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


     
 

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
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Copin, R.
Right arrow Articles by Muraille, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Copin, R.
Right arrow Articles by Muraille, E.
The Journal of Immunology, 2007, 178: 5182-5191.
Copyright © 2007 by The American Association of Immunologists, Inc.

MyD88-Dependent Activation of B220–CD11b+LY-6C+ Dendritic Cells during Brucella melitensis Infection1

Richard Copin*, Patrick De Baetselier{ddagger}, Yves Carlier{dagger}, Jean-Jacques Letesson2,3,* and Eric Muraille2,3,{dagger}

* Unité de Recherche en Biologie Moléculaire, Laboratoire d’Immunologie et de Microbiologie, Faculté Universitaire Notre Dame de la Paix, Namur, Belgium; {dagger} Laboratoire de Parasitologie, Faculté de Médecine, Université Libre de Bruxelles, Bruxelles, Belgium; and {ddagger} Department of Molecular and Cellular Interactions, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Brussels, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IFN-{gamma} is a key cytokine controlling Brucella infection. One of its major function is the stimulation of Brucella-killing effector mechanisms, such as inducible NO synthase (iNOS)/NOS2 activity, in phagocytic cells. In this study, an attempt to identify the main cellular components of the immune response induced by Brucella melitensis in vivo is made. IFN-{gamma} and iNOS protein were analyzed intracellularly using flow cytometry in chronically infected mice. Although TCRbeta+CD4+ cells were the predominant source of IFN-{gamma} in the spleen, we also identified CD11b+LY-6C+LY-6G–MHC-II+ cells as the main iNOS-producing cells in the spleen and the peritoneal cavity. These cells appear similar to inflammatory dendritic cells recently described in the mouse model of Listeria monocytogenes infection and human psoriasis: the TNF/iNOS-producing dendritic cells. Using genetically deficient mice, we demonstrated that the induction of iNOS and IFN-{gamma}-producing cells due to Brucella infection required TLR4 and TLR9 stimulation coupled to Myd88-dependent signaling pathways. The unique role of MyD88 was confirmed by the lack of impact of Toll-IL-1R domain-containing adaptor inducing IFN-beta deficiency. The reduction of IFN-{gamma}+ and iNOS+ cell frequency observed in MyD88-, TLR4-, and TLR9-deficient mice correlated with a proportional lack of Brucella growth control. Taken together, our results provide new insight into how immune responses fight Brucella infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Brucella organisms are facultative intracellular Gram-negative coccobacilli that infect humans and animals. They are causative agents of a worldwide zoonosis. Acute human brucellosis is characterized by undulating fever, which, if untreated, may result in localization of bacteria in various tissues leading to chronic disease with serious clinical manifestations, such as orchitis, osteoarthritis, spondylitis, endocarditis, and several neurological disorders (1, 2, 3, 4). Brucella melitensis is the most frequent cause of human brucellosis (5).

Brucella resides and replicates in a vacuolar compartment within macrophages of the infected host (6, 7, 8). As for other intracellular microbial pathogens, IFN-{gamma}, a Th1 cytokine produced by activated NK, CD4+ T cells, and CD8+ T cells, contributes to control the infection (9, 10, 11). One of its major functions is the stimulation of Brucella-killing effector mechanisms, such as the production of a reactive nitrogen intermediate (RNI)4 and a reactive oxygen intermediate (ROI) in phagocytic cells (12, 13). A previous study (14) has reported that gp91phox–/– mice and iNOS/NOS2–/– mice are both susceptible to Brucella abortus infection but that inducible NO synthase (iNOS) deficiency more drastically affects the resistance, suggesting a predominant role of RNI in Brucella killing. BALB/c mice are less able to control Brucella infections than C57BL/6 mice, having an ~10-fold increase in bacteria in their spleens during the plateau phase of the infection which occurs between 1 and 6 wk postinfection (15, 16). It has been suggested that BALB/c susceptibility is due to a cessation of IFN-{gamma} production that begins after the first week of infection. This has been mainly demonstrated by measuring in vitro IFN-{gamma} secretion by spleen cells isolated from infected mice and cultivated in the presence of Brucella Ag (15, 11). Despite progresses in mouse models of brucellosis, much remains unknown regarding cellular components of the innate and adaptive immune response induced by B. melitensis infection. In particular, the nature and cell surface phenotype of innate immune effectors implicated in Brucella control are largely undetermined.

The initial host defense to infection is stimulated by pathogen-associated molecular patterns (PAMPs) which are common to different groups of pathogens. PAMPs are known to be recognized by a battery of germline-encoded host receptors which rapidly detect and signal microbial infections. Over the past few years, the TLR family has emerged as a major group of signaling receptors for PAMPs (17). Mammalian TLRs comprise a large family consisting of at least 11 members. TLRs detect multiple PAMPs, including LPS—the major glycolipidic component of Gram-negative bacteria (detected by TLR4), bacterial lipoproteins, and lipoteichoic acids (detected by TLR2), and the unmethylated CpG DNA of bacteria (detected by TLR9). TLRs recruit adaptor molecules that subsequently activate downstream signaling pathways and NF-{kappa}B. Four adaptor molecules have been identified: MyD88 (18), Toll-IL-1R domain-containing adaptor protein (TIRAP), Toll-IL-1R domain-containing adaptor inducing IFN-beta (TRIF) (19), and Toll-IL-1R domain-containing adaptor molecule-2 (TICAM-2). Although the role of the various adaptors in the activation of specific signaling pathways still remains controversial, recent studies (17, 20) suggest that Toll-IL-1R domain-containing adaptor protein/MyD88 and TRIF-related adaptor molecule/TRIF regulate independent pathways.

