INFECTION AND IMMUNITY, July 2005, p. 4025–4033
0019-9567/05/$08.00⫹0 doi:10.1128/IAI.73.7.4025–4033.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 7
Identification of Novel Genes in Intestinal Tissue That Are Regulated
after Infection with an Intestinal Nematode Parasite
R. Datta,1 M. L. deSchoolmeester,1 C. Hedeler,2 N. W. Paton,2 A. M. Brass,2 and K. J. Else1*
Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT,
United Kingdom,1 and Department of Computer Science, University of Manchester, Oxford Road,
Manchester M13 9PL, United Kingdom2
Received 8 July 2004/Returned for modification 23 August 2004/Accepted 28 February 2005
Expulsion of the cecum-dwelling nematode parasite Trichuris muris is dependent on the strain of mouse infected and
correlates precisely with the type of T-helper cell provoked.
Resistance requires a dominant Th2 response; susceptible
mice mount a strong but inappropriate Th1 response (13).
There is considerable and detailed literature demonstrating
the immune polarization of the mesenteric lymph node cell
response towards Th2 in resistant mice (e.g., BALB/c) and Th1
in susceptible mice (e.g., AKR) (4, 9, 13, 17, 20, 23). In order
to effect worm expulsion, Th2 cells are then recruited to the
site of infection, where they mediate worm expulsion by some
as-yet-undefined Th2-controlled effector mechanism (7). The
intracellular niche within intestinal epithelial cells occupied by
T. muris presents an intimate association between parasite and
host. Postinfection morphological changes in the gut architecture occur with epithelial cell hyperproliferation and crypt cell
hyperplasia (3). Surprisingly, the cellular infiltrate which develops postinfection has not been characterized in any great
detail, although infection does provoke a mild mastocytosis
and eosinophilia and the recruitment of CD4⫹ T cells and
macrophages (2). Schopf et al. (41) analyzed the levels of
cytokine mRNA in cecal tissue from T. muris-infected
C57BL/6 mice and a variety of cytokine knockout mice by
reverse transcription (RT)-PCR. Their results showed an elevation in the transcripts for gamma interferon (IFN-␥) in gut
tissue from susceptible mice. Beyond those studies (2, 41),
there is a general lack of knowledge surrounding the events
which occur locally in the large intestinal tissue and the mechanisms which prelude either worm expulsion or worm persis-
tence. This prompted us to analyze the broad gene expression
profiles of mucosal tissue from resistant and susceptible mice.
We chose two time points: day 19, a time point when worm
experience is similar but outcome of infection is disparate, with
BALB/c mice having recently resolved infection while worms
persist in AKR mice, and day 60, a time point well into chronic
infection in AKR mice but 40 days beyond the BALB/c host’s
recent experience of worms. mRNA from AKR and BALB/c
gut tissue was analyzed by microarray, and key changes in gene
expression were confirmed by semiquantitative RT-PCR or protein analyses where possible. Data generated present a picture
at the level of the transcriptome of two very different local gut
environments in resistant and susceptible mice at day 19 postinfection, dominated by antimicrobial factors and by IFN-␥-induced genes, respectively. The epithelial cell was identified as
a cellular source of two of the most differentially expressed
genes. Further, analyses at day 60 demonstrate that the chronically infected gut remains a highly active environment in terms
of gene expression, while the resistant gut resolves back to a
resting state.
MATERIALS AND METHODS
Animals. Male AKR and BALB/c mice (Harlan UK) were infected at 6 to 8
weeks of age and killed at various time points postinfection, as described in the
text, in group sizes of five mice unless otherwise indicated. All experiments were
performed under the regulations of the Home Office Scientific Procedures Act
(1986).
Parasite. Mice were infected with 200 embryonated eggs by oral gavage. Worm
burdens were assessed at days 19 and 60 as previously described (14).
Isolation of intestinal epithelial cells. All tissue culture media were purchased
from Invitrogen (Paisley, United Kingdom), and other chemicals were purchased
from Sigma (Poole, United Kingdom). The large intestine (cecum and approximately 5 cm of colon) was recovered, and fat, connective tissue, and cecal patch
lymphoid follicles were removed. The tissue was then slit longitudinally and
rinsed in calcium- and magnesium-free Hanks balanced salt solution containing
2% fetal calf serum (FCS) (CMF2%), cut into 1-cm pieces, and placed into
ice-cold CMF2%. Following vigorous shaking, the supernatant was discarded
* Corresponding author. Mailing address: Faculty of Life Sciences,
University of Manchester, Michael Smith Building, Oxford Road,
Manchester M13 9PT, United Kingdom. Phone: 44 (0) 161-275-5213.
Fax: 44 (0) 161-275-5656. E-mail: kathryn.j.else@manchester.ac.uk.
4025
Downloaded from http://iai.asm.org/ on November 15, 2015 by guest
Infection of resistant or susceptible mice with Trichuris muris provokes mesenteric lymph node responses
which are polarized towards Th2 or Th1, respectively. These responses are well documented in the literature.
In contrast, little is known about the local responses occurring within the infected intestine. Through microarray analyses, we demonstrate that the gene expression profile of infected gut tissue differs according to whether
the parasite is expelled or not. Genes differentially regulated postinfection in resistant BALB/c mice include
several antimicrobial genes, in particular, intelectin (Itln). In contrast, analyses in AKR mice which ultimately
progress to chronic infection provide evidence for a Th1-dominated mucosa with up-regulated expression of
genes regulated by gamma interferon. Increases in the expression of genes associated with tryptophan metabolism were also apparent with the coinduction of tryptophanyl tRNA synthetase (Wars) and indoleamine-2,3dioxygenase (Indo). With the emerging literature on the role of these gene products in the suppression of T-cell
responses in vitro and in vivo, their up-regulated expression here may suggest a role for tryptophan metabolism
in the parasite survival strategy.
4026
DATTA ET AL.
Oligonucleotide microarrays. The mouse “known” gene SGC oligonucleotide
set microarrays were obtained from the Medical Research Council Rosalind
Franklin Centre for Genomics Research (formally the Human Genome Mapping
Project Resource Centre) and consisted of ⬃9,000 genes spotted in duplicate on
glass slides. Analyses of data generated revealed this particular type of array to
be robust and accurate for high-intensity spots. However, these analyses also
revealed a loss of sensitivity for low-abundance transcripts (with an increase in
error measured from technical repeats with decreasing intensity). Microarray
slides were hybridized with fluorescently labeled cDNA targets, prepared as
described above, scanned using a GenePix 4000A Axon scanner, and analyzed
using GenePix Pro 4 and Max D analysis software (http://www.bioinf.man.ac.uk/
microarray/maxd/) and GIMS (8). Each hybridization compared gene expression
levels in infected tissue to those in uninfected tissue of that mouse strain, gut
tissue was pooled from five individual animals, and two independent infection
experiments were run. The average log2-fold changes (infected tissue over uninfected tissue) presented represent values calculated from both infection experiments. Hybridizations included reverse labeling experiments where the fluorescent dyes were incorporated into the alternative cDNA target.
Normalization. The array normalization was based on recent work (16) that
used a simple statistical model to explore the error inherent in using log ratios
from fluor-reversed microarrays and which then optimized the normalization to
minimize that error. We start by defining the measured log ratio (M) and log spot
intensity (A) as follows: M ⫽ logR/G and A ⫽ log[公(RG)], where R and G are
measurements from the red and green channels, respectively (logarithms are to
base 2). Let n represent the number of replicates of an experiment, g represent
the number of the features (probes) on the slide, mj (j ⫽ 1, 2,. . ., g) represent the
true ratio of expression levels for the gene measured by feature j, and Mjk (j ⫽
1, 2,. . ., g; k ⫽ 1, 2,. . ., n) represent the measured ratio of expression levels for
feature j on replicate k.
The measurement Mjk can be modeled as follows: Mjk ⫽ mj ⫹ c ⫹ ck ⫹ e(Fj)
⫹ ek(Ajk) ⫹ ek(Pj) ⫹ ⑀jk, where c represents the expected global measurement
bias between two channels, ck represents the variation of global measurement
bias shown on replicate k, e(Fj) represents feature-specific bias for feature j,
ek(Ajk) represents spot intensity-dependent bias for feature j on replicate k, ek(Pj)
represents spot location-related bias for feature j on replicate k, and ⑀jk represents the zero mean random error introduced to feature j on replicate k. By a
suitable choice of normalization strategies, it is possible to minimize e(Fj),
ek(Ajk), and ek(Pj) and therefore to estimate ⑀jk.
We can therefore create an error matrix ⑀, where the (j,k)th element is the
random error in spot j from the dye flip pair of chips k, ⑀jk. The average value of
(⑀jk)2 down a column, Ek, provides an estimate of the chip error. Chips with a
value for Ek that is significantly different from the value across all chips are
regarded as suspect. Ek therefore provides a useful quality metric for chip quality
control and for eliminating poor-quality chip pairs from further analysis (unpublished data). The average value of (⑀jk)2 along a row, Ej, provides an estimate of
error in the spot reading, i.e., the random error expected from technical repeats.
In the experiments presented here, the root mean squared value of this error was
about 0.1, suggesting that the log ratios being measured on the chip were on
average accurate, ⫾0.1. This value is smaller than the typical variation seen in log
ratio values between the biological repeats.
Semiquantitative RT-PCR. Gut cDNAs used in the microarray hybridizations
were also used for corroborative semiquantitative RT-PCR. Twofold serial dilutions were made in a 25-l reaction volume with Taq DNA polymerase (Promega, Southampton, United Kingdom) for hypoxanthine phosphoribosyltransferase (HPRT) (5⬘-GTAATGATCAGTCAACGGGGGAC-3⬘ and 5⬘-CCAGC
AAGCTTGCAACCTTAACCA-3⬘), indoleamine-2,3-dioxygenase (Indo) (5⬘-C
TGCACGACATAGCTACCAGTCTG-3⬘ and 5⬘-ACATTTGAGGGCTCTTC
CGACTTG-3⬘), intelectin (Itln) (5⬘-GAAGGTAACCCCGTGCAGTGTG-3⬘
and 5⬘-GGAGCCCACAATGGAGAAGTCAG-3⬘), CXCL9 (Mig) (5⬘-CTTCT
GAGGCTCACGTCACCAAG-3⬘ and 5⬘-ATCCCATGGTCTCGAAAGCTAC
G-3⬘), and CXCL11 (I-Tac) (5⬘-GTCTGACTGTGAGCCCTCCA-3⬘ and 5⬘-G
TGCCTCGTGATATTTGGGGAA-3⬘). The starting amounts of cDNA for
indoleamine-2,3-dioxygenase, intelectin, CXCL9, and CXCL11 were determined
by PCR for HPRT. Thus, cDNAs for HPRT were titrated out, and nonsaturated
dilutions giving bands of equivalent brightness were selected. RT-PCR analysis
of gamma interferon was done on individual and pooled gut cDNAs using the
primers 5⬘-AGCTCTTCCTCATGGCTGTTTC-3⬘ and 5⬘-ATGTTGTTGCTGA
TGGCCTGA-3⬘. PCR cycle numbers were 30 cycles for indoleamine-2,3-dioxygenase, 33 cycles for intelectin, 34 cycles for CXCL9, 32 cycles for CXCL11, and
35 cycles for gamma interferon. The expression level of each transcript was
evaluated after ethidium bromide staining on 2% agarose gel.
Total RNA was extracted from 5 ⫻ 106 freshly isolated intestinal epithelial
cells using TRIzol (Invitrogen) according to the manufacturer’s instructions.
Downloaded from http://iai.asm.org/ on November 15, 2015 by guest
and fresh CMF2% was added. This was repeated until the supernatant was clear.
The tissue was then placed into calcium- and magnesium-free Hanks balanced
salt solution containing 10% FCS, 1 mM EDTA, 1 mM dithiothreitol, 100
units/ml penicillin, and 100 g/ml streptomycin (CMF10%) and incubated at
37°C for 20 min. After vigorous shaking, the supernatant was recovered and
placed on ice. This procedure was repeated once more. The supernatant was
then passed through a 100-m cell strainer (Becton Dickinson, Oxford, United
Kingdom) and centrifuged at 200 ⫻ g for 10 min. The cells were resuspended in
ice-cold RPMI medium containing 10% FCS, 2 mM L-glutamine, 100 units/ml
penicillin, 100 g/ml streptomycin, and 60 M monothioglycerol and counted,
and the volume was adjusted to give 5 ⫻ 106 cells/ml. Aliquots were taken for
flow cytometric analysis and total RNA extraction.
Flow cytometry. Cells were stained with rat anti-mouse Ep-CAM (clone G8.8)
which binds to a mouse epithelial cell marker and was kindly donated by G.
Anderson. The percentage of cells binding G8.8 was determined by a secondary
antibody, anti-rat immunoglobulin G2a (IgG2a)-fluorescein isothiocyanate (Serotec, Oxford, United Kingdom). Isotype controls were performed using rat
IgG2a of irrelevant specificity (anti-keyhole limpet hemocyanin; BD Biosciences,
Oxford, United Kingdom) and the same secondary antibody, anti-rat IgG2afluorescein isothiocyanate. Leukocyte contamination of intestinal epithelial cell
preparations was assessed by the use of rat anti-mouse CD45-phycoerythrin
(Serotec) (isotype control, rat IgG2b-phycoerythrin; Caltag, Towcester, United
Kingdom). Cells were stained for 30 min on ice in the dark before being fixed
with 1% paraformaldehyde in phosphate-buffered saline and stored at 4°C in the
dark until analyzed. Results were acquired with a FACSCaliber flow cytometer
and analyzed using CellQuest Pro software (both from BD Biosciences). At all
time points, ⱖ90% of cells were positive for G8.8, whereas ⱕ1% were positive for
CD45.
Serum parasite-specific antibody enzyme-linked immunosorbent assay
(ELISA). Ninety-six-well plates (Dynex, Billingshurst, West Sussex, United Kingdom) were coated with 5 g/ml T. muris excretory/secretory antigen in carbonate
buffer, pH 9.6, at 50 l/well. Sera were serially diluted in phosphate-buffered
saline containing 0.05% Tween 20 from 1:20 to 1:2,560. Antigen-specific antibodies were detected using biotinylated rat anti-IgG1 (Serotec Ltd., Oxford,
United Kingdom) or rat anti-IgG2a (Pharmingen) followed by streptavidinconjugated horseradish peroxidase (Boehringer Mannheim, Germany). 2,2Azino-bis-(3-ethylbenz-thiazoline-6-sulfonic acid) (Sigma) at 1 mg/ml in citrate
buffer with 0.003% H2O2 was used as a substrate, and plates were read at 405 nm
with a 490-nm reference filter.
Serum MMCP-1. Mouse mast cell protease 1 (MMCP-1) was detected in sera
using a commercially available ELISA kit form Moredun Scientific (Penucuik,
Scotland). Briefly, ELISA plates were coated overnight at 4°C with 50 l sheep
anti-MMCP-1 capture antibody at 2 g/ml. Serial dilutions of MMCP-1 standard
or samples were applied followed by a rabbit anti-MMCP-1 horseradish peroxidase conjugate at a 1/1,000 dilution. The substrate 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (Sigma) at 1 mg/ml in citrate buffer with 0.003% H2O2
was added, and plates were read at 405 nm with a 490-nm reference filter.
RNA isolation and cDNA synthesis. One centimeter of colonic tissue immediately adjacent to the cecum was homogenized in TRIZol (Invitrogen), and
RNA was isolated according to the manufacturer’s instructions. RNA samples
were treated with DNase (Ambion) to remove traces of contaminating DNA.
