Skip Navigation


International Immunology Advance Access originally published online on July 3, 2008
International Immunology 2008 20(9):1129-1138; doi:10.1093/intimm/dxn069
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
20/9/1129    most recent
dxn069v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Schulz, S. M.
Right arrow Articles by Alber, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schulz, S. M.
Right arrow Articles by Alber, G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© The Japanese Society for Immunology. 2008. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

IL-17A is produced by Th17, {gamma}{delta} T cells and other CD4– lymphocytes during infection with Salmonella enterica serovar Enteritidis and has a mild effect in bacterial clearance

Silke M. Schulz1, Gabriele Köhler2, Christoph Holscher3, Yoichiro Iwakura4 and Gottfried Alber1

1 Institute of Immunology, College of Veterinary Medicine, University of Leipzig, An den Tierkliniken 11, 04103 Leipzig, Germany
2 Gerhard Domagk Institute of Pathology, University of Münster, Gerhard-Domagk-Str. 17, 48149 Münster, Germany
3 Research Center Borstel, Parkallee 22, 23845 Borstel, Germany
4 Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

Correspondence to: G. Alber; E-mail: alber{at}rz.uni-leipzig.de


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Th17 cells represent a new pro-inflammatory Th cell lineage distinct from Th1 and Th2 cells. Th17 cells have been shown to be involved in extracellular bacterial infection but their role in intracellular infection remains unclear. We found antigen-specific IL-17A production during a systemic infection of mice with the facultative intracellular bacterium Salmonella enterica serovar Enteritidis (S. Enteritidis) and examined the function and cellular source of IL-17A during the adaptive immune response to S. Enteritidis. Infected IL-17A–/– mice survived completely after inoculation with the highest infection dose found to be sub-lethal for wild-type (WT) C57BL/6 mice. However, at 20 and 80 days post-infection (d.p.i.), we repeatedly found mildly elevated bacterial burden in spleen and liver of IL-17A–/– mice as compared with WT mice. Overall, IL-17A–/– mice showed reduced clearance of S. Enteritidis. S. Enteritidis-specific IL-17A production was induced in splenocytes and lymph node cells of infected WT mice at both time points, 20 and 80 d.p.i. Classical CD4+ Th17 cells developed upon infection with Salmonella. CD4– {gamma}{delta} TCR+ and CD4– {gamma}{delta} TCR– cells were found to be additional IL-17A-producing cell populations. In infected IL-17A–/– mice, a normal Th1 cytokine profile was observed consistent with the overall subtle phenotype. Nevertheless, in the absence of IL-17A, recruitment of neutrophils and delayed-type hypersensitivity (DTH) reactivity was significantly compromised. Our data indicate that IL-17A responses are induced by Salmonella and mildly contribute to protective immunity during S. Enteritidis infection. Thus, IL-17A complements the IL-12/IFN-{gamma} axis which is essential for protective immunity against salmonellosis in mice and men.

Keywords: bacteria, cytokine, DTH, mouse, T cell


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
IL-17A is considered a pro-inflammatory cytokine as it induces IL-6, IL-8, granulocyle-colony-stimulating factor (CSF), granulocyte macrophage CSF and other pro-inflammatory factors (1). This has been shown to lead to the recruitment of granulocytes in vitro (2) and in vivo (2, 3). After infection with Klebsiella pneumoniae, IL-17AR–/– mice showed a defect in recruiting neutrophils into the lung tissue (2, 4) and a similar defect was found upon infection with Toxoplasma gondii (5). IL-17A is secreted by activated CD4+ T cells currently characterized as Th17 (6, 7), a distinct effector T cell lineage but it can also be derived by {gamma}{delta} T cells and other CD4– cell fractions upon infection with Mycobacterium tuberculosis (8). Shibata et al. (9) showed IL-17A-mediated recruitment of polymorphonuclear cells (PMN) into the peritoneum that only some hours after intraperitoneal (i.p.) infection with the extracellular bacterium Escherichia coli was controlled by local {gamma}{delta} T cells and was dependent on IL-23.

PMN have also been found to have protective function during immunity to the facultative intracellular bacteria of the genus Salmonella (10–12). Salmonellosis is one of the most common diseases caused by contaminated food world-wide (13). The serovar Salmonella Enteritidis belongs to the species Salmonella enterica. Together with the other serovar, Salmonella Typhimurium, it is the most important serovar for non-typhoid disease in humans (14). After systemic infection, Salmonella Enteritidis causes a typhoid-like disease in mice. IFN-{gamma}-activated macrophages, IL-12-dependent Th1 and B cells contribute to protective innate and adaptive immunity to Salmonella (15). In addition to activated macrophages, PMN have been found to kill Salmonella in vitro (11, 16–18) and during early innate immunity in vivo (12). Interestingly, PMN have a crucial protective role following both intragastrical and intravenous infection of mice (19). While these studies describing the essential role of neutrophils in protection against salmonellosis focused on early time points, investigations on the role of neutrophils in chronic Salmonella infection are rare. It was shown that neutrophils infiltrate chronically Salmonella-infected tissues (10, 19). However, a potential role in bacterial control in these chronically infected tissues could not be directly demonstrated. After intracellular infection of macrophages with Listeria monocytogenes, neutrophils were shown to be also protective during T cell-dependent immunity (20). Moreover, in addition to recruitment of neutrophils, IL-17A seems to contribute to T memory cell responses (21). Also a severely impaired delayed-type hypersensitivity (DTH) response upon immunization with BSA in the absence of IL-23-dependent IL-17 was found (22).

To study the role of IL-17A during the adaptive immune response to S. Enteritidis, we used wild-type (WT) and IL-17A–/– C57BL/6 mice in an i.p. infection model which has been previously established and characterized by us (23). For the first time, we can show subtle phenotypic differences in the absence of IL-17A after infection with an intracellular bacteria as there are significant differences in the organ burden and bacterial clearance between WT and IL-17A–/– mice.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Animals
C57BL/6 WT and IL-17A–/– mice were bred and kept at the animal facility of the Max Planck Institute of evolutionary Anthropology (Leipzig, Germany). IL-17A–/– mice generated from 129Sv embryonic stem cells (21) were backcrossed eight times on the C57BL/6 background. The nramp1 allele was determined (24) in backcrossed C57BL/6 IL-17A–/– mice and found to be the nramp1-resistant allele (nramp1r) as expected from the 129Sv embryonic stem cell origin and the same chromosomal location of the nramp1 and il-17a genes. Since Nramp1 (also termed Ity) is primarily involved in early innate resistance to Salmonella infection (25–27) and this study focuses on the role of IL-17A during adaptive immunity against S. Enteritidis, the nramp1 allele is not expected to play a major role. All mice were housed under specific pathogen-free conditions. Only females were used for experiments at an initial age of 8–12 weeks. All experiments were conducted according to the German Animal Protection Law (approved by the Regierungspräsidium Leipzig, Germany) and the safety guidelines for S1 organisms.