TLR2 (21, 22, 23), TLR4 (21, 22, 23), TLR9 (24), and MyD88 (21, 22) have been found to be involved in the activation of immune response by B. abortus. Although the role of the MyD88 adaptor has been well-established (21, 22), several studies (21, 22, 23) have reported contradictory results about the roles of TLR2 and TLR4. Furthermore, the impact of TLR9 and TRIF adaptor deficiency in the course of infection remains undetermined. In this work, our aim is to characterize the main cellular components of the innate and adaptive immune response induced by mice infected with B. melitensis and to determine which TLRs and adaptor molecules are involved in the activation of these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and reagents

Six- to 8-wk-old female BALB/c and C57BL/6 mice were purchased from Harlan. TLR2–/– (25), TLR4–/– (26), and TLR2,4–/– mice with the BALB/c background and TLR9–/– (27), MyD88–/– (18), TRIF–/– (Ref. 19 ; B. Beutler, The Scripps Research Institute, La Jolla, CA) mice on the C57BL/6 background were bred in the animal facility of the Free University of Brussels (ULB). The maintenance and care of mice complied with the guidelines of the ULB Ethic Committee for the use of laboratory animals.

B. melitensis strain 16M (Biotype1; American Type Culture Collection (ATCC) 23456) was isolated from an infected goat and grown in biosafety level III laboratory facility. Overnight culture grown with shaking at 37°C in 2YT medium (Luria-Bertani broth with double quantity of yeast extract) to stationary phase was washed twice in PBS (3500 x g, 10 min) before use in the inoculation of mice as previously described (28).

Mouse infection

Mice were injected i.p. with 4 x 104 CFU of B. melitensis in 500 µl of PBS. Control animals were injected with the same volume of PBS alone. The infectious doses were validated by plating serial dilutions of the inoculums.

Harvest of spleen and peritoneal exudates

At selected time intervals, mice were sacrificed by cervical dislocation. Immediately after being killed, mice were peritoneally washed with 10 ml of ice-cold HBSS and the spleen harvested. Before cytofluorometric analysis, spleens were cut in very small pieces and incubated (37°C, 30 min) into HBSS (Invitrogen Life Technologies) medium containing 4000 U/ml collagenase IV (Roche) and 0.1 mg/ml DNase I fraction IX (Sigma-Aldrich Chimie SARL). This procedure was performed in all experiments of this work.

Bacteria titers in spleens and peritoneal exudates of infected mice

Spleen cells or peritoneal exudates were recovered in PBS/0.1% Triton X-100 (Sigma-Aldrich). We performed successive serial dilutions in PBS to get the most accurate bacterial count and we plated them onto 2YT medium plates. CFU were counted after 3 days of culture at 37°C.

Statistical analysis

ANOVA I was used for data analysis after testing the homogeneity of variance (Bartlett test). Average comparisons were performed by pairwise Scheffe’s test. Error bars represent the 95% interval of confidence of the mean (computed from residual mean square and Student’s t, 0.95).

Cytofluorometric analysis

Peritoneal cells and spleen cells were first incubated in saturating doses of 2.4G2 (a rat anti-mouse FcR mAb; ATCC) for 10 min to prevent Ab binding to FcR. A total of 3–5 x 106 cells were stained with various fluorescent mAbs combinations in 200 µl of PBS, 0.5% BSA, 0.02% NaN3 (FACS buffer), and further collected on a FACSCalibur cytofluorometer (BD Biosciences). We purchased the following mAbs from BD Pharmingen: fluorescein (FITC)-coupled M1/70 (anti-CD11b), H57-597 (anti-TCRbeta), PE-coupled M5/114.15.2 (anti-IA/IE), HL3 (anti-CD11c), AL-21 (anti-LY-6C), 1A8 (anti-LY-6G), RM4-5 (anti-CD4), 53-6.7 (anti-CD8{alpha}). Cells were gated according to size and scatter to eliminate dead cells and debris from analysis.

Intracellular cytokine staining

Peritoneal and spleen cells were incubated for 4 h in RPMI 1640, 5% FCS with 10 µg/ml Golgi Plug (BD Pharmingen) at 37°C, 5% CO2. The cells were washed with FACS buffer and stained for cell surface markers before fixation in PBS/1% PFA for 15–20 min on ice. These cells were then permeabilized for 30 min using a saponin-based buffer (1x Perm/Wash; BD Pharmingen in FACS buffer) containing one of the following intracellular staining mAbs: allophycocyanin-coupled XMG1.2 (anti-IFN-{gamma}), allophycocyanin-coupled MP6-XT22 (anti-TNF-{alpha}), purified M-19 (anti-NOS2; Santa Cruz Biotechnology) stained with Alexa Fluor 647 goat anti-rabbit (Molecular Probes). After final fixation in PBS/1% PFA, the cells were analyzed on a FACSCalibur cytofluorometer. No signal was detectable with isotype controls.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Analysis of C57BL/6 and BALB/c mice infected with B. melitensis 16M

C57BL/6 and BALB/c mice were infected with 4 x 104 CFU of B. melitensis (strain 16M) by i.p. inoculation. Bacteria titers inside peritoneal cavity and spleen were used to evaluate infection (Fig. 1). As illustrated (Fig. 1 and data not shown), infection peaks in peritoneal cavity and in spleen occurred at 2 and 5 days, respectively. This was followed by a rapid decrease in the peritoneal cavity against a substantial persistent level in the spleen. BALB/c mice susceptibility is therefore confirmed in our experimental model (Fig. 1) with marked susceptibility mainly observed during the plateau phase of infection in spleen. Meanwhile, both C57BL/6 and BALB/c mice could control early infection development with the same efficacy, as evidenced by the similar kinetic of bacteria titer inside the peritoneal cavity and the spleen during 5 days after infection (Fig. 1).


