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. 2010 Nov;131(3):377–385. doi: 10.1111/j.1365-2567.2010.03310.x

Interleukin-17A is required to suppress invasion of Salmonella enterica serovar Typhimurium to enteric mucosa

Hirokazu Mayuzumi 1, Kyoko Inagaki-Ohara 1,2, Catherine Uyttenhove 3, Yuko Okamoto 1, Goro Matsuzaki 1
PMCID: PMC2996558  PMID: 20575990

Abstract

Salmonella enterica serovar Typhimurium (S. typhimurium) causes a localized enteric infection and its elimination is dependent on a T helper type 1 immune response. However, the mechanism of the protective immune response against the pathogen in gut-associated lymphoid tissue (GALT) at an early stage of the infection is not yet clarified. Here, we show that interleukin-17A (IL-17A) was constitutively expressed in GALT; it was also detected on crypt and epithelial cells of the small intestine. Neutralization of the IL-17A in the intestinal lumen exacerbated epithelial damage induced by intestinal S. typhimurium infection at an early stage of the infection. The result suggests that IL-17A has a pivotal role in the immediate early stage of protection against bacterial infection at the intestinal mucosa. As IL-17A neutralization also suppressed the constitutive localization of β-defensin 3 (BD3), an IL-17A-induced antimicrobial peptide, at the apical site of the intestinal mucosa, it is estimated that IL-17A constitutively induces the expression of the antimicrobial peptide to kill invading pathogens at the epithelial surface immediately after the infection. In contrast, interferon-γ is induced around 3 days after S. typhimurium infection, and its expression level increases thereafter. Taken together, the findings lead to the hypothesis that IL-17A participates in the immediate early stage of protection against S. typhimurium intestinal infection whereas interferon-γ is important at a later stage of the infection.

Keywords: interleukin-17A, innate immunity, intestinal infection, Salmonella typhimurium

Introduction

Salmonella enterica infection is a serious public health and veterinary problem worldwide. Salmonella enterica serovar Typhimurium (S. typhimurium) is a Gram-negative bacterium causing enteritis in humans and a typhoid-like systemic disease in mice. After oral infection, S. typhimurium interacts with the intestinal mucosa, invades the mucosal tissue, and triggers pronounced inflammation in the terminal ileum and colon characterized by a massive neutrophil influx.1,2 In pathogenesis of S. typhimurium, type III secretion systems encoded by Salmonella pathogenicity islands 1 and 23,4 are crucial. As the intestinal mucosa is the initial site of the inflammatory response elicited by S. typhimurium and human infection with S. typhimurium induces a rapid development of acute enteritis,5 it is important to elucidate mucosal immunity in the early stage of infection to control infection-induced mucosal damage and eliminate the bacteria.

A variety of cytokines co-operate in protective immunity against S. typhimurium.6 Especially, interferon-γ (IFN-γ), interleukin-17A (IL-17A) and IL-22 have been reported to increase during the early period of enteric S. typhimurium infection.7 The T helper type 1 (Th1) immune response is well known to be essential for the control of Salmonella infection in mice and humans;8,9 indeed, IFN-γ-deficient mice are susceptible to S. typhimurium infection.10 The other host strategy for controlling the colonization and dissemination of the pathogens is to limit their thriving by producing antimicrobial factors to support the host defence mechanism before induction of Th1-acquired immunity. After translocation through the intestinal epithelium, S. typhimurium resides mainly within mononuclear cells, such as macrophages, dendritic cells, neutrophils, in the lamina propria (LP) of the small intestine (SI) and then spreads systemically.11,12 The macrophages and dendritic cells infected with S. typhimurium are a source of cytokines, including IL-12 and IL-23, which stimulate T cells in the intestinal mucosa to produce IFN-γ, IL-17A and IL-22.13