Bacteria and infection model
The attenuated vaccination strain of S. enteritica serovar Enteritidis (S. Enteritidis, ade–/his–; SALMOVAC®SE) (23, 28–30) was kindly provided by J. Selbitz (Impfstoffwerke Dessau-Tornau GmbH, Rosslau, Germany). A single colony of an overnight culture on xylose–lysine–desoxycholate (XLD) agar was grown in Luria–Bertani (LB) medium for 5 h to the log phase. After washing two times in PBS, small aliquots with defined colony-forming units (c.f.u.) of S. Enteritidis were suspended in FCS/10% dimethyl sulfoxide and stored at –70°C. For infection, aliquots were thawed and washed two times in PBS. Mice were i.p. infected with the highest sub-lethal infection dose for WT mice which was determined to be 2.5 x 106 c.f.u. For ex vivo stimulation of splenocytes and induction of DTH reactivity, bacteria were heat-killed (h.k.) by incubation of viable S. Enteritidis at 60°C for 60 min in a water bath and stored in aliquots at –70°C.

DTH response
S. Enteritidis infected and naive mice were tested for a DTH response to h.k. S. Enteritidis 20 days after infection. Thus, 25 µl of h.k. S. Enteritidis (1010 h.k. c.f.u. ml–1) were subcutaneously (s.c.) injected into one hind footpad; the other hind footpad received PBS for negative control. The footpad thickness was 3-fold measured every 24 h for 5 days with a spring-loaded measuring caliper (OditestTM S 5010, Kroeplin Längenmesstechnik, Schlüchtern, Germany). The swelling was expressed as the difference in footpad swelling of the antigen- and the PBS-injected footpad normalized against the footpad thickness before injection.

Survival and bacterial counts in organs
Infected mice were monitored daily for survival. 20 and 80 days after infection, 5–7 mice per group were sacrificed by CO2 asphyxiation, blood was collected by cardiac puncture and spleen and liver were removed under sterile conditions. Organs were weighed and pieces of them were homogenized and diluted 1:10 in PBS (w/v). Log10 serial dilutions were plated onto XLD agar (Sifin, Berlin, Germany) and incubated for 24 h at 37°C. Then the number of c.f.u. was determined and corrected for the whole organ weight. In order to detect low-level persistence, 300 µl of the 1:10 diluted samples were enriched by overnight culture in LB agar prior to plating and a qualitative result (i.e. infected or sterile) was obtained.

Isolation and ex vivo re-stimulation of splenocytes
Single-cell suspensions of the removed spleens were prepared by passing the tissue through a 100-µm-pore size mesh CellstrainerTM (Falcon®, BD Biosciences, Heidelberg, Germany). The suspension was cleared from erythrocytes by treatment with Gey’s solution for 5 min, washed twice with PBS and re-suspended in ISCOVE’s medium (PAA Laboratories GmbH, Germany) supplemented with 10% heat-inactivated FCS, 100 U ml–1 penicillin and 100 µg ml–1 streptomycin. After counting splenocytes from individual mice, the cells of each group were pooled by taking equal cell numbers from individual mice and adjusted to a concentration of 107 cells ml–1. A volume of 500 µl of this splenocyte suspension was dispensed into each well of a 24-well plate and incubated for 12 h at 37°C under a humidified atmosphere containing 5% CO2. Then the cells were re-stimulated for 48 h by addition of 500 µl ConA (final concentration 5 µg ml–1) for polyclonal T cell stimulation, 108 h.k. c.f.u. S. Enteritidis per ml for antigen-specific stimulation or medium for negative control. Cell-free supernatants were harvested and stored at –20°C for cytokine and nitric oxide (NO) measurements.

ELISA for cytokine determination and colorimetric assay for detection of NO
Cytokine concentrations of spleen cell culture supernatants were analyzed by sandwich ELISA. IL-17A and tumor necrosis factor (TNF)-{alpha} were measured with Duo-Set® (R&D Systems, Wiesbaden, Germany) according to recommended standard protocols. IL-12p40 was measured using the mAb 5C3 (5 µg ml–1) as capture antibody and biotinylated polyclonal goat anti-mouse IL-12p40-purified IgG (1:1000) as detection antibody followed by addition of streptavidin-peroxidase (Southern Biotechnology, Birmingham, AL, USA). 3,3',5,5',tetramethylbenzidine KPL substrate was used for the colorimetric development. Recombinant mouse IL-12 was used as standard. IFN-{gamma} was measured by using the mAb AN-18 [5 µg ml–1; American Type Culture Collection (ATCC)] as capture antibody, peroxidase-conjugated mAb XMG1.2 (ATCC) as detection antibody and recombinant mouse IFN-{gamma} was used as standard (kindly provided by G.R. Adolf, Ernst Boehringer Institute, Vienna, Austria; the anti-IFN-{gamma} antibodies were kindly provided by H. Gallati, Hoffmann-La Roche Ltd, Basel, Switzerland; rIL-12 by M. Gately, Hoffmann-La Roche Ltd, Nutley, NJ, USA Ltd). OD measurement of ELISA was performed with an ELISA reader (Spectramax 340 PC, Molecular Devices, Munich, Germany). NO was measured in cell culture supernatants with Griess reagent as described (31). The cytokine and NO concentrations were estimated from the standard curves with SoftmaxPro® (Molecular Devices).

Serum Ig determination
Serum from the cardiac blood was obtained by using serum separators (Microtainer®, BD Biosciences). The concentrations of total Ig isotypes IgE, IgG1 and IgG2c and of S. Enteritidis-specific IgG1 and IgG2c were measured by ELISA as described elsewhere (30). In brief, for detection of the total isotype- or subisotype-specific Ig concentration, polyclonal antibody pairs were used. Antigen-specific IgG1 and IgG2c antibodies were captured by using plate-bound (96 well, Polysorp; Nunc, Wiesbaden, Germany) S. Enteritidis antigen in combination with peroxidase-conjugated anti-mouse IgG1 or anti-mouse IgG2a [cross-reactive with IgG2c (32)], respectively (all antibodies from Southern Biotechnology, diluted 1:2000). The coating antigen was prepared by multiple freeze/thaw cycles of washed log-phase salmonellae.