Figure 1
View larger version (10K):
[in this window]
[in a new window]

  		FIGURE 1. BALB/c and C57BL/6 mice infected with B. melitensis 16M. Wild-type C57BL/6 and BALB/c mice (eight per group) were injected i.p. with PBS alone or 4 x 104 CFU of B. melitensis in PBS as indicated in the figure. At the selected time, mice were sacrificed. Peritoneal exudates and spleens were harvested. Data represent the CFU per organs. Significant differences in relation to C57BL/6 mice are denoted by an asterisk (*) for p < 0.05.

 
IFN-{gamma} (9, 10, 11) and iNOS (11, 14) have been implicated in resistance to B. abortus infection. Using IFN-{gamma}–/– and iNOS–/– C57BL/6 mice, we showed that IFN-{gamma} and iNOS are implicated in the resistance to B. melitensis infection (Fig. 2A). At 5 days postinfection, we found 10-fold increase CFU in the spleen of infected IFN-{gamma}–/– and iNOS–/– mice when compared with wild-type mice. In contrast, in the peritoneal compartment, while iNOS–/– and wild-type mice displayed similar bacteria levels at all test times, IFN-{gamma}–/– mice displayed 10-fold increase CFU. These data suggest that both IFN-{gamma} and iNOS may have a potential role in the control of B. melitensis 16M growth in the spleen whereas only IFN-{gamma} plays a role in the peritoneal cavity.


Figure 2
View larger version (33K):
[in this window]
[in a new window]

  		FIGURE 2. IFN-{gamma} and iNOS are implicated in the control of B. melitensis infection. Wild-type, IFN-{gamma}–/–, iNOS–/– C57BL/6 mice (eight per group) were injected i.p. with PBS or 4 x 104 CFU of B. melitensis, as specified in the figure. Mice were sacrificed at selected time after treatment and peritoneal exudates and spleens harvested. A, Data represent the CFU per organs. These results are representative of two independent experiments. Significant differences in relation to wild-type mice are denoted by an asterisk (*) for p < 0.05. B–F, Pooled peritoneal cells and spleen cells from indicated group of mice were stained with allophycocyanin-labeled anti-IFN-{gamma} mAb or anti-iNOS/NOS2 mAb stained with Alexa Fluor 647 Goat anti-rabbit. In each case, 106 cells per group were collected on a FACSCalibur cytofluorometer. Data represent the number of positive cells per million of acquired total cells. These results are representative of three independent experiments.

 
Flow cytometric analysis of IFN-{gamma} and iNOS cell frequency in peritoneal and splenic compartments in the course of B. melitensis infection was used in the clarification of BALB/c mice susceptibility. It is important to note that cells purified from naive and infected mice were not stimulated in vitro before intracellular staining. Thus, iNOS and IFN-{gamma} detected in these cells derive only from natural in vivo activation during the course of infection. Naive and infected iNOS–/– and IFN-{gamma}–/– C57BL/6 mice were used as internal negative control in these experiments. As expected, significant iNOS and IFN-{gamma} staining were only observed in infected wild-type C57BL/6 mice (Fig. 2, B and C). We observed that the frequency of iNOS+ cells increased slowly during infection of wild-type C57BL/6 mice and peak at 10–15 days in the peritoneal cavity and spleen (Fig. 2, D and E). IFN-{gamma}+ cells were not detected in the peritoneal cavity (data not shown); meanwhile, an impact of this cytokine was observed on the resistance against the bacterial invasion (Fig. 2A). Thus, it is possible that IFN-{gamma} produced in another compartment, such as the spleen, mediated resistance in the peritoneal cavity. In the spleen of C57BL/6 mice, the frequency of IFN-{gamma}+ cells reached its plateau phase between 5 and 15 days postinfection (Fig. 2F). Surprisingly, a similar observation was found for both iNOS- and IFN-{gamma}-producing cells in infected BALB/c mice (Fig. 2, D–F). However, the frequency of iNOS+ cells and mostly IFN-{gamma}+ cells is lower in BALB/c mice. Thus, we found that BALB/c mice kept the ability to produce IFN-{gamma} during the plateau phase of the infection. However, the frequency of IFN-{gamma}+ cells is weak and can explain the lack of resistance of these mice. Cell surface phenotypes of IFN-{gamma}+ and iNOS+ cells during B. melitensis infection in C57BL/6 and BALB/c mice were later characterized.

CD11bhighLY-6ChighLY-6GlowMHC-IIhigh cells are the main iNOS-producing cells during B. melitensis infection

B. melitensis infection induces a recruitment of CD11bhighLY-6Ghigh cells (granulocyte) and CD11bhighLY-6Glow cells in the spleen of infected C57BL/6 mice (Fig. 3A). Both populations contain iNOS+ cells, but the CD11bhighLY-6Glow population appears as the major iNOS-producing cells at 10–15 days of infection. Further analysis of their cell surface phenotype by flow cytometry showed that these later cells display CD11chighMHC-IIhigh, LY-6Chigh and LY-6Clow (Fig. 3, B–E, respectively). INOS+ cells in the peritoneal cavity expressed a very similar phenotype with the exception of CD11c which appeared negative (Fig. 3B). Similar results were obtained in infected BALB/c mice (data not shown).


