The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 6169 -6175
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
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 Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Salazar-Gonzalez, R.-M.
Right arrow Articles by McSorley, S. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Salazar-Gonzalez, R.-M.
Right arrow Articles by McSorley, S. J.

Salmonella Flagellin Induces Bystander Activation of Splenic Dendritic Cells and Hinders Bacterial Replication In Vivo1

Rosa-Maria Salazar-Gonzalez*, Aparna Srinivasan*, Amanda Griffin*, Guruprasaadh Muralimohan{dagger}, James M. Ertelt*, Rajesh Ravindran*, Anthony T. Vella{dagger} and Stephen J. McSorley2,*

* Center for Infectious Diseases and Microbiology Translational Research, Department of Medicine, Division of Gastroenterology, Hepatology, and Nutrition, McGuire Translational Research Facility, University of Minnesota Medical School, Minneapolis, MN 55455; and {dagger} Department of Immunology, University of Connecticut Health Center, Farmington, CT 06030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Bacterial flagellin is a target of innate and adaptive immune responses during Salmonella infection. Intravenous injection of Salmonella flagellin into C57BL/6 mice induced rapid IL-6 production and increased expression of activation markers by splenic dendritic cells. CD11b+, CD8{alpha}+, and plasmacytoid dendritic cells each increased expression of CD86 and CD40 in response to flagellin stimulation, although CD11b+ dendritic cells were more sensitive than the other subsets. In addition, flagellin caused the rapid redistribution of dendritic cells from the red pulp and marginal zone of the spleen into the T cell area of the white pulp. Purified splenic dendritic cells did not respond directly to flagellin, indicating that flagellin-mediated activation of splenic dendritic cells occurs via bystander activation. IL-6 production, increased expression of activation markers, and dendritic cell redistribution in the spleen were dependent on MyD88 expression by bone marrow-derived cells. Avoiding this innate immune response to flagellin is important for bacterial survival, because Salmonella-overexpressing recombinant flagellin was highly attenuated in vivo. These data indicate that flagellin-mediated activation of dendritic cells is rapid, mediated by bystander activation, and highly deleterious to bacterial survival.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Oral infection with Salmonella enterica serovar typhi causes typhoid, a prevalent disease in certain parts of the developing world (1), and responsible for an estimated 200,000 annual deaths (2). Closely related Salmonella serovars cause nontyphoidal salmonellosis, an increasingly important food-borne infection in the U.S. (3). Understanding the interaction of Salmonella with cells of the innate and adaptive immune system is therefore a significant global health concern.

The immune response to Salmonella is usually studied in inbred mouse strains, although larger mammals and avian models are also used (4). Many inbred mouse strains develop fatal infection when injected i.v. with as few as 1–10 virulent Salmonella (5). A large body of literature has demonstrated that IFN-{gamma} production by murine CD4 T cell is critical for vaccine-induced protection against virulent Salmonella and for natural resistance of mice to vaccine strains of Salmonella (6, 7, 8). Somewhat surprisingly, mice lacking {alpha}beta T cells or expression of T-bet, the master-regulator of IFN-{gamma}, are able to contain Salmonella replication for ~3 wk, after which uncontrolled bacterial growth occurs (7, 8). These data suggest that innate immune defenses are responsible for controlling bacterial growth during the first few weeks of Salmonella infection.

Several innate immune receptors involved in early recognition of Salmonella have been identified (9). TLR2, TLR4, and TLR5, and the NOD-like receptor ICE protease activating factor are known to recognize Salmonella lipoproteins, LPS, and flagellin (10, 11, 12, 13, 14, 15, 16). Although the relative contribution of each of these individual receptors during human or murine typhoid is not completely clear, it seems likely that each of these plays an important role during early control of bacterial growth.

Salmonella flagellin has been identified as a mediator of inflammation in vitro and in vivo (17, 18, 19). Bacterial flagellins induce the secretion of TNF-{alpha}, IL-1beta, and IL-6 from monocytic cell lines, epithelial cell lines, or human PBMCs in vitro (20, 21, 22, 23, 24). Furthermore, injection of mice with purified flagellin causes the production of inflammatory cytokines and chemokines (13, 23, 25, 26), acute lung pathology (26, 27, 28), and even toxic shock at high doses (23). This inflammatory activity is mediated by host recognition of conserved flagellin residues by the innate receptors TLR5 and/or ICE protease activating factor (13, 14, 15, 16, 29, 30). Thus, bacterial flagellins have the potential to elicit a robust innate inflammatory response in vitro and in vivo.

Our laboratory and others have reported that flagellin also induces dendritic cell maturation and encourages the development of an adaptive immune response in vivo (28, 31, 32, 33, 34). However, it is not yet clear whether flagellin stimulates splenic dendritic cells directly or indirectly. Didierlaurent et al. (32) reported that purified splenic and bone marrow-derived dendritic cells express TLR5 and increase expression of CD40, CD80, and CD86, and secrete IL-12 p40 and low levels of IL-6 and TNF-{alpha} after exposure to Salmonella flagellin. In contrast, other laboratories report little or no TLR5 expression by splenic dendritic cells (35), and no IL-6 production or increased costimulatory molecule expression following in vitro incubation of splenic dendritic cells or bone marrow-derived dendritic cells with flagellin (35, 36). Therefore, the mechanism by which flagellin causes the activation of splenic dendritic cells following i.v. injection remains unclear. Furthermore, irrespective of the mode of activation, it is also unclear whether certain splenic dendritic cell subsets are more responsive to flagellin activation than others. This issue is particularly important given a recent report demonstrating differential Ag processing and presentation by different dendritic cell subsets to CD4 and CD8 T cells (37).

Expression of activation markers and inflammatory cytokine production are not the only hallmarks of splenic dendritic cell activation. Previous studies have documented the rapid migration of dendritic cells into the T cell area of the white pulp following injection of microbial adjuvants such as LPS or soluble tachyzoite Ag (38, 39), microbial ligands for TLR4 and TLR11 (11, 40). Presumably, such rapid reorganization of splenic dendritic cells increases the likelihood of APCs encountering pathogen-specific T cells and therefore initiating an adaptive response to blood-borne infection. Whether flagellin induces the migration of splenic dendritic cells has not previously been examined.

