Contrasting roles of macrophages and dendritic ... - Wiley Online Library

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Cristel Archambaud et al.

DOI 10.1002/eji.201040497

Eur. J. Immunol. 2010. 40: 3458–3471

Contrasting roles of macrophages and dendritic cells in controlling initial pulmonary Brucella infection Cristel Archambaud1,2,3, Suzana P. Salcedo1,2,3, Hugues Lelouard1,2,3, Elisabeth Devilard1,2,3, Beatrice de Bovis1,2,3, Nico Van Rooijen4, Jean-Pierre Gorvel1,2,3 and Bernard Malissen1,2,3 1 2 3 4

Centre d’Immunologie de Marseille-Luminy, Universite´ de la Me´diterrane´e, Marseille, France INSERM, U631, Marseille, France CNRS, UMR6102, Marseille, France Department of Molecular Cell Biology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands

Control of pulmonary pathogens constitutes a challenging task as successful immune responses need to be mounted without damaging the lung parenchyma. Using immunofluorescence microscopy and flow cytometry, we analyzed in the mouse the initial innate immune response that follows intranasal inoculation of Brucella abortus. Bacteria were absent from parenchymal dendritic cells (DC) but present in alveolar macrophages in which they replicated. When the number of alveolar macrophages was reduced prior to Brucella infection, small numbers of pulmonary DC were infected and a massive recruitment of TNF-a- and iNOS-producing DC ensued. Coincidentally, Brucella disseminated to the lung-draining mediastinal lymph nodes (LN) where they replicated in both migratory DC and migratory alveolar macrophages. Together, these results demonstrate that alveolar macrophages are critical regulators of the initial innate immune response against Brucella within the lungs and show that pulmonary DC and alveolar macrophages play rather distinct roles in the control of microbial burden.

Key words: Brucella . DC . Macrophages . Nasal infection . Pulmonary infection

Supporting Information available online

Introduction The origin of the diverse clinical manifestations in Brucella-infected human beings and animals has not been clearly elucidated. Brucella infection can occur through inhalation and therefore the immune system associated with the lungs play an important role in mediating the initial recognition of infection. The lungs can be grossly divided into two functional compartments: the large conducting airways that are lined with a mucosal pseudostratified

Correspondence: Prof. Bernard Malissen e-mail: [email protected]

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

epithelium and the lung parenchyma that comprises thin-walled alveoli that are dedicated to gas exchange. Among the antigen (Ag) that reach the lung surface, the immune system needs to discriminate between harmless and pathogen-associated Ag. The concerted action of immunosuppressive pulmonary macrophages and immunostimulatory dendritic cells (DC) likely prevents the induction of chronic airway inflammation by innocuous Ag while preserving the ability to mount efficient defence against inhaled pathogens [1]. Under steady-state conditions, the trachea and the large conducting airways contain a dense network of DC [1, 2]. The intraepithelial DC are CD11chiCD11b /lo and express high levels of MHC-encoded class II molecules (MHCII), langerin (CD207) and integrin aE (CD103) b7 [3, 4]. Such intraepithelial DC are denoted

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hereafter as CD11bloCD1031 DC and extend cellular projections into the airway lumen through which they likely sample luminal Ag [4, 5]. Immediately below the basement membrane, the submucosa contains a subset of DC that is CD11chi, CD11bhi MHCIIhi, CD207 , CD103 and is denoted hereafter as CD11bhi CD103 [2]. In the steady state, both DC subsets express low levels of CD80, CD86 and CD40 costimulatory molecules [3, 4]. The alveolar septum contains both CD11bhi CD103 and CD11blo CD1031 DC. Macrophages can be found both within the alveoli and in the interstitium of the lung parenchyma [1, 2]. Lung alveolar macrophages express high amounts of CD11c while lacking CD11b expression whereas interstitial macrophages are CD11c [6, 7]. Alveolar macrophages can be readily distinguished from lung DC due to their high levels of autofluorescence [8]. Brucella abortus is a facultative intracellular Gram-negative bacterium infecting both humans and animals. Human brucellosis can be acquired either by ingestion of contaminated food or, in the case of a disrupted skin barrier, by direct contact with infected animals. Inhalation of aerosolized Brucella from aborted placenta or through laboratory exposures can also lead to infection and therefore Brucella is classified as a class-B bioterrorism agent. Through mechanisms that are still not completely understood, Brucella escapes the bactericidal activities of macrophages and neutrophils and thus persists as a chronic infection resulting in severe clinical manifestations [9, 10]. Until recently, scarce informations were available on the role played by DC during brucellosis. TLR signals are essential for the induction of DC activation and the secretion of pro-inflammatory cytokines. Members of the TLR family (TLR2, TLR4, TLR9) and the adaptor protein MyD88 have been found to be involved in the activation of the immune response by Brucella [11–16]. Brucella is, however, able to replicate within mouse bone marrow-derived DC and to interfere with the process of DC activation [17, 18]. Therefore, DC may constitute for Brucella an important intracellular replication niche that contributes to promote infection. Here, we used a mouse model of infection in which animals were inoculated intranasally with a virulent B. abortus WT strain. We investigated the initial host response against Brucella and determined whether Brucella was able to remain hidden from the innate immune system or capable of inducing a local innate immune response. Our results demonstrate that alveolar macrophages are critical regulators of the initial innate immune response against Brucella within the lung in that they limit both excessive inflammation and Brucella dissemination within the host.