Figure 3
View larger version (54K):
[in this window]
[in a new window]

  		FIGURE 3. CD11b+ LY-6C+ cells are the main iNOS-producing cells during B. melitensis infection. Wild-type C57BL/6 mice (five per group) were injected i.p. with PBS or 4 x 104 CFU of B. melitensis, as specified. At the indicated time after treatment, mice were sacrificed and peritoneal exudates and spleens harvested. A, Pooled spleen cells were incubated with FITC-coupled anti-CD11b, PE-coupled anti-LY-6G, and anti-iNOS/NOS2 mAb stained with Alexa Fluor 647 goat anti-rabbit. Number indicates the percentage of cells in the selected quadrant. B–E, Pooled peritoneal cells an spleen cells from control or infected mice (as indicated) were stained with FITC-labeled anti-CD11b, anti-iNOS/NOS2 mAb stained with Alexa Fluor 647 goat anti-rabbit and with the following PE-coupled mAbs: anti-CD11c (B), anti-IA/IE (C), anti-LY-6C (D), and anti-LY-6G (E). These results are representative of five independent experiments.

 
The majority of iNOS+ cells from both the peritoneal cavity and the spleen expressed CD11bhighLY-6ChighLY-6Glow and MHC-IIhigh cell surface markers similar to inflammatory dendritic cells (DC) recently described during the course of Listeria monocytogenes infection in mouse (28, 29, 30, 31) and in human psoriasis (32). Because these cells have been described to produce TNF-{alpha} (28, 29, 30, 31) and TNF-{alpha} has been associated to the control of Brucella infection (33), we investigated the ability of CD11bhighLY-6Chigh cells to produce TNF-{alpha} during B. melitensis infection (Fig. 4). Infected TNF-{alpha}–/– mice were used as negative control for TNF-{alpha} staining in these experiments. In wild-type C57BL/6 mice, a large fraction of CD11bhighLY-6Chigh peritoneal cells appeared TNF-{alpha} positive. In contrast, splenic CD11bhighLY-6Chigh cells were almost TNF-{alpha} negative.


Figure 4
View larger version (38K):
[in this window]
[in a new window]

  		FIGURE 4. Frequency of iNOS+ cells is dependent of IFN-{gamma}. Wild-type, TNF-{alpha}–/–, iNOS–/–, and IFN-{gamma}–/– C57BL/6 mice (four per group) were injected i.p. with PBS or 4 x 104 CFU of B. melitensis, as specified in the figure. Mice were sacrificed 10 days after treatment and peritoneal cells (A) and spleen cells (B) harvested. Pooled cells in each group were stained with FITC-labeled anti-CD11b, PE-coupled anti-LY-6C, and with allophycocyanin-coupled anti-TNF-{alpha} or anti-iNOS/NOS2 mAb stained with Alexa Fluor 647 goat anti-rabbit. Number under the first line of plot in A and B indicate the percentage of cells in the R1 gate among the total cells acquired. Number in the second and third line of plot indicate the percentage of cells in the quadrant among the cells gated in R1. These results are representative of three independent experiments.

 
It is well-known that IFN-{gamma} positively regulate iNOS production in experimental bacterial infectious model (12, 13) and mediate resistance to Brucella infection (Refs. 9, 10, 11 and Fig. 2A). We therefore analyzed the impact of IFN-{gamma} deficiency on the activation of CD11bhighLY-6Chigh cell population during B. melitensis infection. As shown in Fig. 4, IFN-{gamma} deficiency strongly decreased iNOS+ cell frequency in peritoneal cavity and spleen. In contrast, TNF-{alpha}+ cells frequency was enhanced in peritoneal cavity and in spleen.

Globally, these results suggest that 1) a fraction of the splenic CD11bhighLY-6Chigh cell population can produce both TNF-{alpha} and iNOS during Brucella infection, 2) iNOS synthesis is strictly dependent on IFN-{gamma}, and 3) iNOS and TNF-{alpha} production can be independently regulated.

CD4+ T cells are the main IFN-{gamma}-producing splenic cells at the later stage of B. melitensis infection

IFN-{gamma} appears to be produced by distinct cells during the course of infection. In the early course (5 days), IFN-{gamma} is synthesized mainly by NK cells (TCRbeta–NK1.1+ cells, 38% of producing cells), CD4+ T cells (TCRbeta+CD4+ cells, 21%) and CD8+ T cells (TCRbeta+CD4– cells, 19%) (Fig. 5A). Later on (10 days and above), CD4+ T cells become the major producing cells (74%). IFN-{gamma}+ cells display a similar phenotype during the course of infection in BALB/c mice (data not shown). It is interesting to observe that the frequency of IFN-{gamma}+ cells at 5 days among CD4+ T cells and CD8+ T cells appear to be very close (0.23 and 0.39%, respectively) (Fig. 5B). However, at 12 days, only the CD4+ T cells frequency increase to 1.79%.


Figure 5
View larger version (43K):
[in this window]
[in a new window]

  		FIGURE 5. CD4+ T cells are the main IFN-{gamma}-producing splenic cells at the later stage of B. melitensis infection. Wild-type C57BL/6 mice (five per group) were injected i.p. with PBS or 4 x 104 CFU of B. melitensis, as specified in the figure. Mice were sacrificed at selected time after treatment and spleen cells were harvested. Pooled spleen cells in each group were stained with FITC-labeled anti-TCRbeta, allophycocyanin-coupled anti-IFN-{gamma} and with one of the following PE-coupled mAb: anti-CD4 or anti-NK1.1. Number indicates the percentage of cells in the selected quadrant (A) or gate (B). These results are representative of four independent experiments.

 
TLR-4, TLR-9 and MyD88, but not TLR-2 and TRIF, are implicated in the control of brucellosis

To gain insight into the role of TLRs and their adaptor proteins during Brucella infection, we compared in the same experiment the susceptibility of mice genetically deficient for these molecules. Mice were infected and bacteria count of bacteria examined at 5 and 21 days in peritoneal exudates and spleen (Fig. 6). We observed that TLR4 and TLR9 deficiency moderately affected the resistance of mice to infection. This was mostly apparent in the spleen and less in the peritoneal cavity. TLR2 deficiency, alone or in combination with TLR4 deficiency, has no effect. MyD88- but not TRIF-deficient mice displayed higher susceptibility.


Figure 6
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 6. TLR-4, TLR-9, and MyD88, but not TLR-2 and TRIF, are implicated in the control of B. melitensis infection. Wild-type, MyD88–/–, TRIF–/–, TLR9–/– C57BL/6 mice and wild-type, TLR2–/–, TLR4–/–, TLR2/4–/– BALB/c mice (eight per group) were injected i.p. with PBS or 4 x 104 CFU of B. melitensis, as specified in the figure. Mice were sacrificed at selected time after treatment and peritoneal exudates and spleens harvested. Data represent the CFU per organs. These results are representative of four independent experiments. Significant differences in relation to wild-type mice are denoted by an asterisk (*) for p < 0.05.

 
In the same experiment, we analyzed the impact of TLRs and adaptor protein deficiency on the activation of iNOS and IFN-{gamma}-producing cells (Fig. 7). As expected, deficiencies that affect resistance to infection proportionally reduce the frequency of effectors cells observed in peritoneal exudates and spleen. MyD88 and TLR4 deficiency strongly affects the frequency of iNOS+ and IFN-{gamma}+ cells at 5 and 21 days in both compartments tested. In contrast, TLR-9 deficiency reduces iNOS+ cell frequency only in peritoneal cavity.


Figure 7
View larger version (29K):
[in this window]
[in a new window]

  		FIGURE 7. TLR-4, TLR-9, and MyD88, but not TLR-2 and TRIF, regulate frequency of iNOS+ and IFN-{gamma}+ cell during B. melitensis infection. Wild-type, MyD88–/–, TRIF–/–, TLR9–/– C57BL/6 mice and wild-type, TLR2–/–, TLR4–/–, TLR2/4–/– BALB/c mice (five per group) were injected i.p. with PBS or 4 x 104 CFU of B. melitensis, as specified in the figure. Mice were sacrificed at selected time after treatment and peritoneal exudates and spleens harvested. Cells of each animals were stained with allophycocyanin-labeled anti-IFN-{gamma} or anti-iNOS/NOS2 mAb stained with Alexa Fluor 647 goat anti-rabbit. Data represent the number of positive cells per 106 total cells acquired. These results are representative of three independent experiments. Significant differences in relation to wild-type mice are denoted by an asterisk (*) for p < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
IFN-{gamma} is a key cytokine regulating the protective immune response against Brucella. This has been demonstrated by showing that administering of rIFN-{gamma} enhances mice resistance resulting in a 10-fold decrease in the number of bacteria (9). Also, it has been shown that depleting IFN-{gamma} by the administration of mAbs which neutralize IFN-{gamma} (10) or by the use of IFN-{gamma}–/– mice (Ref. 11 and data not shown) results in a 10-fold increase in the number of bacteria. Here, for the first time, we characterized by flow cytometry the cell surface phenotype of spleen cells producing IFN-{gamma} in vivo during the course of Brucella infection. At the peak of infection (5 days), IFN-{gamma} is produced mainly by TCRbeta–NK1.1+ NK cells, TCRbeta+CD4+ T cells, and TCRbeta+CD8+ T cells. During the plateau phase of infection (10–21 days), CD4+ T cells become the major producing cells (74% of IFN-{gamma}+ cells).

The respective contribution of CD4+ T cells and CD8+ T cells to protective immune response against Brucella is controversial (reviewed in Ref. 34). Transfer experiments showed an equal rule for both subsets. In contrast, beta2-microglobulin–/– mice (deficient for CD8+ T cell activation) appeared more susceptible that MHC-II–/– mice (deficient for CD4+ T cell activation) suggesting that CD8+ T cells play a crucial role in protection. However, it is well-known that beta2-microglobulin–/– mice are deficient for both MHC-I and CD1d Ag-presentation pathways rendering complex the interpretation of the results in this model. The relatively equal role of NK, CD4+, and CD8+ T cells in IFN-{gamma} secretion at the peak of infection could explain the results of transfer experiments. In addition, the predominant role of CD4+ T cells in IFN-{gamma} secretion during the plateau phase of infection could explain why RAG1 (35) but not beta2-microglobulin-deficient mice (11) and NK-depleted mice (36) drastically fail to control Brucella infection.

Experiments in genetically deficient mice have showed that MyD88 adaptor protein is implicated in the resistance to B. abortus infection (21, 22). We demonstrated that MyD88 deficiency strongly reduces the frequency of IFN-{gamma}+ cells at all time of B. melitensis infection. The unique role of MyD88-dependent signaling pathways is confirmed by the fact that TRIF-deficient mice do not have a reduced frequency of IFN-{gamma}+ cells and display normal resistance to infection. Identification of TLRs coupled to MyD88 and detecting B. melitensis infection appear complex. Contradictory results have been reported concerning the implication of TLR2 and TLR4 in resistance to B. abortus infection. TLR2 has been involved in heat-killed B. abortus-induced TNF-{alpha} (22) but not in the resistance to infection (21, 23). TLR4 deficiency has been reported to reduce (23) or have no affect (21) on resistance in B. abortus. In our experimental model of B. melitensis infection, we found that TLR4, but not TLR2, significantly decrease resistance to infection. The reasons for these discrepancies are not known but could be due to the different strain of Brucella used. In addition, we reported for the first time that TLR9 deficiency also reduces resistance. This result is in agreement with previous work (24) showing the role of TLR9 in the induction of inflammatory cytokines by heat-killed B. abortus. Collectively, our data suggest that both TLR4 and TLR9 cooperate in the immune system to detect B. melitensis infection. A logical consequence of multiple TLR signaling is that the reduction of IFN-{gamma}+ cell frequency is moderately or not affected by the deficiency of TLR4 or TLR9 alone.