Both IL-17A and IL-22 are prominently induced during S. typhimurium infection.7,14 Interleukin-17A was originally reported as an inflammatory cytokine, which is produced by CD4+ T-cell receptor (TCR) -αβ T cells. It induces the differentiation and chemotaxis of neutrophils15 and contributes to the elimination of pathogens or induces inflammatory autoimmune diseases.16–18 However, in wild-type unimmunized mice kept under specific pathogen-free conditions, CD4+ IL-17-producing Th17 cells are present almost exclusively in the small intestinal LP and other mucosal tissues.19 This suggests the involvement of IL-17A in mucosal immune surveillance against the invasion of pathogens at the intestine. Consistent with this, IL-17A induces not only neutrophil migration but also the expression of various antimicrobial molecules, including β-defensins (BDs), S100A8/9 protein, a chelator that deletes ions essential for bacterial activity,20 and lipocalin-2, an inhibitor of bacterial iron acquisition.7 The IL-17A-mediated expression of antimicrobial molecules is further enhanced in the presence of IL-22.20 However, the role of IL-17A in host defence against S. typhimurium infection in the intestinal tract at an early stage of infection is not fully understood. We therefore focused on the role of IL-17A in the gut-associated lymphoid tissue (GALT) and intestinal mucosa during the early stage of intestinal S. typhimurium infection.

Materials and methods

Mice

Male C57BL/6 mice were purchased at the age of 7 weeks from Japan SLC (Hamamatsu, Japan). All mice were used between 8 and 12 weeks of age, and the protocols were approved by the institutional review board for animal experiments of the University of the Ryukyus.

Bacterial strains and culture condition

Salmonella enterica serovar Typhimurium strain LT2, a kind gift from Dr Takaaki Akaike (Kumamoto University, Japan) was used in all experiments. The bacteria were grown in brain–heart infusion broth (Difco Laboratories, Detroit, MI). The bacterial suspension was prepared in 10 mm phosphate-buffered saline (PBS; pH 7·4) and stored at − 80° until used.

Infection of S. typhimurium and antibody treatment

Mice were fasted overnight before S. typhimurium inoculation. To orally infect mice with S. typhimurium, mice were pre-treated with 5% NaHCO3 for neutralization of gastric juice, and 10 min later, were infected with 109 colony-forming units (CFU) of S. typhimurium. To infect the ligated ileal loop of mice with S. typhimurium, mice were anaesthetized with Nembutal, stretches of the ileum almost 5 cm long were ligated with surgical thread to prepare ligated ileal loop and 5 × 105 CFU of S. typhimurium was injected into the loop in 200 μl PBS. Two hundred microgrammes of anti-IL-17A monoclonal antibody (mAb) (MM17F3)21 or mouse immunoglobulin G (ImmunoResearch Laboratories Inc., West Grove, PA) as a control antibody were administered to mice on day 8 and day 1 before S. typhimurium infection for systemic antibody treatment. In other experiments, 20 μg/mouse of anti-IL-17A mAb or control antibody was administrated into ileal loops with S. typhimurium and 1 or 3 hr later, mice were killed to collect the loops.

Measurement of bacterial growth in intestine

The Peyer's patches (PP), PP-removed SI, and mesenteric lymph nodes (MLN) were collected from mice at the indicated times after S. typhimurium infection. The number of bacteria was quantified by the following method: the tissues were washed and incubated in RPMI-1640 medium containing 0·25% gentamycin in a CO2 incubator at 37° for 1·5 hr to kill extracellular bacteria. After washing them with ice-cold PBS, they were homogenized in ice-cold PBS using a homogenizer (EYELA, Tokyo, Japan). In some experiments, SI was washed with PBS but not treated with gentamycin to avoid killing the S. typhimurium colonized on the surface of intestinal epithelial cells (EC), and then homogenized. The homogenates were then serially diluted and plated on Brilliant Green Agar. The Brilliant Green Agar plates were incubated for 18 hr at 37° and the colony-forming assay was carried out.

Histological analysis

Paraffin-embedded sections of 10% formalin-fixed tissues were stained with haematoxylin & eosin. Immunohistochemical analysis was carried out using the paraffin-embedded sections. For antigen retrieval, deparaffinized and rehydrated specimens were microwaved in a Retrieval kit (BD PharMingen, San Jose, CA). The slides were then incubated with the primary antibody (anti-IL-17A antibody; Santa Cruz Biotechnology, Santa Cruz, CA), anti-BD3 antibody (Santa Cruz) and anti-IL-22 antibody (Abcam, Cambridge, UK) at 4° overnight. Subsequently, these were incubated at room temperature with a biotinylated secondary antibody, streptavidin labelled with peroxidase and 3, 3′-diaminobenzidine, followed by counterstaining with haematoxylin.