Salmonella express flagellin when replicating in vitro, but reduce expression of this protein during intracellular growth in the mammalian host (41, 42). Such coordinated flagellin regulation has led to the idea that induction of innate inflammation by flagellin expression might be deleterious to Salmonella survival in vivo, although this has not been tested directly. Salmonella lacking flagellin expression were initially reported to have reduced virulence (43, 44), but this finding was later refuted (45, 46). The most recent data indicate that nonflagellated bacteria have reduced virulence in cell culture experiments, but are virulent, or only have slightly reduced virulence in vivo (47, 48). However, the effect of Salmonella flagellin overexpression has not been examined.

In this study, we examined splenic dendritic cell activation in response to flagellin administration. We demonstrate that injection of purified flagellin induces rapid production of IL-6, increased expression of activation markers, and dendritic cell redistribution in the spleen. CD11b+ dendritic cells were more sensitive to the effect of flagellin injection than CD8{alpha}+ or plasmacytoid dendritic cells. The effects of flagellin on splenic dendritic cells were not due to direct activation, but instead are mediated by flagellin activation of another bone marrow-derived cell and required MyD88 expression. Furthermore, we demonstrate that overexpression of flagellin in Salmonella severely limits bacterial growth in vivo. These data therefore demonstrate the importance of bacterial flagellin regulation for avoiding host innate immune activation in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mouse strains

C57BL/6 mice were purchased from National Cancer Institute and were used at 8–16 wk of age. MyD88-deficient mice were a gift from L. Lefrancois (University of Connecticut Health Center, Farmington, CT). All mice were housed in specific pathogen-free conditions and cared for in accordance with Research Animal Resources practices at the University of Minnesota and University of Connecticut Health Center.

Bacterial strains

Flagellar phase-fixed Salmonella enterica serovar typhimurium BC116 (49) was a gift from B. Cookson (University of Washington, Seattle, WA). S. typhimurium X4700 has a galE deletion and cannot incorporate galactose into the O-Ag or outer core. It is thus a type of deep rough LPS-deficient mutant and was provided by R. Curtiss (Arizona State University, Tempe, AZ). Escherichia coli strain F18 was a gift from A. Gewirtz (Emory University, Atlanta, GA). BRD509 AroA–D– (50) was provided by D. Xu (University of Glasgow, Glasgow, U.K.). BRD509-Flag was constructed by inserting the Salmonella flagellin (FliC) gene into plasmid pTrc, electroporating into BRD509, and selecting with 100 µg/ml ampicillin (Sigma-Aldrich). BRD509-E{alpha}RFP was constructed in our laboratory by introducing a previously described plasmid construct into BRD509 (51).

Flagellin purification

Flagellin was purified from S. typhimurium (BC116 or X4700) or E. coli using a modified acid-shock protocol (52). Bacteria were grown overnight at 37°C without shaking, washed, and resuspended in PBS/HCl (pH 2) for 30 min at room temperature. Supernatants were collected and flagellin was harvested by ultracentrifugation and ammonium sulfate precipitation. Silver-stained SDS-PAGE gels and antiflagellin Western blots were used to confirm the purity and identity of flagellin preparations. Residual LPS was removed by serial passage through detoxigel columns (Pierce Biotechnology). To confirm LPS removal, an aliquot from each batch of flagellin was digested with proteinase K, and 30–50 µg of undigested and digested flagellin was injected i.v. into C57BL/6 mice. The expression of CD80 and CD86 was determined on splenic dendritic cells 6 h later, and only batches that failed to induce any dendritic cell activation after digestion were used in this study. This in vivo assay was found to be more sensitive and reliable than the in vitro Limulus assay, as previously reported (31).

Production of bacterial flagellin in S2 cells

Flagellin was cloned from E. coli by PCR and inserted into plasmid pMT/BiP/V5, and Drosophila S2 cells were cotransfected with a hygromycin selection plasmid using calcium phosphate. Transfected S2 cells were selected using 200 µg/ml hygromycin B in complete Schneider’s Drosophila medium, and individual colonies were picked and expanded for several weeks. Individual lines were grown without serum, and flagellin expression was induced by adding 500 µM copper sulfate. Western blots using Abs against the V5 epitope and flagellin were used to confirm flagellin production in the supernatant. Flagellin was purified from insect cell supernatants using the 6-HIS tag and Ni columns before being concentrated and stored at –80°C.

Flagellin injections and Salmonella infection

Mice were injected with different doses of purified Salmonella flagellin, purified E. coli flagellin, insect cell flagellin, or ultrapure LPS (Alexis). At various times later, mice were sacrificed to obtain serum and spleens for dendritic cell isolation or histology. For infection, S. typhimurium AroA–D– (BRD509) or BRD509-Flag was grown overnight in LB broth without shaking and diluted in PBS following estimation of bacterial concentration using a spectrophotometer. Mice were infected i.v. in the lateral tail vein with 5 x 105 bacteria and monitored for signs of infection. In all experiments, the actual bacterial dose administered was confirmed by plating serial dilutions onto MacConkey agar plates. To determine bacterial colonization in vivo, spleens from infected mice were homogenized in PBS and serial dilutions were plated onto MacConkey agar plates. After overnight incubation at 37°C, bacterial plates were counted and bacterial burdens were calculated for each individual organ.

Splenic dendritic cell isolation

Spleens were harvested from mice and sequentially incubated with collagenase D (37°C for 20 min) and EDTA to liberate dendritic cells. CD11c+ cells were harvested by positive selection using magnetic anti-CD11c microbeads and multiple passes through selection columns (Miltenyi Biotec). Cells eluted from the columns were typically 85–95% pure CD11c+ cells. In some experiments, these dendritic cells were washed and placed in culture with various concentrations of bacterial flagellins overnight, and supernatants and cells were collected 24 and 48 h later for analysis. In other experiments, dendritic cells were immediately surface stained and examined by flow cytometry.

Cytokine measurement

IL-6, IL-1{alpha}, TNF-{alpha}, and IL-12p70 were measured in serum and supernatants by sandwich ELISA following the manufacturer’s recommendations.

Flow cytometric analysis

Purified dendritic cells or spleen cells were incubated for 20–45 min at 4°C in Fc block (spent culture supernatant from the 24G2 hybridoma, 2% rat serum, 2% mouse serum, and 0.01% sodium azide) in the presence of primary Abs. FITC-, PE-, CyChrome-, PE-Cy5-, or allophycocyanin-conjugated Abs specific for CD4, CD8{alpha}, CD11b, CD11c, CD40, CD80, CD86, and isotype control Abs were purchased from eBioscience and BD Biosciences. After staining, cells were fixed and analyzed by flow cytometry using a FACS Canto (BD Biosciences). Data were analyzed using FlowJo software (Tree Star).

Bone marrow chimeras

Femurs and tibias were harvested from wild-type (Wt)3 or MyD88-deficient C57BL/6 mice, bone marrow was recovered, and a single-cell suspension was generated. RBC were lysed, and the remaining cells were suspended in HBSS supplemented with HEPES, L-glutamine, Pen/Strep, and gentamicin. Mature T cells were removed by incubation with anti-CD90 ascites fluid (T24), followed by Low-Tox-M rabbit complement (Cedarlane Laboratories) for 45 min at 37°C. Recipient C57BL/6 mice were irradiated (1000 rad) and bone marrow cells were transferred by i.v. injection (2 x 106 to 5 x 106/mouse). Mice were rested for 8 wk before use in experimental protocols.

Immunohistology

Whole spleens were harvested and embedded in OCT (Sakura Finetek) before snap freezing in liquid nitrogen. Sections (10 µM) were cut onto slides, dehydrated in acetone, and stored at –80°C. Slides were thawed and incubated with 3% H2O2, Fc block, and avidin/biotin block (Vector Laboratories), before staining with Abs specific for CD11c and B220. Abs were detected using Vectastain ABC Elite (Vector Laboratories) and developed with diaminobenzidine or Vector SG (Vector Laboratories). Tissues were counterstained with 5% methyl green and mounted before images were collected using standard bright-field microscopy.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Injection of mice with Salmonella flagellin induces the production of inflammatory cytokines (13, 23, 25, 26). We injected purified S. typhimurium flagellin into C57BL/6 mice and collected serum at various time points. Intravenous flagellin induced production of serum IL-6 and low levels of TNF-{alpha} within 1 h, although this disappeared from circulation by 4 h postinjection (Fig. 1A and data not shown). Serum IL-1{alpha} or IL-12 p70 was not detected at any time point following flagellin injection (data not shown). As with any biologically active microbial product, it was possible that bacterial contamination contributed to this effect. We therefore produced E. coli flagellin in S2 insect cells and compared the activity of this eukaryotic product with purified Salmonella flagellin. Flagellin produced in insect cells induced similar levels of serum IL-6, and with identical kinetics, compared with purified flagellin (Fig. 1B). Although bone marrow-derived cells are thought to be important mediators of innate responses, stromal cells can also respond to TLR ligands (53). We therefore examined whether signaling through MyD88 by bone marrow-derived cells was required for flagellin-induced IL-6. Wt bone marrow chimeras produced IL-6 in response to flagellin injection (Fig. 1C). In contrast, flagellin-induced serum IL-6 production was ablated in MyD88-deficient bone marrow chimeras (Fig. 1C). Therefore, bone marrow-derived cells respond to bacterial flagellin in a MyD88-dependent manner, and rapidly produce serum IL-6.


Figure 1
View larger version (14K):
[in this window]
[in a new window]

  		FIGURE 1. Bacterial flagellin induces serum IL-6 and requires bone marrow cell expression of MyD88. C57BL/6 mice were injected i.v. with 5 µg of purified Salmonella flagellin (A) or recombinant E. coli flagellin (B) produced in insect cells. At various time points later, mice were sacrificed and serum was collected. C, Wt, Myd88-deficient bone marrow chimeras and C57BL/6 mice were injected i.v. with 5 µg of purified Salmonella flagellin, and serum was collected 1 h later. IL-6 in serum samples was determined by sandwich ELISA. Each data point shows mean ± SD for three mice per group, and each graph is representative of two separate experiments.

 
Next, we examined the activation of splenic dendritic cells following injection of Salmonella flagellin. Flagellin induced a substantial increase in the surface expression of CD86, and a smaller increase in CD80 and CD40, on CD11b+ dendritic cells (Fig. 2, Flagellin). This activity was lost if flagellin was pretreated with proteinase K (Fig. 2, PK-Flagellin). Flagellin had only a modest effect on CD8{alpha}+ dendritic cells, causing a small increase in the expression of CD40, CD80, and CD86 (Fig. 2, Flagellin). In contrast, Salmonella LPS induced substantial increases in CD80, CD86, and CD40 expression on both CD11b+ and CD8{alpha}+ dendritic cells (Fig. 2, LPS). This difference in the activation of CD11b+ and CD8{alpha}+ dendritic cell subsets by flagellin was detected at several different doses of flagellin. Examination of various doses of flagellin demonstrated consistent activation of CD11b+ dendritic cells, but significantly reduced activity on CD8{alpha}+ dendritic cells, across a 100-fold range (Fig. 3). In contrast, LPS had similar effects on both subsets (Fig. 3). Flagellin also caused a small dose-dependent increase in the expression of CD40 and CD86 by plasmacytoid dendritic cells (data not shown). Thus, plasmacytoid dendritic cells also respond to flagellin injection by increasing expression of surface activation markers, although the magnitude of this response was considerably lower than CD11b+ dendritic cells. Together these data demonstrate that bacterial flagellin can activate splenic dendritic cells in vivo, but has a more pronounced effect on CD11b+ dendritic cells.