In various experimental models of infection by intracellular pathogen, a major function of IFN-{gamma} is the stimulation of killing effector mechanisms, such as the production of RNI and ROI in phagocytic cells (12, 13). A previous study (14) reported that both gp91phox–/– mice and iNOS/NOS2–/– mice are susceptible to B. abortus infection but that iNOS deficiency more drastically affects the resistance, suggesting a predominant role of RNI in Brucella killing. Based on these data, we selected iNOS as a marker of protective innate immune response against Brucella. We showed that IFN-{gamma} positively regulates the frequency of iNOS+ cells in the peritoneal cavity and spleen. Infected IFN-{gamma}–/– mice display a 10-fold reduction of iNOS+ cells frequency. In contrast, TNF-{alpha} deficiency moderately affects this frequency during Brucella infection. This suggests a key role of CD4+ T cells, the main IFN-{gamma}+ cells, in the regulation of Brucella-killing effector mechanisms.

Characterization of cell surface marker expressed by peritoneal and splenic iNOS+ cells showed that the great majority of these cells expressed the following phenotype: CD11bhighLY-6ChighLY-6GlowMHC-IIhigh. They are negative for T cell (TCRbeta, CD8{alpha}), B cell (IgD, IgM, B220), mastocyte (c-kit), and NK (NK1.1) cell surface markers (data not shown). Splenic iNOS+ cells also express CD11c and thus display a phenotype very similar to inflammatory DC recently described during L. monocytogenes infection in mice (28, 29, 30, 31) and psoriasis in human (32). These cells, termed TNF/iNOS-producing DC by the authors, appear as a major inflammatory and effectors cell type in these pathologies. In our infectious model, peritoneal iNOS+ cells are CD11c negative but the absence of CD11c expression on DC is frequent in nonlymphoid organs (37). Their very similar cell surface phenotype suggests that they might be related to splenic inflammatory DC.

DC populations (38) are highly heterogeneous and distinct subsets have been discriminated on the basis of differential expression of various cell surface markers and specialized functions. However, relatively little is known about DC recruitment and function in response to pathogen infection. In addition to B220–LY-6C–NK1.1– "classical" DC, various specialized DC subsets have been recently defined. Plasmacytoid DC, expressing B220+CD11b–LY-6C+NK1.1– markers, are specialized in IFN-{alpha} production and antiviral response (39). NK DC, expressing CD11b–DX5+NK1.1+ markers, displayed both cytotoxicity and APC functions (40). The implication of B220–CD11b+LY-6C+NK1.1– iNOS+ DC in both Listeria (28, 29, 30, 31) and Brucella infection (this study) allows us to propose that these cells can be major effectors in murine Th1 immune response against intracellular bacteria. Moreover, as CD11c+CD11b+LY-6C+ DC isolated during Listeria infection display APC function in vitro (30), it is possible that these cells also play an important regulatory role by stimulating and programming T cell responses during these infections. Interestingly, their recent finding in human psoriasis (32) suggests that they can also play an important role in human Th1 immune response. As demonstrated in the Listeria model (31), we also showed that inflammatory DC activation during Brucella infection is strongly dependent on MyD88-signaling pathways. In contrast, TRIF pathways do not appear to be required. As for IFN-{gamma}+ cell, we observed a reproducible but weak impact of TLR-4 and TLR-9 deficiency on iNOS+ cell frequencies.

Finally, BALB/c mice, previously described as susceptible to Brucella infection (15, 16), display few differences with C57BL/6 mice. The kinetic and the type of IFN-{gamma} and iNOS cells producing seem similar in both types of mice. Only the frequency of these cells is reduced. This suggests that the immune response developed by C57BL/6 and BALB/c mice differ only by its intensity. This might be explained by the well-known reduced ability of BALB/c mice to mount Th1 response.

In summary, our study confirms the predominant role of MyD88 adaptor molecule in the development of protective immune response against Brucella. We showed that MyD88-dependent signaling pathways appear strictly necessary to the development of IFN-{gamma}+ and iNOS+ cells during infection. We also demonstrated that B220–CD11b+LY-6C+ DC are recruited and activated during infection and constitute the main iNOS-producing cells in all compartments analyzed. Taken together, our results provide new insight into how innate and adaptive immune response fight Brucella infection.


   Acknowledgments
 
We thank Dr. B. Beutler (The Scripps Research Institute) for giving us TRIF mice. We are indebted to Grégoire Lauvau for his critical review of the manuscript and to Christian Didembourg and Matthieu Terwagne for their diligent technical assistance. We thank Eric Depiereux for statistical analysis of our results and Bernard Nkengfac for relevant English corrections.