Cell preparation

Mucosal lymphocytes were isolated and prepared according to a modification of previously published methods.22,23 Dissected small segments of the intestines were incubated at 37° for 30 min in an RPMI-1640 medium (Sigma-Aldrich, St Louis, MO) containing 10% fetal calf serum and 1 mm dithiothreitol with vigorous shaking. The tissue suspension was passed through a nylon mesh to remove debris and centrifuged through a 25/40/75% discontinuous Percoll (Sigma-Aldrich) gradient at 600 g at 20° for 20 min. The cell fraction collected from the interface of 40/75% was intestinal intraepithelial T lymphocytes (IEL). To isolate lamina propria lymphocytes (LPL), after removal of intestinal EC and IEL, tissues were incubated for 30 min at 37° in RPMI-1640 containing collagenase type VIII (Sigma-Aldrich). The cell suspension was centrifuged through a 40/75% discontinuous Percoll gradient, and the cells at the interface were used as LPL.

Flow cytometry

Cells were stained, as described previously.22 The mAbs used in this study were as follows: fluorescein isothiocyanate-conjugated anti-CD4 (RM4-5) and anti-TCR-γδ (GL3) mAb; phycoerythrin-conjugated anti-CD8α (53-6.7) and anti-TCR-β (H57-597) mAb and allophycocyanin-conjugated anti-CD3ε (145-2C11) mAb (BD Biosciences, San Jose, CA). Intracellular flow cytometry of IFN-γ and IL-17A in the small intestinal IEL and LPL was performed using a Cytofix/Cytoperm Plus kit (BD Biosciences), according to the manufacturer's instructions. After permeabilization, IEL and LPL were stained with fluorescein isothiocyanate-conjugated anti-IFN-γ (XMG1·2; e-Bioscience, San Diego, CA) and phycoerythrin-conjugated IL-17A (eBio17B7; e-Bioscience). Flow cytometry analysis was performed on a FACSCalibur flow cytometer (BD Biosciences).

Western blot analysis

Lysates of ileal mucosa were prepared and analysed by Western blotting according to a published method.24 Quantitative analysis of the blotting was performed using Image J software (http://rsb.info.nih.gov/ij/docs/menus/analyze.html).

Statistics

Student's t-test was used to determine significant differences. A P-value < 0·05 was considered significant.

Results

Kinetics of colony number in GALT during S. typhimurium infection

Before investigating the T-cell responses in GALT after intragastric inoculation of S. typhimurium, we determined the kinetics of bacterial numbers in GALT after intragastric inoculation of S. typhimurium. Following the infection, a significant increase of the bacterial load was evident in PP from day 3 to day 7 (Fig. 1). In the SI and MLN, the number of S. typhimurium achieved a peak on day 5. This result showed that S. typhimurium emerges in GALT on days 3–5 post-infection during the early phase of the infection.

Figure 1.

Figure 1

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Bacterial load in gut-associated lymphoid tissue following Salmonella enterica serovar Typhimurium (S. typhimurium) oral infection. C57BL/6 mice were orally inoculated with 109 colony-forming units (CFU) of S. typhimurium and the bacterial burdens in infected organs were determined on days 1, 3, 5, 7, and 10 post-infection. Five mice were used at each point. Values represent the mean ± SD of three individual experiments. MLN, mesenteric lymph nodes; PP, Peyer's patches; SI, small intestine; UD, undetectable.

