il-23 orchestrates mucosal responses to serotype ... · interferon (ifn)-γ, interleukin (il)-22...
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IL-23 orchestrates mucosal responses to Salmonella enterica serotype
Typhimurium in the intestine
Running title: IL-23 orchestrates mucosal responses to Salmonella
Ivan Godinez1, Manuela Raffatellu1, Hiutung Chu1, Tatiane A. Paixão1,2, Takeshi
Haneda1, Renato L. Santos2, Charles L. Bevins1, Renée M. Tsolis1 and Andreas J.
Bäumler1,*
1 Department of Medical Microbiology and Immunology, School of Medicine, University
of California at Davis, One Shields Ave., Davis, CA
2 Departamento de Clínica e Cirurgia Veterinárias, Escola de Veterinária, Universidade
Federal de Minas Gerais, Belo Horizonte, MG, Brasil
* Correspondence: E-mail: [email protected]
Fax: 530-754-7240
Phone: 530-754-7225
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Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.00933-08 IAI Accepts, published online ahead of print on 27 October 2008
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ABSTRACT
Salmonella enterica serotype Typhimurium causes an acute inflammatory reaction in the
cecum of streptomycin pre-treated mice that involves T cell-dependent induction of
interferon (Ifn)-γ, interleukin (Il)-22 and Il-17 expression. Here we investigated the role of
IL-23 in initiating these inflammatory responses using the streptomycin pre-treated
mouse model. Compared to wild type mice, expression of Il-17 was abrogated, Il-22
expression was markedly reduced but Ifn-γ expression was normal in the cecum of IL-23
p19 deficient mice during serotype Typhimurium infection. IL-23 p19 deficient mice also
exhibited a markedly reduced expression of regenerating islet-derived 3 gamma
(Reg3g), keratinocyte-derived cytokine (Kc), and reduced neutrophil recruitment into the
cecal mucosa during infection. Analysis of CD3+ lymphocytes in the intestinal mucosa by
flow cytometry revealed that αβ T cells were the predominant cell type expressing the IL-
23 receptor in naïve mice. However, a marked increase in the number of IL-23 receptor
expressing γδ T cells was observed in the lamina propria during serotype Typhimurium
infection. Compared to wild type mice, γδ T cell receptor deficient mice exhibited blunted
expression of Il-17 during serotype Typhimurium infection while Ifn-γ expression was
normal. These data suggested that γδ T cells are a significant, but not the sole source of
IL-17 in the acutely inflamed cecal mucosa of mice. Collectively our results point to IL-23
as an important player in initiating a T cell-dependent amplification of inflammatory
responses in the intestinal mucosa during serotype Typhimurium infection.
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INTRODUCTION
Salmonella enterica serotype Typhimurium elicits an acute inflammatory
response in the intestinal mucosa of humans that can be modeled using streptomycin
pre-treated mice (2). This inflammatory reaction is initiated by direct contact of serotype
Typhimurium with host cells, such as epithelial cells, macrophages or dendritic cells,
followed by an amplification of inflammatory responses in tissue (31). Responses that
are effectively amplified in tissue give rise to the most prominent changes in gene
expression observed in the intestinal mucosa during serotype Typhimurium infection,
including markedly increased mRNA levels of Ifn-γ, Il-17, and Il-22 (9, 24, 25). T cells
play an important role in amplifying inflammatory responses in the cecal mucosa,
because depletion of CD3+ cells causes in a dramatic reduction in cecal inflammation
and neutrophil recruitment (9). T cell depletion also results in a markedly blunted
induction of Ifn-γ, Il-17, and Il-22 in the intestinal mucosa during serotype Typhimurium
infection (9, 25). IL-17 and IL-22 help to orchestrate intestinal inflammation by inducing
the production of neutrophil chemoattractants (e.g. KC or IL-8), dendritic cell
chemoattractants (e.g. CCL20), and antimicrobials (e.g. Lipocalin-2 and iNOS) in the
mucosa (9, 25, 42). However, the mechanisms by which T cell-dependent amplification
of responses to serotype Typhimurium infection is initiated in the intestinal mucosa have
not been explored experimentally.
In other models of infection, cytokines released by macrophages or dendritic
cells have been implicated in stimulating cytokine production by T cells. For example,
detection of bacterial flagellin by cytosolic pattern recognition receptors in macrophages
activates caspase 1, resulting in the release of mature IL-18 (6, 20, 21, 34). IL-18 can
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stimulate antigen experienced T cells to rapidly secrete IFN-γ during bacterial infection
by an antigen-independent mechanism, thereby significantly amplifying early effector
responses in vivo (32). In a mouse model of Klebsiella pneumoniae lung infection,
bacterial stimulation of Toll-like receptor 4 on dendritic cells results in IL-23 production
(13). IL-23 in turn triggers the rapid production of IL-17 and IL-22 by T cells (1, 12),
which is required for efficient neutrophil recruitment in this model (39, 40). IL-23 has also
been implicated in enhancing inflammatory responses elicited by other bacterial
pathogens, including Citrobacter rodentium, Pseudomonas aeruginosa, Mycoplasma
pneumoniae and Mycobacterium bovis (5, 36, 38, 42). The goal of this study was to
determine whether IL-23 contributes to an amplification of inflammatory responses in the
cecal mucosa during serotype Typhimurium infection of streptomycin pre-treated mice.
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MATERIALS AND METHODS
Bacterial strains and culture conditions. Serotype Typhimurium strain IR715
is a fully virulent, nalidixic acid resistant derivative of isolate ATCC14028 and was used
in all experiments (33). Bacteria were cultured aerobically at 37°C in Luria-Bertani (LB)
broth.
Animal experiments. Mice deficient in p19 (IL-23 p19-/- mice) were generated by
breeding B6.129S5-ll23p19tm1Lex mice with C57BL/6 mice under specific pathogen-free
conditions in a barrier facility. IL-23 p19-/- mice and wild-type littermates were bred and
genotyped at the Mutant Mouse Regional Resource Center at the University of
California, Davis. Mice deficient for Tcd, the gene encoding the δ T cell receptor chain,
were obtained from Jackson laboratory (B6.129P2-Tcrdtm1Mom/J).
To study inflammation in the cecum, streptomycin-pretreated mice were orally
infected with serotype Typhimurium as described previously (2). In brief, mice were
inoculated with streptomycin (0.1 ml of a 200mg/ml solution in sterile water)
intragastrically. IL-23 p19-/- mice (N = 16) and wild-type littermates (N = 9) were
inoculated intragastrically 24 hours later with bacteria (0.1ml containing approximately
5x108 colony forming units [CFU]). As a control, IL-23 p19-/- mice (N = 6) and wild-type
littermates (N = 8) were inoculated with 0.1ml of sterile LB broth (mock infection). Trd -/-
(N = 6) and wild-type C57/B6 (N = 5) mice were infected as described above with
serotype Typhimurium. As a control, wild-type (N = 6) and Trd -/- mice (N = 4) were
inoculated with 0.1ml LB broth (mock infection). At 48 hours after infection, mice were
euthanized and samples of the cecum collected for isolation of mRNA and for
histopathological analysis. For bacteriologic analysis, cecal contents and Peyer’s
patches were homogenized and serial 10-fold dilutions spread on agar plates containing
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the appropriate antibiotics. For isolation of intra-epithelial and lamina propria
lymphocytes from infected mice, three groups of two 8-10 week old mice (C57BL/6,
Jackson lab) were inoculated with streptomycin (0.1 ml of a 200mg/ml solution in sterile
water) intragastrically. Twenty-four hours later, mice were inoculated with bacteria (0.1ml
containing approximately 5x108 CFU). IEL and LPL were isolated 48 hours post
infection.
Real-time PCR. For analysis of changes in gene expression after serotype
Typhimurium infection in the mouse cecum, tissue was collected and immediately snap-
frozen in liquid nitrogen at the site of surgery, and store at -80○C until processing. RNA
was then extracted from snap-frozen tissue with TriReagent (Molecular Research
Center) according to instruction by the manufacturer. Next, 1µg from each sample was
reverse transcribed in 50 µl volume (Taqman reverse transcription reagent; Applied
Biosystems) and 4 µl of cDNA was used for each real-time reaction. Real-time PCR was
performed using SYBR Green (Applied Biosystems) and the 7900HT Fast Real-Time
PCR System. The data were analyzed using a comparative threshold cycle method
(Applied Biosystems). Increases in cytokine expression in infected mice were calculated
relative to the average level of the respective cytokine in 8 mock-infected wild type mice.
A list of genes analyzed in this study with the respective primers is provided in Table 1.
For analysis of absolute copy number expression for Il-17 and Reg3g, real-time
PCR was performed using 1µl of cDNA (as described above) for each reaction in a
temperature cycler equipped with a fluorescence detection monitor (LightCycler, Roche
Diagnostics, Mannheim, Germany). Thus, cDNA corresponding to 20ng RNA served as
a template in a 10µl reaction containing 4mM MgCl2, 0.5µM of each primer and 1X
LightCycler-Fast Start DNA Master SYBR Green I mix (Roche Diagnostics). A negative
control reaction without cDNA template was included with each set of reaction to check
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for possible contamination. The PCR conditions were: initial denaturation at 95 °C for 10
min, followed by 45 cycles with each cycle consisting of denaturation, 95 °C for 15 s;
annealing at 60 °C for 5 s; and extension at 72 °C for 10 s. The cycle-to-cycle
fluorescence emission was monitored at 530 nm and analyzed using LightCycler
Software (Roche Diagnostics). Gene-specific plasmid standards were included with
every set of reactions and standard curves generated for each gene product was used to
quantify expression of Il-17 and Reg3g. All reactions were run in duplicate and inter-
sample variation was <10%.
Histopathology. Tissue samples were fixed in formalin, processed according to
standard procedures for paraffin embedding, sectioned at 5 µm, and stained with
hematoxylin and eosin. A veterinary pathologist scored inflammatory changes using a
blind-sample analysis. Neutrophil counts were determined per high (x400)-magnification
microscopy, and numbers were averaged from 10 microscopic fields for each animal.
Isolation of intestinal lymphocytes. Intra-epithelial lymphocytes (IEL) and
lamina propria lymphocytes (LPL) were isolated from C57BL/6 mice by standard
procedures (4, 19, 30). Briefly, three groups of two naïve mice and three groups of two
serotype Typhimurium infected mice were euthanized and organs from each group of
mice were combined. Intestines were removed beginning from the duodenum and
ending at the proximal colon. Intestines were dissected by removing the remaining
mesentery and vasculature. Each segment was then opened longitudinally, the luminal
content was removed, and the tissue was cut into approximately 5 mm sections with a
scalpel. The sections were subsequently washed in cold 1x HBSS (Gibco catalog no.
14185) containing 0.015M HEPES (Gibco catalog no.15630) a total of six times to
remove mucus and remaining fecal matter.
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For IEL isolation, tissue was placed and stirred for 15 minutes at room
temperature in pre-warmed (37°C) 1x HBSS containing 10% FBS (Gibco catalog no.
10082), 0.015M HEPES, and 5mM EDTA and stirred for 15 minutes 37°C followed by
three 15-minute washes with buffer adjusted to room temperature. The supernatant from
each wash was pooled and poured through a nylon wool column to enrich for T cells and
remove mucus. The resulting cell suspension was used to analyze IEL.
To isolate LPL, the tissue remaining after IEL isolation was stirred in pre-warmed
(37°C) 1x RPMI (Sigma R1145) containing 10% FBS, penicillin/streptomycin (Gibco
catalog no.15240-062) and 0.015M HEPES, and 1.6mg/mL collagenase (Sigma-Aldrich
C6885) for 45 minutes in a 37°C incubator. The resulting cell suspension was washed
twice with 1x HBSS containing 0.015M HEPES, enriched for T cells using a nylon wool
column and used to analyze LPL.