Results Alveolar macrophages constitute the main cellular target of Brucella We first characterized the cells of the immune system that are present in the lungs and that constitute the target of B. abortus


Immunity to infection

following intranasal infection with a WT strain of DsRedexpressing B. abortus (strain 2308). Using confocal microscopy of lung sections, we observed that Brucella was internalized by CD11c1 cells 2 h after intranasal instillation (Fig. 1A). Although both lung DC and alveolar macrophages express high levels of CD11c [8], they could be distinguished by confocal microscopy using several cell surface markers [6, 8, 19, 20]. For instance, pulmonary DC do not engulf intranasally delivered microspheres and express high levels of MHCII molecules and no lysozyme, whereas alveolar macrophages display a strong phagocytic activity and express high levels of lysozyme, CD68 and F4/80 and no detectable MHCII molecules (Supporting Information Fig. 1). Brucella-containing cells expressed F4/80 suggesting that they correspond to alveolar macrophages (Fig. 1B). At 1.5 days post infection (p.i.), 178 Brucella-containing cells were analyzed and found to express CD11c, of which 99% were negative for MHCII molecules (Fig. 1C). Moreover, Brucella-containing CD11c1 cells expressed high levels of lysozyme confirming that they correspond to alveolar macrophages (Fig. 1D). Therefore, at early time points of infection, alveolar macrophages, phenotypically characterized as CD11c1F4/801MHCII Lyso1 cells, constitute the main cell type capable of capturing Brucella within the lungs. The possibility for Brucella to survive and replicate inside macrophages is thought to be critical for the establishment, the development and the chronicity of brucellosis [21]. Consistent with this view, 2 days p.i. the number of Brucella per infected alveolar macrophage started to increase (over 60 infected cells were observed and found to contain 1.770.7 and 3.272.0 bacteria 1 day and 2 days p.i., respectively; Fig. 1E). This was even more obvious at 5 days p.i., a time point at which some infected macrophages already contained more than 20 bacteria (Fig. 1E). Therefore, between 2 and 5 days p.i., microscopic analysis indicated that Brucella survives and replicates within alveolar macrophages.

Lung-draining LN contain migratory alveolar macrophages and DC that are infected by Brucella We determined next whether Brucella-infected cells could be found in the mediastinal LN (MdLN) that drain the lungs. Infected cells were detected in MdLN first at 1.5 days p.i. (data not shown) and were still present at day 5 p.i., Brucella was found in both CD11c1Lyso and CD11c1Lyso1 cells (Fig. 2A). To determine whether the former cells corresponded to DC, MdLN sections were stained with Ab directed against MHCII and CD103. All the infected CD11c1Lyso cells expressed MHCII molecules and variable levels of CD103 (Fig. 2B and C), a phenotype consistent with that of migratory lung DC (see below). It is most often assumed that migration of pulmonary DC is the only mechanism by which Ag are transported from lungs to the MdLN and along that line alveolar macrophages are not commonly thought to contribute to adaptive immunity due to their putative inability to migrate from the alveolar spaces to the MdLN.

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Figure 1. Brucella are mainly found in alveolar macrophages in which they replicate. (A–D) Lung sections of mice infected with a DsRed-expressing Brucella (red) were stained 2 h p.i. with Ab recognizing the Brucella LPS (green), CD11c (blue in panel A) or F4/80 (blue in panel B) and after 24 h with an anti-MHCII (green in panel C) and anti-lysozyme (green in panel D) and imaged by confocal microscopy. (E) Lung sections of mice infected for 2, 24, 48 or 120 hours with DsRed-expressing Brucella (red) were stained for CD11c (blue) and nuclei (grey). Data are representative from two (2 h p.i.) to six (24 h p.i.) intranasally infected mice. Scale bars, 20 mm.

However, a recent study demonstrated that following intranasal exposure to Streptococcus pneumoniae, alveolar macrophages can rapidly transport such bacteria from lung to MdLN [22]. To determine whether the Brucella-infected CD11c1Lyso1 cells present in the MdLN corresponded to migratory alveolar macrophages, we attempted to track their migration from the lungs to the MdLN by using fluorescent microspheres that were delivered intranasally 3 h prior to intranasal exposure to Brucella. Considering that microspheres cannot cross the lung epithelium and reach the MdLN in a free form, the presence of microspheres


in the Brucella-infected CD11c1Lyso1 cells should indicate that those cells correspond to migratory alveolar macrophages. As expected, analysis of the lungs showed that most alveolar macrophages, but none of the DC, were loaded with microspheres (Supporting Information Fig. 1), and 5 days p.i., the presence of microspheres in the Brucella-infected CD11c1Lyso1 cells present in MdLN indicated that they have migrated from the lung (Fig. 2D). Thus, alveolar macrophages participate, together with migratory lung DC, in the transport of B. abortus from the lung to the draining MdLN.

