) (B). The arrows indicate intact crypts exhibiting reduced PAS staining and
numerous hypotrophic goblet cells. The asterisks indicate crypts magnified in the insets. Note that reduced PAS staining in PBS-treated mice
(B) was associated mainly with crypts that were severely disrupted due to bacterial overload (arrowheads), which was not observed in infected
reconstituted mice. Original magnification, 100. Bar 100 m. (C and D) Anti-CD3 staining showing greater numbers of infiltrating
CD3-positive cells in the lamina propria and submucosa of reconstituted Rag1 KO mice (arrows) (C) than in the lamina propria and submucosa
of PBS-treated mice (D). Original magnification, 200. Bar 50 m. (E and F) Quantitative RT-PCR analysis of expression of genes encoding
Muc2 and Tff3 (E) or TNF- and IL-17 (F) in distal colons of reconstituted or nonreconstituted (PBS-treated) mice at day 10 p.i. All samples were
normalized to -actin. The bars indicate the means for seven reconstituted mice and three nonreconstituted mice from two independent infections.
The error bars indicate the standard errors of the means. Asterisk, P 0.05, as determined by the Mann-Whitney t test.
VOL. 76, 2008
of colonic hyperplasia during infection, consistent with previ-
ous reports by workers in our lab (66) and other groups (55).
We also found that the numbers of epithelial cells within elon-
gated crypts were not significantly different in the two mouse
strains following infection (unpublished observations). Indeed,
the relationship between crypt hyperplasia and goblet cell de-
pletion phenotypes observed in other models of intestinal in-
flammation appears to be complex: for example, the immune
system-mediated pathology that occurs during murine hel-
minth infections results in both crypt and goblet cell hyperpla-
sia (2, 34, 35). Moreover, in SAMP1/YitFc mice which develop
spontaneous Crohns disease-like ileitis (72), inflammation-
induced crypt elongation is associated with the expansion of
secretory lineages, including Paneth and goblet cells (72).
Taken together, these studies suggest that the mechanisms
underlying the depletion of mature goblet cells during C. ro-
dentium infection may involve processes that are independent
of, but coordinated with, the induction of crypt hyperplasia.
The concept of immunomodulation of goblet cells during
enteric bacterial infection is intriguing in light of the role that
the immune system plays in modulating goblet cell function
when it is faced with other intestinal challenges. For example,
the robust Th2 response that typically accompanies intestinal
nematode infections induces goblet cell hyperplasia (34) and
goblet cell-specific gene expression, which are thought to con-
tribute to host defense (3, 34). In contrast, we observed loss of
the mature goblet cell phenotype. Given that Th1 responses
(26) and, more recently, Th17 responses (42) have been linked
to C. rodentium infection, it is possible that these polarized
T-helper-type responses are specifically responsible for medi-
ating the loss of the goblet cell phenotype. In this regard, while
we observed increased IL-17 expression during infection in
immunocompetent mice compared to Rag1 KO mice, we have
in previous studies observed a similar trend for gamma inter-
feron expression (66). Therefore, further studies are needed to
address which T-cell subsets and potentially which cytokine(s)
are specifically responsible for the modulation of goblet cell
function during C. rodentium infection.
In addition, our report of direct infection of goblet cells in
vivo by C. rodentium reflects a novel and intriguing host-patho-
gen interaction in the intestine that may have consequences for
local colonization by this pathogen. Given that C. rodentium,
like EPEC and EHEC, can subvert enterocyte function, it is
tempting to speculate that these pathogens can also subvert the
function of intestinal goblet cells; however, this possibility has
yet to be explored in detail. Furthermore, the evidence of
possible bacterial internalization within goblet cells is intrigu-
ing, although its significance and frequency of occurrence re-
main unclear. While it seems counterintuitive that cells as
specialized for secretion as goblet cells could be involved in
uptake of luminal contents, it is interesting to note that rodent
colonic goblet cells have been observed to internalize their
apical membrane along with experimentally injected luminal
cationic ferritin via endocytosis (9). Clearly, the interactions
between enteric pathogens like C. rodentium and goblet cells
reflect a dynamic host-bacterium interaction, with the goblet
cells exposed to A/E effectors and C. rodentium directly ex-
posed to proteins secreted by the goblet cells.
In conclusion, we demonstrate here that although goblet
cells are infected by C. rodentium, they are also subject to
GOBLET CELLS AND C. RODENTIUM INFECTION 809
functional modulation by the host immune system during in
vivo infection by this A/E bacterial pathogen. As the host can
utilize goblet cells for protection against an array of challenges,
understanding how the host modulates goblet cells during A/E
and other bacterial challenges should help us determine what
role these important cell types play during infectious colitis and
during maladaptive responses against normal microflora, such
as those observed during human inflammatory bowel disease.
ACKNOWLEDGMENTS
We gratefully acknowledge Jan Dekker for providing the anti-mu-
rine Muc2 antisera, Daniel Podolsky for providing the anti-Tff3 anti-
sera, and Wanyin Deng for providing the anti-C. rodentium Tir anti-
sera. We also thank Mehran Ghoreishi for help with the reconstitution
experiments and Sharon Edwards for help with the immunohistochem-
istry.
This study was supported by operating grants from the Canadian
Institutes for Health Research and the Crohns and Colitis Foundation
of Canada to B.A.V. B.A.V. is the Children with Intestinal and Liver
Disorders (CHILD) Foundation Research Scholar, a Michael Smith
Foundation for Health Research Scholar, and the Canada Research
Chair in Pediatric Gastroenterology. K.S.B.B. was supported by an
MSFHR graduate scholarship. J.A.G. is a CAG/CIHR/AstraZeneca
and MSFHR Postdoctoral Fellow.
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