Constitutive expression of IL-17A and induced expression of IFN-γ in the GALT during S. typhimurium infection

The importance of IFN-γ in protective immunity against S. typhimurium is well established.8–10 Recently, the role of IL-17A in protective immunity against Salmonella and other intracellular bacteria has been reported.7,14,25,26 Therefore, to clarify the mechanism of the early protective response against intestinal S. typhimurium infection, we examined the kinetics of IFN-γ-producing and IL-17A-producing T cells in the GALT during S. typhimurium infection. The total cell number of LPL and MLN increased following S. typhimurium infection, whereas numbers of IEL and PP did not (Fig. 2a). The IFN-γ-producing IEL increased to a peak on day 3 and then decreased until day 10 post-infection The T cells in LP and PP achieved a peak of IFN-γ production on day 7, but the MLN showed a delayed peak of IFN-γ-positive cells on day 10 (Fig. 2b). The TCR-αβ T cells mainly produced IFN-γ, whereas IL-17A production was hardly detected in IEL. As previously reported,19 IL-17A was constitutively produced in LP, PP and MLN. After S. typhimurium infection, the number of IL-17A-producing T cells decreased in the PP and MLN. Interleukin-17A production in T cells in LP transiently increased on day 7 (Fig. 2b,c). These results suggested that IL-17A is constitutively produced in the GALT and several factors induced by IL-17A-induced factors may play a protective role against S. typhimurium infection at an early phase of infection. Interferon-γ may participate in the protective immunity at a later phase of S. typhimurium infection. Hence, IL-17A and IFN-γ are important because of their protective ability against S. typhimurium infection at different stages.

Figure 2.

Figure 2

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Kinetics of interferon-γ (IFN-γ) and interleukin-17A (IL-17A) -producing cells in gut-associated lymphoid tissue following Salmonella enterica serovar Typhimurium oral infection. Intestinal intraepithelial T lymphocytes (IEL), lamina propria lymphocytes (LPL), Peyer's patches (PP) and mesenteric lymph nodes (MLN) were stained with monoclonal antibodies against T-cell receptor (TCR) -β, TCR-γδ, IFN-γ and IL-17A. Five mice were used at each point. (a) Total cell number of IEL, LPL, PP and MLN after the infection. (b) Absolute number of TCR-αβ± or TCR-γδ± T cells expressing IFN-γ or IL-17A. Values represent the mean ± SD of three individual experiments. Closed squares, TCR-αβ cells; open squares, TCR-γδ cells. (c) Representative flow cytometry profiles of TCR-β± cells in IEL and LPL on day 0 and day 7 of the infection.

Expression of IL-17A by intestinal EC

To further analyse the localization of IL-17A-producing cells, immunohistochemical analysis of the SI was carried out. To analyse the immediate early stage of S. typhimurium-infected intestine, we inoculated S. typhimurium into the ligated loop of the SI of the mice. Unexpectedly, in uninfected mice, IL-17A was detected in the crypt and villus EC (Fig. 3). In contrast, in S. typhimurium-infected mice, IL-17A expression in the crypts was diminished. The results suggest that the crypt and villus EC of the SI also express IL-17A and they secrete IL-17A in response to S. typhimurium infection.

Figure 3.

Figure 3

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Interleukin-17A (IL-17A) expression in the small intestine of mice with a ligated loop. C57BL/6 mice were underwent operation to create ligated loops in the small intestine and were infected with 5 × 104 colony-forming units of Salmonella enterica serovar Typhimurium for 3 hr. Five mice were used in each group. Magnification, × 400; Ab, antibody.

Severe damage of intestinal epithelium caused by S. typhimurium infection in anti-IL-17A antibody-treated mice

The observation of the constitutive IL-17A production in the SI before S. typhimurium infection led us to examine the role of IL-17A in the protective response against S. typhimurium infection in the immediate early stage of host defence at the intestinal mucosal surface. To address this issue, mice were injected with anti-IL-17A mAb or control antibody and infected with S. typhimurium into the intestinal loop, and the load of S. typhimurium invading the cells of the SI and PP was analysed. In the experiments, the bacterial number invading the cells was analysed 3 hr after infection by inoculation of the tissues with a gentamicin-containing medium to kill extracellular bacteria. No significant difference of the colony number in SI and PP was observed in the mice given the anti-IL-17A mAb intraperitoneally (Fig. 4a). Nor did injection of anti-IL-17A mAb into the lumen affect the number of invading bacteria in the PP and intestine (Fig. 4b).

Figure 4.