Figure 2
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 2. Bacterial flagellin induces activation of CD11b+ and CD8{alpha}+ splenic dendritic cells in vivo. C57BL/6 mice were injected i.v. with 10 µg of purified Salmonella flagellin, proteinase K-treated Salmonella flagellin, or LPS, and 6 h later dendritic cells were purified and stained with Abs specific for CD11c, CD11b, and CD8{alpha} to identify dendritic cell subsets. Dendritic cells were also stained with Abs specific for CD80 (A), CD86 (B), and CD40 (C) to monitor activation. All histograms show plots after gating on CD11c+CD11b+CD8{alpha}– (left column) or CD11c+CD11b–CD8{alpha}+ (right column). Gray shading shows surface staining on dendritic cells from naive C57BL/6 mice, and black lines show staining in mice injected with flagellin, PK-flagellin, or LPS. Plots are similar to three separate experiments.

 

Figure 3
View larger version (35K):
[in this window]
[in a new window]

  		FIGURE 3. Dose response of splenic CD11b+ and CD8{alpha}+ dendritic cells to flagellin in vivo. C57BL/6 mice were injected i.v. with 50, 5, or 0.5 µg of purified Salmonella flagellin, or 10 µg of LPS, and 6 h later dendritic cells were purified and stained with Abs specific for CD11c, CD11b, and CD8{alpha} to identify dendritic cell subsets. Dendritic cells were stained with Abs specific for CD80, CD86, and CD40 to monitor activation. All histograms show plots after gating on CD11c+CD11b+CD8{alpha}– (left column) or CD11c+CD11b–CD8{alpha}+ (right column). Gray shading shows surface staining on dendritic cells from naive C57BL/6 mice, and black lines show staining in mice injected with flagellin or LPS. Plots are similar to two separate experiments.

 
Next, we examined the importance of MyD88 expression by bone marrow-derived cells for dendritic cell responsiveness to flagellin. CD11b+ and CD8{alpha}+ dendritic cells in Wt > Wt bone marrow chimeras increased expression of CD80, CD86, and CD40 in response to flagellin injection (Fig. 4). However, this response was reduced in MyD88-deficient > Wt bone marrow chimeras, although increased expression of CD80, CD86, and CD40 on dendritic cells was still observed (Fig. 4). These data suggest that flagellin responsiveness by stromal cells may contribute to splenic dendritic cell activation in response to flagellin.


Figure 4
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 4. Activation of CD11b+ and CD8{alpha}+ splenic dendritic cells in response to flagellin is reduced in MyD88-deficient bone marrow chimeras. Wt or MyD88-deficient bone marrow chimeras were injected i.v. with 5 µg of purified Salmonella flagellin, and 6 h later dendritic cells were purified and stained with Abs specific for CD11c, CD11b, and CD8{alpha} to identify dendritic cell subsets. Dendritic cells were also stained with Abs specific for CD80, CD86, and CD40 to monitor in vivo activation. Histograms show plots after gating on CD11c+CD11b+CD8{alpha}– (left column) or CD11c+CD11b–CD8{alpha}+ (right column). Gray shading shows surface staining on dendritic cells from naive C57BL/6 mice, and black lines show staining in mice injected with flagellin. Plots are similar to two separate experiments.

 
It has been reported that murine splenic dendritic cells respond directly to bacterial flagellin, producing inflammatory cytokines and increasing expression of CD80 and CD86 (32). In contrast, other reports claim that murine splenic dendritic cells do not respond directly to bacterial flagellin (35, 36). We purified splenic CD11c+ dendritic cells and examined the response to purified Salmonella and E. coli flagellin in vitro. As previously reported (36), CD11c+ splenic dendritic cells did not increase surface expression of CD80, CD86, or CD40, and did not secrete IL-6, TNF-{alpha}, or IL-12 p70 in response to either Salmonella or E. coli flagellin, despite the fact that these preparations were clearly biologically active when injected in vivo (data not shown). These data suggest that dendritic cells are activated by flagellin in a bystander fashion rather than by direct activation.

We also examined the distribution of dendritic cells in the spleen of mice injected with flagellin. In naive C57BL/6 mice, CD11c+ dendritic cells were found scattered throughout the red pulp, the T cell area of the white pulp, and the marginal zone (Fig. 5A). Six hours after flagellin injection, CD11c+ dendritic cells were found exclusively within the T cell area of the white pulp and were strikingly absent from the red pulp or marginal zone (Fig. 5B). However, in MyD88-deficient bone marrow chimeras, CD11c+ dendritic cells did not undergo significant redistribution in response to flagellin injection (Fig. 5, C and D). Thus, injection of bacterial flagellin induces the redistribution of splenic dendritic cells to the T cell area of the white pulp in a MyD88-dependent manner.


Figure 5
View larger version (81K):
[in this window]
[in a new window]

  		FIGURE 5. Bacterial flagellin injection induces the redistribution of splenic dendritic cells. (A, B) C57BL/6 mice or (C, D) MyD88-deficient bone marrow chimeras were (B, D) injected i.v. with 5 µg of purified Salmonella flagellin, and 6 h later spleens were harvested and tissue sections were stained to detect CD11c (brown) and B220 (blue). Images are representative of several other sections from multiple mice and experiments.

 
Although Salmonella express flagella in culture, they markedly decrease expression of flagellin during intracellular growth (41, 42). Our data demonstrate that flagellin is an effective activator of dendritic cells, suggesting that flagellin regulation by bacteria may be orchestrated to avoid innate immune activation. If this is correct, flagellin overexpression should be detrimental to Salmonella survival in vivo, but this has not been examined directly. We overexpressed Salmonella flagellin in BRD509, a flagellated, attenuated strain of S. typhimurium, effectively removing the ability of bacteria to modulate flagellin expression in vivo. Despite the fact that both these strains grow well in vitro, BRD509-Flag were absent from the spleen 3 or 10 days after infection, whereas increasing numbers of BRD509 were detected over this period (Fig. 6). We also compared the colonization of BRD509-Flag vs BRD509 expressing an irrelevant protein, E{alpha}RFP. Although similar numbers of both strains could be detected in the spleen at early time points, BRD509-Flag was eliminated by 12 days after infection (Fig. 6B). In marked contrast, BRD509-E{alpha}RFP was found in greater numbers at this time point (Fig. 6B).


Figure 6
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 6. Salmonella-overexpressing flagellin is highly attenuated in vivo. A, C57BL/6 mice were infected i.v. with 5 x 105 BRD509 or BRD509-Flag, and spleens were harvested 3 and 10 days later. Bacterial colonization was determined by plating serial dilutions of spleen aliquots on McConkey agar and counting Salmonella colonies the following day. Data are representative of two individual experiments. B, C57BL/6 mice were infected i.v. with 5 x 105 BRD509-Flag or BRD509-E{alpha}RFP, and spleens were harvested 1, 2, and 12 days later. Bacterial colonization and in vivo plasmid stability were determined by plating serial dilutions of spleen aliquots on McConkey agar with or without ampicillin, and counting Salmonella colonies the following day.

 
Furthermore, the splenomegaly that normally accompanies Salmonella infection was significantly reduced in mice infected with BRD509-Flag. The number of splenocytes in mice infected with BRD509-Flag and the proportions of various cell populations were similar to uninfected mice (Fig. 7). In contrast, mice infected with BRD509 displayed a considerable increase in spleen cells, an increase in the proportion of CD11b+ and Gr1+ cells, and a corresponding decrease in the proportion of B and T lymphocytes (Fig. 7B). These data demonstrate that flagellin expression is highly detrimental to bacterial survival and disease progression in vivo.


Figure 7
View larger version (43K):
[in this window]
[in a new window]

  		FIGURE 7. Mice infected with Salmonella-overexpressing flagellin do not develop splenomegaly. C57BL/6 mice were infected i.v. with 5 x 105 BRD509 or BRD509-Flag; spleens were harvested 3 and 10 days later; and total cells were counted (A) or stained (B) using Abs specific for lymphocyte or macrophage populations. Data in B are from the day 10 time point and are representative of two individual experiments and four to five mice per group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Salmonella are known to down-regulate expression of flagellin during intracellular growth in the mammalian host (41, 42). However, the reason for this regulation of flagellin expression has not been apparent. It seems likely that the innate immune response to flagellin may be deleterious to the bacterial growth in vivo, although this has not been conclusively demonstrated.

Splenic dendritic cells were activated following flagellin injection of mice, but did not respond to direct flagellin stimulation in vitro. Therefore, in vivo activation of splenic dendritic cells most likely occurs through a bystander process requiring activation of other TLR5-expressing cells. Similar bystander activation of dendritic cells has been described for LPS (54). From our bone marrow chimera experiments, it seems clear that this flagellin-responsive cell population is bone marrow derived. We have considered the possibility that marginal zone macrophages can respond directly to injected flagellin and are responsible for the bystander activation of splenic dendritic cells. However, we did not detect any in vitro response to flagellin by unfractionated spleen cells (data not shown), suggesting that flagellin responsiveness may occur outside the spleen and that splenic dendritic cells are activated by circulating inflammatory mediators. Indeed, this hypothesis would concur with the recent finding that intestinal dendritic cells are enriched for TLR5 expression and can respond to flagellin directly (35). Whether intestinal dendritic cells are responsible for the bystander activation of splenic dendritic cells following i.v. injection of flagellin will require further investigation.

The conclusion that splenic dendritic cells are activated indirectly by flagellin is in broad agreement with previous reports describing the inability of splenic dendritic cells to respond directly to flagellin in vitro (35, 36), but conflicts with another report (32). It is possible that contamination with other active bacterial products was responsible for the direct dendritic cell activation noted in this study. Our laboratory tests every batch of flagellin using an in vivo dendritic cell assay, due to concerns with the sensitivity of current in vitro assays for LPS detection. Our data showing equivalent IL-6 responses using insect cell and purified flagellin indicate that our flagellin preparations do not have significant biologically active contaminants.

Our data also indicate heightened activation by CD11b+ vs CD8{alpha}+, and plasmacytoid, dendritic cells in response to flagellin, a finding consistent with differential expression of TLR5 by these subsets (55). This pronounced activation of CD11b+ vs CD8{alpha}+ dendritic cells was also observed when insect cell, or purified E. coli, flagellin was used as the in vivo stimulus (data not shown). Given their anatomical distribution, CD11b+ dendritic cells are also likely to be the CD11c+ population that migrates rapidly to the white pulp following flagellin injection. Given the indirect and undefined nature of the activating stimulus, it is not clear why CD11b+ dendritic cells respond more strongly to flagellin injection in vivo. Because a recent report has indicated that CD11b+ dendritic cells preferentially process and present Ag to CD4 T cells (37), and that CD4 T cells are required for resistance to Salmonella infection (7, 56, 57), this may indicate an adaptation of the innate immune system to target dendritic cell activation to a subset that will induce a protective adaptive response.

Although it has been known for some time that Salmonella down-regulate the expression of flagellin during intracellular growth, the effect of flagellin overexpression has not been previously examined. Our data show that in the absence of flagellin regulation Salmonella are highly attenuated in vivo and recruit fewer phagocytes to the spleen. The reduced splenomegaly is most likely a consequence of the inability of the bacteria to survive due to innate recognition of bacterial flagellin. Thus, virulent Salmonella most likely modulate flagellin expression to avoid the detrimental effect of innate immune recognition in vivo. It may be possible to modulate the virulence of current live attenuated vaccine candidate strains in vivo by altering flagellin regulation, although whether this would actually increase the immunogenicity of such strains is not clear.

Overall, our data indicate that flagellin activates splenic dendritic cells in a bystander manner via a bone marrow-derived, flagellin-responsive cell population, through MyD88. This activation leads to rapid expression of activation markers and dendritic cell redistribution in the spleen. This innate immune response to flagellin is highly deleterious to the growth of Salmonella in vivo, and suggests an explanation for bacterial modulation of flagellin expression in vivo.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the National Institutes of Health AI056172 (to A.T.V. and S.J.M.). Back

2 Address correspondence and reprint requests to Dr. Stephen J. McSorley, Center for Infectious Diseases and Microbiology Translational Research, Department of Medicine, Division of Gastroenterology, Hepatology, and Nutrition, McGuire Translational Research Facility, University of Minnesota Medical School, Minneapolis, MN 55455. E-mail address: mcsor002{at}umn.edu Back

3 Abbreviation used in this paper: Wt, wild type. Back

Received for publication April 10, 2007. Accepted for publication August 21, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Jones, B. D., S. Falkow. 1996. Salmonellosis: host immune responses and bacterial virulence determinants. Annu. Rev. Immunol. 14: 533-561. [Medline]
  2. Crump, J. A., S. P. Luby, E. D. Mintz. 2004. The global burden of typhoid fever. Bull. W. H. O. 82: 346-353. [Medline]
  3. Mead, P. S., L. Slutsker, V. Dietz, L. F. McCaig, J. S. Bresee, C. Shapiro, P. M. Griffin, R. V. Tauxe. 1999. Food-related illness and death in the United States. Emerg. Infect. Dis. 5: 607-625. [Medline]
  4. Santos, R. L., S. Zhang, R. M. Tsolis, R. A. Kingsley, L. G. Adams, A. J. Baumler. 2001. Animal models of Salmonella infections: enteritis versus typhoid fever. Microbes Infect. 3: 1335-1344. [Medline]
  5. Plant, J., A. A. Glynn. 1976. Genetics of resistance to infection with Salmonella typhimurium in mice. J. Infect. Dis. 133: 72-78. [Medline]
  6. Nauciel, C., F. Espinasse-Maes. 1992. Role of {gamma} interferon and tumor necrosis factor {alpha} in resistance to Salmonella typhimurium infection. Infect. Immun. 60: 450-454. [Abstract/Free Full Text]
  7. Hess, J., C. Ladel, D. Miko, S. H. Kaufmann. 1996. Salmonella typhimurium aroA– infection in gene-targeted immunodeficient mice: major role of CD4+ TCR-{alpha}beta cells and IFN-{gamma} in bacterial clearance independent of intracellular location. J. Immunol. 156: 3321-3326. [Abstract]
  8. Ravindran, R., J. Foley, T. Stoklasek, L. H. Glimcher, S. J. McSorley. 2005. Expression of T-bet by CD4 T cells is essential for resistance to Salmonella infection. J. Immunol. 175: 4603-4610. [Abstract/Free Full Text]
  9. Delbridge, L. M., M. X. O’Riordan. 2007. Innate recognition of intracellular bacteria. Curr. Opin. Immunol. 19: 10-16. [Medline]
  10. Weiss, D. S., B. Raupach, K. Takeda, S. Akira, A. Zychlinsky. 2004. Toll-like receptors are temporally involved in host defense. J. Immunol. 172: 4463-4469. [Abstract/Free Full Text]
  11. Poltorak, A., X. He, I. Smirnova, M. Y. Liu, C. V. Huffel, X. Du, D. Birdwell, E. Alejos, M. Silva, C. Galanos, et al 1998. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282: 2085-2088. [Abstract/Free Full Text]
  12. Hoshino, K., O. Takeuchi, T. Kawai, H. Sanjo, T. Ogawa, Y. Takeda, K. Takeda, S. Akira. 1999. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J. Immunol. 162: 3749-3752. [Abstract/Free Full Text]
  13. Hayashi, F., K. D. Smith, A. Ozinsky, T. R. Hawn, E. C. Yi, D. R. Goodlett, J. K. Eng, S. Akira, D. M. Underhill, A. Aderem. 2001. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410: 1099-1103. [Medline]
  14. Gewirtz, A. T., T. A. Nava, S. Lyons, P. J. Godowski, J. L. Madera. 2001. Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. J. Immunol. 167: 1882-1885. [Abstract/Free Full Text]
  15. Franchi, L., A. Amer, M. Body-Malapel, T. D. Kanneganti, N. Ozoren, R. Jagirdar, N. Inohara, P. Vandenabeele, J. Bertin, A. Coyle, et al 2006. Cytosolic flagellin requires Ipaf for activation of caspase-1 and interleukin 1beta in Salmonella-infected macrophages. Nat. Immunol. 7: 576-582. [Medline]
  16. Miao, E. A., C. M. Alpuche-Aranda, M. Dors, A. E. Clark, M. W. Bader, S. I. Miller, A. Aderem. 2006. Cytoplasmic flagellin activates caspase-1 and secretion of interleukin 1beta via Ipaf. Nat. Immunol. 7: 569-575. [Medline]
  17. Salazar-Gonzalez, R. M., S. J. McSorley. 2005. Salmonella flagellin, a microbial target of the innate and adaptive immune system. Immunol. Lett. 101: 117-122. [Medline]
  18. Honko, A. N., S. B. Mizel. 2005. Effects of flagellin on innate and adaptive immunity. Immunol. Res. 33: 83-101. [Medline]
  19. Gewirtz, A. T.. 2006. Flag in the crossroads: flagellin modulates innate and adaptive immunity. Curr. Opin. Gastroenterol. 22: 8-12. [Medline]
  20. Ciacci-Woolwine, F., I. C. Blomfield, S. H. Richardson, S. B. Mizel. 1998. Salmonella flagellin induces tumor necrosis factor {alpha} in a human promonocytic cell line. Infect. Immun. 66: 1127-1134. [Abstract/Free Full Text]
  21. Ciacci-Woolwine, F., P. F. McDermott, S. B. Mizel. 1999. Induction of cytokine synthesis by flagella from Gram-negative bacteria may be dependent on the activation or differentiation state of human monocytes. Infect. Immun. 67: 5176-5185. [Abstract/Free Full Text]
  22. Wyant, T. L., M. K. Tanner, M. B. Sztein. 1999. Salmonella typhi flagella are potent inducers of proinflammatory cytokine secretion by human monocytes. Infect. Immun. 67: 3619-3624. [Abstract/Free Full Text]
  23. Eaves-Pyles, T., K. Murthy, L. Liaudet, L. Virag, G. Ross, F. G. Soriano, C. Szabo, A. L. Salzman. 2001. Flagellin, a novel mediator of Salmonella-induced epithelial activation and systemic inflammation: I{kappa}B{alpha} degradation, induction of nitric oxide synthase, induction of proinflammatory mediators, and cardiovascular dysfunction. J. Immunol. 166: 1248-1260. [Abstract/Free Full Text]
  24. Zeng, H., A. Q. Carlson, Y. Guo, Y. Yu, L. S. Collier-Hyams, J. L. Madara, A. T. Gewirtz, A. S. Neish. 2003. Flagellin is the major proinflammatory determinant of enteropathogenic Salmonella. J. Immunol. 171: 3668-3674. [Abstract/Free Full Text]
  25. Liaudet, L., K. G. Murthy, J. G. Mabley, P. Pacher, F. G. Soriano, A. L. Salzman, C. Szabo. 2002. Comparison of inflammation, organ damage, and oxidant stress induced by Salmonella enterica serovar Muenchen flagellin and serovar Enteritidis lipopolysaccharide. Infect. Immun. 70: 192-198. [Abstract/Free Full Text]
  26. Honko, A. N., S. B. Mizel. 2004. Mucosal administration of flagellin induces innate immunity in the mouse lung. Infect. Immun. 72: 6676-6679. [Abstract/Free Full Text]
  27. Liaudet, L., C. Szabo, O. V. Evgenov, K. G. Murthy, P. Pacher, L. Virag, J. G. Mabley, A. Marton, F. G. Soriano, M. Y. Kirov, et al 2003. Flagellin from Gram-negative bacteria is a potent mediator of acute pulmonary inflammation in sepsis. Shock 19: 131-137. [Medline]
  28. Feuillet, V., S. Medjane, I. Mondor, O. Demaria, P. P. Pagni, J. E. Galan, R. A. Flavell, L. Alexopoulou. 2006. Involvement of Toll-like receptor 5 in the recognition of flagellated bacteria. Proc. Natl. Acad. Sci. USA 103: 12487-12492. [Abstract/Free Full Text]
  29. Eaves-Pyles, T. D., H. R. Wong, K. Odoms, R. B. Pyles. 2001. Salmonella flagellin-dependent proinflammatory responses are localized to the conserved amino and carboxyl regions of the protein. J. Immunol. 167: 7009-7016. [Abstract/Free Full Text]
  30. Smith, K. D., E. Andersen-Nissen, F. Hayashi, K. Strobe, M. A. Bergman, S. L. Barrett, B. T. Cookson, A. Aderem. 2003. Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility. Nat. Immunol. 4: 1247-1253. [Medline]
  31. McSorley, S. J., B. D. Ehst, Y. Yu, A. T. Gewirtz. 2002. Bacterial flagellin is an effective adjuvant for CD4 T cells in vivo. J. Immunol. 169: 3914[Abstract/Free Full Text]
  32. Didierlaurent, A., I. Ferrero, L. A. Otten, B. Dubois, M. Reinhardt, H. Carlsen, R. Blomhoff, S. Akira, J. P. Kraehenbuhl, J. C. Sirard. 2004. Flagellin promotes myeloid differentiation factor 88-dependent development of Th2-type response. J. Immunol. 172: 6922-6930. [Abstract/Free Full Text]
  33. Sanders, C. J., Y. Yu, D. A. Moore, III, I. R. Williams, A. T. Gewirtz. 2006. Humoral immune response to flagellin requires T cells and activation of innate immunity. J. Immunol. 177: 2810-2818. [Abstract/Free Full Text]
  34. Huleatt, J. W., A. R. Jacobs, J. Tang, P. Desai, E. B. Kopp, Y. Huang, L. Song, V. Nakaar, T. J. Powell. 2007. Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands induces rapid cellular and humoral immunity. Vaccine 25: 763-775. [Medline]
  35. Uematsu, S., M. H. Jang, N. Chevrier, Z. Guo, Y. Kumagai, M. Yamamoto, H. Kato, N. Sougawa, H. Matsui, H. Kuwata, et al 2006. Detection of pathogenic intestinal bacteria by Toll-like receptor 5 on intestinal CD11c+ lamina propria cells. Nat. Immunol. 7: 868-874. [Medline]
  36. Means, T. K., F. Hayashi, K. D. Smith, A. Aderem, A. D. Luster. 2003. The Toll-like receptor 5 stimulus bacterial flagellin induces maturation and chemokine production in human dendritic cells. J. Immunol. 170: 5165-5175. [Abstract/Free Full Text]
  37. Dudziak, D., A. O. Kamphorst, G. F. Heidkamp, V. R. Buchholz, C. Trumpfheller, S. Yamazaki, C. Cheong, K. Liu, H. W. Lee, C. G. Park, et al 2007. Differential antigen processing by dendritic cell subsets in vivo. Science 315: 107-111. [Abstract/Free Full Text]
  38. Reis e Sousa, C., S. Hieny, T. Scharton-Kersten, D. Jankovic, H. Charest, R. N. Germain, A. Sher. 1997. In vivo microbial stimulation induces rapid CD40 ligand-independent production of interleukin 12 by dendritic cells and their redistribution to T cell areas. J. Exp. Med. 186: 1819-1829. [Abstract/Free Full Text]
  39. Reis e Sousa, C., R. N. Germain. 1999. Analysis of adjuvant function by direct visualization of antigen presentation in vivo: endotoxin promotes accumulation of antigen-bearing dendritic cells in the T cell areas of lymphoid tissue. J. Immunol. 162: 6552-6561. [Abstract/Free Full Text]
  40. Yarovinsky, F., D. Zhang, J. F. Andersen, G. L. Bannenberg, C. N. Serhan, M. S. Hayden, S. Hieny, F. S. Sutterwala, R. A. Flavell, S. Ghosh, A. Sher. 2005. TLR11 activation of dendritic cells by a protozoan profilin-like protein. Science 308: 1626-1629. [Abstract/Free Full Text]
  41. Eriksson, S., S. Lucchini, A. Thompson, M. Rhen, J. C. Hinton. 2003. Unravelling the biology of macrophage infection by gene expression profiling of intracellular Salmonella enterica. Mol. Microbiol. 47: 103-118. [Medline]
  42. Cummings, L. A., W. D. Wilkerson, T. Bergsbaken, B. T. Cookson. 2006. In vivo, fliC expression by Salmonella enterica serovar Typhimurium is heterogeneous, regulated by ClpX, and anatomically restricted. Mol. Microbiol. 61: 795-809. [Medline]
  43. Carsiotis, M., D. L. Weinstein, H. Karch, I. A. Holder, A. D. O’Brien. 1984. Flagella of Salmonella typhimurium are a virulence factor in infected C57BL/6J mice. Infect. Immun. 46: 814-818. [Abstract/Free Full Text]
  44. Weinstein, D. L., M. Carsiotis, C. R. Lissner, A. D. O’Brien. 1984. Flagella help Salmonella typhimurium survive within murine macrophages. Infect. Immun. 46: 819-825. [Abstract/Free Full Text]
  45. Carsiotis, M., B. A. Stocker, D. L. Weinstein, A. D. O’Brien. 1989. A Salmonella typhimurium virulence gene linked to flg. Infect. Immun. 57: 3276-3280. [Abstract/Free Full Text]
  46. Lockman, H. A., R. Curtiss, III. 1990. Salmonella typhimurium mutants lacking flagella or motility remain virulent in BALB/c mice. Infect. Immun. 58: 137-143. [Abstract/Free Full Text]
  47. Schmitt, C. K., J. S. Ikeda, S. C. Darnell, P. R. Watson, J. Bispham, T. S. Wallis, D. L. Weinstein, E. S. Metcalf, A. D. O’Brien. 2001. Absence of all components of the flagellar export and synthesis machinery differentially alters virulence of Salmonella enterica serovar Typhimurium in models of typhoid fever, survival in macrophages, tissue culture invasiveness, and calf enterocolitis. Infect. Immun. 69: 5619-5625. [Abstract/Free Full Text]
  48. Robertson, J. M., N. H. McKenzie, M. Duncan, E. Allen-Vercoe, M. J. Woodward, H. J. Flint, G. Grant. 2003. Lack of flagella disadvantages Salmonella enterica serovar Enteritidis during the early stages of infection in the rat. J. Med. Microbiol. 52: 91-99. [Abstract/Free Full Text]
  49. McSorley, S. J., B. T. Cookson, M. K. Jenkins. 2000. Characterization of CD4+ T cell responses during natural infection with Salmonella typhimurium. J. Immunol. 164: 986-993. [Abstract/Free Full Text]
  50. Chatfield, S. N., I. G. Charles, A. J. Makoff, M. D. Oxer, G. Dougan, D. Pickard, D. Slater, N. F. Fairweather. 1992. Use of the nirB promoter to direct the stable expression of hetrologous antigens in Salmonella oral vaccine strains: development of a single-dose oral tetanus vaccine. Biotechnology 10: 888-892. [Medline]
  51. Ravindran, R., L. Rusch, A. Itano, M. K. Jenkins, S. J. McSorley. 2007. CCR6-dependent recruitment of blood phagocytes is necessary for rapid CD4 T cell responses to local bacterial infection. Proc. Natl. Acad. Sci. USA 104: 12075-12080. [Abstract/Free Full Text]
  52. Ibrahim, G. F., G. H. Fleet, M. J. Lyons, R. A. Walker. 1985. Method for the isolation of highly purified Salmonella flagellins. J. Clin. Microbiol. 22: 1040-1044. [Abstract/Free Full Text]
  53. Sato, A., A. Iwasaki. 2004. Induction of antiviral immunity requires Toll-like receptor signaling in both stromal and dendritic cell compartments. Proc. Natl. Acad. Sci. USA 101: 16274-16279. [Abstract/Free Full Text]
  54. Yadav, R., D. J. Zammit, L. Lefrancois, A. T. Vella. 2006. Effects of LPS-mediated bystander activation in the innate immune system. J. Leukocyte Biol. 80: 1251-1261. [Abstract/Free Full Text]
  55. Edwards, A. D., S. S. Diebold, E. M. Slack, H. Tomizawa, H. Hemmi, T. Kaisho, S. Akira, C. Reis e Sousa. 2003. Toll-like receptor expression in murine DC subsets: lack of TLR7 expression by CD8{alpha}+ DC correlates with unresponsiveness to imidazoquinolines. Eur. J. Immunol. 33: 827-833. [Medline]
  56. Nauciel, C.. 1990. Role of CD4+ T cells and T-independent mechanisms in acquired resistance to Salmonella typhimurium infection. J. Immunol. 145: 1265-1269. [Abstract]
  57. Mastroeni, P., B. Villarreal-Ramos, C. E. Hormaeche. 1993. Adoptive transfer of immunity to oral challenge with virulent Salmonella in innately susceptible BALB/c mice requires both immune serum and T cells. Infect. Immun. 61: 3981-3984. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
A. Griffin, D. Baraho-Hassan, and S. J. McSorley
Successful Treatment of Bacterial Infection Hinders Development of Acquired Immunity
J. Immunol., July 15, 2009; 183(2): 1263 - 1270.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. T. Bates, S. Uematsu, S. Akira, and S. B. Mizel
Direct Stimulation of tlr5+/+ CD11c+ Cells Is Necessary for the Adjuvant Activity of Flagellin
J. Immunol., June 15, 2009; 182(12): 7539 - 7547.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. J. Sanders, D. A. Moore III, I. R. Williams, and A. T. Gewirtz
Both Radioresistant and Hemopoietic Cells Promote Innate and Adaptive Immune Responses to Flagellin
J. Immunol., June 1, 2008; 180(11): 7184 - 7192.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
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 Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Salazar-Gonzalez, R.-M.
Right arrow Articles by McSorley, S. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Salazar-Gonzalez, R.-M.
Right arrow Articles by McSorley, S. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS