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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 Godinez 1 , Manuela Raffatellu 1 , Hiutung Chu 1 , Tatiane A. Paixão 1,2 , Takeshi Haneda 1 , Renato L. Santos 2 , Charles L. Bevins 1 , Renée M. Tsolis 1 and Andreas J. Bäumler 1, * 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 ACCEPTED 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 on August 27, 2019 by guest http://iai.asm.org/ Downloaded from

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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

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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

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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’

    AC

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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) 48

    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

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  • 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

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  • 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–/–

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  • 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

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  • 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

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  • 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

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  • 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–/–ACCE

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  • 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

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