Figure 4

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Bacterial numbers in the small intestine (SI) and Peyer's patches (PP) of mice with ligated loops. (a) Mice were administered 200 μg of anti-interleukin-17A (IL-17A) monoclonal antibody (mAb) or control antibody intraperitoneally on day 8 and day 1 before Salmonella enterica serovar Typhimurium (S. typhimurium) infection into the ileal loops. (b) Mice were treated with 20 μg of anti-IL-17A mAb or control antibody and simultaneously challenged with 5 × 104 colony-forming units (CFU) of S. typhimurium into the ileal loops. Three hours after the infection, bacterial numbers in the tissue were determined in PP-removed SI and PP after killing extracellular bacteria with gentamicin. (c) Mice were treated with 20 μg of anti-IL-17A mAb or control antibody and simultaneously challenged with 5 × 104 CFU of S. typhimurium. One hour after the infection, the bacterial number both in the tissue and on the surface of SI was determined. We examined samples from at least five mice and obtained reproducible results in three independent experiments. Values represent the mean ± SD of a representative analysis. *P ≤ 0·05.

In the next experiments, we intended to analyse the number of S. typhimurium colonized on the surface of EC at an earlier time after S. typhimurium infection into the intestinal loop. Killing of S. typhimurium on the epithelial surface was avoided by preparing tissue homogenates after washing the intestinal lumen with PBS but without a treatment of gentamicin-containing medium. One hour after infection, the colony number of intestinal loops co-injected with anti-IL-17A mAb showed a significant increase compared with that of the control antibody-treated mice (Fig. 4c).

Histological analysis of the intestinal loop revealed that S. typhimurium-infected mice showed a thickening LP (Fig. 5a) and a lower ratio of villi/crypt than the uninfected mice (Fig. 5b), both of which suggest an inflammatory response of the intestine. Intraperitoneal treatment with anti-IL-17A mAb before intestinal loop infection with S. typhimurium had no influence on the histological characteristics. In contrast, the intestinal mucosa of the mice simultaneously injected with S. typhimurium and anti-IL-17A mAb exhibited erosions and severe ablation of the villi (Fig. 5b). The villus : crypt ratio was significantly decreased relative to that of control S. typhimurium-infected mice. These results suggested that IL-17A secreted into the intestinal lumen suppresses enteric mucosal damage caused by S. typhimurium infection.

Figure 5.

Figure 5

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Severe intestinal damage in mice treated with anti-interleukin-17A (IL-17A) monoclonal antibody (mAb) after Salmonella enterica serovar Typhimurium (S. typhimurium) infection. (a) Mice were administered 200 μg of anti-IL-17A mAb or control antibody intraperitoneally on day 8 and day 1 before administration of 5 × 104 colony-forming units (CFU) of S. typhimurium into ileal loops (systemic). Mice were also treated with 20 μg of anti-IL-17A mAb or control antibody and simultaneously challenged with 5 × 104 CFU of S. typhimurium into ileal loops (loop). Three hours later, mice were killed, and their small intestine were fixed with formalin for haematoxylin & eosin (H&E) staining. (b) The ratio of the villi to crypt was determined from H&E-stained sections, and the values were demonstrated as the mean ± SD of five mice per group from three independent experiments. *P ≤ 0·05. We examined samples from five mice, and obtained reproducible results in three independent experiments.

Constitutive expression of BD at the intestinal mucosal surface depended on IL-17A secreted into the intestinal lumen

It has been reported that BD3 is induced by IL-17A in collaboration with IL-22,20 and it is thought to be an effector molecule against S. typhimurium induced by IL-17A. We therefore examined the expression of BD3 and IL-22 in the intestine. Immunohistochemical analysis revealed that BD3 expression was prominently observed at the villus tip of enteric the mucosa before and after S. typhimurium infection (Fig. 6a). Anti-IL-17A mAb treatment into the intestinal loop resulted in a remarkable decrease of the expression of BD3 relative to that in control mice (Fig. 6a). Interleukin-22 expression was detected in LP, and crypt and villus EC, which is similar to that of IL-17A before and after the infection in the control and anti-IL-17A mAb-treated mice. We further quantitatively analysed IL-22 expression by Western blotting analysis. Expression of IL-22 increased after the infection, but the increase was not detected in the anti-IL-17A mAb-treated mice, which sustained the IL-22 expression level (Fig. 6b,c). These results suggested that the expression of BD3 on the surface of intestinal EC depends on constitutively expressed IL-17A and/or IL-17A-dependent expression of IL-22 and BD3 secreted into the lumen effectively kills S. typhimurium before its invasion into enteric EC, resulting in the suppression of mucosal epithelial damage caused by S. typhimurium infection.

Figure 6.

Figure 6

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Enhanced β-defensin-3 (BD3) expression but not interleukin-22 (IL-22) expression in the small intestine (SI) by anti-IL-17A monoclonal antibody (mAb) treatment. Mice were treated with 20 μg of anti-IL-17A mAb or control antibody and simultaneously challenged with 5 × 104 colony-forming units (CFU) of Salmonella enterica serovar Typhimurium (S. typhimurium) into ileal loops. Three hours later, mice were killed and SI were fixed with formalin for immunohistochemical analyses. (a) BD3 and IL-22 expression in uninfected and infected mice with or without treatment of anti-IL-17A mAb. (b) Western blot analysis of IL-17 and IL-22 in uninfected (U) and infected (I) mice with or without treatment of anti-IL-17A mAb. (c) The data of Western blot shown in (b) was quantified and the values are demonstrated as mean ± SD of six mice per group. *P ≤ 0·05. We examined samples from at least five mice and obtained reproducible results in three independent experiments.

Discussion

Although Th1-type immunity is well known to be critically important for host defence against S. typhimurium infection, the mechanism of innate immunity at the epithelial surface against the infection is not well defined. In this study, we show that IL-17A is produced by LPL, PP and MLN but not by IEL in the SI. Furthermore, the constitutively expressed IL-17A has an important role in initial protection at the epithelial surface, possibly through the induction of antimicrobial molecules on the enteric EC.

Interleukin-17A exerts multiple roles in both acquired and innate immunity. It elicits pro-inflammatory cytokines, which induces the differentiation and migration of neutrophils.27,28 In an arthritis encephalitis and colitis model, IL-17A has been shown to be a pivotal pathogenic factor in T-cell-mediated inflammatory diseases.29 In contrast, IL-17A has been recently reported to play an important role in protective immunity against various pathogens.27,30–32 Experiments with IL-17Rα−/− mice demonstrated that defective IL-17 responses accelerate Salmonella translocation to MLN and the spleen.14 Recently, IL-17A has been reported to be produced by Paneth cells in the intestine33 and by dendritic cells in Langerhans cell histiocytosis,34 which strongly suggests that IL-17A plays a pivotal role in preventing host responses against pathogens in mucosal sites. As IL-17A is constitutively expressed by LPL (Fig. 2 and reference19), and by crypt and villus EC, IL-17A-producing cells may participate in immunosurveillance in an enteric environment. Interleukin-17A elicits the expression of antimicrobial factors, such as BD3, lopocalin-2 and S100A8/9 proteins in collaboration with IL-22,7,20 suggesting that IL-17A-producing cells in LP enable the suppression of the invasion of pathogens through the induction of antimicrobial factors. Beta-defensin-3 is a candidate of the IL-17A-dependent immediate early protective effector because it is constitutively expressed at the epithelial surface of intestinal mucosa in an IL-17A-dependent manner, and S. typhimurium was reported to be resistant to lipocalin-2.35

Direct injection of anti-IL-17A mAb into the intestinal lumen showed a drastic increase of bacterial colonization to the intestinal epithelial surface (Fig. 4c) and tissue destruction (Fig. 5a) after intestinal S. typhimurium infection, although systemic treatment with anti-IL-17A mAb did not show any effect on the bacterial burden and tissue damage. Furthermore, immunohistochemical analysis revealed the constitutive IL-17A expression in the LP and crypt, presumably in Paneth cells and villus EC (Fig. 3). Constitutively expressed IL-17A is indispensable in the continuous expression of BD3 at the surface of intestinal epithelia. After S. typhimurium infection, IL-17A-positive cells decreased, suggesting the possibility that IL-17A is immediately released from its intracellular storage into the intestinal tract. The BD3 is also strongly expressed at the luminal site of EC, implying that constitutively expressed IL-17A elicits BD3 production and enhances the immunosurveillance system in the SI. The prominent decrease of BD3 expression after the injection of anti-IL-17A mAb into the intestinal lumen supports this (Fig. 6).