The concentration of RNA was estimated using a spectrophotometer at a
260-nm wavelength (Phamacia Biotech), and the quality was assessed by gel
electrophoresis. First-strand cDNA synthesis was performed using oligo(dT)15 as
a primer and Im Prom II RT enzyme (Promega, United Kingdom) following the
protocol standardized by the Human Genome Mapping Project Resource Centre, Harwell, United Kingdom (http://www.hgmp.mrc.ac.uk). cDNA mix was
treated with RNase (Sigma) to eliminate traces of RNA and was purified using
Amicon Mirocon-PCR filters (Millipore). The quantity of cDNA was estimated
by a spectrophotometer at 260 nm, and the quality was assessed by visualizing on
1% agarose gel. cDNA from five mice per group was pooled, and 2.5 g of the
pooled purified target cDNA was labeled with the fluorescent dyes Cye5-dCTP
and Cye3-dCTP (Amersham Biosciences, United Kingdom) using a Bioprime
labeling kit (Invitrogen, United Kingdom). Labeled cDNAs were purified using
Probequant Sephadex G50 columns (Amersham Biosciences, United Kingdom)
to remove unincorporated nucleotides and were pooled together within a group.
Poly(dA) (5.0 l; Amersham Biosciences, United Kingdom) and mouse cot DNA
(10.0 l; Invitrogen, United Kingdom) were added to the pooled DNA mix to
block nonspecific targets. The resulting mix was concentrated to 10 to 15 l using
a vacuum centrifuge and diluted with 10.0 l of sterile water and 25.0 l of
hybridization solution to make the volume 40 to 45 l. Prior to use, the mix was
heated at 85°C for 5 min and then incubated at 42°C for 30 to 60 min.
INFECT. IMMUN.
VOL. 73, 2005
RESPONSES OF HELMINTH-INFECTED GUT TISSUE
4027
RNA was assessed by spectrometry and gel electrophoresis and stored at ⫺80°C.
Oligo(dT)18-primed cDNA was synthesized from 0.5 g of RNA using Superscript II reverse transcriptase (Invitrogen). Twofold serial dilutions were analyzed by PCR in 25-l reaction volumes with Taq DNA polymerase (Promega,
Southampton, United Kingdom) for HPRT, indoleamine-2,3-dioxygenase, and
intelectin primers as described above. The starting amounts of cDNA for indoleamine-2,3-dioxygenase and intelectin were determined by PCR for HPRT.
Thus, cDNAs for HPRT were titrated out, and nonsaturated dilutions giving
bands of equivalent brightness were selected. The number of PCR cycles was 28
for HPRT, 30 for indoleamine-2,3-dioxygenase, and 32 for intelectin.
Statistical analyses. Significance analysis of microarrays (SAM) (44) was used
to analyze the data from the AKR and BALB/c mice at day 19 and day 60. It was
observed that genes with a log2 ratio ⱖ0.9 had q values of 2.5%. (two-class
unpaired data, 100 permutations; delta ⫽ 0.55). In all experiments, we therefore
used a log2 ratio cutoff of 0.9 for genes to include for further analysis. Significant
differences (P ⬍ 0.05) in MMCP-1 levels were determined using analysis of
variance with Bonferroni’s multiple comparison test.
FIG. 1. Transcriptome analyses of Trichuris-infected gut tissue
from resistant and susceptible mice at day 19 postinfection. The scatter
plot shows the average log2-fold changes in gene expression in gut
tissue from AKR mice at day 19 compared to that from uninfected
mice (x axis) versus the average log2-fold changes in gene expression in
gut tissue from BALB/c mice at day 19 compared to that from uninfected mice (y axis). Genes up-regulated in AKR and/or BALB/c gut
tissue are labeled by gene symbol: indoleamine-2,3-dioxygenase
(Indo), pancreatitis-associated protein (Pap), Ia-associated invariant
chain (Ii), CXCL11 (Cxcl11), tryptophan tRNA synthase (Wars), Tcell-specific GTPase (Tgtp), Z-DNA binding protein 1 (Zbp1),
CXCL9 (Cxcl9), coagulation factor VIII (F8), keratin-associated protein 5-4 (Krtap5-4), interferon alpha-inducible protein (G1p2), mouse
mast cell protease 1 (Mcpt1), angiogenin like (Angl), intelectin (Itln),
pancreatic lipase-related protein 2 (Pnliprp2), chloride channel calcium-activated 3 (Clca3), angiogenin-related protein (Angrp), and somatostatin (Sst). Genes regulated by an average log2-fold change of
ⱖ⫹0.9 or ⱕ⫺0.9 have a false discovery rate at this threshold of 2.9%
by SAM analysis (44). Gut tissue was pooled from five individual
animals within a group, and two independent infection experiments
were run. The average log2-fold changes presented represent values
calculated from both infection experiments.
expression of 12 known genes and down-regulated (average
log2-fold change of ⱕ⫺0.9) 33 genes. In the BALB/c host, only
7 known genes were up-regulated and 11 were down-regulated.
Surprisingly, of the known genes which showed altered levels
of expression (up or down), only six genes were common to
both mouse strains, including mouse mast cell protease 1 (Mcpt1
[up-regulated in both strains]). Qualitatively, the genes which
were regulated postinfection in the AKR mouse were very
different from those regulated in the BALB/c host. Interestingly, transcripts for a number of IFN-␥-regulated genes, including indoleamine-2,3-dioxygenase (Indo), tryptophantRNA synthetase (Wars), the chemokines CXCL9 (Cxcl9) and
CXCL11 (Cxcl11), T-cell-specific GTPase (Tgtp), and alpha
interferon-inducible protein (G1p2), were elevated in AKR,
but not BALB/c, mice. AKR mice also had increased levels of
transcripts for Ia-associated invariant chain (Ii), Z-DNA binding protein 1 (Zbp1), coagulation factor VIII (F8), and keratin-associated protein 5-4 (Krtap5-4). Gene expression of pancreatitis-associated protein (Pap) was up-regulated in AKR
mice and down-regulated in BALB/c mice in the context of
infection. However, when hybridizations using naı̈ve gut tissue
Downloaded from http://iai.asm.org/ on November 15, 2015 by guest
RESULTS
Adult worms chronically infected the AKR mouse strain to
at least day 60 postinfection (one of two experiments [data not
shown]). Although some resolution of worm burden was evident from the levels at day 19, all mice harbored fecund adult
worms at a time point over 3 weeks beyond the time point
previously used to define chronic infection (day 35). Microarray analyses at day 60 postinfection thus allow insights at the
level of the transcriptome into the mechanisms of persisting
chronic infection in the AKR mouse. In contrast, mice of the
BALB/c strain expelled the parasite by day 19 (worm counts 0,
0, 1, 0, and 0 [one of two experiments]), providing a time point
postinfection when the transcriptional responses within the gut
are associated with worm expulsion. Analyses of the parasitespecific IgG1 and IgG2a antibody responses at day 60 postinfection revealed a strong IgG1 response in both resistant and
susceptible mouse strains but an IgG2a response associated
uniquely with worm persistence (one of two experiments [data
not shown]). The same profile was seen at day 19, although
optical densities were lower (data not shown). These data thus
conform to the results of previous publications (4, 13) reporting serological analyses up to day 35 postinfection and linking
these IgG isotype profiles to a dominant Th1 response in susceptible mice and a dominant Th2 response in resistant mice.
Transcriptional responses of infected gut tissue. Microarray
analyses were performed on gut tissue taken at day 19 and day
60 postinfection from AKR and BALB/c mice. Each hybridization compared gene expression levels in infected tissue to
those of uninfected tissue of the same mouse strain, gut tissue
was pooled from five individual animals, and two independent
infection experiments were performed. The log2-fold changes
presented represent average values calculated from both infection experiments. In some cases, individual gut tissues were
also analyzed to confirm the robustness of the gene expression
data.