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Figure 2. Brucella disseminates to mediastinal draining LN where they reside in DC and in migratory alveolar macrophages. (A–D) 5 days p.i. of mice with a DsRed-expressing Brucella (red), MdLN sections were stained for lysozyme (green) and the specified markers (blue) and analyzed by confocal microscopy. In (D), mice received fluorescent microspheres (blue) intranasally 3 h prior to infection with Brucella. (A) Both Lyso1 (arrowheads) and Lyso (arrows) CD11c1 cells contained bacteria. (B and C) Lyso cells that are infected by Brucella expressed MHCII (B) and CD103 (C) molecules. (D) Cells present in the MdLN and expressing lysozyme (green) can contain both fluorescent microspheres (blue) and bacteria (red, arrowheads). Data are representative of three independent experiments. Scale bars, 20 mm.

Brucella infection neither modifies macrophage and DC distribution nor activate them We investigated next by flow cytometry whether the establishment of a Brucella intracellular replication niche within alveolar macrophages had an impact on the activation and on the relative distribution of alveolar macrophages and of lung DC subsets. Mice were infected intranasally with the WT B. abortus strain 2308 and single-cell suspensions generated following enzymatic digestion of lungs were analyzed at 1.5 and 5 days p.i. In uninfected mice, combining autofluorescence and side-scatter (SSC) allows to split the CD11c1 population into DC, which show a low autofluorescence and a low SSC, and alveolar macrophages that are characterized by a high autofluorescence and a high SSC (Fig. 3A and B, left panels). Moreover, DC can be further segregated into CD11bloCD1031 and CD11bhiCD103 subsets. In


the lungs of Brucella-infected mice, both the relative distribution and the absolute numbers of alveolar macrophages and of CD11blo CD1031 and CD11bhi CD103 DC subsets were identical to those of uninfected mice that were analyzed (Fig. 3A and B, middle panels and Fig. 3C). We also analyzed the level of expression of molecules involved in T-cell activation such as CD80 and CD86 (Fig. 3D–F). After Brucella infection, both lung DC subsets expressed low levels of CD80 and CD86 that were identical to those found in control mice and were characteristic of immature DC (Fig. 3D and E). Similarly, the levels of CD80 and CD86 expressed on macrophages did not change upon infection (Fig. 3F). Taken together, these results suggest that after intranasal infection, the presence of Brucella within the alveolar macrophages neither modifies the relative numbers of alveolar macrophages and of lung DC nor increases the expression of molecules associated with maturation.

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Figure 3. Brucella infection neither modifies the relative representation of alveolar macrophages and lung DC subsets nor induces their activation. (A and B) Cell suspensions from lungs of mice injected with PBS or infected with a WT B. abortus strain or with a WT Salmonella strain were analyzed 1.5 (A) or 5 days (B) p.i. by flow cytometry. Cells were first gated for expression of CD11c1 and autofluorescence and SSC were then used to discriminate DC with a low autofluorescence from alveolar macrophages with a high autofluorescence. DC were then subdivided according to expression of CD103 and CD11b. The percentage of cells within each gate is indicated. (C) CD11bloCD1031 (CD1031) DC, CD11bhiCD103 (CD11b1) DC and macrophages isolated from lungs of mice injected with PBS or infected with a WT B. abortus strain or with a WT Salmonella strain were enumerated 1.5 days p.i. (D–F) CD80 and CD86 expression was analyzed on CD11bloCD1031 (CD1031) DC (D), CD11bhiCD103 (CD11b1) DC (E) and on alveolar macrophages (MP) (F) 1.5 and 5 days p.i. with WT B. abortus or WT Salmonella. Expression level of CD80 and CD86 was plotted as geometric mean (Geo mean). Data are represented as mean7SEM. po0.05, po0.01; two-tailed Student’s t-test. Experiments have been performed at least twice with three mice per group.

We next analyzed whether the absence of pulmonary DC maturation was due to the capacity of Brucella to remain hidden to the host immune system or resulted from the fact that the


conditions we used to infect the mice were rather inefficient. Accordingly, we infected mice with Salmonella enterica serovar Typhimurium strain 12023 using the same dose and the same

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conditions as those adopted for Brucella infection. Salmonella is a Gram-negative bacterium that expresses multiple virulence factors responsible for the induction of a strong inflammatory response [23]. Following intranasal infection with Salmonella, a dramatic change occurred in the relative distribution of the CD11bloCD1031 and CD11bhiCD103 DC subsets. At 1.5 and 5 days after infection, the lungs of Salmonella-infected mice showed a lower percentage of CD11bloCD1031 DC and a higher percentage of CD11bhiCD103 DC as compared with those of Brucella-infected mice (Fig. 3A and B). Such major change corresponded to a 480-fold decrease in the numbers of CD11bloCD1031 DC and to a 14-fold increase in the numbers of CD11bhiCD103 DC (Fig. 3C). The alveolar macrophages and the DC responded to Salmonella infection by upregulating the expression of CD80 at their surface (Fig. 3D–F). At 1.5 days after infection, CD11bloCD1031 DC from Salmonella-infected mice also expressed higher levels of CD86 (Fig. 3D). Taken together, these results show that Salmonella infection induces robust signs of activation of the lung immune system upon intranasal instillation. Therefore, the lack of detectable effects observed in the case of Brucella infection cannot be accounted for by the use of an inefficient protocol of lung infection but rather by the capability of Brucella to remain hidden from the host.