Interleukin-22 and IL-17A synergistically act on intestinal EC to induce not only expression of antimicrobial peptides such as BD3 and S100A8/9,20,36 but also migration and cell turnover.37,38 These cytokines also modulate intestinal pro-inflammatory responses.37,39 Raffatellu et al.14 reported increases of IL-17A, IL-22 and several downstream genes of the IL-17A/IL-22 activation pathway by S. typhimurium infection in the ileal mucosa of macaques in vivo. Interestingly, simian immunodeficiency virus-infected macaques showed marked decrease of Th17 cells, resulting in increased susceptibility to S. typhimurium infection and its dissemination. The importance of IL-22 in innate immunity at the intestinal mucosal site was also reported on another intestinal bacterial pathogen, Citrobacter rodentium.40,41 Therefore, both IL-17A and IL-22 would have important roles in innate protective immunity against intestinal bacterial pathogens. Furthermore, our data showed that IL-22 is constitutively expressed in enteric mucosa and its expression is not affected by the administration of anti-IL-17A mAb, suggesting that IL-22 expression is not dependent on IL-17A. Further analysis will be necessary to elucidate the role of IL-22 for mucosal defence during S. typhimurium infection.

Our analysis also demonstrated the induction of IFN-γ production by IEL after S. typhimurium infection. Interferon-γ is well established as a central mediator of immunity to S. typhimurium in mice.10 Although IEL, which reside at a basolateral site of EC, are considered to be critical T cells that participate in the first line of host defence to intestinal infection, the role of IFN-γ produced by the IEL during S. typhimurium infection is not yet clarified. The IFN-γ produced by the IEL may play an important role in innate protection against S. typhimurium infection as in the acquired immune response, which inhibits the dissemination of the pathogen to other peripheral tissues, such as the spleen.10 Host resistance genes play a critical role in suppressing the pathogenicity of S. typhimurium infection. Nramp-1 is an important innate host resistance factor to S. typhimurium42 and may act as a co-stimulatory molecule in IFN-γ-mediated signals in resistant macrophages.43–45 It is interesting to investigate the interaction between cytokines and host resistant genes against Salmonella infection in the intestine. We hypothesize that collaboration of IL-17A released by EC and LPL with IFN-γ produced by IEL has an important role in early protection against intestinal infection by S. typhimurium, which the escape immediate early protection at the surface of intestinal epithelia. Further analysis is required to clarify the role of the IEL against S. typhimurium infection.

Acknowledgments

This work is supported by the Program of Founding Research Centers for Emerging and Re-emerging Infectious Diseases from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (G.M.) and by Grants-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (20599015) (K.I-O.). We thank Mr Kensho Tanaka, Dr Takeshi Arakawa and Dr Takeshi Miyata (University of the Ryukyus) for constructive discussion. We are grateful to Prof. Yasuhiko Minokoshi (National Institute for Physiological Sciences), Prof. Hirosuke Oku and Prof. Hisami Watanabe (University of the Ryukyus) for their steadfast encouragement. The authors declare that they have no competing financial interests.

Glossary

Abbreviations:

BD3

β-defensin 3

EC

epithelial cells

GALT

gut-associated lymphoid tissue

IEL

intestinal intraepithelial T lymphocytes

IFN-γ

interferon-γ

IL

interleukin

LPL

lamina propria lymphocytes

mAb

monoclonal antibody

MLN

mesenteric lymph nodes

PBS

phosphate-buffered saline

PP

Peyer's patches

S. typhimurium

Salmonella enterica serovar Typhimurium

SI

small intestine

TCR

T-cell receptor

Th1

T helper type 1

Disclosures

The authors have no potential conflict of interest.

References

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