FACS analysis of splenocyte and lymph node cell subsets
Single-cell suspensions of the spleens and the inguinal lymph node cells were prepared as described above. A total of 105 cells per staining were plated in 96-well plates, pre-treated with anti-CD16/CD32 Fc block (clone 2.4G2, BD Biosciences) and subsequently stained with the indicated antibodies and their isotype controls coupled to PE, FITC or allophycocyanin (APC). Cells were washed twice with FACS buffer (3% FCS, 0.1% NaN3 in PBS) and fixed with PBS/4% formaldehyde (v/v). Fluorescence was detected by cytometry (FACSCaliburTM BD Biosciences). A total of 10 000 cells was measured. Analyses were performed with the software CellQuestProTM (BD Biosciences Immunocytometry Systems).

Intracellular IL-17A staining
Inguinal lymph node cells from infected (20 days) and naive mice were re-stimulated for 22 h with either ConA or h.k. S. Enteritidis or left unstimulated as described above for splenocytes. One microliter of GolgiPlugTM (BD Biosciences) per 1 x 106 cells was then added for the last 5 h of incubation. Cells were stained for surface CD4 and {gamma}{delta} TCR with biotinylated anti-mouse CD4 mAb (BD Biosciences) combined with streptavidin-conjugated APC (0.2 mg ml–1, BD Biosciences) and FITC-conjugated anti-mouse {gamma}{delta} TCR mAb (eBiosciences, San Diego, CA, USA) or isotype controls for 30 min at 4°C. Fixed cells were washed twice and then incubated for 20 min with permeabilization buffer [0.55% (w/v) saponin (Sigma–Aldrich, Taufkirchen, Germany), 2 mM HEPES (PAA) and 5% (v/v) FCS (PAA) in PBS] at 4°C. Intracellular staining was performed according to manufacturer's instructions using PE-conjugated rat anti-mIL-17A mAb (BD Biosciences) or the isotype control (PE rat IgG1 isotype control, BD Biosciences). For FACS analysis of IL-17-producing cells, a total of 100 000 cells was gated.

Histology
Footpads from sacrificed mice were fixed in 4% bufferd formalin, decalcified in EDTA solution, embedded in paraffin and processed routinely for light microscopy and were stained with hämatoxilin-eosin (H&E). Additional naphtol-AS-D-chloracetate esterase staining was performed for the detection of PMN. PMN appear red in the stainings. All studies were done with an Olympus BX51 microscope (Hamburg, Germany).

Statistical analyses
For comparison of two independent groups, the Mann–Whitney rank sum test was used. For statistical analyses of differences between more than two independent groups, a Kruskal–Wallis statistics followed by Dunn's post-test was performed. The Fisher's Exact test was used for comparison of two independent groups in 2 x 2 contingency tables with categorical variables. Differences were considered to be significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
IL-17A is not required for survival but for bacterial control and clearance
To characterize the function of IL-17A in adaptive immunity to S. Enteritidis, we focused on an early (day 20) as well on a late time point (day 80) during the period of specific immunity. IL-17A–/– mice and age- and sex-matched WT C57BL/6 control mice were i.p. infected with the highest sub-lethal dose (2.5 x 106 c.f.u., S. M. Schulz and G. Alber, unpublished data) of a vaccine strain of S. Enteritidis. Hundred percentage of both groups survived until the end of the experiment (day 80 after infection, data not shown) and did not show any clinical symptoms. However, the infection course of the IL-17A–/– mice resulted in higher organ burden recovered from the spleen and the liver at day 20 and day 80 post-infection (p.i.). The bacterial load was only mildly elevated (around 1 log) but was statistically significant in most cases (Fig. 1). Since at 80 days p.i. (d.p.i.) the limit of detection was reached in some cases, we did overnight enrichment of the homogenized organs in liquid media (Table 1). Again, S. Enteritidis was recovered from the cultures of all IL-17A–/– mice but only in less than half of WT spleens and livers (P = 0.0046). These data indicate that IL-17A is not essential for survival during systemic Salmonella infection but it contributes to bacterial clearance.


Figure 1
View larger version (10K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. Reduced bacterial clearance in spleen (A) and liver (B) of IL-17A–/– mice as compared with WT mice. Mice were i.p. infected with 2.5 x 106 c.f.u. S. Enteritidis and sacrificed at day 20 or 80 after infection. Shown is one of two independent experiments (5–7 mice per group) with similar results.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Reduced bacterial clearance by IL-17A–/– mice

 
Salmonella infection induces IL-17A production by classical Th17 and {gamma}{delta} T cells
To determine whether IL-17A was induced in response to Salmonella infection, splenocytes from infected WT mice were obtained 20 and 80 days after infection, cultured in medium or re-stimulated with either h.k. S. Enteritidis or ConA for 48 h. At both time points of the infection, IL-17A was detected upon antigen-specific stimulation with h.k. S. Enteritidis and after polyclonal stimulation with ConA. At day 20, even slightly higher amounts of antigen-specific IL-17A were found (Fig. 2A). No S. Enteritidis-induced IL-17A production was found in splenocytes of naive mice. These findings suggest the induction of a Th17 response upon infection with the facultative intracellular bacterium Salmonella as it has been reported before in response to infection with the extracellular bacterium Klebsiella (33).


Figure 2
View larger version (11K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. Strong IL-17A secretion by re-stimulated splenocytes and lymph node cells after S. Enteritidis infection. (A) WT mice were i.p. infected with S. Enteritidis and sacrificed at days 20 and 80 after infection. For negative control, naive mice were used (0 d.p.i.). Splenocytes of each group were pooled, plated (5 x 106 cells per well) and stimulated with either h.k. S. Enteritidis (h.k. SE) or ConA for 48 h. Culture supernatants were tested for IL-17A by ELISA. The data represent a triplicate sample ± standard error of mean for each group. Shown is one representative of three independent experiments (pooled cells of 5–7 mice per experiment). (B) Inguinal lymph node cells of individual mice infected for 20 days were plated (5 x 106 cells per well) and stimulated with either h.k. SE or ConA for 17 h (same experiment as in Fig. 3). Culture supernatants were tested for IL-17A by ELISA. Open symbols represent naive mice, filled symbols infected mice. Pooled data from two independent experiments are shown (4–6 mice per group). Each symbol represents an individual mouse. Differences between naive and infected mice were statistically tested by Mann–Whitney test. Similar data as shown in Fig. 2B for lymph node cells were obtained for splenocyte responses of individual mice (data not shown).

 
We wanted to clarify if mainly Th17 or other cell populations were the producers of IL-17A in response to S. Enteritidis. It has been reported before that upon pulmonary infection with M. tuberculosis and also after i.p. infection with E. coli, {gamma}{delta} T cells contribute strongly to the IL-17A production (8, 9). Therefore, we performed intracellular IL-17A staining after overnight re-stimulation of inguinal lymph node cells of infected and naive mice 20 days after infection. For comparison, we also determined the concentration of IL-17A in the cell culture supernatants of spleens and lymph nodes. IL-17A was found in the cell culture supernatants already after overnight re-stimulation of splenocytes (data not shown) and lymph node cells (Fig. 2B). Lymph node cells of naive mice produced some IL-17A upon stimulation with ConA but not in response to S. Enteritidis. Cells of infected mice, however, produced IL-17A nicely upon stimulation with antigen or ConA (Fig. 2B). In accordance with the IL-17A concentrations of the cell culture supernatants, intracellular IL-17A was found in antigen-stimulated CD4+ (Th17) as well as CD4– lymph node lymphocytes of infected mice and in ConA-stimulated lymphocytes of naive and infected mice. In response to S. Enteritidis, an average of 0.2% (±0.06%, n = 6) of all lymphocytes isolated from Salmonella-infected mice were found to produce IL-17A in two different experiments in a total of six individually studied mice (Fig. 3A). We did not observe IL-17A production in S. Enteritidis-activated lymph node cells from naive mice (Fig. 3C and D). IL-17A-producing lymphocytes comprised Th17, characterized by co-expression of CD4 (Fig. 3A and C), and CD4– cells (Fig. 3A). The latter included {gamma}{delta} TCR+ and {gamma}{delta} TCR– cells (Fig. 3B). Around 50% of the IL-17A+CD4– cells occurred to be {gamma}{delta} TCR+ cells (Fig. 3D). Therefore, classical Th17 develop upon infection with Salmonella but there are also CD4– {gamma}{delta} TCR+ and CD4– {gamma}{delta} TCR– IL-17-producing cells during the period of adaptive immunity to Salmonella. Overall, each of these three populations made up about one-third of the total IL-17A-producing lymphocytes. IL-17A-producing cells outside the lymphocyte gate could not be detected (data not shown).


Figure 3
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. CD4+, CD4– {gamma}{delta} TCR+ and CD4– {gamma}{delta} TCR– lymphocytes produce IL-17A upon ex vivo re-stimulation following infection with S. Enteritidis. Twenty days after infection, single-cell preparations of inguinal lymph node cells of WT mice were plated and stimulated for 17 h with medium, h.k. S. Enteritidis or ConA (same experiment as in Fig. 2). GolgiPlug was added to the cultures for 5 h. Cells were then analyzed for intracellular IL-17A staining. The forward/sideward scatter was used to gate on the lymphocytes. For analysis of IL-17-producing cells, a total of 100 000 cells was gated. (A) Representative IL-17A/CD4 staining for S. Enteritidis-stimulated lymph node cells. (B) Representative staining for {gamma}{delta} TCR. Shown are percentages of the CD4–/IL-17+ population. In (C) and (D), the pooled data (corrected by the isotype controls) of two independently performed experiments are displayed (n = 6) and statistically tested by Kruskal–Wallis followed by Dunn's post-test. Open bars represent the cells of naive mice and filled bars represent the cells of infected mice. (C) Shown are percentages of Th17 (IL-17+ CD4+) out of total lymphocytes. (D) Shown are percentages of IL-17A-producing {gamma}{delta} TCR+ CD4– cells out of total IL-17A-producing CD4– cells.

 
No difference in the Th1-related cytokine profile and NO production of IL-17A–/– mice
Protective immunity to Salmonella infection is known to be Th1-mediated with IFN-{gamma} and IL-12 as key cytokines (34) and the effector molecule NO (35, 36). IL-17 can induce the expression of inducible NO synthase (iNOS) in many different cell types [reviewed in ref. (37)]. To determine whether the differences in organ burden correlate with differences in the Th1 cytokine profile or NO secretion, splenocytes were re-stimulated with h.k. S. Enteritidis or ConA for 48 h and supernatants were analyzed by ELISA. As shown in Fig. 4A, both mouse genotypes showed equally strong pro-inflammatory responses 20 and even stronger 80 days after infection with S. Enteritidis. The IFN-{gamma} responses were similarly elevated in both groups (between 5 and 13 ng ml–1) and showed maximal antigen-specific responses 80 days after infection. The IL-6 and the TNF-{alpha} production did not reproducibly differ between infected WT and IL-17A–/– mice at either time point studied. Also, there was no difference in IL-12p40 (data not shown) and NO production (Fig. 4B). This indicates that IL-17A–/– mice do mount a normal Th1 response after infection and that IL-17A contributes to pathogen control by other mechanisms.


Figure 4
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. Infection with S. Enteritidis induces a Th1-related cytokine profile and does not affect Th1-dependent effector mechanisms. WT and IL-17A–/– mice were i.p. infected with S. Enteritidis and sacrificed at days 20 and 80 after infection. Splenocytes of each group were pooled, plated (5 x 106 cells per well), and stimulated with either h.k. S. Enteritidis or ConA or left unstimulated (medium). Culture supernatants were tested for IFN-{gamma}, IL-6 and TNF-{alpha} by ELISA. (A) No differences in the Th1-related cytokine production were found in IL-17A–/– splenocytes. (B) NO was measured by colorimetric assay. The data represent a triple sample ± standard error of mean for each group. Shown is one representative of two independent experiments (pooled cells of 5–7 mice per experiment).