Depletion of alvelor macrophages retargets Brucella towards DC and enhances Brucella dissemination It is likely that by capturing invading Brucella, the highly phagocytic macrophages that crawl over the alveoli surface prevent them from reaching with a high efficiency the deeper alveoli layer and from being internalized by interstitial DC. To analyze whether reducing the number of alveolar macrophages renders the DC present in the interstitium prone to Brucella infection, we limited the number of lung alveolar macrophages at the time of Brucella infection using a method that relies on intranasal delivery of clodronate-loaded liposomes (Cl2MDP-LIP). Such a method that generates little discernible inflammation results in the transient depletion of alveolar macrophages without affecting interstitial macrophages and DC [6, 20, 24–26]. Mice were thus injected intranasally with Cl2MDP-LIP and infected with Brucella 2.5 days later, a time point that corresponds to the greatest decrease achieved in terms of alveolar macrophage number (Supporting Information Fig. 2). Analysis of lung sections 1.5 days p.i. showed the presence of Brucella in the remaining CD11c1MHCII Lyso1 alveolar macrophages, in CD11c MHCII1Lyso DC and in CD11c cells, the identity of which remains to be defined (Fig. 4A). Among the infected CD11c1 cells, 85% corresponded to alveolar macrophages and 15% to DC. Therefore, those alveolar macrophages that escaped treatment with Cl2MDP-LIP remained the primary target of Brucella. Moreover, some of them contained over ten bacteria, whereas the number of bacteria present in the few infected DC never exceeded five bacteria (Fig. 4B). Without prior treatment with Cl2MDP-LIP, most alveolar macrophages contained only one & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


or two bacteria (Fig. 4B). At 5 days p.i., replication of bacteria was observed in all infected cell types including CD11c (data not shown). Interestingly, infected DC can still be observed in the lung at 5 days p.i. (Fig. 4C). Therefore, partial alveolar macrophage depletion enables the uptake of Brucella by DC. However, the alveolar macrophages that escaped depletion were still the most efficient cell type capable of capturing Brucella. To analyze the consequences of partial alveolar macrophage depletion on Brucella dissemination, we determined the number of live bacteria present in the lungs, liver and spleen of mice treated or not with Cl2MDP-LIP and intranasally infected with the WT Brucella 2308 strain. Consistent with our immunofluorescence microscopy studies, we observed in lungs of untreated mice an increase in the number of CFUs between 1.5 and 5 days p.i. (Fig. 4D). Within 5 days p.i., Brucella replicated within the lungs and coincidently disseminated to the spleen and the liver, two organs known to constitute sites of Brucella replication. When the lungs of mice treated with Cl2MDP-LIP were analyzed at 1.5 days p.i., the number of Brucella was approximately 30-fold higher than in control mice and reached up to 107 bacteria per lung at 5 days p.i. Such number corresponded to approximately 100 times the number of bacteria present in the initial inoculum (Fig. 4D). At 1.5 days p.i., higher numbers of bacteria were also detectable in the liver and the spleen of Cl2MDP-LIP-treated mice (Fig. 4D). Moreover, at 5 days p.i., the numbers of Brucella found in the liver and spleen of Cl2MDP-LIP-treated mice were approximately 55-fold higher than in untreated mice (Fig. 4D). Therefore, following intranasal delivery, Brucella can reach more efficiently the liver and the spleen in the presence of a reduced number of alveolar macrophages, suggesting that the latter cells play a key role in limiting Brucella dissemination within the host.

After alveolar macrophage depletion Brucella infection has a major impact on the lung DC subsets We next analyzed the phenotype of the DC present in mice pretreated with Cl2MDP-LIP and infected with Brucella. Analysis of single-cell suspensions obtained from lungs at 1.5 days p.i. showed that both the percentage and the absolute numbers of CD11bloCD1031 and CD11bhiCD103 DC subsets were almost similar to those of control mice (Fig. 5A and C). Moreover, the expression of CD80 and CD86 on both DC subsets was also similar to that of control mice (Fig. 5D and E). At 5 days p.i., the absolute number of CD11bloCD1031 DC was similar to that of control mice and there was an 11-fold increase in the absolute numbers of CD11bhiCD103 DC (Fig. 5B and C). Interestingly, this increase is reminiscent of that observed in Salmonellainfected mice without pretreatment with Cl2MDP-LIP (Fig. 3). The expanded populations of CD11bhiCD103 DC showed, however, levels of CD80 and CD86 almost similar to those of the CD11bhiCD103 DC that are present in control mice whereas