Flow Cytometry. The IEL and LPL cell suspensions containing approximately
4x106 cells each were resuspended in cold PBS and stained with Aqua LIVE/DEAD cell
discriminator (Invitrogen #L34597) as per manufacturer protocol. Cells were then stained
for one hour in the dark at 4°C with optimized concentrations of anti-CD3 Alexa750-APC
(eBioscience clone 17A2), anti-CD8 Alexa700 (eBioscience clone 53-6.7), anti-CD4
Pacific Blue (eBioscience clone RM4-5), anti-TCR GD R-PE (BD Pharmingen clone
GL3), and biotinylated polyclonal anti-IL-23R (R&D systems BAF1686). Cells were
washed twice with PBS containing 1% bovine serum albumin (FACS buffer). Cells were
then stained for one hour with streptavidin conjugated Qdot 605 (Invitrogen Q10101MP).
Stained cells were washed once in FACS buffer and subsequently fixed in 4% formalin
for one hour. Cells were then washed once and resuspended in FACS buffer and
analyzed using an LSR II (Becton-Dickinson, San Jose, CA) flow cytometer. Data were
analyzed using Flowjo software (Treestar, inc. Ashland, OR). Gates were set on singlets
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then on live lymphocytes. Subsequent gates were based on Fluorescence-Minus-One
and unstained controls.
Statistical analysis. Fold changes in mRNA levels measured by real-time PCR
underwent logarithmic transformation, and percentage values underwent angular
transformation prior to analysis by Student’s t test.
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RESULTS
IL-23 is required for induction of Il-17, Il-22 and Reg3g expression, but not for Ifn-γγγγ
expression, in the cecal mucosa during serotype Typhimurium infection
We have recently shown that Ifn-γ, Il–17 and Il–22 are among the genes whose
transcript levels are increased most prominently in the cecum of streptomycin pre-
treated mice during serotype Typhimurium infection (9). To study the contribution of IL-
23 in triggering cytokine production in the cecal mucosa, we compared the mRNA levels
of Ifn-γ, Il–17 and Il–22 in IL-23 deficient mice and their wild-type littermates in response
to inoculation with serotype Typhimurium or sterile LB broth (mock infection). IL-23 is a
heterodimer composed of p19 and p40. The p40 subunit is shared with IL-12, a
heterodimer of p40 and p35. We used IL-23p19–/– mice to determine the role of IL-23 in
amplifying inflammatory responses in the intestine. We recently established the time
course of cytokine production in the streptomycin pre-treated mouse model, which
shows that pro-inflammatory cytokines are strongly induced in the cecal mucosa by 48
hours after serotype Typhimurium infection (9). We therefore chose the 48-hour time
point for experiments described in this study.
We compared mRNA levels of cytokines to levels detected in mock-infected wild
type mice (Figure 1). As expected, Il-23p19 mRNA was detected neither in mock-
infected nor in serotype Typhimurium-infected IL-23p19–/– mice (Figure 1A). Compared
to mock-infected wild type mice, Il-23p19 mRNA levels were increased approximately 10
fold in wild type mice infected with serotype Typhimurium. Importantly, while Il–17 mRNA
levels were markedly increased in serotype Typhimurium-infected wild type mice, no
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induction of Il–17 expression was observed in IL-23p19–/– mice (Figure 1B). These data
suggested that induction of Il–17 expression in the cecal mucosa of mice 48 hours after
serotype Typhimurium infection was fully dependent on the presence of IL-23. While Il–
22 mRNA levels were increased in response to serotype Typhimurium infection in both
wild type mice and IL-23p19–/– mice, induction was significantly greater (p<0.001) in wild
type mice (Figure 1C). These data suggested that IL-23 dependent mechanisms
contributed to an induction of Il–22 expression in the cecal mucosa of mice. However,
the increase in Il-22 mRNA levels observed in serotype Typhimurium-infected IL-23p19–
/– mice suggested that IL-23-independent mechanisms also contributed to IL-22
production in vivo. Serotype Typhimurium infection induced Ifn-γ mRNA to similar levels
in both wild type mice and IL-23p19–/– mice (Figure 1D), indicating that IFN-γ production
is induced by IL-23-independent mechanisms in the cecal mucosa. The lower degree of
Il–17 and Il–22 expression in the ceca in IL-23p19–/– mice was not due to differences in
bacterial load, because similar bacterial numbers were recovered from intestinal
contents and intestinal tissue of serotype Typhimurium-infected wild type mice and IL-
23p19–/– mice (Figure 1E).
Next, we determined the absolute number of Il-17 transcripts using quantitative
real-time PCR (Figure 2). In wild type mice, the absolute number of Il-17 transcripts was
markedly increased during serotype Typhimurium infection compared to mock infection.
In contrast, mock-infected and serotype Typhimurium IL-23p19–/– mice had almost
identical Il-17 transcript levels (Figure 2A). These data further supported the idea that
increases in Il–17 transcript levels were entirely IL-23-dependent. We also determined
absolute transcript levels of Reg3g, a gene encoding an antimicrobial, whose production
is induced by IL-22 in the cecal mucosa of mice during inflammation (42). During
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serotype Typhimurium infection, Reg3g was produced at high levels in the cecal mucosa
of wild type mice, averaging 11,000 copies/ng RNA compared to 470 copies/ng in mock-
infected mice (Figure 2B). Induction of Reg3g was largely IL-23 dependent, since IL-
23p19–/– mice exhibited markedly reduced transcript levels during serotype Typhimurium
infection. In summary, our results supported the idea that IL-23 helps to amplify
inflammatory responses in the cecal mucosa by inducing expression of IL-17 and by
contributing to a full induction of IL-22 expression during serotype Typhimurium infection.
IL-23 contributes to neutrophil recruitment in the cecal mucosa during serotype
Typhimurium infection
Since IL-17 and IL-22 play a major role in orchestrating inflammatory responses
in the intestinal mucosa of mice (25, 42), we investigated the consequences of the IL-
17/IL-22 deficiency in IL-23p19–/– mice (Figure 3). As these cytokines orchestrate a
mucosal inflammatory response resulting in neutrophil influx at the site of infection, we
hypothesized that p19 deficient mice would exhibit reduced expression of neutrophil
chemoattractants and reduced neutrophil influx in the cecal mucosa after infection with
serotype Typhimurium. We first investigated expression of the neutrophil
chemoattractant KC in the cecum of wild type mice and IL-23p19–/– mice 48 hours after
infection with serotype Typhimurium. Compared to mock-infected wild type mice, Kc
mRNA levels were markedly elevated (approximately 200 fold) in serotype Typhimurium-
infected wild type mice (Figure 3A). Induction of Kc expression was notably blunted in
the ceca of serotype Typhimurium-infected IL-23p19–/–mice compared to serotype
Typhimurium-infected wild type mice (P < 0.001). These data suggested that IL-23 is
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required for the full induction of neutrophil chemoattractants in the cecal mucosa during
serotype Typhimurium infection.
Next, we quantified neutrophil recruitment in the cecal mucosa by determining
counts per microscopic field at high magnification. Few neutrophils were detected in the
cecal mucosa of mock-infected mice while infection with serotype Typhimurium was
accompanied by marked neutrophil recruitment. However, there were significantly (P =
0.02) less neutrophils per field in serotype Typhimurium-infected ceca of IL-23p19–/–
mice compared serotype Typhimurium-infected ceca of wild type mice (Figure 3B).
These results were in good agreement with the lower Kc expression observed in
serotype Typhimurium-infected IL-23p19–/–mice (Figure 3A). The severity of
inflammatory changes was reduced in serotype Typhimurium-infected IL-23p19–/–mice
(Figure 3C) compared to serotype Typhimurium-infected wild type mice (Figure 3D).
However, compared to mock-infected mice (Figure 3E and 3F), serotype Typhimurium
infection was associated with marked inflammatory changes (Figure 3C and 3D). In
summary, our data suggested that IL-23 contributed to the recruitment of neutrophils into
the cecal mucosa during serotype Typhimurium infection.
The IL-23 receptor (IL-23R) is expressed by a subset of intestinal T cells
We have recently shown that depletion of T cells results in a marked reduction in
the expression levels of IL-17 and IL-22 in the cecum of streptomycin pre-treated mice
during serotype Typhimurium infection (9). These data suggest that the IL-17/IL-22
deficiency observed in IL-23p19–/– mice (Figure 1) could be explained by hypothesizing
that IL-23 stimulates a subset of intestinal T cells to produce IL-17 and IL-22. This
hypothesis would predict that a subset of intestinal T cells expresses the receptor for IL-
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23. To test this prediction, we isolated intra-epithelial lymphocytes (Figure 4A) and
lamina propria lymphocytes (Figure 4B) from the intestine of mice and analyzed
expression of surface markers by flow cytometry. Intra-epithelial CD3+ cells were divided
into cells expressing the γδ T cell receptor (γδ T cells) and γδ T cell receptor negative
cells (representing αβ T cells) (Figure 4C). Finally, αβ T cell subsets were defined based
on expression of CD4 and CD8 (Figure 4D). The same procedure was applied to lamina
propria CD3+ cells (Figure 4E and F).
Approximately 40% of the intra-epithelial CD3+ cells expressed the γδ T cell
receptor in naïve mice (i.e. CD8+ γδ+ T cells and CD8- γδ
+ T cells constituted
approximately 40% of intra-epithelial CD3+ T cells) (Figure 5A). In contrast, γδ T cells
were only a minor population (approximately 10%) of the lamina propria CD3+ cell
population, which was dominated by CD4+ T cells. That is, approximately 50% of CD3+
cells in the lamina propria were CD4+ CD8- γδ- T cells (Figure 5B). These data were
consistent with previous studies on the composition of intestinal intra-epithelial and
lamina propria CD3+ cell populations in the mouse (8). No significant differences in the
relative proportions of T cell subsets were observed during analysis of tissue collected
from naïve mice compared to tissue collected from serotype Typhimurium-infected mice
(Figure 5).
We next investigated expression of the IL-23 receptor by T cell subsets isolated
from the intestinal mucosa (Figure 6). In naïve mice, the overall fraction of CD3+ intra-
epithelial lymphocytes or CD3+ lamina propria lymphocytes that expressed the IL-23
receptor was approximately 10%. In the intra-epithelial CD3+ lymphocyte population, the
majority of cells expressing the IL-23 receptor were CD4-CD8- γδ- cells, regardless of
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whether tissue had been collected from naïve mice or from serotype Typhimurium-
infected mice (Figure 6A). Previous studies suggest that CD4-CD8- γδ- cells in the gut
mucosa of mice comprise natural killer T (NKT) cells and CD4-CD8- T cells (15). In the
lamina propria CD3+ lymphocyte population of naïve mice, the majority of cells
expressing the IL-23 receptor were either CD4-CD8- γδ- cells or CD4+CD8- γδ- cells
(potentially representing TH17 cells) (Figure 6B).
Importantly, in serotype Typhimurium-infected tissue, we observed a marked
increase in the lamina propria CD3+ lymphocyte population of CD8- γδ+ cells expressing
the IL-23 receptor (Figure 6C). This notable increase in IL-23 receptor expressing γδ T
cells during serotype Typhimurium infection raised the overall fraction of CD3+ lamina
propria lymphocytes that expressed the receptor for IL-23 above 20%. In summary, our
results show that a fraction (10-20%) of CD3+ lymphocytes in the intestinal mucosa of
mice express the receptor for IL-23. Furthermore, 48 hours after serotype Typhimurium
infection, we observed a marked increase in IL-23 receptor expressing γδ T cells.
γδγδγδγδ T cells contribute to Il-17 expression in the inflamed cecal mucosa of mice
Since an increase in IL-23 receptor expressing γδ T cells was the only notable
change in mucosal T cell populations observed during serotype Typhimurium infection
(Figure 6B and C), we investigated whether γδ T cells contribute to cytokine production
in the inflamed murine cecum. To this end, we compared the mRNA levels of Ifn-γ and
Il–17 in γδ T cell receptor deficient (Trd-/-) mice and wild-type controls (C57BL/6 mice) in
response to inoculation with serotype Typhimurium or sterile LB broth (mock infection)
(Figure 7). There was a compensatory increase in Il-23p19 mRNA expression in
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serotype Typhimurium-infected γδ T cell receptor deficient mice (Figure 7A). Il–17 mRNA
levels induced during serotype Typhimurium infection were significantly lower in γδ T cell
receptor deficient mice than in wild type (P < 0.05) (Figure 7B). In contrast, serotype
Typhimurium infection induced Ifn-γ mRNA (Figure 7C) and Il-22 mRNA (Figure 7D) to
similar levels in both wild type mice and γδ T cell receptor deficient mice. Similar
bacterial numbers were recovered from intestinal contents of infected mice (Figure 7E).
These data suggested that γδ T cells contributed to Il-17 expression in the inflamed cecal
mucosa. However, unlike IL-23p19–/– mice, γδ T cell receptor deficient mice still exhibited
increased Il–17 mRNA levels in response to serotype Typhimurium infection, suggesting
that γδ T cells are not the sole source of IL-17 in the inflamed cecum.
The absolute number of Il-17 transcripts in the cecal mucosa was quantified
using real-time PCR (Figure 8). Mice with γδ T cell deficiency induced Il-17 expression in
response to S. Typhimurium infection (P = 0.003), but transcript levels were markedly
reduced compared to those measured in wild type mice infected with serotype
Typhimruium (P = 0.03) (Figure 8A). These data further supported the idea that γδ T
cells contribute Il–17 expression in the cecal mucosa. Although absolute transcript levels
of Reg3g were reduced (Figure 8B) and lower numbers of neutrophils were observed in
cecal tissue of γδ T cell receptor deficient mice during serotype Typhimurim infection
(Figure 8C), these differences were not statistically significant. Our data suggest that the
partial inhibition of Il-17 expression (Figure 7B and 8A), which was accompanied by
normal expression of Il-22 (Figure 7D), was not sufficient to significantly reduce Reg3g
expression or neutrophil recruitment in γδ T cell receptor deficient mice (Figure 8B and
8C). Thus, while γδ T cells contribute to Il-17 expression, our data suggest that there
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must be additional cellular sources to fully account for the increased IL-17 and IL-22
production in the inflamed cecum of the mouse.
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DISCUSSION
Acute intestinal inflammation characterized by a massive neutrophil influx is a
hallmark of serotype Typhimurium infection (27, 35, 41). However, the precise
mechanisms by which this host response is orchestrated in tissue have not been fully
worked out. We have recently shown that depletion of CD3+ lymphocytes markedly
reduces the ability of mice to recruit neutrophils into the cecal mucosa and to produce
KC, IFN-γ, IL-22 and IL-17, which are among the most prominently induced cytokines in
serotype Typhimurium-infected tissue (9). These data suggest that T cells are an
important component of mechanisms that help to amplify inflammatory responses to
serotype Typhimurium infection in the intestinal mucosa. However, little is known about
how serotype Typhimurium infection initiates these T cell-dependent amplification
mechanisms. Here we show that diverse subsets of T cells in the intestinal mucosa
expresses the receptor for IL-23, a cytokine important for initiating the production of KC,
RegIIIγ, IL-22 and IL-17 in response to serotype Typhimurium infection. IFN-γ production
was not affected by IL-23 deficiency, suggesting that the early expression of this
important cytokine by T cells (i.e. at 2 days after infection) is triggered through other
pathways, perhaps involving IL-12 or IL-18 production. For example, IL-18 has recently
been implicated in amplifying inflammatory responses early after serotype Typhimurium
infection in the spleen of mice by triggering IFN-γ production in antigen experienced CD4
T cells by an antigen-independent mechanism (32).
Two important questions arise from the results of our study. First, which intestinal
T cell subsets contribute to IL-17 and IL-22 production during serotype Typhimurium
infection. In the lung mucosa, γδ T cells have been implicated as an important source of
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IL-17 during M. tuberculosis and M. bovis infection (18, 22, 36). Similarly, injection of
Escherichia coli into the peritoneal cavity of naive mice triggers IL-23 production in a
TLR4 signaling-dependent manner and the resulting IL-17 production originates largely
from γδ T cells (28). Our results suggest that γδ T cells are also one of the cellular
sources of IL-17 in the serotype Typhimurium infected mouse. However, γδ T cell
deficient mice were still able to produce Il-17 mRNA, albeit at reduced levels, during
serotype Typhimurium infection, suggesting that additional cell types contributed to
production of this cytokine in the inflamed cecum. In addition to γδ T cells, the receptor
for IL-23 was expressed predominantly by CD3+CD4-CD8- γδ
- cells (NKT cells and/or
CD4-CD8- T cells) in the intestinal epithelium and by CD4+ T cells and CD3+CD4-CD8- γδ
-
cells in the lamina propria. Each of these cell types has been implicated as a source of
IL-17 production in different animal models of inflammation (3)(13)(17)(16)(23)(25). A
distinct subset of CD4- NKT cells produces IL-17, contributing to infiltration of neutrophils
in a galactosylceramide-induced model of airway inflammation (17). NKT cells
constitutively express IL-23R and rapidly produce IL-17 upon stimulation with IL-23 (23).
IL-17 mRNA has been shown to be specifically expressed by a subset of murine CD4-
CD8- T cells (16). In contrast, IL-17 is mainly derived from CD4+ T cells during M.
pneumoniae lung infection (38). Both CD4+ T cells and CD8+ T cells are a source of IL-
17 during infection of mice with H. pylori (3) or K. pneumoniae (13). Finally, depletion of
memory CD4+ T cells by simian immunodeficiency virus blunts IL-17 responses elicited
early (i.e. 5 hours) after serotype Typhimurium infection of the ileal mucosa in rhesus
macaques (25), pointing to an innate induction of these T cell responses. Thus, TH17
cells contribute to IL-17 production in the ileal mucosa of a relevant animal species.
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The second question arising from this study relates to the mechanisms that
initiate IL-23 production during serotype Typhimurium infection. Electron microscopic
analysis of serotype Typhimurium infection shows that all bacteria detected in the
intestinal mucosa have an intracellular location, either within mononuclear phagocytes
(macrophages and/or dendritic cells) or within neutrophils (7, 26). Since only a very
small fraction of cells in infected tissue contain bacteria, the sum-total capacity for
cytokine production by these cells may be limited in scope. However, macrophages and
dendritic cells infected with serotype Typhimurium are a potential source of IL-23 and
our data suggest that this cytokine helps to amplify a subset of inflammatory responses
in tissue. During K. pneumoniae infection, release of IL-23 by dendritic cells in the lung
mucosa is triggered through stimulation of TLR4 by lipopolysaccharide (13). Similarly,
production of IL-23 by murine bone marrow-derived dendritic cells in response to S.
enterica serotype Enteritidis infection is TLR4-dependent (29). However,
CD11c+CX3CR1+ mucosal dendritic cells do not generate MyD88-dependent responses
in the ceca of serotype Typhimurium infected mice (10), suggesting that the signals
produced at mucosal sites are not mimicked adequately by bone marrow-derived cells. A
recent finding that points to macrophages as possible sources of IL-23 is the observation
that the inflamed human intestine contains a unique subset of CD14+ intestinal
macrophages, which produces larger amounts of IL-23 than the resident CD14-
macrophages (14). Alternatively, serotype Typhimurium may stimulate mucosal dendritic
cells or mucosal macrophages to produce IL-23 through MyD88-independent
mechanisms, which have been proposed to contribute to cecal inflammation (11). One
possible MyD88-independent mechanism leading to IL-23 production by dendritic cells is
the activation of the intracellular bacterial sensor NOD2. IL-23 produced by this NOD2-
dependent, MyD88-independent mechanism results in IL-17 production in human
memory T cells (37). However, additional work is needed to understand the precise
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mechanisms by which the IL-23/IL-17 axis is triggered in the intestinal mucosa during
serotype Typhimurium infection.
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ACKNOWLEDGEMENTS
We would like to thank Sebastian Winter, Maria Winter, and Sean-Paul Nuccio
for their help with animal experiments. We would also like to thank Monica Macal and
Carol Oxford for their input in designing flow cytometry panels.
This investigation was conducted in a facility constructed with support from
Research Facilities Improvement Program Grant Number C06 RR12088-01 from the
National Center for Research Resources, National Institutes of Health. Work in AJB's
laboratory was supported by Public Health Service grants AI040124, AI044170 and
AI079173. TAP and RLS are recipient of fellowships from CNPq (Conselho Nacional de
Desenvolvimento Científico e Tecnológico, Brasília, Brazil). I.G. was supported by Public
Health Service grant AI060555.
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FIGURE LEGENDS
Figure 1: Cytokine expression elicited by serotype Typhimurium in streptomycin pre-
treated wild type mice (black bars) and streptomycin pre-treated IL-23 deficient mice
(gray bars) 48 hours after infection measured by quantitative real-time PCR. (A-D) Bars
represent fold changes in mRNA levels of Il-23 (A), Il-17 (B), Il-22 (C) and Ifn-γ (D)
compared to mRNA levels detected in a group of mock-infected wild type mice (N = 8).
Data are shown as geometric means of fold-changes ± standard error determined for
RNA from individual mice. (E) Average bacterial numbers (CFU) recovered 48 hours
after serotype Typhimurium infection from colon contents or Peyer’s patch tissue of wild
type mice (black bars) or IL-23 deficient mice (gray bars). Statistical significance of
differences is indicated by P values above brackets. NS, not significant.
Figure 2: Absolute transcript levels of Il-17 (A) and Reg3g (B) in IL-23 p19 deficient
mice (IL-23p19-/-, gray bars) or wild type littermates (black bars) determined by
quantitative real-time PCR 48 hours after mock infection or infection with serotype
Typhimurium. Data represent mean mRNA copy numbers per 20ng of RNA ± standard
error. Statistically significant differences are indicated by P values.
Figure 3: Neutrophil recruitment into the cecal mucosa. (A) Expression of the neutrophil
chemoattractant Kc elicited by serotype Typhimurium in streptomycin pre-treated wild
type mice (black bars) and streptomycin pre-treated IL-23 deficient mice (gray bars) 48
hours after infection measured by quantitative real-time PCR. Bars represent fold
changes in mRNA levels compared to mRNA levels detected in a group of mock-infected
wild type mice (N = 8). Data are shown as geometric means of fold-changes ± standard
error determined for RNA from individual mice. (B) The numbers of neutrophils per
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microscopic field were determined by a veterinary pathologist during a blinded
examination of slides from the cecal mucosa. Data represent means±standard error.
Statistical significance of differences is indicated by P values. (C to F) Histopathological
appearance of the murine cecum of serotype Typhimurium-infected IL-23 deficient mice
(C), serotype Typhimurium-infected wild type mice (D), mock-infected IL-23 deficient
mice (E) or mock infected wild type mice (F). All images were taken from hematoxylin
and eosin stained cecal sections at the same magnification (100x).
Figure 4: Isolation of T cells from the intestinal epithelium and lamina propria. Total
number of live CD3+ cells present in preparations of intra-epithelial lymphocytes (A) and
lamina propria lymphocytes (B) from naïve mice (N = 6, gray bars) or serotype
Typhimurium infected mice (N = 6, black bars). (C) Representative example of the
gating strategy used to define γδ+ and γδ- T cell populations among live intra-epithelial
lymphocytes. (D) Representative example of the gating strategy used to separate intra-
epithelial γδ- T cells into different subsets. (E) Representative example of the gating
strategy used to define γδ+ and γδ- T cell populations among live lamina propria
lymphocytes. (F) Representative example of the gating strategy used to separate lamina
propria γδ- T cells into different subsets. (C-F) Axis represent the fluorescence intensity
produced by fluorescent antibody conjugates recognizing the γδ T cell receptor (γδTCR),
CD3, CD4 or CD8.
Figure 5: Characterization of T cell subsets in the intestine of naïve mice (N = 6, gray
bars) or serotype Typhimurium-infected mice (N = 6, black bars). (A) T cell subsets in
the intra-epithelial lymphocyte population are shown as percentage of the total number
of intra-epithelial T cells (CD3+ intra-epithelial lymphocytes). (B) T cell subsets in the
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lamina propria lymphocyte population are shown as percentage of the total number of
lamina propria T cells (CD3+ lamina propria lymphocytes). Data are shown as
mean±standard error.
Figure 6: Expression of IL-23R by intra-epithelial T cells (A) and lamina propria T cells
(B) in the intestine of naïve mice (N = 6, gray bars) or serotype Typhimurium-infected
mice (N = 6, black bars). (A) IL-23R expressing cells expressing the indicated markers
(CD4, CD8 and/or γδ TCR) are shown as a percentage of the total number of intra-
epithelial T cells (CD3+ intra-epithelial lymphocytes). (B) IL-23R expressing cells
expressing the indicated markers (CD4, CD8 and/or γδ TCR) are shown as a percentage
of the total number of lamina propria T cells (CD3+ lamina propria lymphocytes). Data
are shown as mean±standard error. Statistical significance of differences is indicated by
P values. (C) Representative example of IL-23 receptor expression by CD8- γδ+ lamina
propria T cells pooled from the intestine of two naïve mice (left panel) or two serotype
Typhimurium infected mice (right panel).
Figure 7: Cytokine expression elicited by serotype Typhimurium in streptomycin pre-
treated wild type mice (C57BL/6, black bars) or streptomycin pre-treated T cell receptor
δ chain deficient mice (Trd-/-, gray bars) 48 hours after infection measured by quantitative
real-time PCR. (A-D) Bars represent fold changes in mRNA levels of Il-23 (A), Il-17 (B),
Ifn-γ (C) and Il-22 (D) compared to mRNA levels detected in a group of mock-infected
wild type mice (N = 8). Data are shown as geometric means of fold-changes ± standard
error determined for RNA from individual mice. (E) Average bacterial numbers (CFU)
recovered 48 hours after serotype Typhimurium infection from colon contents or Peyer’s
patch tissue of wild type mice (black bars) or γδ T cell receptor deficient mice (gray bars).
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Statistical significance of differences is indicated by P values above brackets. NS, not
significant.
Figure 8: Absolute transcript levels of Il-17 (A) and Reg3g (B) in γδ T cell receptor
deficient mice (Trd-/-, gray bars) or wild type mice (C57BL/6, black bars) determined by
quantitative real-time PCR 48 hours after mock infection or infection with serotype
Typhimurium. Data represent mean mRNA copy numbers per 20ng of RNA ± standard
error. (C) Numbers of neutrophils per microscopic field were determined by a veterinary
pathologist during a blinded examination of slides from the cecal mucosa. Data
represent means±standard error. Statistically significant differences are indicated by P
values above brackets. NS, not significant.
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Table 1: Primers for real-time PCR.
Gene Primer pairs
Gapdh 5’-TGTAGACCATGTAGTTGAGGTCA-3’
5’-AGGTCGGTGTGAACGGATTTG-3’
Il23 p19 5’-TGTGCCTAGGAGTAGCAGTCCTGA-3’
5’-TTGGCGGATCCTTTGCAAGCAGAA-3’
Il-17
(relative)
5’-GCTCCAGAAGGCCCTCAGA-3’
5’-AGCTTTCCCTCCGCATTGA-3’
Il-22 5’-GGCCAGCCTTGCAGATAACA-3’
5’-GCTGATGTGACAGGAGCTGA -3’
Kc 5’-TGCACCCAAACCGAAGTCAT-3’
5’-TTGTCAGAAGCCAGCGTTCAC-3’
Ifn-γ 5’- TCAAGTGGCATAGATGTGGAAGAA-3’
5’-TGGCTCTGCAGGATTTTCATG-3’
Reg3g 5’-CCTCAGGACATCTTGTGTC-3’
5’-TCCACCTCTGTTGGGTTCA-3’
Il-17
(absolute)
5’-AACCCCCACGTTTCTCAGCAAAC-3’
5’-GGACCCCTTTACACCTTCTTTTCATTG -3’
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A
500
1000
1500
2000
2500
3000
3500B
IL-23p19–/– wild type
S. Typhimurium
infection
Mock
infection
IL-23p19–/–
Il–17
mRNA
(fold
increase)
P < 0.001
IL-23p19–/– wild type
S. Typhimurium
infection
Mock
infection
IL-23p19–/–
Ifn-γ
mRNA
(fold
increase)
500
1000
1500
2000
2500
3000
IL-23p19–/– wild type
S. Typhimurium
infection
Mock
infection
IL-23p19–/–
P < 0.001CIl–22
mRNA
(fold
increase)
200
400
600
800
1000
1200
IL-23p19–/– wild
type
S. Typhimurium
infection
Mock
infection
IL-23p19–/–
Il–23
mRNA
(fold
increase) 4
8
12
16
D
IL-23p19–/– wild
type
IL-23p19–/– wild
type
Peyer’s patchesColon contents
100
101
102
103
104
105
106
107
108E
CFU
Figure 1
NS
NS
NS
0
0
00
ACCEPTED
on August 27, 2019 by guest
http://iai.asm.org/
Dow
nloaded from
Figure 2
0
100
200
300
400
500
600
700
800
IL-23p19–/– wild
type
IL-23p19–/– wild
type
S. Typhimurium
infection
Mock infection
AIl–17
mRNA
copy
number/
20ng
RNA
P < 0.001
IL-23p19–/– wild
type
IL-23p19–/– wild
type
S. Typhimurium
infection
Mock infection
BReg3g
mRNA
copy
number/
20ng
RNA
0
1x105
2x105
3x105
P < 0.001
ACCEPTED
on August 27, 2019 by guest
http://iai.asm.org/
Dow
nloaded from
0
50
100
150
200
250
P = 0.001
IL-23p19–/– wild
type
S. Typhimurium
infection
Mock
infection
IL-23p19–/–
Kc
mRNA
(fold
increase)
ANeutrophils
(counts
per
microscopic
field)
B
IL-23p19–/– wild
type
Mock
infection
IL-23p19–/– wild
type
S. Typhimurium
infection
0
40
80
120 P = 0.02
Figure 3
C D
E F
Mock
infection
S. Typhimurium
infection
Wild typeIL-23p19–/–
ACCEPTED
on August 27, 2019 by guest
http://iai.asm.org/
Dow
nloaded from
C
E
D
F
CD8
CD8
CD4
CD4
CD3
CD3
γδ
TCR
γδ
TCR
Lamina propria lymphocytes γδ- T cells
Intra-epithelial lymphocytes γδ- T cells
γδ+ T cells
γδ+ T cells
CD4-CD8- CD4-CD8+
CD4+CD8- CD4+CD8+
CD4-CD8- CD4-CD8+
CD4+CD8- CD4+CD8+
live
CD3+
lymphocytes/
106 events
A
Figure 4
B
0
10,000
20,000
30,000
40,000
50,000
0
20,000
40,000
60,000
80,000
naive S. Typhimurium
infection
Intra-epithelial lymphocytes
naive S. Typhimurium
infection
Lamina propria lymphocytes
live
CD3+
lymphocytes/
105 events
ACCEPTED
on August 27, 2019 by guest
http://iai.asm.org/
Dow
nloaded from
0
10
20
30
40
50
CD4+CD8- CD4-CD8+ CD4-CD8- CD4+CD8+ CD8+ CD8-
0
10
20
30
40
50
60
70
γδ- T cells γδ+ T cells
% of
total
CD3+
cells
Intra-epithelial lymphocytes
naïve S. Typhimurium infection
CD4+CD8- CD4-CD8+ CD4-CD8- CD4+CD8+ CD8+ CD8-
γδ- T cells γδ+ T cells
% of
total
CD3+
cells
Lamina propria lymphocytes
naïve S. Typhimurium infection
A
B
Figure 5
ACCEPTED
on August 27, 2019 by guest
http://iai.asm.org/
Dow
nloaded from
0
2
4
6
8
10
0
2
4
6
8
10
12
14
CD4+CD8- CD4-CD8+ CD4-CD8- CD4+CD8+ CD8+ CD8-
γδ- T cells γδ+ T cells
IL-23R+
cells as
% of total
CD3+ cells
Intra-epithelial lymphocytes
naïve S. Typhimurium infection
CD4+CD8- CD4-CD8+ CD4-CD8- CD4+CD8+ CD8+ CD8-
γδ- T cells γδ+ T cells
IL-23R+
cells as
% of total
CD3+ cells
Lamina propria lymphocytes
naïve S. Typhimurium infection
A
B P = 0.002
C
CD3
IL23R
S. Typhimurium infection
CD8-γδ+ lamina propria T cells
CD3
IL23R
Naïve
CD8-γδ+ lamina propria T cells
Figure 6
ACCEPTED
on August 27, 2019 by guest
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Dow
nloaded from
0
200
400
600
800
1000
1200
1400B
Trd–/– wild type
S. Typhimurium
infection
Mock
infection
Trd–/–
Il–17
mRNA
(fold
increase)
P < 0.05A
Trd–/– wildtype
S. Typhimurium
infection
Mock
infection
Trd–/–
Il–23
mRNA
(fold
increase)
0
5
10
15
20
25
30
35
40
Figure 7
0
500
1000
1500
2000
Trd–/– wild type
S. Typhimurium
infection
Mock
infection
Trd–/–
Ifn-γ
mRNA
(fold
increase)
CNS
P < 0.05
Peyer’s patchesColon contents
Trd–/– wild type
Trd–/– wild type
NS
NS
100
101
102
103
104
105
106
107
CFU
E
0
200
400
600
800
1000
1200
1400Il-22
mRNA
(fold
increase)
DNS
Trd–/– wild
type
S. Typhimurium
infection
Mock
infection
Trd–/–
ACCEPTED
on August 27, 2019 by guest
http://iai.asm.org/
Dow
nloaded from
0
100
200
300
400
500
600
Trd–/– wild
type
Trd–/– wild
type
S. Typhimurium
infection
Mock infection
B
Reg3g
mRNA
copy
number/
20ng RNA
P = 0.003
0
100
200
Neutrophils
(counts
per
microscopic
field)
C
Trd–/– wild
type
Trd–/– wild
type
S. Typhimurium
infection
Mock infection
0
1x106
2x106
Trd–/– wildtype
Trd–/– wildtype
S. Typhimurium
infection
Mock infection
AIl–17
mRNA
copy
number/
20ng RNA
NS
NS
Figure 8
P = 0.03
ACCEPTED
on August 27, 2019 by guest
http://iai.asm.org/
Dow
nloaded from