Figure 1 shows a scatter plot representing the average log2fold changes for genes with up-regulated or down-regulated
expression postinfection in AKR and BALB/c mice at day 19
postinfection. Genes regulated by an average log2-fold change
of ⱖ0.9 or ⱕ⫺0.9 have a false discovery rate of 2.9% by SAM
analysis (44), and thus, these thresholds are used to identify
genes with increased or decreased expression. Quantitatively,
the gene expression profile of gut tissue from a susceptible
mouse was very different from that of a resistant mouse. Thus,
AKR mice up-regulated (average log2-fold change of ⱖ0.9) the
4028
DATTA ET AL.
from the two mouse strains were conducted to address strainspecific differences in gene expression irrespective of infection,
Pap showed a strain-specific difference, with uninfected
BALB/c mice having much lower levels of expression of this
gene than uninfected AKR mice (data not shown). None of the
other genes represented in Fig. 1 were significantly different
between the two uninfected mouse strains. Relatively more
genes were down-regulated in infected AKR gut tissue than
up-regulated, and these included the transcript for carbonic
anhydrase 2 (average log2-fold change, ⫺1.0).
The gene expression profile of gut tissue from resistant
BALB/c mice was quite distinct from that of susceptible AKR
mice (Fig. 1). Here, the expression levels of the antimicrobial
factors intelectin (Itln), angiogenin-like protein (Angl), and
angiogenin-related protein (Angrp), plus other genes including
chloride channel calcium-activated 3 (Clca3) and pancreatic
lipase-related protein 2 (Pnliprp2), were up-regulated. These
genes were not significantly up-regulated in the AKR mouse
(Fig. 1).
The expression levels of genes in gut tissue of AKR and
BALB/c mice at day 60 postinfection are depicted by a scatter
plot in Fig. 2. In AKR mice which still harbor parasites, Indo
and Tgtp remain elevated. In addition, the expression levels of
FIG. 3. Serum mast cell protease 1 protein levels precisely mirror
changes in mRNA levels revealed by microarray. MMCP-1 levels in
the sera of infected AKR and BALB/c mice at day 19 and day 60
postinfection compared to uninfected (naı̈ve) levels are shown. Significantly higher levels of MMCP-1 were seen in BALB/c mice at day 19
postinfection (**P ⬍ 0.001) than in AKR mice at this time point, with
levels declining by day 60 postinfection. Levels of MMCP-1 in AKR
mice show a gradual rise postinfection. Levels are means ⫾ standard
deviations for five mice per time point.
other genes have increased, including ones seen only in the
BALB/c mice at day 19 (Angl and Clca3) (Fig. 1). Mcpt1
expression levels at day 60 are higher than at day 19 in the
AKR mouse, although not as high as those seen in BALB/c
mice at day 19. Transcripts for other genes not apparent at day
19 are also elevated, including mucin 2 (Muc2), Von HippelLindau binding protein 1 (Vbp1), and desmoyokin (Ahnak).
Down-regulated genes include carbonic anhydrase 2 (average
log2-fold change, ⫺1.1) and aquaporin 8 (average log2-fold
change, ⫺1.1). In the absence of an infection for over 40 days,
the BALB/c gut tissue is largely unchanged from naı̈ve expression levels, represented by dots clustering around the 0 value of
the y axis (Fig. 2), although Angl remains elevated above a log2
0.9-fold change and transcripts for a potassium channel are
raised (Kcnmb1, K large conductance-calcium). Quantitatively, the transcripts for 12 known genes are up-regulated
(average log2-fold change of ⱖ0.9), and 28 are down-regulated
(average log2-fold change, ⱕ⫺0.9) in the AKR gut tissue a day
60, compared to just 2 up-regulated and 4 down-regulated in
gut tissue from BALB/c mice and with just one gene (Angl)
common to both mouse strains.
Protein levels of mouse mast cell protease 1 mirror the
changes in gene expression determined by microarray. To
substantiate the microarray data, five genes were chosen,
Mcpt1 (expressed by both infected AKR and BALB/c mice)
indoleamine-2,3-dioxygenase, CXCL9 and CXCL11 (expressed
by infected AKR mice), and intelectin (expressed by infected
BALB/c mice). Protein levels of MMCP-1 were analyzed in the
sera of AKR and BALB/c mice on day 19 and day 60 postinfection. Serum MMCP-1 levels are known to correlate well
with an ongoing intestinal mastocytosis (2, 27, 29). Although
the intestinal mast cell response is not an essential component
of the anti-T. muris effector mechanism (6), the MMCP-1 analysis does provide a useful way to corroborate the gene expression data at the level of a protein. The results are shown in Fig.
3 and mirror those shown at the mRNA level by microarray
Downloaded from http://iai.asm.org/ on November 15, 2015 by guest
FIG. 2. Altered levels of gene expression are still evident in chronically infected mice but not resistant mice at day 60 postinfection. The
scatter plot shows the average log2-fold changes in gene expression in
gut tissue from AKR mice at day 60 compared to that from uninfected
mice (x axis) versus the average log2-fold changes in gene expression in
gut tissue from BALB/c mice at day 60 compared to that from uninfected mice (y axis). Genes up-regulated in AKR and/or BALB/c gut
tissue are labeled by gene symbol: angiogenin-like (Angl), pancreatitisassociated protein (Pap), mouse mast cell protease 1 (Mcpt1), indoleamine-2,3-dioxygenase (Indo), phospholipase A2 group IIA
(Pla2g2a), mucin 2 (Muc2), von Hippel-Lindau binding protein 1
(Vbp1), nonagouti (a), chloride channel calcium-activated 3 (Clca3),
T-cell-specific GTPase (Tgtp), desmoyokin (Ahnak), K-large-conductance calcium (Kcnmb1), and phosphatidylinositol membrane associated (Pitnm). Genes regulated by an average log2-fold change of
ⱖ⫹0.9 or ⱕ⫺0.9 have a false discovery rate at this threshold of 2.9%
by SAM analysis (44). Gut tissue was pooled from five individual
animals within a group, and two independent infection experiments
were run. The average log2-fold changes presented represent values
calculated from both infection experiments.
INFECT. IMMUN.
VOL. 73, 2005
(Fig. 1 and 2). Thus, both mouse strains up-regulate MMCP-1
postinfection when measured by ELISA, with significantly
higher levels (P ⬍ 0.001) in the BALB/c mouse at day 19
compared to levels in AKR mice. By day 60 postinfection,
MMCP-1 levels were still rising in the AKR mouse strain,
although they had not reached the levels seen in BALB/c mice
at day 19. In contrast, levels of this mast cell protease were
returning to normal in BALB/c mice. Correlating with this, in
BALB/c mice, the average log2-fold change in gene expression
from naı̈ve levels at day 19 was 1.7 (Fig. 1), falling to below 0.9
at day 60 (Fig. 2). In AKR mice, the log2-fold change in gene
expression from naı̈ve levels at day 19 was 0.9 (Fig. 1), rising to
1.4 at day 60 (Fig. 2). Thus, for this particular gene, protein
levels determined by ELISA precisely mirror the pattern of
change in gene expression determined by microarray.
Gene expression profiles of indoleamine-2,3-dioxygenase,
CXCL9, CXCL11, and intelectin are supported by semiquantitative RT-PCR. Using the same cDNAs used for the microarray analyses, semiquantitative RT-PCR was carried out for
naı̈ve pooled gut tissue and infected pooled gut tissue from five
mice for each strain. The results are shown in Fig. 4 (day 19
postinfection) for the pooled gut tissue samples from both
strains. cDNA for each sample was titrated from 1:2 to 1:32. As
can be seen in Fig. 4A, uninfected gut tissue from AKR and
BALB/c mice expressed indoleamine-2,3-dioxygenase, although
expression levels are higher in uninfected AKR mice with
transcripts clearly detectable at the 1:32 dilution. However,
upon infection, it is only AKR mice which show up-regulated
expression of this gene. Similarly, CXCL11 (Fig. 4B) shows an
increased level of expression postinfection in AKR mice compared to those of uninfected mice, with brighter bands clearly
4029
FIG. 5. RT-PCR detects an up-regulation in gamma interferon
transcripts postinfection that is not detected by microarray. (A and B)
Gamma interferon expression in whole gut tissue from (A) individual
infected AKR mice at day 19 postinfection (lanes 1 to 3) and naı̈ve
AKR mice (lanes 4 to 8) and (B) infected BALB/c mice (lanes 1 to 2)
and naı̈ve BALB/c mice (lanes 3 to 4). (C) Gamma interferon expression in whole gut tissue using the same pools of tissue from infected
and naı̈ve AKR and BALB/c mice as those used for microarray analysis. Lane 1, infected AKR; lane 2, naı̈ve AKR; lane 3, infected
BALB/c; lane 4, naı̈ve BALB/c. The starting amount of cDNA was
determined by PCR for HPRT.
detected in infected mice at the lower dilutions (1:16 and 1:32).
In contrast, the BALB/c host did not up-regulate expression of
CXCL11 postinfection. Although the increase in expression
postinfection in AKR mice of CXCL9 (Fig. 4C) is less obvious
than that for indoleamine-2,3-dioxygenase and CXCL11, it is
clear that the BALB/c mouse does not up-regulate expression
of this gene postinfection. Thus, the RT-PCR analyses corroborated the up-regulated expression of genes in the AKR mouse.
The analyses, however, also revealed an apparent down-regulation in expression of indoleamine-2,3-dioxygenase, CXCL11,
and CXCL9 postinfection in BALB/c mice. Using an average
log2-fold change threshold of ⱕ⫺0.9, the expression levels of
these genes were not significantly altered by microarray analyses. The results from RT-PCR study do not, however, contradict the microarray data. Rather, they suggest that the differences in gene expression levels in the susceptible mouse
upon infection compared to those of the resistant host upon
infection are even more pronounced than what was revealed by
microarray. Thus, resistant mice actually down-regulate the
expression of genes which appeared unchanged by microarray
but which susceptible mice clearly up-regulate, as determined
by microarray and RT-PCR. RT-PCR analyses of intelectin
mRNA confirmed the large differences observed in expression
levels of this gene in susceptible AKR and resistant BALB/c
mice in response to infection (Fig. 4D). Thus, no transcripts for
intelectin were detected in uninfected mice of either strain or
in infected AKR mice at day 19. In contrast, a small but clear
up-regulation in gene expression was seen in BALB/c mice at
this time point.
Although it is not clear why the microarray analyses failed to
reveal a significant down-regulation of indoleamine-2,3-dioxygenase, CXCL11, and CXCL9 in infected BALB/c mice compared that to uninfected animals, it may reflect a lack of sensitivity of the microarray technique in detecting significant
Downloaded from http://iai.asm.org/ on November 15, 2015 by guest
FIG. 4. Semiquantitative RT-PCR confirms the mRNA expression
profiles of indoleamine-1,2-dioxygenase, intelectin, CXCL9, and
CXCL11 as revealed by microarray. RT-PCR analyses of the expression levels of (A) indoleamine-1,2-dioxygenase (Indo), (B) CXCL11,
(C) CXCL9, and (D) intelectin (Itln) at day 19 postinfection in gut
tissue from AKR and BALB/c mice are shown. cDNA for HPRT was
titrated out, and nonsaturated dilutions giving bands of equivalent
brightness, shown as a single band per sample in the figure, were
selected as the starting dilution for the serial dilutions. cDNA was
serially diluted (1:2) and amplified by PCR as described in Materials
and Methods. A, AKR; B, BALB/c. N, naı̈ve; 19, day 19 postinfection.
RESPONSES OF HELMINTH-INFECTED GUT TISSUE
4030
DATTA ET AL.
changes in genes of low abundance. Certainly, for this particular glass oligonucleotide array, we observed a loss in sensitivity for low-abundance transcripts. The relative sensitivity of
microarray compared to that of RT-PCR is addressed in Fig.
5A, B, and C. Transcripts for gamma interferon are clearly
detectable in the gut tissue of individual infected AKR mice at
day 19 postinfection (5A, lanes 1 to 3) compared to those of
uninfected animals (Fig. 5B, lanes 4 to 8) and BALB/c mice
(Fig. 5B, lanes 1 and 2, infected BALB/c mice, lanes 3 and 4
uninfected BALB/c mice). Transcripts were also detected in
the pool of infected gut tissue used directly for the microarray
analyses (5C, lane 1). However by microarray, the log2-fold
change at day 19 postinfection for interferon gamma in AKR
mice was less than 0.9, at only 0.22.
Intestinal epithelial cells express indoleamine-2,3-dioxygenase and intelectin. To begin to dissect out the cellular sources
of the differentially expressed genes, we carried out semiquantitative RT-PCR for indoleamine-2,3-dioxygenase and intelectin on epithelial cells stripped from the intestine of infected
mice (day 21 postinfection) and naı̈ve mice (routinely, 90%
were G8.8 positive and ⬍1% were CD45 positive). These studies were performed on groups of mice separate from those
used in the microarray experiments. The results are shown in
Fig. 6. Expression levels of indoleamine-2,3-dioxygenase are
up-regulated from naı̈ve levels in epithelial cells from AKR
mice postinfection but not in epithelial cells from BALB/c mice
postinfection. In contrast, transcripts for intelectin are upregulated from naı̈ve levels in epithelial cells from infected
BALB/c mice but not in epithelial cells from infected AKR
mice. Thus, using whole gut tissue, where the epithelial cell
represents just a fraction of the material, RT-PCR revealed a
weak but clear band for intelectin in infected BALB/c mice
(Fig. 4). Using purified epithelial cells, intelectin transcripts
can also be seen in naı̈ve BALB/c mice with a marked upregulation of expression postinfection. Transcripts for intelectin can also be seen in epithelial cells from naı̈ve AKR mice.
However, in AKR mice, there is a clear and profound downregulation of expression of the intelectin gene in epithelial cells
postinfection. Thus, importantly, the trend in the changes of
intelectin gene expression is the same whether the analyses are
conducted on whole gut tissue or epithelial cells: BALB/c mice
FIG. 7. Goblet cell hyperplasia correlates with worm expulsion in
resistant mice. Goblet cell hyperplasia in AKR and BALB/c mice at
day 22 and day 34 postinfection were compared to uninfected (naı̈ve)
levels. Values are means ⫾ standard deviations for mice per time point
from an experiment run separately from the microarray analyses. Goblet cell numbers are significantly elevated (*P ⬍ 0.05) in BALB/c mice
at day 22 postinfection compared to those of AKR mice, a time when
transcripts for three antimicrobial proteins (intelectin, angiogenin-like
protein, and angiogenin-related protein) show large severalfold increases in this mouse strain.
up-regulate expression of intelectin postinfection, while AKR
mice do not.
The time course of goblet cell hyperplasia correlates with
worm expulsion and intelectin expression. The intestinal epithelium consists of enterocytes, enteroendocrine cells, Paneth
cells, and goblet cells. Intelectin is considered within the literature as a predominantly Paneth-cell-derived molecule (28),
with Paneth cells residing at the base of crypts of the small
intestine. Although no Paneth cells are thought to exist in the
large intestine, at least under resting uninfected conditions,
there are sporadic reports of Paneth cells occurring within the
colon in inflammatory conditions (26). However, using tissue
from a separate infection experiment, histological staining for
Paneth cells in gut sections from AKR and BALB/c mice at day
22 and day 34 postinfection failed to reveal any Paneth cells at
the base of the large intestinal crypts, while Trichinella spiralisinfected small intestinal tissue, used as a positive control, was
rich in Paneth cells (data not shown). Goblet cells are also rich
sources of intelectin (39), and thus, groups of AKR and
BALB/c mice were infected to examine the time course of
goblet cell hyperplasia postinfection in these mouse strains.
Although only correlative, Fig. 7 shows the presence of significantly more goblet cells in the gut tissue of BALB/c mice by
day 22 postinfection (P ⬍ 0.05) than in the gut tissue of AKR
mice. No increase from naı̈ve levels in goblet cells is observed
in AKR mice at this time point.
DISCUSSION
The polarization of lymph node cells towards Th1- or Th2like phenotypes postinfection is well documented in the literature for a variety of parasites and is based primarily on cytokine secretion profiles from mesenteric lymph node cells (11,
13, 20). The intestinal nematode parasite Trichuris muris in the
mouse is a model system which exemplifies polarized lymph
node responses. Thus, a Th2 response (interleukin-4 [IL-4],
Downloaded from http://iai.asm.org/ on November 15, 2015 by guest
FIG. 6. Epithelial cells are a source of indoleamine-2,3-dioxygenase in susceptible mice and intelectin in resistant mice. Indoleamine2,3-dioxygenase (A) and intelectin (B) expression in intestinal epithelial cells was measured by RT-PCR. Epithelial cells were isolated from
the large intestine of naı̈ve or T. muris-infected AKR and BALB/c
mice, and total RNA was extracted. cDNA for HPRT was titrated out,
and nonsaturated dilutions giving bands of equivalent brightness,
shown as a single band per sample in the figure, were selected as the
starting dilution for the serial dilutions. cDNA was serially diluted
(1:2) and amplified by PCR as described in Materials and Methods. A,
AKR; B, BALB/c. N, naı̈ve; 21, day 21 postinfection.
INFECT. IMMUN.
VOL. 73, 2005
4031
strain, is under the control of Th2 cytokines (25). The detection of changes in typical Th2-associated transcripts may require analyses to be performed on gut tissue taken at earlier
time points or the purification of T cells from gut tissue for
gene expression profiling.
The much-documented Th1 response that dominates the
mesenteric lymph node of susceptible mice (4, 9, 13, 17, 20, 23)
is also reflected in the gene expression profiles of gut tissue,
with the expression of a variety of IFN-␥-regulated genes increasing at day 19 postinfection. These include the chemokines
CXCL11 and CXCL9 and T-cell-specific GTPase. Surprisingly,
by microarray, we did not see a significant increase in expression of the IFN-␥ gene itself. However, RT-PCR analyses
showed the presence of IFN-␥ transcripts in gut tissue taken
from infected AKR mice at this time postinfection (Fig. 5), and
thus, our inability to detect any up-regulation by microarray
may simply reflect a difference in the sensitivity of the two
assays when whole gut tissue is used. Certainly, microarray
analyses of mesenteric lymph node responses in AKR mice
revealed a significant increase (log2 1.3) in the transcripts for
IFN-␥ at day 19 postinfection (data not shown). The data
presented in this paper are based on the use of glass oligonucleotide arrays. Through their use, it emerged that despite
providing strong, robust data for up-regulated, abundant transcripts, they did lack sensitivity for low-abundance transcripts.
Thus, to further evaluate the key candidate genes highlighted
by their prominence on the glass arrays, a third biological
repeat (i.e., a new infection) in AKR and BALB/c mice was
performed, and the gene expression profiles were analyzed at
day 19 postinfection using a completely different microarray
platform, the Affymetrix chip. The results of this confirmed the
original analyses, and in all cases, genes represented on both
arrays and which form the core of the paper followed entirely
similar patterns. Thus, by Affymetrix, expression of the genes
for intelectin, mouse mast cell protease 1, pancreatic lipaserelated protein 2, chloride channel calcium-activated 3, and
somatostatin were more highly up-regulated from uninfected
levels in resistant BALB/c gut tissue than in AKR gut tissue, as
also seen in Fig. 1. As before, of these genes, only mouse mast
cell protease 1 was also elevated in the AKR host. Thus, the
log2-fold changes for these genes were as follows: intelectin,
6.5 for BALB/c and ⫺0.6 for AKR mice; mouse mast cell
protease 1, 6.0 for BALB/c and 2.5 for AKR mice; pancreatic
lipase-related protein 2, 5.0 for BALB/c and 0.1 for AKR mice;
chloride channel calcium-activated 3, 1.8 for BALB/c and ⫺0.7
for AKR mice; and somatostatin, 1.4 for BALB/c and ⫺0.1 for
AKR mice. Figure 1 also shows the genes which were more
highly up-regulated from uninfected levels in susceptible AKR
gut tissue than in the BALB/c host. These changes were also
mirrored on the Affymetrix chip, with log2-fold increases in
expression postinfection relative to naı̈ve levels of indoleamine-2,3-dioxygenase of 6.5 for AKR and ⫺0.04 for BALB/c
mice; pancreatitis-associated protein of 1.9 for AKR and ⫺1.7
for BALB/c mice; CXCL11 of 4.8 for AKR and 0.0 for BALB/c
mice; tryptophan tRNA synthase of 3.9 for AKR and 0.14 for
BALB/c mice; T-cell-specific GTPase of 5.3 for AKR and 0.7
for BALB/c mice; Z-DNA binding protein 1 of 1.3 for AKR
and ⫺0.3 for BALB/c mice; Ia-associated invariant chain of 4.2
for AKR and 1.8 for BALB/c mice; and CXCL9 of 5.4 for AKR
and ⫺0.2 for BALB/c mice. Interestingly, in susceptible mice,
Downloaded from http://iai.asm.org/ on November 15, 2015 by guest
IL-5, IL-9, and IL-13) as seen in BALB/c mice underlies the
host’s ability to expel the parasite, and a Th1 response (IFN-␥)
as seen in AKR mice results in chronic infection (4, 11, 13, 17,
20, 23). Less attention has been paid to the responses that
occur locally in gut tissue in mouse strains resistant or susceptible to this helminth. We report here the first study to analyze
the broad transcriptional responses of gut tissue during acute
and chronic helminth infection. The data reveal that at the
level of the gut, a mouse that is ultimately susceptible to infection responds very differently to the insult of infection with
respect to its gene expression profile compared to that of a
mouse that is able to expel the parasite.
BALB/c mice, which expelled the parasite by day 19 postinfection, responded to infection by up-regulating the expression
of a variety of genes encoding potential antiparasitic proteins,
namely intelectin, angiogenin-like protein, and angiogenin-related protein (24, 39, 43). Susceptible AKR mice showed an
up-regulation of the gene coding angiogenin-like protein by
day 60. However, expression levels of the other two genes were
low at both day 19 and day 60. The intestinal epithelial cell
forms the host-parasite interface and represents the niche
within which Trichuris parasites burrow (37). We present data
showing that epithelial cells from BALB/c mice represent one
cellular source of intelectin within the gut. Within the epithelial cell compartment, Paneth cells and goblet cells are known
sources of intelectin (28, 39). In the absence of any Paneth cells
in the large intestine post-Trichuris infection, it is likely that the
goblet cell (36, 39) is the cellular source of this antimicrobial
activity, and indeed, goblet cell hyperplasia correlated with the
kinetics of worm expulsion. Thus, BALB/c mice developed
elevated numbers of goblet cells by day 22 postinfection. In
AKR mice, no increase in goblet cell numbers from uninfected
levels was seen until day 34, when numbers were starting to
rise. However, by this time, adult parasites which are less
readily expelled had developed. Thus, early stages of T. muris
are immunogenic and can be expelled by appropriate (but
as-yet-undefined) effector mechanisms. Later, larval stages
modulate immunity to promote their own survival, and potential effector mechanisms become less effective (12). Hence, the
rate at which an effector response develops in relation to the
parasite growth rate is at least as, if not more, important as the
level of that response.
Thus, our data suggest that one component of the host
protective immune response to Trichuris may involve the local
release of innate antimicrobial factors such as intelectin by
cells within the gut. Supporting this, it has recently been suggested that goblet cell-derived intelectin, in this case intelectin
2, may play an important role in the innate immune response
to Trichinella spiralis (39). Since the recent description of
mouse intelectin 2 in the literature (38), and using the differential PCR for intelectin 1 and intelectin 2 described previously (39), we have extended our own data to show that
BALB/c mice infected with T. muris express both intelectin 1
and intelectin 2 (R. Datta and K. J. Else, unpublished data).
Precisely how these factors might be involved in worm expulsion is not clear, although they may recognize carbohydrates
on the surface of nematodes (43). At the time points analyzed,
we did not detect a significant up-regulation of genes encoding
Th2 cytokines in gut tissue from resistant mice, although goblet
cell hyperplasia, which proceeds more potently in the resistant
RESPONSES OF HELMINTH-INFECTED GUT TISSUE
4032
DATTA ET AL.
nated model but for the first time also places the gut tissue
responses in the context of resistance and susceptibility to the
pathogen, highlighting candidate genes which may play roles in
both worm elimination and worm survival.
ACKNOWLEDGMENTS
This work was supported by the BBSRC (34/S15449) and the Wellcome Trust (044494 and 068639).
We acknowledge the MRC Rosalind Franklin Centre for Genomics
Research (formally the UK Human Genome Mapping Project Resource Centre) for support and supply of the microarrays and Andy
Hayes for use of the microarray facility at Manchester. We also thank
G. Anderson for the G8.8 monoclonal antibody.
REFERENCES
1. Aleksandersen, M., L. Kai-Inge, B. Gjerde, and T. Landsverk. 2002. Lymphocyte depletion in ileal Peyer’s patch follicles in lambs infected with
Eimeria ovinoidalis. Clin. Diagn. Lab. Immunnol. 9:83–91.
2. Artis, D., N. E. Humphreys, C. S. Potten, N. Wagner, W. Müller, J. R.
McDermott, R. K. Grencis, and K. J. Else. 2000. 7 integrin-deficient mice:
delayed leukocyte recruitment and attenuated protective immunity in the
small intestine during enteric helminth infection. Eur. J. Immunol. 30:1656–
1664.
3. Artis, D., C. S. Potten, K. J. Else, F. D. Finkelman, and R. K. Grencis. 1999.
Trichuris muris: host intestinal epithelial cell hyperproliferation during
chronic infection is regulated by interferon-␥. Exp. Parasitol. 92:144–153.
4. Bancroft, A. J., A. N. J. McKenzie, and R. K. Grencis. 1998. A critical role
for IL-13 in resistance to intestinal nematode infection. J. Immunol. 160:
3453–3461.
5. Barceló-Batllori. S., M. André, C. Servis, N. Lévy, O. Takikawa, P. Michetti,
M. Reymond, and E. Felley-Bosco. 2002. Proteomic analysis of cytokine
induced proteins in human intestinal epithelial cells: implications for inflammatory bowel diseases. Proteomics 2:551–560.
6. Betts, C. J., and K. J. Else. 1999. Mast cells, eosinophils and antibody
mediated cytotoxicity are not critical in resistance to Trichuris muris. Parasite
Immunol. 21:45–52.
7. Betts, C. J., M. L. deSchoolmeester, and K. J. Else. 2000. Trichuris muris:
CD4⫹ T cell mediated protection in reconstituted SCID mice. Parasitology
121:631–637.
8. Cornell, M., N. W. Paton., C. Hedeler, P. Kirby, D. Delneri, A. Hayes, and
S. G. Oliver. 2003. GIMS: an integrated data storage and analysis environment for genomic and functional data. Yeast 20:1291–1296.
9. deSchoolmeester, M. L., M. C. Little, B. J. Rollins, and K. J. Else. 2003.
Absence of CC chemokine ligand 2 results in an altered Th1/Th2 cytokine
balance and failure to expel Trichuris muris infection. J. Immunol. 170:4693–
4700.
10. Dieckgraefe, B. K., W. F. Stenson, J. R. Korzenik, P. E. Swanson, and C. A.
Harrington. 2000. Analysis of mucosal gene expression in inflammatory
bowel disease by parallel oligonucleotide arrays. Phys. Genom. 4:1–11.
11. Else, K. J., and F. D. Finkelman. 1998. Intestinal nematode parasites, cytokines and effector mechanisms. Int. J. Parasitol. 28:1145–1158.
12. Else, K. J., D. Wakelin, and T. I. A. Roach. 1989. Host predisposition to
trichuriasis: the mouse-T. muris model. Parasitology 89:275–282.
13. Else, K. J., F. D. Finkelman, C. R. Maliszewski, and R. K. Grencis. 1994.
Cytokine-mediated regulation of chronic intestinal helminth infection. J.
Exp. Med. 179:347–351.
14. Else, K. J., D. Wakelin, D. L. Wassom, and K. M. Hauda. 1990. The influence of genes mapping within the major histocompatibility complex on
resistance to Trichuris muris infections in mice. Parasitology 101:61–67.
15. Fallarino, F., U. Grohmann, K. W. Hwang, C. Orabona, C. Vacca, R. Bianchi, M. L. Belladonna, M. C. Fioretti, M. L. Alegre, and P. Puccetti. 2003.
Modulation of tryptophan catabolism by regulatory T cells. Nat. Immunol.
4:1206–1212.
16. Fang, Y., A. Brass, D. C. Hoyle, A. Hayes, A. Bashein, S. G. Oliver, D.
Waddington, and M. Rattray. 2003. A model-based analysis of microarray
experimental error and normalisation. Nucleic Acids Res. 31:e96.
17. Faulkner, H., J.-C. Renauld, J. Van Snick, and R. K. Grencis. 1998. Interleukin-9 enhances resistance to the intestinal nematode Trichuris muris.
Infect. Immun. 66:3832–3840.
18. Flach, C.-F., S. Lange, E. Jennische, and I. Lonnroth. 2004. Cholera toxin
induces expression of ion channels and carriers in rat small intestinal mucosa. FEBS Lett. 561:122–126.
19. Fujigaki, S., K. Saito, M. Takemura, N. Maekawa, Y. Yamada, H. Wada, and
M. Seishima. 2002. L-Tryptophan–L-kynurenine pathway metabolism accelerated by Toxoplasma gondii infection is abolished in gamma interferongene-deficient mice: cross-regulation between inducible nitric oxide synthase
and indoleamine-2,3-dioxygenase. Infect. Immun. 70:779–786.
20. Grencis, R. K. 2001. Cytokine regulation of resistance and susceptibility to
Downloaded from http://iai.asm.org/ on November 15, 2015 by guest
we identified two IFN-␥-induced genes which are part of the
tryptophan metabolism pathway: indoleamine-2,3-dioxygenase
(Indo) and tryptophan tRNA synthetase (Wars). Wars is thought
to be induced in cells to allow their survival in low-tryptophan
concentrations. Indoleamine-2,3-dioxygenase is the rate-limiting enzyme in the metabolism of tryptophan through the
kynurenine pathway. Indoleamine-2,3-dioxygenase is known to
be up-regulated in tissue infected with a variety of parasites,
but its function in these instances is thought to be in the
depletion of tryptophan and thus inhibition of parasite growth
(19, 22, 42). Its presence here in susceptible mice where the
worms are thriving, and absence from resistant mice, suggests
a more interesting role relating to the recent literature on the
role of indoleamine-2,3-dioxygenase in immunoregulation and
T-cell suppression demonstrated in vitro and in vivo (15, 30–
32, 35). Thus, its dominance in local tissues of chronically
infected mice may represent a survival strategy of the parasite
either by suppressing the functions of effector T cells or by
dampening down potentially damaging host inflammation. Its
induction by IFN-␥ and the knowledge that T. muris expresses
an IFN-␥-like homologue (21) make this hypothesis even more
attractive. Coinduction of the transcripts for indoleamine-2,3dioxygenase and Wars has previously been demonstrated in a
proteomic analysis of the proteins present in the mucosa of
humans with inflammatory bowel disease and a T-cell-regulatory role proposed previously (5). As far as we are aware, their
coinduction has not been described before for intestinal nematode parasites in the context of resistance and susceptibility to
infection.
In addition to genes with up-regulated expression postinfection, we identified genes with down-regulated expression
postinfection. Interestingly, at day 19 postinfection, there were
almost three times as many genes down-regulated in expression in susceptible AKR mice compared to that in resistant
BALB/c mice. Although the significance of this is not clear as
yet, it is possible that the absence of gene expression products
rather than their presence is contributing to the failure to clear
the parasite. Equally, some of these down-regulated genes may
simply be markers of persisting infection. For instance, the
expression of carbonic anhydrase (Car2) is reduced in gut
tissue of AKR mice at both day 19 and day 60 postinfection
and has also been shown to be reduced postinfection in other
models of intestinal parasite infections (1). Equally, the decrease in transcripts for the water channel aquaporin 8 may be
a response by the host to decrease fluid transport across the
epithelium during chronic infection. Indeed, cholera toxin has
been shown to induce similar changes in the intestinal mucosa
of rats (18). Here, the down-regulation of transcripts for aquaporin 8 was suggested to play a role in diminishing water
transport during cholera.
Microarray analyses have been used to characterize the gene
expression profiles of a variety of infections and diseases of the
intestine including inflammatory bowel disease (10) and Helicobacter infection (33, 34). Using microarray, Sandler et al.
(40) have recently described distinct gene expression profiles
of Th1- and Th2-type granulomas. Here, transcripts up-regulated in Th1-type granulomas were associated with tissue damage, while the gene expression profiles of Th2 granulomas were
more related to fibrosis and wound healing. Our study also
reveals distinct gene expression profiles in a Th1/Th2-domi-
INFECT. IMMUN.
VOL. 73, 2005
21.
22.
23.
24.
25.
26.
28.
29.
30.
31.
32.
33.
Editor: J. F. Urban, Jr.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
4033
unique transcriptional signature. Proc. Natl. Acad. Sci. USA 100:12289–
12294.
Mueller, A., J. O’Rourke, J. Grimm, K. Guillemin, M. F. Dixon, A. Lee, and
S. Falkow. 2003. Distinct gene expression profiles characterize the histopathological stages of disease in Helicobacter-induced mucosa-associated
lymphoid tissue lymphoma. Proc. Natl. Acad. Sci. USA 100:1292–1297.
Munn, D. H., E. Shaftzadeh, J. T. Attwood, I. Bondariec, A. Pashine, and
A. L. Mellor. 1999. Inhibition of T cell proliferation by macrophage tryptophan catabolism. J. Exp. Med. 189:1363–1372.
Ouellette, A. J. 1999. IV. Paneth cell antimicrobial peptides and the biology
of the mucosal barrier. Am. J. Physiol. 277:G257–G261.
Panesar, T. S. 1981. The early phase of tissue invasion by Trichuris muris
(Nematoda: Trichuroidea). Zetschrifte Parasitenkunde 66:163–166.
Pemberton, A. D., P. A. Knight, S. H. Wright, and H. R. P. Miller. 2004.
Proteomic analysis of mouse jejunal epithelium and its response to infection
with the intestinal nematode, Trichinella spiralis. Proteomics 4:1101–1108.
Pemberton, A. D., P. A. Knight, J. Gamble, W. H. Colledge, J.-K. Lee, M.
Pierce, and H. R. P. Miller. 2004. Innate BALB/c enteric epithelial response
to Trichinella spiralis: inducible expression of a novel goblet cell lectin,
intelectin-2, and it natural deletion in C57BL/10 mice. J. Immunol. 173:
1894–1901.
Sandler, N. G., M. M. Mentink-Kane, A. W. Cheever, and T. A. Wynn. 2003.
Global gene expression profiles during acute pathogen-induced pulmonary
inflammation reveal divergent roles for Th1 and Th2 responses in tissue
repair. J. Immunol. 171:3655–3667.
Schopf, L. R., K. F. Hoffmann, A. W. Cheever, J. F. Urban, Jr., and T. A.
Wynn. 2002. IL-10 is critical for host resistance and survival during gastrointestinal helminth infection. J. Immunol. 168:2383–2392.
Silva, N. M., C. V. Rodrigues, M. M. Santoro, L. F. L. Reis, J. I. AlvarezLeite, and R. T. Gazzinelli. 2002. Expression of indoleamine 2,3-dioxygenase, tryptophan degradation, and kynurenine formation during in vivo infection with Toxoplasma gondii: induction by endogenous gamma interferon
and requirement of interferon regulatory factor 1. Infect. Immun. 70:859–
868.
Tsuji. S., J. Yehori, M. Matsumoto, Y. Suzuki, A. Matsuhisa, K. Toyoshima,
and T. Seya. 2001. Human intelectin is a novel soluble lectin that recognizes
galactofuranose in carbohydrate chains of bacterial cell wall. J. Biol. Chem.
276:23456–23463.
Tusher, V. G., R. Tibshirani, and G. Chu. 2001. Significance analysis of
microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci.
USA 98:5116–5121.
Downloaded from http://iai.asm.org/ on November 15, 2015 by guest
27.
intestinal nematode infection—from host to parasite. Vet. Parasitol. 100:45–
50.
Grencis, R. K., and G. M. Entwistle. 1997. Production of an interferongamma homologue by an intestinal nematode: functionally significant or
interesting artefact? Parasitology 115:S101–S106.
Grohmann, U., F. Fallarino, and P. Puccetti. 2003. Tolerance, DCs and
tryptophan: much ado about IDO. Trends Immunol. 24:242–248.
Helmby, H., K. Taleda, S. Akira, and R. K. Grencis. 2001. Interleukin
(IL)-18 promotes the development of chronic gastrointestinal helminth infection by down regulating IL-13. J. Exp. Med. 194:355–364.
Hooper, L. V., T. S. Stappenbeck, C. V. Hond, and J. I. Gordon. 2003.
Angiogenins: a new class of microbicidal proteins involved in innate immunity. Nat. Immunol. 4:269–273.
Ishikawa, N., D. Wakelin, and Y. R. Mahida. 1997. Role of T helper 2 cells
in intestinal goblet cell hyperplasia in mice infected with Trichinella spiralis.
Gastroenterology 113:542–549.
Kelly, P., R. Feakins, P. Domizio, J. Murphy, C. Bevins, J. Wilson, G.
McPhail, R. Poulsom, and W. Dhaliwal. 2004. Paneth cell granule depletion
in the human small intestine under infective and nutritional stress. Clin. Exp.
Immunol. 135:303–309.
Knight, P. A., S. H. Wright, J. K. brown, X. Huang, D. Sheppard, and
H. R. P. Miller. 2002. Enteric expression of the integrin ␣v6 is essential for
nematode-induced mucosal mast cell hyperplasia and expression of the granule chymase, mouse mast cell protease 1. Am. J. Pathol. 161:771–779.
Komiya, T., Y. Tanigawa, and S. Hirohashi. 1998. Cloning of the novel gene
intelectin, which is expressed in intestinal paneth cells in mice. Biochem.
Biophys. Res. Commun. 251:759–762.
McDermott, J. R., R. E. Bartram, P. A. Knight, H. R. P. Miller, D. R. Garrod,
and R. K. Grencis. 2003. Mast cells disrupt epithelial barrier function during
enteric nematode infection. Proc. Natl. Acad. Sci. USA 100:7761–7766.
Mellor, A. L., B. Baban, P. Chandler, B. Marshall, K. Jhaver, A. Hansen,
P. A. Koni, M. Iwashima, and D. H. Munn. 2003. Cutting edge: induced
indoleamine 2,3 dioxygenase expression in dendritic cell subsets suppresses
T cell clonal expansion. J. Immunol. 171:1652–1655.
Mellor, A. L., and D. H. Munn. 1999. Tryptophan catabolism and T-cell
tolerance: immunosuppression by starvation? Immunol. Today 20:469–473.
Mellor, A. L., and D. H. Munn. 2003. Tryptophan catabolism and regulation
of adaptive immunity. J. Immunol. 170:5809–5813.
Mueller, A., J. O’Rourke, P. Chu, C. C. Kim, P. Sutton, A. Lee, and S.
Falkow. 2003. Protective immunity against Helicobacter is characterized by a
RESPONSES OF HELMINTH-INFECTED GUT TISSUE