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Figure 4. Depletion of alveolar macrophages increases Brucella uptake by DC and promotes bacterial dissemination. (A) Mice pretreated with Cl2MDP-LIP were infected with a DsRed-expressing B. abortus strain and their lungs analyzed by confocal microscopy 1.5 days p.i.. The upper panel shows bacteria (red) that have been internalized by cells expressing both lysozyme (green) and CD11c (blue). The lower panel shows bacteria that have been internalized by a cell expressing both MHCII (green) and CD11c (blue). Data are representative of three independent experiments. Scale bar, 20 mm. (B) Lungs of mice that have been treated with or without Cl2MDP-LIP prior to infection with Brucella were analyzed 1.5 days p.i. for the numbers of bacteria contained in CD11c1 MHCII Lyso1 and CD11c1MHCII1Lyso infected cells. Cells containing a single bacterium were excluded from the analysis of mice treated with Cl2MDP-LIP since many different cell types, including CD11c cells, were found to contain one bacterium. (C) 5 days after infection, CD11c1 MHCII1 infected cells are still present in the lungs of mice that were treated with Cl2MDP-LIP prior to infection with WT B. abortus strain. (D) The number of Brucella present in the lungs, liver and spleen of infected mice was determined at 18 h, 1.5 and 5 days p.i. using a CFU assay. For each time point, bacteria were enumerated in mice injected with (grey dots) or without (black dots) Cl2MDP-LIP 2.5 days prior to Brucella infection. The mean (horizontal bar) is indicated for each condition (n 5 7).

CD11bloCD1031 DC showed higher expression levels of both CD80 and CD86 molecules (Fig. 5D and E). Therefore, reducing the number of alveolar macrophages at the time of intranasal Brucella delivery has a major impact on the DC that are found in the interstitium of the lung parenchyma.


Depletion of alveolar macrophages is accompanied by the recruitment of inflammatory DC in the lung Following intraperitoneal Brucella melitensis infection, monocytederived inflammatory DC are recruited to the spleen and the

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Figure 5. DC activation occurs in Brucella-infected mice following alveolar macrophage depletion. (A and B) The lungs of mice pretreated with Cl2MDP-LIP were infected with Brucella and analyzed by flow cytometry 1.5 (A) and 5 days (B) p.i. Relative representation of the CD11bloCD1031 and CD11bhiCD103 DC subsets is shown and compared with that present in mice that were injected with PBS. The percentage of cells within each gate is indicated. (C) Numbers of CD11bloCD1031 (CD1031) DC, CD11bhiCD103 (CD11b1) lung DC and of alveolar macrophages determined 1.5 and 5 days p.i. (D–F) CD80 and CD86 expression on CD11bloCD1031 (CD1031) DC (D), CD11bhiCD103 (CD11b1) lung DC (E) and alveolar macrophages (F) analyzed 1.5 or 5 days p.i. Expression level of the costimulatory molecules was plotted as geometric mean (Geo mean). Data show mean7SEM. po0.05, po0.01; two-tailed Student’s t-test. Experiments have been done at least twice with three mice per group.

peritoneal cavity and constitute the main iNOS-producing cells [16]. Such DC display a CD11b1Ly-6C1Ly-6G– phenotype reminiscent of the TNF-a- and iNOS-producing (Tip) DC originally described during Listeria monocytogenes infection [27]. Therefore, we determined whether the CD11bhiCD103 DC that accumulate in the lungs of mice that were treated with Cl2MDP-LIP prior to infection with Brucella corresponded to Tip DC. After gating on CD451 cells and excluding the CD11b1Ly-6G1 granulocytes using


a CD11b versus Ly-6G dot plot, we focused on the CD11b1Ly-6G monocytic cells and determined the levels of CD11c and MHCII molecules they express as well as their capacity to produce TNF-a and iNOS. In uninfected mice, less than 10% of the CD451 CD11b1Ly-6G cells present in the lung produced TNF-a and none of them produced iNOS (Fig. 6A). In addition, those TNF-aproducing CD11b1Ly-6G cells did not express CD11c and MHCII molecules. Mice infected with Brucella and containing normal

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numbers of alveolar macrophages showed no recruitment of Tip DC (Fig. 6A). In contrast, when the CD451CD11b1Ly-6G cells present in the lungs of Salmonella-infected mice were analyzed 1.5 days p.i., 25% of them produced TNF-a alone, 14.3% expressed iNOS alone and 13.7% produced both TNF-a and iNOS (Fig. 6A). In addition, those cells also showed an upregulation of CD11c and MHCII molecules at their surface, indicating that they likely correspond to monocyte-derived inflammatory DC. In the absence of Brucella infection, the phenotype of these CD11b1Ly-6G cells showed the same pattern of TNF-a and iNOS expression as the one observed in untreated mice (data not shown). Therefore, treatment with Cl2MDP-LIP by itself did not trigger the appearance of Tip DC. However, when the lungs of mice treated with Cl2MDP-LIP and infected with Brucella were analyzed at day 5 p.i., CD451CD11b1Ly-6G cells producing TNF-a and iNOS, alone or in combination, and expressing CD11c and MHCII molecules were readily detectable (Fig. 6B). Therefore, these results suggest that Tip DC can be recruited in the lungs of Brucella-infected mice, provided that they contain a reduced number of alveolar macrophages.

After alveolar macrophage depletion Brucella infection affects the DCs present in the MdLN The MdLN of mice that received Cl2MDP-LIP and were infected 5 days prior to analysis showed a 20-fold increase in the numbers of lung-derived DC as compared with control mice (Fig. 7A). However, in contrast to the lung, such change can be accounted for by an increase in the absolute numbers of both CD11bloCD1031 and CD11bhiCD103 DC (Fig. 7B). Therefore, reducing the number of alveolar macrophages at the time of intranasal Brucella delivery has also a major impact on the DC that are found in the lung-draining MdLN.

Discussion In the present study, we showed that shortly after intranasal infection B. abortus were internalized by pulmonary alveolar macrophages and, consistent with a recent study based on S. pneumoniae [22], such alveolar macrophages transported B. abortus from lungs to the MdLN. Although we did not detect any infected DC in the alveolar septum, Brucella-containing DC were readily found in the MdLN. It was only when alveolar macrophages were depleted prior to pulmonary Brucella infection that we observed bacterial uptake by the DC found in the lung alveolar parenchyma. Therefore, since the presence of alveolar macrophages prevents the infection of those DC that reside deeper in the alveoli septa, it is likely that the large conducting airways that contain a dense network of DC but the surface of which is deprived of macrophages constitute the predominant source of the Brucella-infected DC that are found in the MdLN [3]. We also showed that at early time points of infection, the number of CFU present in lung extracts was six-fold higher in


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mice in which alveolar macrophages were partially depleted. Since we showed that Brucella mainly resides in alveolar macrophages, it is likely that their antimicrobial activity accounts for the efficient killing of Brucella that occurs during the early phase of infection. However, at later time points of infection and in agreement with previous in vitro data [28], Brucella was able to replicate in alveolar macrophages in vivo. Therefore, by participating to bacterial killing, alveolar macrophages prevent the capture of Brucella by the DC residing in the alveolar septa and contain the dissemination of Brucella in the organism. However, Brucella is capable of establishing an intracellular replicative niche in infected macrophages and such strategy likely contributes to pathogenesis by allowing Brucella to ‘‘silently’’ multiply hidden from the host immune system. Accordingly, upon intranasal infection, Brucella did not induce an inflammatory response in the lungs comparable to that resulting from Salmonella infection. Therefore, following pulmonary infection, Brucella can persist and disseminate in the whole organism. Corroborating the view that the ability of Brucella to dampen innate immune responses is critically dependent on alveolar macrophages, a strong local inflammatory response was restored by depleting alveolar macrophages immediately prior to Brucella infection. Therefore, despite its low endotoxic LPS, intranasal instillation of Brucella can induce a substantial inflammatory response in mice with reduced numbers of alveolar macrophages. Although a lot of work has been done to address the role of DC in the immune response to pathogens [29], the respective role of the CD11bhi CD103 and CD11bloCD1031 DC subsets that reside in the lung under steady-state conditions and of the monocyte-derived DC that are recruited upon inflammation has not been systematically analyzed yet. We showed that when alveolar macrophages were partially depleted, Brucella infection resulted in the massive recruitment of inflammatory DC that, owing to their ability to secrete TNF-a and iNOS, likely correspond to Tip DC [27, 30]. The role of the Tip DC that are recruited into Brucella-infected lung following partial depletion of alveolar macrophages remains, however, to be determined. For instance, by conveying Brucella from the lung to the MdLN, Tip DC may contribute to bacterial dissemination and thus provide a detrimental effect. Alternatively, Tip DC recruitment may play a protective role [31]. Those innate immune responses to a bacterial infection resemble those described in the case of viral infection. For instance, it has been shown that mice infected with the influenza type A virus display a selective accumulation of Tip DC within the lungs [32]. Moreover, upon Respiratory Syncytial Virus infection a massive influx of CD11bhiCD103 DC was observed while CD1031CD11blo DC disappeared from the lungs [33]. Following Brucella infection of mice with partial depletion of alveolar macrophages, we observed that MdLN contained increased numbers of both CD11bloCD1031 and CD11bhiCD103 DC, suggesting that both DC subsets can migrate to the MdLN. This is consistent with previous reports showing that both CD11bloCD1031 and CD11bhiCD103 DC subsets are capable of migrating from the airways to the MdLN to

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Figure 6. Inflammatory DC are recruited to the lungs of Brucella-infected mice following alveolar macrophage depletion. (A and B) Mice were infected with a WT B. abortus strain or a Salmonella WT strain and the CD45.21 cells present in the lungs 1.5 and 5 days p.i. were analyzed for the expression of CD11b and Ly6G (upper panel). CD11b1 Ly6G cells were analyzed for iNOS and TNF-a production (middle panel) and for CD11c and MHCII expression (bottom panel). The percentage of cells within each gate is indicated. Data are representative of three independent experiments.

present Ag to T cells [34–36]. It should be noted, however, that at later time points of infection the migratory CD11bhiCD103 DC likely comprise migratory Tip DC. Francisella infection also results in trafficking of CD11bhi DC from the respiratory tract to the MdLN [37]. A wealth of studies has attempted to understand the complex relationships that exist between alveolar macrophages and the DC that reside in the lung parenchyma [25, 38, 39]. It has been suggested that alveolar macrophages inhibit DC function and that discernible pulmonary innate immune responses are generated only when the protective phagocytic capacities of the alveolar macrophages are overwhelmed, allowing the incoming pathogens to interact with the DC present in the alveolar septa [39, 40]. Consistent with this last view, we showed that capture of Brucella by alveolar macrophages prevents the activation of pulmonary DC and that upon macrophage depletion, Brucella uptake by pulmonary DC is enhanced and stronger inflammatory responses against the pathogen are induced. Our results have focused on the initial host response prior to the onset of adaptive immunity. Analysis of the later stages of Brucella pulmonary infection will allow to further determine whether the lack of activation of the DC in the lung parenchyma that limits the damage inflicted to the lung parenchyma is also compatible with the generation of an appropriate adaptive immune response against Brucella.


Materials and methods Bacterial strains Bacterial strains used in this study were S. enterica serovar Typhimurium strain 12023 and the smooth virulent B. abortus strain 2308. DsRed-expressing B. abortus was kindly provided by Xavier de Bolle (Namur, Belgium). To construct a DsRedexpressing B. abortus strain, a 500 bp-long fragment located upstream of the initiation codon corresponding to the secE promoter was amplified by PCR and inserted into a mobilizable version of the pBluescriptKS vector. A DsRed-coding sequence was then inserted downstream of the secE promoter and a kanamycin resistance gene subsequently inserted between the end of the DsRed-coding sequence and the lac promoter. The resulting plasmid was amplified into a S17-1 Escherichia coli strain and then mobilized into B. abortus 2308 NalR. Bacterial colonies resistant to both nalidixic acid and kanamycin were selected. The expression of DsRed is stable and had no effect on the virulence of Brucella (data not shown). Brucella was grown in tryptic soy broth medium and Salmonella in Luria Bertani medium. All the experiments with B. abortus were performed at biosafety level 3.

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Figure 7. CD11c1 MHCIIhi to int DC accumulate in the MdLN of Brucella-infected mice following alveolar macrophage depletion. After gating on large cells using the FCS and SSC parameters, the percentages (A) and the absolute numbers (B) of CD11c1MHCIIhi to int, CD11bloCD1031 and CD11bhiCD103 DC were determined in mice that have been either injected with PBS or infected for 5 days with Brucella. The percentage of cells within each gate is indicated. po0.01; two-tailed Student’s t-test.

Mouse infection C57BL/6 WT mice were housed under specific pathogen-free conditions and handled in accordance with French and European directives. INSERM guidelines have been followed regarding animal experimentation and CIML has been credited by the French government with authorization No. 02875 for mouse experimentation. Mice were used at 6–8 wk of age. For Brucella infection, bacteria were inoculated in 2 mL of tryptic soy broth and grown for 16 h at 371C and up to an OD (OD600 nm) of approximately 1.8. For Salmonella infection, overnight cultures of Salmonella were diluted in Luria Bertani medium and bacteria were grown until OD600nm of 1.8. Mice were infected intranasally with 5 105 bacteria diluted in 30 mL of PBS 1 . Serial dilutions of the inoculum were plated to control the number of Brucella or Salmonella that were injected in mice. To deplete macrophages, mice were injected intranasally with 30 mL of clodronate containing liposomes (Cl2MDP-LIP), 2.5 days before infection. Cl2MDP-LIP were synthesized using clodronate (Cl2MDP) that was a gift of Roche Diagnostics, Mannheim, Germany [41]. When specified 0.4 mm dark red fluorescent carboxylate-modified microspheres (Duke Scientific Corporation) were diluted in PBS (1:40) and administered intranasally 3 h before infection.

digestion for 20 min at room temperature with a mixture of Collagenase type 2 (Serlabo) and of DNase I (Sigma). During digestion, samples were vigorously pipetted. Cell suspensions were treated for 5 min with EDTA and filtered trough cell strainers. Light density cells from the lungs were enriched by centrifugation using a Nycoprep solution (density 5 1.068) as previously described [42]. Although the initial bronchoalveoloar lavage steps remove a large number of alveolar macrophages, they did not dislodge those that are strongly adherent to the surface of the alveoli. Accordingly, the later are still found in the cell suspension resulting from whole lung digestion [7]. Cells were preincubated at 41C for 10 min with the 2.4G2 Ab to block Fc receptors. Single-cell suspensions were stained for 20 min with combinations of FITC-, PE-, Percp-Cy5-5, APC-, APC-Cy7-, Alexa700-, PE-Cy7-, Pacific Blue- or biotin-conjugated Ab. Biotinylated Ab were visualized with APC-conjugated streptavidin. Anti-CD11b (M1/70), anti-CD103 (M290) and anti-CD11c (HL3) Ab were purchased from BD Biosciences Pharmingen. AntiCD80 (16-10-A1), anti-CD86 (GL1) and anti-MHCII (M5/114) Abs were purchased from eBioscience. Flow cytometry was performed on a BD FACSCanto system (BD Biosciences) after gating out dead cells by using forward and side scatters. Analysis was performed with a FlowJo (Tree Star) software.

Cell preparation and flow cytometry Intracellular staining The lungs were first flushed four times with 800 mL of PBS 1 via a tracheal canula. Following thoracotomy, the whole lung tissue and the MdLN were separately removed and cut in pieces before


For intracellular staining, cells were activated in vitro in 96-well plate in presence of brefeldin A (GolgiPlug; BD PharMingen).

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After 4 h at 371C, cells were recovered, washed and first stained with Ab for surface markers as described above. For intracellular cytokine staining, cells were fixed using the cytofix/ cytoperm kit (BD Pharmingen) and stained with a FITCconjugated anti-TNF-a Ab (MP6-XT22, BD Pharmingen). A purified anti-iNOS Ab (M19, Santa Cruz) and a goat Alexa-647conjugated anti-rabbit Ab were used to detect the iNOS production (A21244, Molecular Probes).

Immunofluorescence staining and confocal microscopy Lungs and MdLN were fixed with 4% paraformaldehyde for 2 h, washed in PBS, infused overnight in 35% sucrose and frozen in Tissue-Teks O.C.T. (Optimal Cutting Temperature compound). After permeabilization with 0.5% saponin for 5 min and unspecific binding site blockade with a cocktail of 2% BSA, 1% FCS and 1% donkey or goat serum, cryostat tissue sections were labeled overnight at 41C with primary Ab or control Ab (Jackson Immunoresearch). Polyclonal rabbit anti-lysozyme was from Dako. Polyclonal anti-B. abortus LPS was a gift from E. Moreno (Universidad Nacional, Heredia, Costa Rica). Monoclonal Ab anti-CD11c (N418) and anti-MHCII (2G9) were from Biolegend and BD Biosciences, respectively. After washing, slides were incubated for 1 h at room temperature with secondary Ab and SYTOX Blue (Invitrogen) when nuclear staining was required. Alexa Fluor 488 anti-rat and anti-rabbit secondary Ab were from Invitrogen. Cy5 Goat anti-hamster was from Jackson Immunoresearch. Slides were mounted in Prolong Gold (Invitrogen) and observed with a Zeiss LSM 510 confocal microscope. Images were analyzed using Adobe Photoshop 7.0 and Imaris 6.1. Number of bacteria per infected cell was determined at 1 (n 5 6 mice), 1.5 (n 5 3 mice) and 2 days (n 5 4 mice) p.i. in untreated mice or at 1.5 days p.i. in mice (n 5 3) pretreated with Cl2MDP-LIP. At 5 days p.i., the number of bacteria per cell was too high to differentiate individual bacteria. Data were expressed as the mean number of bacteria per cell7SD.


Acknowledgements: The authors would like to thank Xavier de Bolle and Richard Copin for DsRed-expressing Brucella. We thank our colleagues from the animal facilities and the cytometry platform and Alexandre Muller for technical assistance and Martin Guilliams and Javier Pizarro-Cerda for technical advices and helpful discussions. This work was supported by CNRS, INSERM, ANR (DC in vivo) and by a postdoctoral fellowship from FRM (CA). Conflict of interest: The authors declare no financial or commercial conflict of interest.

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Abbreviations: Cl2MDP-LIP: clodronate-loaded liposomes Lyso: lysozyme MdLN: mediastinal LN MHCII: MHC-encoded class II molecules p.i.: post infection SSC: side-scatter Tip DC: TNF-a- and iNOS-producing DC

Current address: Dr. Cristel Archambaud, Unite´ des Interactions Bacte´ries-Cellules, INSERM, U604, INRA USC2020, Institut Pasteur, France

Full correspondence: Prof. Bernard Malissen, Centre d’Immunologie de Marseille-Luminy, Case 906, 13288 Marseille Cedex 09, France Fax: 133-4-91-26-94-30 e-mail: [email protected]

Received: 15/3/2010 Revised: 4/8/2010 Accepted: 21/9/2010 Accepted article online: 5/10/2010

Additional correspondence: Dr. Jean-Pierre Gorvel, Centre d’Immunologie de Marseille-Luminy, Case 906, 13288 Marseille Cedex 09, France Fax: 133-4-91-26-94-30 e-mail: [email protected]


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