   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 grants from the Fond National de la Recherche Scientifique (FNRS; Convention Fonds de la Recherche Scientifique Médicale FNRS 3.4.600.06.F, Belgium), the Fond Emile Defay (Belgium), and the Fond Van Buuren (Belgium). E.M. is "Chercheur Qualifié du FNRS." Back

2 J.-J.L. and E.M. should be considered equally as last authors. Back

3 Address correspondence and reprint requests to Dr. Eric Muraille, Laboratoire de Parasitologie, Faculté de médecine, Route de Lennik 808, Université Libre de Bruxelles, 1070 Bruxelles, Belgium; E-mail address: emuraille{at}hotmail.com or Dr. Jean-Jacques Letesson, Unité de Recherche en Biologie Moléculaire, Laboratoire d’Immunologie et de Microbiologie, Université de Namur, Rue de Bruxelles 61, 5000 Namur, Belgium; E-mail address: jean-jacques.letesson{at}fundp.ac.be Back

4 Abbreviations used in this paper: RNI, reactive nitrogen intermediate; ROI, reactive oxygen intermediate; iNOS, inducible NO synthase; PAMP, pathogen-associated molecular pattern; TIR, Toll-IL-1R; TRIF, TIR domain-containing adaptor inducing IFN-beta; DC, dendritic cell. Back