 
Inhibited recruitment of neutrophils into the spleen in IL-17A–/– mice
IL-17A has been shown to induce recruitment of neutrophils (3). To determine whether the increased organ burden of IL-17A–/– mice was accompanied by differences of the influx of neutrophils, the percentage of CD11b+Gr1+ splenocytes was measured by FACS analysis 20 and 80 days after infection and compared with WT controls. Baseline levels of CD11b+Gr1+ splenocytes in either mouse genotype lay between 2 and 5% before infection (data not shown). Twenty days after infection, the percentage of CD11b+Gr1+ was increased significantly in each of both genotypes (Fig. 5A). Interestingly, WT mice were found to have a higher relative portion of neutrophils (CD11b+Gr1+) than IL-17A–/– mice (Fig. 5A and B). Total numbers of neutrophils showed a similar trend in WT and IL-17A–/– mice (Fig. 5B) as shown in two independent experiments. Eighty days after infection, the neutrophil fraction was back to baseline levels in both groups (Fig. 5A and B). In contrast, the relative portions of CD4+ and CD8+ cells (data not shown), of macrophages (F4/80+CD11c–) and dendritic cells (CD11c+F4/80–), did not differ between infected WT and IL-17A–/– mice (Fig. 5B). Therefore, IL-17A significantly enhances the recruitment of PMN into the spleen during the period of the adaptive immune response.


Figure 5
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. Reduced percentages of CD11b+Gr1+ cells in the spleen of IL-17A–/– mice as compared with WT mice at 20 days after infection. (A) Representative dot plots of CD11b+Gr1+ cells derived from spleen of WT and IL-17A–/– mice 20 and 80 days after infection with S. Enteritidis. At 80 d.p.i., percentages of CD11b+Gr1+ cells are back to the level of uninfected mice. (B) Relative portions and absolute numbers of neutrophils (PMN, CD11b+Gr1+), macrophages (Mph, F4/80+CD11c–) and dendritic cells (DC, CD11c+F4/80–). (B) Shows medians with interquartile range. Mice were i.p. infected with 2.5 x 106 c.f.u. S. Enteritidis and sacrificed at days 20 and 80 after infection. Shown are the data of one from two independent experiments (5–7 mice per group).

 
Impaired DTH reactivity when IL-17A is absent
IL-17A has been shown to contribute to the development of a Th1-mediated DTH response (21). Therefore, 20 days after infection, WT and IL-17A–/– mice received S. Enteritidis antigen s.c. into one hind footpad while the other one was treated with PBS for negative control. WT as well as the IL-17A–/– mice developed a significant DTH response compared with uninfected control mice, indicating an influx of specifically primed T cells in both groups. However, the IL-17A–/– mice showed a significantly lower swelling at 2 and 3 days after antigen application as compared with WT mice (Fig. 6A). In WT mice, the histopathological analysis showed a widespread diffuse infiltration of the footpad with inflammatory cells. They contained increased numbers of PMN in the subcutis, occasionally even reaching the musculature layer of the footpad (Fig. 6B and C). In 17A–/– mice, however, we found inflammatory cell infiltration only in the s.c. region with only few PMN. Therefore, IL-17A is not essential for but contributes to the DTH response after infection with Salmonella.


Figure 6
View larger version (74K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6. Compromised DTH reactivity in the absence of IL-17A. Seven mice per group were i.p. infected with 2.5 x 106 c.f.u. S. Enteritidis or injected with PBS instead (naive mice). Twenty days later, 109 h.k. S. Enteritidis were administered in the right hind footpad. PBS was administered in the left footpad (negative control). Swelling of the footpads was monitored daily for 5 days. (A) Shown are the changes of the swelling relative to PBS-treated left footpad. To exclude natural differences in thickness of left and right footpad, data were normalized as described in Materials and methods. Shown is one representative of two independently performed experiments (7 mice per group). (B) Shows representative sections of the footpads of infected mice 5 days after antigen application stained with H&E, magnification bar = 1 mm. (C) Footpad section stained with H&E and naphtol-AS-D-chloracetate esterase, magnification bar = 0.1 mm. Lower numbers of naphtol-AS-D-chloracetate esterase+ PMN were found in IL-17A–/– mice.

 
Similar percentage of splenic B cells and similar levels of serum IgG2c, IgG1 and IgE in the absence of IL-17A
B cells play a role in protection against Salmonella infection since mice without functional B cells show impaired immunity to S. Typhimurium (38, 39). Thus, the humoral antibody response also contributes to protective immunity against Salmonella. Th1-dependent isotypes such as IgG2a represent the dominant opsonizing isotypes that increase the phagocytosis of Salmonella via Fc{gamma}RI (40). IgG2a is predominately induced by IFN-{gamma} and therefore associated with a typical Th1-dominated immune response. This appears to hold true also for IgG2c (41) detectable in C57Bl/6 mice which lack IgG2a (32). IgG1 in contrast is induced by IL-4 and contributes to the clearance of Salmonella (30). According to this, we first determined the percentage of B cells in the spleen and measured the total IgG2c, IgG1 and IgE concentrations in sera 20 and 80 days after infection. We found no differences in the percentage of B220+MHC-II+ B cells in IL-17A–/– mice (data not shown). S. Enteritidis-specific IgG1 and IgG2c did not differ between WT and IL-17A–/– mice (Fig. 7A). Both groups showed increased IgG2c but not IgG1 levels 20 d.p.i. At 80 d.p.i., both, IL-17A–/– mice and WT mice, showed equally strong elevated IgG2c levels. The levels of IgG1 increased only slightly. The total IgG2c levels but not IgG1 levels strongly increased between day 20 and 80 p.i. unaffected by the genotype. The IgE levels were slightly elevated in both groups on day 80 after infection (Fig. 7B). Both groups of mice mounted an IgG2c- and therefore Th1-dominated antibody profile which apparently is independent of IL-17A.


Figure 7
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7. Equal serum levels of antigen-specific and total IgG1, IgG2c and IgE in WT and IL-17A–/– mice at day 20 and day 80 after infection with S. Enteritidis. (A) S. Enteritidis-specific serum IgG2c and IgG1 titers are shown as median OD ± range. The ODs of serum from naive WT and IL-17A–/– mice were similar. (B) Total serum IgG2c, IgG1 and IgE concentrations of WT and IL-17A–/– mice are shown. The symbols represent individual animals from one representative of two independent experiments. The horizontal lines indicate the medians.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
Presently, IL-17A and Th17 cells are in the center of studies related to their role in organ-dependent autoimmunity. Therapeutic strategies targeting IL-17A for treatment of autoimmune diseases are being developed. This leaves open what the physiological function of IL-17A is during host defense. Thus, our approach addressed this question in the context of a facultative intracellular bacterial infection with S. Enteritidis. Indeed, we found that Th17 are induced during experimental systemic salmonellosis in mice. Moreover, IL-17A complements protective adaptive immunity against S. Enteritidis associated with neutrophil recruitment and supporting DTH reactivity.

In other experimental murine infection models, IL-17A has been shown to play a critical role for a protective immune response to different types of pathogens (mostly extracellular) at early time points p.i. After pulmonary infection with the gram-negative bacterium K. pneumoniae, 100% of IL-17R–/– mice died within 48 h as compared with 40% dying mice of the WT controls (4). Reduced survival combined with elevated organ burden shortly after infection was also reported upon systemic infection of IL-17AR–/– mice with Candida albicans (42). In both models, the protective role was addressed to an IL-17A-mediated recruitment of PMN to the site of infection as a first line of host defense. Similar results were found after infection with the intracellular protozoan T. gondii (5). We were interested in the role of IL-17A in a chronic infection. Therefore, in the present study, we focused on the potential role of IL-17A at late time points during systemic salmonellosis. We found elevated bacterial load in spleen and liver 20 and 80 days after infection in IL-17A–/– mice. Since neutrophils contribute to the host resistance to S. Enteritidis (3, 10), we determined the frequencies of PMN in the spleen and found them to be elevated in both, WT and IL-17A–/– mice, but with significantly lower proportions in IL-17A–/– mice, whereas the other cell subsets did not differ. Therefore, in line with others (3, 5, 9), our data confirm the role of IL-17A in PMN recruitment and demonstrate furthermore that this is the case even at later time points of an infection when adaptive immunity takes place.

It is likely that the reduced recruitment of PMN to the Salmonella-infected sites found at 20 d.p.i. contributes to the elevated organ burden in liver and spleen since the IFN-{gamma}-dominated Th1 cytokine profile (as a major protective strategy against Salmonella infection) was still intact in the absence of IL-17A. Re-stimulated splenocytes of WT and IL-17A–/– mice produced about the same amounts of IFN-{gamma}, IL-12p40 and TNF-{alpha}. As a result of IFN-{gamma}-activated macrophages, NO is important for the killing of intracellular Salmonella (35, 36). It is known that IL-17A can induce the expression of iNOS in many different cell types although not in macrophages (37). To find out whether the absence of IL-17A influences the NO production after S. Enteritidis infection, we measured the NO secretion in the supernatants of the re-stimulated splenocytes but did hardly find any differences between WT and IL-17A–/– mice. In addition, IL-6 production which can be induced by IL-17A was not affected. Therefore, our findings point to similar IL-17A-mediated effector mechanisms during the adaptive immune response against S. Enteritidis as were found for the innate immune response in other infection models.

For the first time, we show that Salmonella infection is able to induce antigen-specific IL-17A production by CD4+ cells termed classical Th17 and by {gamma}{delta} T cells and other CD4– lymphocytes. Interestingly, IL-17A can be induced in the spleen of Salmonella-infected WT mice even at very late time points after infection when the infected organs are mostly cleared of bacteria. It has very recently been reported that {gamma}{delta} T cells can produce IL-17 during early stages of E. coli infection by Shibata et al. (9) who identified resident {gamma}{delta} T cells as the dominant IL-17A producers in the peritoneum upon i.p. infection. In addition, Lockhart et al. (8) found {gamma}{delta} T cells to be the major producers of IL-17A in the lung during the early immune response to M. tuberculosis. Our results demonstrate that {gamma}{delta} T cells contribute strongly to IL-17A production not only at the early innate stages of infection but also at later time points during adaptive immunity. It is yet to be determined if the IL-17A-producing {gamma}{delta} T cells we have found differ from those observed by others at early stages of infection.

It was shown that IL-17A deficiency results in a compromised DTH response following immunization with a model antigen (21). Our findings reveal the function of IL-17A in DTH responses during infection with S. Enteritidis. Twenty days after S. Enteritidis infection, IL-17A–/– mice mount a compromised DTH response with reduced swelling and shorter duration as compared with WT mice. This is associated with reduced infiltration of PMN at the site of antigen application in IL-17A–/– mice as compared with WT mice. These results indicate that specifically primed Th17 cells contribute to the inflammatory response at the site of DTH induction in particular to the influx of PMN. Moreover, in addition to the PMN defect found in the spleen in the absence of IL-17A, this may be another critical mechanism of protection which contributes to control of Salmonella infection. The IL-17A-dependent DTH defect may reflect a weaker effector and/or memory T cell activity in the absence of IL-17A required for optimal control of S. Enteritidis.

For adaptive immunity against Salmonella, not only Th1 but also B cells contribute to protection (15). We observed no differences of B cell frequencies in the spleen and equally elevated total and antigen-specific IgG2c concentrations in the serum 80 days after infection of WT and IL-17A–/– mice. It has been shown before that IL-23 is essential for an efficient T cell-dependent humoral response after immunization with ovalbumin peptide (22). In contrast to these findings, we found no differences in the humoral responses between WT and IL-17A–/– mice, indicating that IL-23 does not contribute to the humoral response via the IL-23/IL-17A axis, but does influence the humoral response by other mechanisms.

S. Enteritidis induces IL-17A production in different cell populations. In the absence of IL-17A though, systemically infected mice still mount normal Th1 response associated with normal macrophage activation. Therefore, IL-17A is not involved in regulation of a type 1 immune response but appears to contribute to control of S. Enteritidis by other mechanisms such as neutrophil recruitment and/or DTH-associated mechanisms. In particular, the presence of IL-17A seems to contribute to bacterial clearance since in contrast to WT mice none of the IL-17A–/– mice completely cleared the infection until 80 d.p.i. In WT mice, antigen-specific IL-17A-producing spleen cells were present 80 days after infection when the organs were largely cleared of the infection. This suggests that memory Th17 which are present even after clearance of Salmonella may contribute to the elimination of the pathogen. A protective role of IL-17A may become relevant under conditions of immunosuppression, e.g. to avoid reactivation of latent enteric salmonellosis. In conclusion, Th17 are induced during experimental systemic salmonellosis in mice. Moreover, IL-17A complements protective adaptive immunity against S. Enteritidis by contributing to bacterial control and clearance.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Research grant Al 371/3-3 from the Deutsche Forschungsgemeinschaft (G.A.).


   Acknowledgements
 
We thank Nicole Schütze, Sabine Schöneberger and Juliane Richter for substantial technical assistance and we appreciate the help of Uwe Müller, Eva Marquardt, Norbert Kirchhoff and Rowina Voigtländer in the animal facility and their friendly cooperation. We are grateful to Petra Meier for preparing the histological slides and we thank Thomas Kamradt for critically reading the manuscript.


   Abbreviations
 
ATCC, American Type Culture Collection
c.f.u., colony-forming units
CSF, colony-stimulating factor
d.p.i., days post-infection
DTH, delayed-type hypersensitivity
h.k., heat-killed
iNOS, inducible NO synthase
i.p., intraperitoneal
LB, Luria–Bertani
NO, nitric oxide
p.i., post-infection
PMN, polymorphonuclear cells
s.c., subcutaneously
S. Enteritidis, Salmonella enterica serovar Enteritidis
TNF, tumor necrosis factor
WT, wild type
XLD, xylose–lysine–desoxycholate

   Notes
 
Transmitting editor: T. Hirano

Received 29 February 2008, accepted 3 June 2008.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

  1. Alber G, Kamradt T. Regulation of protective and pathogenic Th17 responses. Curr. Immunol. Rev. (2007) 3:3.
  2. Fossiez F, Djossou O, Chomarat P, et al. T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines. J. Exp. Med. (1996) 183:2593.[Abstract/Free Full Text]
  3. Schwarzenberger P, La RV, et al. IL-17 stimulates granulopoiesis in mice: use of an alternate, novel gene therapy-derived method for in vivo evaluation of cytokines. J. Immunol. (1998) 161:6383.[Abstract/Free Full Text]
  4. Ye P, Garvey PB, Zhang P, et al. Interleukin-17 and lung host defense against Klebsiella pneumoniae infection. Am. J. Respir. Cell Mol. Biol. (2001) 25:335.[Abstract/Free Full Text]
  5. Kelly MN, Kolls JK, Happel K, et al. Interleukin-17/interleukin-17 receptor-mediated signaling is important for generation of an optimal polymorphonuclear response against Toxoplasma gondii infection. Infect. Immun. (2005) 73:617.[Abstract/Free Full Text]
  6. Harrington LE, Hatton RD, Mangan PR, et al. Interleukin 17-producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat. Immunol. (2005) 6:1123.[CrossRef][Web of Science][Medline]
  7. Park H, Li Z, Yang XO, et al. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat. Immunol. (2005) 6:1133.[CrossRef][Web of Science][Medline]
  8. Lockhart E, Green AM, Flynn JL. IL-17 production is dominated by gammadelta T cells rather than CD4 T cells during Mycobacterium tuberculosis infection. J. Immunol. (2006) 177:4662.[Abstract/Free Full Text]
  9. Shibata K, Yamada H, Hara H, Kishihara K, Yoshikai Y. Resident Vdelta1+ gammadelta T cells control early infiltration of neutrophils after Escherichia coli infection via IL-17 production. J. Immunol. (2007) 178:4466.[Abstract/Free Full Text]
  10. Johansson C, Ingman M, Jo WM. Elevated neutrophil, macrophage and dendritic cell numbers characterize immune cell populations in mice chronically infected with Salmonella. Microb. Pathog. (2006) 41:49.[CrossRef][Web of Science][Medline]
  11. Vassiloyanakopoulos AP, Okamoto S, Fierer J. The crucial role of polymorphonuclear leukocytes in resistance to Salmonella dublin infections in genetically susceptible and resistant mice. Proc. Natl Acad. Sci. USA. (1998) 95:7676.[Abstract/Free Full Text]
  12. Wick MJ. Living in the danger zone: innate immunity to Salmonella. Curr. Opin. Microbiol. (2004) 7:51.[CrossRef][Web of Science][Medline]
  13. Thorns CJ. Bacterial food-borne zoonoses. Rev. Sci. Tech. (2000) 19:226.[Web of Science][Medline]
  14. Rabsch W, Tschape H, Baumler AJ. Non-typhoidal salmonellosis: emerging problems. Microbes. Infect. (2001) 3:237.[CrossRef][Web of Science][Medline]
  15. Mastroeni P. Immunity to systemic Salmonella infections. Curr. Mol. Med. (2002) 2:393.[CrossRef][Medline]
  16. Steigbigel RT, Lambert LH Jr, Remington JS. Phagocytic and bacterial properties of normal human monocytes. J. Clin. Invest. (1974) 53:131.[CrossRef][Web of Science][Medline]
  17. Mandell GL. Bactericidal activity of aerobic and anaerobic polymorphonuclear neutrophils. Infect. Immun. (1974) 9:337.[Abstract/Free Full Text]
  18. Shiloh MU, Ruan J, Nathan C. Evaluation of bacterial survival and phagocyte function with a fluorescence-based microplate assay. Infect. Immun. (1997) 65:3193.[Abstract]
  19. Conlan JW. Neutrophils prevent extracellular colonization of the liver microvasculature by Salmonella typhimurium. Infect. Immun. (1996) 64:1043.[Abstract]
  20. Rakhmilevich AL. Neutrophils are essential for resolution of primary and secondary infection with Listeria monocytogenes. J. Leukoc. Biol. (1995) 57:827.[Abstract]
  21. Nakae S, Komiyama Y, Nambu A, et al. Antigen-specific T cell sensitization is impaired in IL-17-deficient mice, causing suppression of allergic cellular and humoral responses. Immunity (2002) 17:375.[CrossRef][Web of Science][Medline]
  22. Ghilardi N, Kljavin N, Chen Q, Lucas S, Gurney AL, De Sauvage FJ. Compromised humoral and delayed-type hypersensitivity responses in IL-23-deficient mice. J. Immunol. (2004) 172:2827.[Abstract/Free Full Text]
  23. Lehmann J, Bellmann S, Werner C, Schroder R, Schutze N, Alber G. IL-12p40-dependent agonistic effects on the development of protective innate and adaptive immunity against Salmonella enteritidis. J. Immunol. (2001) 167:5304.[Abstract/Free Full Text]
  24. Medina E, Rogerson BJ, North RJ. The Nramp1 antimicrobial resistance gene segregates independently of resistance to virulent Mycobacterium tuberculosis. Immunology (1996) 88:479.[CrossRef][Web of Science][Medline]
  25. Benjamin WH Jr, Hall P, Roberts SJ, Briles DE. The primary effect of the Ity locus is on the rate of growth of Salmonella typhimurium that are relatively protected from killing. J. Immunol. (1990) 144:3143.[Abstract]
  26. Oswald IP, Lantier F, Moutier R, Bertrand MF, Skamene E. Intraperitoneal infection with Salmonella abortusovis is partially controlled by a gene closely linked with the Ity gene. Clin. Exp. Immunol. (1992) 87:373.[Web of Science][Medline]
  27. Vidal S, Tremblay ML, Govoni G, et al. The Ity/Lsh/Bcg locus: natural resistance to infection with intracellular parasites is abrogated by disruption of the Nramp1 gene. J. Exp. Med. (1995) 182:655.[Abstract/Free Full Text]
  28. Springer S, Lehmann J, Lindner T, Thielebein J, Alber G, Selbitz HJ. A new live Salmonella enteritidis vaccine for chickens–experimental evidence of its safety and efficacy. Berl Munch. Tierarztl. Wochenschr. (2000) 113:246.[Web of Science][Medline]
  29. Martin G, Methner U, Steinbach G, Meyer H. Immunization with potential Salmonella enteritidis mutants—2. Investigations on the attenuation and immunogenicity for mice and young hens. Berl Munch. Tierarztl. Wochenschr. (1996) 109:369.[Web of Science][Medline]
  30. Lehmann J, Springer S, Werner CE, et al. Immunity induced with a Salmonella enterica serovar Enteritidis live vaccine is regulated by Th1-cell-dependent cellular and humoral effector mechanisms in susceptible BALB/c mice. Vaccine (2006) 24:4779.[CrossRef][Web of Science][Medline]
  31. Boudard F, Vallot N, Cabaner C, Bastide M. Chemiluminescence and nitrite determinations by the MALU macrophage cell line. J. Immunol. Methods. (1994) 174:259.[CrossRef][Web of Science][Medline]
  32. Martin RM, Brady JL, Lew AM. The need for IgG2c specific antiserum when isotyping antibodies from C57BL/6 and NOD mice. J. Immunol. Methods. (1998) 212:187.[CrossRef][Web of Science][Medline]
  33. Happel KI, Dubin PJ, Zheng M, et al. Divergent roles of IL-23 and IL-12 in host defense against Klebsiella pneumoniae. J. Exp. Med. (2005) 202:761.[Abstract/Free Full Text]
  34. Jouanguy E, Doffinger R, Dupuis S, Pallier A, Altare F, Casanova JL. IL-12 and IFN-gamma in host defense against mycobacteria and salmonella in mice and men. Curr. Opin. Immunol. (1999) 11:346.[CrossRef][Web of Science][Medline]
  35. Eisenstein TK. Implications of Salmonella-induced nitric oxide (NO) for host defense and vaccines: NO, an antimicrobial, antitumor, immunosuppressive and immunoregulatory molecule. Microbes Infect. (2001) 3:1223.[CrossRef][Web of Science][Medline]
  36. Vazquez-Torres A, Jones-Carson J, Mastroeni P, Ischiropoulos H, Fang FC. Antimicrobial actions of the NADPH phagocyte oxidase and inducible nitric oxide synthase in experimental salmonellosis. I. Effects on microbial killing by activated peritoneal macrophages in vitro. J. Exp. Med. (2000) 192:227.[Abstract/Free Full Text]
  37. Miljkovic D, Trajkovic V. Inducible nitric oxide synthase activation by interleukin-17. Cytokine Growth Factor Rev. (2004) 15:21.[CrossRef][Web of Science][Medline]
  38. Mastroeni P, Simmons C, Fowler R, Hormaeche CE, Dougan G. Igh-6(-/-) (B-cell-deficient) mice fail to mount solid acquired resistance to oral challenge with virulent Salmonella enterica serovar typhimurium and show impaired Th1 T-cell responses to Salmonella antigens. Infect. Immun. (2000) 68:46.[Abstract/Free Full Text]
  39. Mittrucker HW, Raupach B, Kohler A, Kaufmann SH. Cutting edge: role of B lymphocytes in protective immunity against Salmonella typhimurium infection. J. Immunol. (2000) 164:1648.[Abstract/Free Full Text]
  40. Uppington H, Menager N, Boross P, et al. Effect of immune serum and role of individual Fcgamma receptors on the intracellular distribution and survival of Salmonella enterica serovar Typhimurium in murine macrophages. Immunology (2006) 119:147.[CrossRef][Web of Science][Medline]
  41. Karachunski PI, Ostlie NS, Monfardini C, Conti-Fine BM. Absence of IFN-gamma or IL-12 has different effects on experimental myasthenia gravis in C57BL/6 mice. J. Immunol. (2000) 164:5236.[Abstract/Free Full Text]
  42. Huang W, Na L, Fidel PL, Schwarzenberger P. Requirement of interleukin-17A for systemic anti-Candida albicans host defense in mice. J. Infect. Dis. (2004) 190:624.[CrossRef][Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Immunol.Home page
H. M. S. Algood, S. S. Allen, M. K. Washington, R. M. Peek Jr., G. G. Miller, and T. L. Cover
Regulation of Gastric B Cell Recruitment Is Dependent on IL-17 Receptor A Signaling in a Model of Chronic Bacterial Infection
J. Immunol., November 1, 2009; 183(9): 5837 - 5846.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
S. Siegemund, N. Schutze, S. Schulz, K. Wolk, K. Nasilowska, R. K. Straubinger, R. Sabat, and G. Alber
Differential IL-23 requirement for IL-22 and IL-17A production during innate immunity against Salmonella enterica serovar Enteritidis
Int. Immunol., May 1, 2009; 21(5): 555 - 565.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
L. W. Kappel, G. L. Goldberg, C. G. King, D. Y. Suh, O. M. Smith, C. Ligh, A. M. Holland, J. Grubin, N. M. Mark, C. Liu, et al.
IL-17 contributes to CD4-mediated graft-versus-host disease
Blood, January 22, 2009; 113(4): 945 - 952.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. M. Schulz, G. Kohler, N. Schutze, J. Knauer, R. K. Straubinger, A. A. Chackerian, E. Witte, K. Wolk, R. Sabat, Y. Iwakura, et al.
Protective Immunity to Systemic Infection with Attenuated Salmonella enterica serovar Enteritidis in the Absence of IL-12 Is Associated with IL-23-Dependent IL-22, but Not IL-17
J. Immunol., December 1, 2008; 181(11): 7891 - 7901.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
20/9/1129    most recent
dxn069v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Schulz, S. M.
Right arrow Articles by Alber, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schulz, S. M.
Right arrow Articles by Alber, G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?