Received for publication July 31, 2006. Accepted for publication January 23, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Pappas, G., N. Akritidis, M. Bosilkovski, E. Tsianos. 2005. Brucellosis. N. Engl. J. Med. 352: 2325-2324. [Free Full Text]
  2. Godfroid, J., A. Cloeckaert, J. P. Liautard, S. Kohler, D. Fretin, K. Walravens, B. Garin-Bastuji, J. J. Letesson. 2005. From the discovery of the Malta fever’s agent to the discovery of a marine mammal reservoir, brucellosis has continuously been a re-emerging zoonosis. Vet. Res. 36: 313-326. [Medline]
  3. Cutler, S. J., A. M. Whatmore, N. J. Commander. 2005. Brucellosis–new aspects of an old disease. J. Appl. Microbiol. 98: 1270-1281. [Medline]
  4. Pappas, G., P. Papadimitriou, N. Akritidis, L. Christou, E. V. Tsianos. 2006. The new global map of human brucellosis. Lancet Infect. Dis. 6: 91-99. [Medline]
  5. Corbel, M. J.. 1997. Brucellosis: an overview. Emerg. Infect. Dis. 3: 213-221. [Medline]
  6. Celli, J., J. P. Gorvel. 2004. Organelle robbery: Brucella interactions with the endoplasmic reticulum. Curr. Opin. Microbiol. 7: 93-97. [Medline]
  7. Roop, R. M., B. H. Bellaire, M. W. Valderas, J. A. Cardelli. 2004. Adaptation of the brucellae to their intracellular niche. Mol. Microbiol. 52: 621-630. [Medline]
  8. Celli, J.. 2006. Surviving inside a macrophage: the many ways of Brucella. Res. Microbiol. 157: 93-98. [Medline]
  9. Stevens, M. G., G. W. Pugh, Jr, L. B. Tabatabai. 1992. Effects of {gamma} interferon and indomethacin in preventing Brucella abortus infections in mice. Infect. Immun. 60: 4407-4409. [Abstract/Free Full Text]
  10. Zhan, Y., C. Cheers. 1993. Endogenous {gamma} interferon mediates resistance to Brucella abortus infection. Infect. Immun. 61: 4899-4901. [Abstract/Free Full Text]
  11. Murphy, E. A., J. Sathiyaseelan, M. A. Parent, B. Zou, C. L. Baldwin. 2001. Interferon-{gamma} is crucial for surviving a Brucella abortus infection in both resistant C57BL/6 and susceptible BALB/c mice. Immunology 103: 511-518. [Medline]
  12. MacMicking, J., Q. W. Xie, C. Nathan. 1997. Nitric oxide and macrophage function. Annu. Rev. Immunol. 15: 323-350. [Medline]
  13. Chakravortty, D., M. Hensel. 2003. Inducible nitric oxide synthase and control of intracellular bacterial pathogens. Microbes Infect. 5: 621-627. [Medline]
  14. Ko, J., A. Gendron-Fitzpatrick, G. A. Splitter. 2002. Susceptibility of IFN regulatory factor-1 and IFN consensus sequence binding protein-deficient mice to brucellosis. J. Immunol. 168: 2433-2440. [Abstract/Free Full Text]
  15. Fernandes, D. M., X. Jiang, J. H. Jung, C. L. Baldwin. 1996. Comparison of T cell cytokines in resistant and susceptible mice infected with virulent Brucella abortus strain 2308. FEMS Immunol. Med. Microbiol. 16: 193-203. [Medline]
  16. Montaraz, J. A., A. J. Winter. 1986. Comparison of living and nonliving vaccines for Brucella abortus in BALB/c mice. Infect. Immun. 53: 245-251. [Abstract/Free Full Text]
  17. Akira, S., K. Takeda. 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4: 499-511. [Medline]
  18. Kawai, T., O. Adachi, T. Ogawa, K. Takeda, S. Akira. 1999. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11: 115-122. [Medline]
  19. Hoebe, K., X. Du, P. Georgel, E. Janssen, K. Tabeta, S. O. Kim, J. Goode, P. Lin, N. Mann, S. Mudd, et al 2003. Identification of Lps2 as a key transducer of MyD88-independent TIR signalling. Nature 424: 743-748. [Medline]
  20. Palsson-McDermott, E. M., L. A. O’Neill. 2004. Signal transduction by the lipopolysaccharide receptor, Toll-like receptor-4. Immunology 113: 153-162. [Medline]
  21. Weiss, D. S., K. Takeda, S. Akira, A. Zychlinsky, E. Moreno. 2005. MyD88, but not Toll-like receptors 4 and 2, is required for efficient clearance of Brucella abortus. Infect. Immun. 73: 5137-5143. [Abstract/Free Full Text]
  22. Huang, L. Y., J. Aliberti, C. A. Leifer, D. M. Segal, A. Sher, D. T. Golenbock, B. Golding. 2003. Heat-killed Brucella abortus induces TNF and IL-12p40 by distinct MyD88-dependent pathways: TNF, unlike IL-12p40 secretion, is Toll-like receptor 2 dependent. J. Immunol. 171: 1441-1446. [Abstract/Free Full Text]
  23. Campos, M. A., G. M. Rosinha, I. C. Almeida, X. S. Salgueiro, B. W. Jarvis, G. A. Splitter, N. Qureshi, O. Bruna-Romero, R. T. Gazzinelli, S. C. Oliveira. 2004. Role of Toll-like receptor 4 in induction of cell-mediated immunity and resistance to Brucella abortus infection in mice. Infect. Immun. 72: 176-186. [Abstract/Free Full Text]
  24. Huang, L. Y., K. J. Ishii, S. Akira, J. Aliberti, B. Golding. 2005. Th1-like cytokine induction by heat-killed Brucella abortus is dependent on triggering of TLR9. J. Immunol. 175: 3964-3970. [Abstract/Free Full Text]
  25. Takeuchi, O., K. Hoshino, T. Kawai, H. Sanjo, H. Takada, T. Ogawa, K. Takeda, S. Akira. 1999. Differential roles of TLR2 and TLR4 in recognition of Gram-negative and Gram-positive bacterial cell wall components. Immunity 11: 443-451. [Medline]
  26. 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]
  27. Hemmi, H., O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M. Matsumoto, K. Hoshino, H. Wagner, K. Takeda, S. Akira. 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408: 740-745. [Medline]
  28. Tam, M. A., M. J. Wick. 2006. Differential expansion, activation and effector functions of conventional and plasmacytoid dendritic cells in mouse tissues transiently infected with Listeria monocytogenes. Cell. Microbiol. 8: 1172-1187. [Medline]
  29. Tam, M. A., M. J. Wick. 2004. Dendritic cells and immunity to Listeria: TipDCs are a new recruit. Trends Immunol. 25: 335-339. [Medline]
  30. Serbina, N. V., T. P. Salazar-Mather, C. A. Biron, W. A. Kuziel, E. G. Pamer. 2003. TNF/iNOS-producing dendritic cells mediate innate immune defense against bacterial infection. Immunity 19: 59-70. [Medline]
  31. Serbina, N. V., W. Kuziel, R. Flavell, S. Akira, B. Rollins, E. G. Pamer. 2003. Sequential MyD88-independent and -dependent activation of innate immune responses to intracellular bacterial infection. Immunity 19: 891-901. [Medline]
  32. Lowes, M. A., F. Chamian, M. V. Abello, J. Fuentes-Duculan, S. L. Lin, R. Nussbaum, I. Novitskaya, H. Carbonaro, I. Cardinale, T. Kikuchi, et al 2005. Increase in TNF-{alpha} and inducible nitric oxide synthase-expressing dendritic cells in psoriasis and reduction with efalizumab (anti-CD11a). Proc. Natl. Acad. Sci. USA 102: 19057-19062. [Abstract/Free Full Text]
  33. Zhan, Y., C. Cheers. 1998. Control of IL-12 and IFN-{gamma} production in response to live or dead bacteria by TNF and other factors. J. Immunol. 161: 1447-1453. [Abstract/Free Full Text]
  34. Ko, J., G. A. Splitter. 2003. Molecular host-pathogen interaction in brucellosis: current understanding and future approaches to vaccine development for mice and humans. Clin. Microbiol. Rev. 16: 65-78. [Abstract/Free Full Text]
  35. Izadjoo, M. J., Y. Polotsky, M. G. Mense, A. K. Bhattacharjee, C. M. Paranavitana, T. L. Hadfield, D. L. Hoover. 2000. Impaired control of Brucella melitensis infection in Rag1-deficient mice. Infect. Immun. 68: 5314-5320. [Abstract/Free Full Text]
  36. Fernandes, D. M., R. Benson, C. L. Baldwin. 1995. Lack of a role for natural killer cells in early control of Brucella abortus 2308 infections in mice. Infect. Immun. 63: 4029-4033. [Abstract]
  37. Austyn, J. M., D. F. Hankins, C. P. Larsen, P. J. Morris, A. S. Rao, J. A. Roake. 1994. Isolation and characterization of dendritic cells from mouse heart and kidney. J. Immunol. 152: 2401-2410. [Abstract]
  38. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392: 245-252. [Medline]
  39. O’Keeffe, M., H. Hochrein, D. Vremec, I. Caminschi, J. L. Miller, E. M. Anders, L. Wu, M. H. Lahoud, S. Henri, B. Scott, et al 2002. Mouse plasmacytoid cells: long-lived cells, heterogeneous in surface phenotype and function, that differentiate into CD8+ dendritic cells only after microbial stimulus. J. Exp. Med. 196: 1307-1319. [Abstract/Free Full Text]
  40. Pillarisetty, V. G., S. C. Katz, J. I. Bleier, A. B. Shah, R. P. DeMatteo. 2005. Natural killer dendritic cells have both antigen presenting and lytic function and in response to CpG produce IFN-{gamma} via autocrine IL-12. J. Immunol. 174: 2612-2618. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
G. S. Deepe Jr, R. S. Gibbons, and A. G. Smulian
Histoplasma capsulatum manifests preferential invasion of phagocytic subpopulations in murine lungs
J. Leukoc. Biol., September 1, 2008; 84(3): 669 - 678.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
E. Billard, J. Dornand, and A. Gross
Interaction of Brucella suis and Brucella abortus Rough Strains with Human Dendritic Cells
Infect. Immun., December 1, 2007; 75(12): 5916 - 5923.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
E. Billard, J. Dornand, and A. Gross
Brucella suis Prevents Human Dendritic Cell Maturation and Antigen Presentation through Regulation of Tumor Necrosis Factor Alpha Secretion
Infect. Immun., October 1, 2007; 75(10): 4980 - 4989.
[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
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Copin, R.
Right arrow Articles by Muraille, E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Copin, R.
Right arrow Articles by Muraille, E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS