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The Journal of Immunology, 2005, 174: 3570-3579.
Copyright © 2005 by The American Association of Immunologists

Influence of ESAT-6 Secretion System 1 (RD1) of Mycobacterium tuberculosis on the Interaction between Mycobacteria and the Host Immune System1

Laleh Majlessi2,*, Priscille Brodin{dagger}, Roland Brosch{dagger}, Marie-Jésus Rojas*, Huot Khun{ddagger}, Michel Huerre{ddagger}, Stewart T. Cole{dagger} and Claude Leclerc*

* Unité de Biologie des Régulations Immunitaires, Institut Pasteur, Institut National de la Santé et de la Recherche Médicale Equipe 352, Paris, France; and {dagger} Unité de Génétique Moléculaire Bactérienne and {ddagger} Unité de Recherche et d’Expertise Histotechnologie et Pathologie, Institut Pasteur, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The chromosomal locus encoding the early secreted antigenic target, 6 kDa (ESAT-6) secretion system 1 of Mycobacterium tuberculosis, also referred to as "region of difference 1 (RD1)," is absent from Mycobacterium bovis bacillus Calmette-Guérin (BCG). In this study, using low-dose aerosol infection in mice, we demonstrate that BCG complemented with RD1 (BCG::RD1) displays markedly increased virulence which albeit does not attain that of M. tuberculosis H37Rv. Nevertheless, phenotypic and functional analyses of immune cells at the site of infection show that the capacity of BCG::RD1 to initiate recruitment/activation of immune cells is comparable to that of fully virulent H37Rv. Indeed, in contrast to the parental BCG, BCG::RD1 mimics H37Rv and induces substantial influx of activated (CD44highCD45RB–CD62L–) or effector (CD45RB–CD27–) T cells and of activated CD11c+CD11bhigh cells to the lungs of aerosol-infected mice. For the first time, using in vivo analysis of transcriptome of inflammatory cytokines and chemokines of lung interstitial CD11c+ cells, we show that in a low-dose aerosol infection model, BCG::RD1 triggered an activation/inflammation program comparable to that induced by H37Rv while parental BCG, due to its overattenuation, did not initiate the activation program in lung interstitial CD11c+ cells. Thus, products encoded by the ESAT-6 secretion system 1 of M. tuberculosis profoundly modify the interaction between mycobacteria and the host innate and adaptive immune system. These modifications can explain the previously described improved protective capacity of BCG::RD1 vaccine candidate against M. tuberculosis challenge.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The bacille Calmette-Guérin (BCG)3 is a live, attenuated vaccine that was initially obtained in vitro from serial passages of virulent Mycobacterium bovis, closely related to Mycobacterium tuberculosis. The exact molecular bases of the attenuation of BCG remain to be elucidated. Comparative genomics identified >100 open reading frames absent from BCG substrains compared with M. tuberculosis (1, 2, 3). These genes are spread over 14–16 distinct chromosomal regions of M. tuberculosis which are referred to as regions of difference (RD) 1–16. RD have been deleted during the divergence of M. bovis from M. tuberculosis or during the continuous in vitro subcultures of the progenitor of BCG (1, 2, 3).

Of particular interest, RD1 is the only region absent from all BCG substrains and the related live vaccine Mycobacterium microti, but present in virulent M. bovis and M. tuberculosis (2, 3, 4). RD1 is a {approx}10-kb deletion that removes, from BCG, genes encoding early secreted antigenic target, 6 kDa (ESAT-6) secretion system 1 (5) which is required for secretion of a 10-kDa culture filtrate protein (CFP-10) (6) and the ESAT-6 (7), two potent immunogens in humans and rodents. Although a correlation exists between the presence of RD1 and in vitro cytolytic activity of mycobacteria against an alveolar epithelial cell line (8), the exact function of CFP-10 and ESAT-6, or other products of RD1, remains to be elucidated. We and others recently provided experimental evidence showing that the products of these flanking genes encode elements of a secretory apparatus necessary for the export, secretion, and immunogenicity of ESAT-6 and CFP-10 proteins (9, 10, 11).

Reintroduction of RD1 into BCG (BCG::RD1) leads to: 1) marked modifications in colonial morphology, 2) increased virulence in immunodeficient mice monitored by bacterial proliferation, and 3) increased persistence in the immunocompetent host revealed by the presence of bacteria in target organs at late time points (12). As a corollary, targeted deletion of the RD1 from M. tuberculosis resulted in 1) severely reduced in vivo growth and dissemination and 2) increased survival of immunocompetent mice (8, 13).

We recently described the improved capacity of BCG::RD1 (9) or M. microti::RD1 (14), respectively, compared with BCG or to M. microti, to protect mice and guinea pigs against M. tuberculosis challenge. The basis of this improved protective potential has not been elucidated. In the present investigation, we focused on the influence of RD1 on interactions between the immunocompetent host and mycobacteria. We performed comparative analyses between parental BCG and BCG::RD1 in a low-dose aerosol infection model which allowed us to study in vivo the role of RD1 in virulence, dissemination, and pathogenicity as well as in recruitment/activation of different cell subsets of the adaptive or innate immune system. We demonstrate that BCG::RD1 displays increased virulence compared with parental BCG, without reaching the full level virulence of M. tuberculosis H37Rv. However, BCG::RD1 was as efficient as H37Rv in triggering the recruitment of activated CD11c+CD11bhigh cells and of activated CD4+ and CD8+ T cells to the site of infection and inflammation, i.e., lung parenchyma. Moreover, investigation of the profile of expression of inflammatory cytokines, chemokines, and their receptors in dendritic cells (DC) infected in vitro with parental BCG, BCG::RD1, or H37Rv or in lung parenchymal CD11c+ cells from mice infected with these mycobacterial strains by aerosol, showed that the presence of RD1 essentially influences the in vivo interaction between mycobacteria and the host immune system. Despite the relative virulence of BCG::RD1, its enhanced capacity to trigger antimycobacterial innate and adaptive immunity could explain its improved protective potential against infection with M. tuberculosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Aerosol infection of mice with mycobacterial strains

Recombinant BCG::RD1 was obtained by electroporation of BCG Pasteur 1173P2 with the integrative cosmid clone RD1-2F9 (12). As control, BCG carries the shuttle cosmid pYUB412 alone (BCG::pYUB412). One-milliliter cultures of recombinant BCG and M. tuberculosis H37Rv were grown in parallel in Dubos medium (Difco) supplemented with albumin-dextrose-catalase (Difco) and, when necessary, with 50 µg/ml hygromycin (Invitrogen Life Technologies). Bacteria were then harvested, washed twice, and resuspended in 50 mM sodium phosphate buffer (pH 7.0). Bacteria were sonicated briefly and allowed to stand for 1 h to allow residual aggregates to settle. The bacterial suspensions (108 CFU/ml) were finally aliquoted at –80°C. Six-week-old female C57BL/6 (H-2b) mice (Charles River Breeding Laboratories) were infected via the aerosol route using a customized apparatus with recombinant BCG::pYUB412, BCG::RD1, or M. tuberculosis H37Rv to obtain a retained inhaled dose of 100 ± 10 CFU. At different time points after aerosol challenge, mice were sacrificed and spleen and lungs were homogenized using an MM300 apparatus (Qiagen) and 2.5-mm diameter glass beads. Serial 5-fold dilutions in medium were plated on 7H11 medium supplemented with oleic acid-albumin-dextrose-catalase (Difco) and 50 µg/ml hygromycin when necessary. CFU were ascertained after 2 wk of growth at 37°C for H37Rv and 3 wk of growth for BCG::RD1.

Immunohistological analyses

To study the recruitment of inflammatory cells within the lungs of infected mice, the following procedure was performed. Lungs were incubated with zinc acetate-containing buffer and embedded in Paraffin 37. Staining with H&E or with Ziehl-Neelsen was performed according to standard protocols. Immunohistochemistry was achieved by use of anti-ESAT-6 (HYB 076-08; Statens Serum Institute, Copenhagen, Denmark), anti-CD3{epsilon} (AB-1; Neomarkers), or anti-CD11c (HL3; BD Pharmingen) mAbs. Detection was conducted with Envision System HRP (DakoCytomation) and revealed with 3-amino-9-ethylcarbazole (Sigma-Aldrich).

Infection of bone marrow-derived DC

To generate bone marrow-derived DC (BM-DC), femur marrows from C57BL/6 mice were flushed out with RPMI 1640 Glutamax (Invitrogen Life Technologies) and the recovered cells were cultured at 2 x 105 cell/ml in antibiotic-free RPMI 1640 complemented with 10% FCS (ICN Pharmaceuticals), 5 x 10–5 M 2-ME, and 10 ng/ml recombinant GM-CSF. The cultures were fed at day 3 with this medium. BM-DC were then infected at day 7 with briefly sonicated mycobacteria at a multiplicity of infection of 4 bacilli per cell. After a 16-h incubation at 37°C in 5% CO2, cells were harvested and stained with magnetic microbeads conjugated to anti-CD11c mAb (Miltenyi Biotec) in the presence of Fc{gamma}II/III receptor blocking CD16/32 mAb (2.4G2; BD Pharmingen). CD11c+ cells were positively selected by two successive passages through OctoMACS magnetic cell separators (Miltenyi Biotec). The eluted cell suspensions contained at least 98% CD11c+ cells as judged by FACS analysis.

Preparation of lung-derived cells

To obtain bronchoalveolar lavage (BAL) fluids, mice were sacrificed by CO2 inhalation and their lungs were washed by four instillations with 500 µl of PBS following cannulation of their trachea. BAL cells were used for FACS analysis. Lungs were then removed aseptically and were disaggregated by treatment with 400 U/ml type IV collagenase and DNase I (Roche). Following a 45-min incubation at 37°C, single-cell suspensions of pneumocytes were prepared by lung dissociation and passage through 100-µm nylon filters (Cell Strainer; BD Falcon). Such pneumocyte suspensions were either used for preparation of lymphocytes or DC. Pneumocytes were enriched in lymphocytes by 15-min centrifugation at 3000 rpm on Ficoll gradient medium (Lympholyte M; Cedarlane Laboratories). Lung DC were prepared using of iodixanol gradient medium (OptiPrep; Axis-Shield). Briefly, pneumocytes were suspended in dense medium (15% iodixanol) and layered with low-density medium (11.5% iodixanol). Subsequent to 15-min centrifugation at 2000 rpm, low-density cells floated to the top of the 11.5% iodixanol solution (15). These cells were either used for FACS analyses or were further purified using magnetic microbeads conjugated to anti-CD11c mAb, as described above for BM-DC. Such purified lung DC contained at least 96% of CD11c+ cells.

FACS analyses

Allophycocyanin-conjugated anti-CD4 (RM4-5), anti-CD8{alpha} (53-6.7), anti-CD11c (HL3), anti-CD62L (L-selectin) (MEL-14), PE-conjugated anti-CD44 (IM7), anti-CD27 (LG.3A40), anti-CD11b (5C6), and FITC-conjugated anti-CD45RB (16A) mAbs were all from BD Pharmingen. Cells stained with appropriate dilutions of mAbs were washed and fixed overnight with 4% paraformaldehyde and were analyzed after setting gates on forward vs side scatter in a FACSCalibur system using CellQuest software (BD Biosciences).

T cell assays

To measure the T cell proliferative responses to mycobacterial Ags, lung lymphocytes or splenocytes were cultured (0.5–1 x 106 cells/well) in 96-well flat-bottom plates in synthetic HL-1 medium (BioWhittaker) as previously detailed (9) in the presence of various concentrations of appropriate Ags. Cultures were pulsed 72 h later with 1 µCi [methyl-3H]thymidine (ICN Pharmaceuticals) for 16 h and the incorporated cpm were counted in an LKB beta plate counter. IFN-{gamma} production by lymphocytes was assessed subsequent to in vitro stimulation with 10 µg/ml purified protein derivative (Serum Institute), recombinant ESAT-6, CFP-10, or maltose binding protein from Escherichia coli (MalE) proteins or synthetic peptides from ESAT-6 (ESAT-6:1–20) (16), Ag 85A (Ag85A:241–260) (17), or MalE (MalE:100–114) (18). After a 72-h incubation, amounts of IFN-{gamma} were quantified in culture supernatants by an ELISA with a detection limit of 500 pg/ml as described elsewhere (9).

Macroarray analyses

BM-DC or lung DC purified by magnetic cell separation (purity CD11c+ ≥96%, see above) were lysed and total RNA was extracted using an RNeasy kit subsequent to treatment with DNase I (Qiagen). The absence of DNA contamination or of RNA degradation was confirmed for each preparation by RNA electrophoresis in 1% agarose gels in RNase-free Tris-borate-EDTA buffer in the presence of 0.15 µg/ml ethidium bromide. Gene expression of inflammatory cytokines, chemokines, and their receptors was investigated by using GEArray Q Series high-density membrane arrays (SuperArray), allowing the study of the following genes: IL-1{alpha}, IL-1{beta}, IL-1R1, IL-1R2, putative transmembrane receptor IL-1Rrp, IL-2, IL-2R{alpha}, IL-2R{beta}, IL-2R{gamma}, IL-4, IL-5, IL-5R, IL-6, IL-6R{alpha}, IL-6sig trans, IL-9, IL-9R, IL-10, IL-10R{alpha}, IL-10R{beta}, IL-11, IL-11R{alpha}1, IL-12{alpha}(p35), IL-12{beta}(p40), IL-12R{beta}1, IL-12R{beta}2, IL-13, IL-13R{alpha}1, IL-13R{alpha}2, IL-15, IL-15R{alpha}, IL-16, IL-17, IL-17R, IL-17{beta}, IL-18, IL-20, IL-21, IL-25, CCR1 to CCR9, CXCR1–CXCR5, CX3CR-1, CCL1–CCL9, CCL11, CCL12, CCL17, CCL19–CCL22, CCL24, CCL25, CXCL2, CXCL5, CXCL9–CXCL11, CXCL13–CXCL15, migration inhibitory factor, lymphotactin, fractalkine, stromal cell-derived factor 1a, stromal cell-derived factor 2, IFN-{gamma}, TGF{alpha}, TGF{beta}1, TGF{beta}2, TGF{beta}3, TNF-{alpha}, TNF-{beta}, TNFR1, TNFR2, lymphotoxin {beta}, and lymphotoxin {beta} receptor.

Reverse transcription to cDNA and hybridization to membrane arrays were performed according to the instructions provided by SuperArray. Briefly, RNA (2.5–5.0 µg) was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase SuperScript(Invitrogen Life Technologies) in the presence of primers specific for the above-mentioned genes or several housekeeping genes, 30 IU RNase inhibitor, 500 µM of each dNTP, except for dCTP which was used at 5 µM, and 2.5 µCi [{alpha}-32P]dCTP (3000 Ci/mmol; Amersham Pharmacia Biotech. The GEArrays were prehybridized with 100 µg/ml heat-denatured salmon sperm DNA (Invitrogen, Life Technologies) for 2 h at 58°C. Denatured 32P-cDNA were hybridized for 16 h on GEArrays at 58°C. The latter were then washed twice with 2x SSC/1% SDS and twice with 0.1x SSC/0.5% SDS at 58°C. A STORM imaging system (Amersham Pharmacia Biotech) was used to record the image of the arrays. The acquired signals were converted to digital data by use of ImageQuant software (Molecular Dynamics) for quantification of GEArray tetra-spots. For each array, cpm were obtained for individual tetra-spots after subtracting local background. Data were then normalized by dividing individual cpm by cpm obtained for the housekeeping gene GAPDH. For a given gene, the fold change consisted of the ratio of the normalized value for infected cells over that obtained for uninfected control cells. Fold changes were considered as significant when the gene expression decrease or increase was at least 2-fold in two independent experiments. Gene expression variations were defined as "intense," "marked," or "down-regulated" when the fold change was respectively >10, comprised between 2 and 10 or <1. For a given gene, in two totally independent experiments, fold change remains always in the same category because the variation never exceeded 15% from one experiment to another.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
RD1-mediated virulence in mice

Following a low-dose infection (100 CFU/mouse) via the aerosol route, BCG complemented with the complete RD1 region (BCG::RD1) grew actively in the lungs of C57BL/6 mice with the same kinetics as M. tuberculosis H37Rv, whereas the control BCG (BCG::pYUB412) did not proliferate (Fig. 1a). Bacteria were present in the spleen of BCG::RD1- or H37Rv-infected mice, but not in their BCG-infected counterparts (Fig. 1b). Neither replication in the lungs nor dissemination to the spleen was detected with 100-fold increased initial doses of BCG (data not shown). When a lower initial challenge dose (10 CFU/mouse) was given by the aerosol route, again active multiplication of BCG::RD1 occurred in the lungs while dissemination to the spleen was delayed (Fig. 1c). BCG::RD1 was detected up to 100 days after infection, in both organs, pointing to strong persistence of this strain during the chronic phase of infection. During this chronic phase, BCG::RD1 loads in the organs remained {approx} 100-fold lower than M. tuberculosis H37Rv and never reached those of the latter irrespective of the initial infecting dose (Fig. 1, a and b, and data not shown).



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  		FIGURE 1. Virulence, persistence, and dissemination of M. bovis BCG::RD1 compared with control BCG (BCG::pYUB412) or M. tuberculosis H37Rv in C57BL/6 mice infected via the aerosol route. CFU of these three mycobacterial strains in lungs (a) or in spleen (b) at different time points following infection with 100 CFU/mouse. c, Persistence of BCG::RD1 in the lungs and its dissemination to the spleen following low-dose aerosol infection, i.e., 10 CFU/mouse. Data are representative of three independent experiments. d, CFU of BCG::pYUB412, BCG::RD1 or M. tuberculosis H37Rv in different cell subsets during the chronic phase of infection, i.e., at 12 wk after infection with 100 CFU/mouse. Three to four mice were studied individually per group and per time point.

 
During the chronic phase of infection, BCG::RD1 was not detectable in cells from BAL fluids but was found in comparable levels in CD11c+ or CD11c– cells isolated from lung interstitium. In parallel, only low CFU numbers of M. tuberculosis H37Rv were detected in cells from BAL fluids (Fig. 1d). In accordance with the higher virulence of M. tuberculosis H37Rv compared with that of BCG::RD1 (Fig. 1a), parenchymal CD11c+ or CD11c– cell subsets contain 2 logs more CFU than the same preparations from BCG::RD1-infected mice (Fig. 1d).

RD1-induced pathogenicity

By the end of the acute phase, the lungs of BCG::pYUB412-infected mice were negative by Ziehl-Neelsen staining and displayed only a few scanty and diffuse infiltrates containing mainly macrophages and T cells. No granuloma was observed. In contrast, the lungs of mice infected with BCG::RD1 were positive for Ziehl-Neelsen staining and contained granulomatous inflammation around the bronchia within alveoli with focalized infiltrates containing large macrophages and cells with aggregated cytoplasmic vacuoles and lymphocytes (Fig. 2). The pathology caused by BCG::RD1 displayed the typical patterns of chronic granulomatous inflammation induced by M. tuberculosis H37Rv as large and diffuse granuloma contained numerous activated cells (Fig. 2). Immunohistochemistry demonstrated that these granulomas mainly contained F4/80+ macrophages around bronchioles and vessels, CD11c+ leukocytes, and CD3+ T cells (data not shown). Characteristic vasculitis and necrosis in the perivascular and peribronchial areas were detected. The total surface of these granulomas did not exceed one-fifth of the total surface of the lung.



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  		FIGURE 2. Presence of granulomatous lesions in the lungs of BCG::RD1-infected C57BL/6 mice, as detected by hematoxylin staining as detected at 40- (a), 400- (b), and 100- (c) fold magnification. Results were obtained at 6 wk following infection with 100 CFU/mouse.

 
Taken together, these results highlighted the preferential tropism of BCG::RD1 for the lungs and showed that this aerosol experimental setup is appropriate for the investigation of the host innate and acquired immune responses induced by these bacilli.

Capacity of BCG::RD1 to induce strong CD4+ and CD8+ T cell recruitment and activation

We then investigated lymphocytes present in the lungs of mice untreated or aerosol-infected with 100 CFU/mouse of BCG::pYUB412, BCG::RD1 or M. tuberculosis H37Rv for their activation or migratory phenotype. At 3 wk after infection, lung CD4+ or CD8+ T subsets from untreated, BCG::pYUB412- or BCG::RD1-infected mice displayed comparable profiles in terms of their percentages of CD44highCD45RB– or CD44highCD62L– cells (Table I). In contrast, at this time point, in H37Rv-infected mice, CD4+ and CD8+ T cell compartments contained 1.5 and 5.0 times higher percentages of CD44highCD45RB– cells (Table I), respectively. In contrast to the other experimental groups, H37Rv-infected mice also contained two times higher percentages of CD44highCD62L– within their CD8+ T cell subset. Comparable data were obtained at 4 wk postinfection (data not shown). Therefore, following aerosol challenge with 100 CFU/mouse, at 3 or 4 wk postinfection, only the fully virulent H37Rv strain was able to induce T cell recruitment/activation.


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  		Table I. Comparison of recruitment of activated T cells in the lungs of C57BL/6 mice, aerosol-infected with BCG::pYUB412, BCG::RD1, or M. tuberculosis H37Rva

 
At 6 wk after infection, CD4+ or CD8+ T cells from untreated or BCG::pYUB412-infected mice still contained comparable percentages of CD44highCD45RB– cells. In contrast, like in H37Rv-infected mice, markedly increased percentages of CD44highCD45RB– cells were detected within CD4+ and notably within CD8+ T cells in BCG::RD1-infected mice (Fig. 3a). Compared with untreated controls, in BCG::pYUB412-infected mice, slightly increased percentages of CD44highCD62L– were detected in both CD4+ and CD8+ T cell subsets. Nevertheless, the proportions of CD44highCD62L– cells were higher in BCG::RD1- or H37Rv-infected counterparts (Fig. 3a). Such T subset status was maintained in the different experimental groups until the end of the experiment, i.e., 100 days postinfection (data not shown). From the early to the chronic phase of infection, the number of total lymphocytes recovered from the lungs of BCG::RD1- or H37Rv-infected mice raised reproducibly from 0.7–0.8 x 106 to 1.5–2.0 x 106 cells/mouse while it was unchanged in BCG::pYUB-412-infected mice. Comparable T cell recruitment/activation also took place in lungs of mice infected initially with {approx}10 CFU/mouse (data not shown). Thus, during the chronic phase of infection, BCG::RD1 is as efficient as M. tuberculosis H37Rv in inducing recruitment/activation of T cells to the site of inflammation. It seems likely that, during the first phase of infection, the kinetics of T cell recruitment/activation by BCG::RD1 or H37Rv reflects the bacterial burden and dissemination of these mycobacteria.



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  		FIGURE 3. Recruitment of activated T cells in the lungs of BCG::RD1- or H37Rv-aerosol-infected C57BL/6 mice. FACS analyses of lung T cells, from mice untreated or aerosol-infected with 100 CFU/mouse of BCG::pYUB412, BCG::RD1, or H37Rv. Cells were gated on CD4+ or CD8+ T cell subsets and studied for their expression of (a) CD44 and CD45RB or CD44 and CD62L or for (b) CD45RB and CD27 markers. Data were obtained at 6 wk postinfection and are representative of three independent experiments performed on pools of cells from four mice.

 
It is noteworthy that, compared with the CD4+ T cell compartment, the CD8+ T cell subset in the lungs of both BCG::RD1 or H37Rv aerosol-infected mice reproducibly contained strikingly higher percentages of CD44highCD45RB– or CD44highCD62L–. Even if the role of CD8+ T cells in antimycobacterial immunity is now admitted and if a dramatic CD8+ T cell recruitment has been observed in the lungs shortly after mycobacterial challenge (19), the basis for this striking CD8+ T cell recruitment/activation is not elucidated.

It has been recently reported that Ag-experienced CD4+ T cells accumulating within mycobacteria-infected lungs can be divided into CD27+ or CD27– subsets and that only the CD27– subset constitutes highly differentiated IFN-{gamma}-producing effector cells (20). Accordingly, we detected substantially increased percentages of CD45RB–CD27– cells within both CD4+ and CD8+ T cell compartments of mice aerosol-infected with BCG::RD1 or H37Rv compared with those infected with control BCG (Fig. 3b). The latter were identical to those of the untreated controls (data not shown).

In vivo expression of RD1-encoded Ag and induction of specific T cell responses

To assess the in vivo expression of the RD1-encoded Ags, immunohistochemistry analysis was performed. Substantial amounts of ESAT-6 were specifically detected within the lesions of the lung of BCG::RD1-, but not of BCG::pYUB412-infected, mice during the acute (data not shown) and chronic (6 wk after challenge) phases of infection (Fig. 4), showing that the recombinant ESAT-6 Ag is actively expressed in the lung tissue. In parallel, we investigated the presence of T cells specific for RD1-encoded immunogens in BCG::RD1- or H37Rv-infected mice. At 6 wk after infection, T lymphocytes recovered from the lungs of these mice released high amounts of IFN-{gamma} in response to in vitro stimulation with the peptide ESAT-6:1–20 containing an immunodominant epitope and, to a lesser extent, in response to the Ag85A:241–260 immunodominant peptide, but not to unrelated MalE:100–114 peptide (Fig. 5a). In accordance with the dissemination of BCG::RD1 or H37Rv to the spleen of aerosol-infected mice (Fig. 1b), the splenocytes of these mice specifically produced substantial levels of IFN-{gamma} in response to the ESAT-6:1–20 peptide or to the recombinant ESAT-6 or CFP-10 proteins (Fig. 5b) and proliferated markedly in response to the ESAT-6:1–20 peptide (Fig. 5c). Cells from untreated mice or BCG::pYUB412-infected mice displayed no responses to RD1-specific Ags (data not shown). Thus, BCG::RD1 is as efficient as H37Rv in priming T cells specific to RD1-encoded Ags.



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  		FIGURE 4. Expression of ESAT-6 in the lungs of BCG::RD1-aerosol-infected C57BL/6 mice. Immunohistochemistry of lungs of BCG::pYUB412- or BCG::RD1-infected mice, performed at 6 wk post-aerosol challenge (100 CFU/mouse), by use of an anti-ESAT-6 mAb as detected at 200- (top) or 400- (bottom) fold magnification.

 


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  		FIGURE 5. Induction of T cell immunity to RD1-encoded Ags in lungs and spleen of BCG::RD1- or H37Rv-aerosol-infected C57BL/6 mice. IFN-{gamma} production by lung interstitial T cells (a) or splenocytes (b) and proliferative responses of splenocytes (c) of BCG::RD1- or H37Rv-aerosol-infected C57BL/6 mice after in vitro stimulation with PPD, ESAT-6:1–20, Ag85A:241–260, or MalE:100–114 (negative control) peptides (10 µg/ml), or ESAT-6, CFP-10, or MalE (negative control) recombinant proteins (5 µg/ml). Results were obtained with pools of cells from four mice at 6 wk following infection with 100 CFU/mouse.

 
Increased capacity of BCG::RD1 to stimulate in vivo innate immunity

Considering the enhanced capacity of BCG::RD1, compared with BCG::pYUB412, to recruit T cells in lungs of aerosol-infected mice, we sought to determine whether BCG::RD1 possesses a stronger capacity to stimulate the innate immunity which provides essential chemoattractants for T cells. To address this issue, using macroarray technology, we first determined the profile of inflammatory cytokines, chemokines, and their receptors expressed by BM-DC infected in vitro with BCG::pYUB412, BCG::RD1 or M. tuberculosis H37Rv at a multiplicity of infection of 4 for 16 h. Comparable gene expression profiles were obtained for BM-DC infected with these three mycobacterial strains (Fig. 6). Indeed, for all experimental groups, we detected intense up-regulation (fold change compared with uninfected cells >10) of IL-1{alpha}, IL-1{beta}, IL-6, IL-12{beta}(p40), and CCL5; marked up-regulation (fold change compared with uninfected cells between 2 and 10) of IL-2R{alpha}, CCL2, CCL3, CCL4, CCL22, CXCL2, and CCR7; and down-regulation (fold change compared with uninfected cells <1) of CCR2 and CCR5. CCL6, CCL9, and CCL17 were constitutively expressed by these BM-DC and their expression was unchanged upon infection (Fig. 6). Thus, subsequent to short-term in vitro infection, in our experimental conditions, comparable activation/inflammation programs were induced by BCG::pYUB412, BCG::RD1, or M. tuberculosis H37Rv.



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  		FIGURE 6. Comparable expression of inflammatory cytokines, chemokines, or their receptors by BM-DC infected with BCG::pYUB412, BCG::RD1, or M. tuberculosis H37Rv. Results were obtained by macroarray analysis of total RNA from purified CD11c+ cells. For each gene, fold changes are ratios between the signal obtained for mycobacteria-infected cells over the one obtained for uninfected control BM-DC. Expression of the other genes studied (see Materials and Methods) was undetectable. Results are representative of two independent experiments.

 
We then investigated in vivo the capacity of BCG::RD1 to recruit/activate cells of the innate immune system. During the chronic phase of infection, i.e., 6 wk postinfection, more CD11c+ cells were detected by immunohistochemistry in the lungs of BCG::RD1-infected mice compared with a few scattered CD11c+ cells in the lungs of their BCG::pYUB412-infected counterparts (Fig. 7a). BAL fluids from BCG::RD1- or H37Rv-infected mice contained increased percentages of CD11c+CD11bhigh cells relative to untreated or BCG::pYUB412-infected mice (Fig. 7b). At the level of BAL CD11c+ cells, a matured phenotype was detected, i.e., substantial up-regulation of H-2Kb and significant up-regulation of CD86 costimulatory molecule (data not shown). Interstitial low-density cells recovered from the lung parenchyma of BCG::RD1-infected mice contained also increased percentages of CD11c+CD11bhigh cells (Fig. 7b). Indeed, in BCG::RD1-infected mice, this population represented 6.1% of the total low-density cells which corresponded to 38.8% of total parenchymal CD11c+ cells. During the chronic phase of infection, typically {approx}0.5 x 106 low-density cells/mouse were recovered from BCG::RD1- or H37Rv-infected mice compared with {approx}0.1 x 106 cells in untreated or BCG::pYUB412-infected controls. Compared with BCG::pYUB412-infected controls, weak but reproducible shifts in expression of H-2Kb, I-Ab, CD40, CD80, and CD86 molecules were also detected notably in CD11c+CD11bhigh cells and, to a lesser extent, in CD11c+CD11bdim cells of BCG::RD1-infected mice showing their mature/activated phenotype (data not shown).



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  		FIGURE 7. Massive recruitment of CD11c+ cells to the lungs of BCG::RD1-aerosol-infected C57BL/6 mice. a, CD11c+ cells detected by immunohistochemistry in lungs of BCG::pYUB412- or BCG::RD1-infected mice, at 6 wk post-aerosol challenge with 100 CFU/mouse. b, FACS analyses of CD11c+ cells isolated from BAL fluids or from interstitial low density cells from lung parenchyma of mice untreated or aerosol-infected with 100 CFU/mouse of BCG::pYUB412, BCG::RD1, or M. tuberculosis H37Rv. Data, obtained with pools of cells from four mice, are representative of two independent experiments. BAL cells are gated on CD11c+ cells. Values indicated in parentheses are percentages of CD11bhigh cells within the CD11c+ cell population.

 
We further compared the pattern of expression of inflammatory cytokines, chemokines, and their receptors in total CD11c+ cells isolated from the lung parenchyma of mice infected with BCG::pYUB412, BCG::RD1 or M. tuberculosis H37Rv during the chronic phase of the infection. Of note were the differences observed between the profile of activation in BM-DC infected in vitro (Fig. 6) or in lung parenchymal CD11c+ cells from infected mice (Fig. 8). Parenchymal CD11c+ cells from BCG::pYUB412-infected mice displayed a profile comparable to the one constitutively expressed by resident cells in untreated mice (fold change compared with cells from uninfected cells {approx}1; Fig. 8). The expression profile of CD11c+ cells from BCG::RD1- or H37Rv-infected mice was similar and displayed marked up-regulation (fold change included between 2 and 10) of IL-1{alpha}, CCL3, CCL4, CCL5, CCL6, CCL9, and TNFR1 substantial up-regulation (fold change >10) of CXCL9 and down-regulation of CCR7 (fold change <1). IL1{beta}, IL15R{alpha}, CXCL2, CXCL13, TNF-{alpha}, and TNF-{beta} were also detectable but the level of their expression was comparable to those of resident CD11c+ cells from untreated controls or BCG::pYUB412-infected mice (Fig. 8). Therefore, during the chronic phase of infection with BCG::RD1, the parenchymal CD11c+ cells display a pattern of expression of cytokines, chemokines, and their receptors distinct from the resident lung CD11c+ cells of uninfected mice. This pattern mimics that of H37Rv-infected mice and suggests the involvement of parenchymal CD11c+ cells in recruitment of other cells of innate and adaptive immunity. Therefore, the intermediately virulent BCG::RD1 is able to recruit/activate innate immune cells in vivo with the same efficiency as fully virulent M. tuberculosis H37Rv.



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  		FIGURE 8. Expression of inflammatory cytokines, chemokines or their receptors by CD11c+ cells isolated from interstitial low density cells from lungs of C57BL/6 mice infected by 100 CFU/mouse of BCG::pYUB412, BCG::RD1, or M. tuberculosis H37Rv. Results, representative of two independent experiments, were obtained by macroarray analysis of total RNA from CD11c+ cells purified from a pool of lung cells from 10 mice at 8 wk post challenge. For each gene, fold changes are ratios between the signal obtained for mycobacteria-infected mice over the one obtained for uninfected mice. Expression of the other genes studied (see Materials and Methods) was undetectable.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The present study demonstrates the role of the ESAT-6 secretion system 1 of M. tuberculosis (RD1) in the virulence and interaction between mycobacteria and the host innate and adaptive immune system. We recently reported that s.c. vaccination of mice with BCG::RD1, but not with control BCG, generates strong T cell responses against ESAT-6 and CFP-10, which correlates with the significantly enhanced capacity of BCG::RD1 to protect mice and guinea pigs against challenge with M. tuberculosis (9). This increased protective efficacy can be due to 1) the secretion of ESAT-6 and CFP-10, leading to the generation of T cell responses to these strong immunogens and/or 2) to increased virulence/persistence of BCG::RD1 with potential impact on the host-pathogen interactions leading to the induction of stronger and longer lasting innate and adaptive antimycobacterial immune responses. It is noteworthy that s.c. immunization of mice with BCG or BCG::RD1 gives rise to comparable MHC class I- or II-restricted T cell responses against reporter Ags shared by these mycobacterial strains with no significant changes in the expression of activation markers by peripheral T cells (our unpublished observations). Thus, following s.c. immunization it was not possible to detect any difference between the immune responses induced by BCG or BCG::RD1. Nevertheless, in the present investigation, based on the pronounced tropism of tubercle bacilli for lungs, the use of an aerogenic infection model allowed us to appreciate better differences between parental BCG and BCG::RD1 in terms of their interaction with the host immune system. Indeed, this study shows that BCG::RD1 displays a degree of virulence intermediate between those of BCG and H37Rv. In contrast to parental BCG, following low-dose aerosol challenge, BCG::RD1 induced 1) chronic infection of lungs, 2) bacterial dissemination to the spleen, 3) marked mobilization of activated T cells to the lung parenchyma, and 4) substantial influx of matured CD11c+CD11bhigh cells to the BAL fluids and lung parenchyma.

In our previous study, proof of partial reversal of attenuation was observed in SCID mice, with a 3 log increase in bacterial growth in the lungs for BCG::RD1 compared with BCG following i.v. infection (12). Changing the route of administration to an aerosol model of infection led to a similar increase in virulence in the immunocompetent mouse model, thus confirming the role of RD1 in active bacterial replication in vivo. Importantly, BCG::RD1 displays increased virulence which, however, does not attain that of M. tuberculosis H37Rv. This result is in accordance with the very recent observation that M. tuberculosis H37Rv deleted for the whole RD1 region is more virulent than BCG in long-term mouse infection (21), pointing out the contribution of other M. tuberculosis-specific genes or genomic regions in virulence. This increased growth of BCG::RD1 seems to be intimately linked to in vivo conditions since no difference in growth rate was observed in vitro for BCG::RD1 compared with BCG in the conditions tested. Just like in vitro grown cells, BCG::RD1 actively expressed the potent immunogen ESAT-6 at the site of infection, regardless of the phase of infection, and this suggests its involvement in the virulence/pathogenicity of mycobacteria. This correlates with the fact that an M. tuberculosis H37Rv mutant deleted for ESAT-6 is severely attenuated in the mouse model and induces only a few lesions (11). Comparative in vitro analysis of the mycobacterial transcriptome of BCG and BCG::RD1 did not reveal other differentially expressed genes than those of RD1 (our unpublished observation). However, the ESAT-6 secretion system 1 apparatus (9) could also be involved in the export of other Ags or virulence factors which remain to be identified.

The spatial distribution and kinetics of recruitment of CD4+ and CD8+ T lymphocytes to the lung following aerogenic or i.v. infection of mice with M. tuberculosis have been well characterized (22, 23). Indeed, percentages of both CD4+ and CD8+ T cells peak at 3–4 wk postinfection, which correlates with the end of the acute phase of infection. Even if the expression of the adhesion molecule CD44 is not essential for the proper trafficking of T cells into the lungs (24), the presence of both CD4+ and CD8+ T cells with CD44high phenotype in the lungs is a hallmark of activated antimycobacterial T cells (22, 23). The CD44high phenotype of T cells is further associated with low expression of CD62L and CD45RB. CD62L, leukocyte adhesion molecule, mediates lymphocyte migration to the organs and leukocyte rolling on vascular endothelium during inflammation and is rapidly cleaved from the cell surface after cellular activation (25). Reduction in the expression of CD45RB is associated with effector phenotype in the T cell compartment. Accordingly, like M. tuberculosis H37Rv and in contrast to the parental BCG, BCG::RD1, with its intermediary degree of virulence, induced comparable influx of CD4+ and CD8+ T cells in the lungs with a characteristic activated CD44highCD45RB–CD62L– cells as well as CD45RB–CD27– T cells that may essentially constitute IFN-{gamma}-producing T cells (20). BCG::RD1 aerosolization efficiently primed T cells specific for RD1-encoded immunogens. In line with the persistence of BCG::RD1 and the effective expression of ESAT-6 within the lungs of BCG::RD1-infected mice, this infection leads to the generation of T cell responses (lymphoproliferation or IFN-{gamma} production) to strong RD1-encoded immunogens at the site of inflammation (lungs) and in peripheral lymphoid organs (spleen). Therefore, complementation with RD1 confers to BCG a potential comparable to that of M. tuberculosis to influence lung T cell phenotype, composition, and functions upon aerosol infection.

We further detected in BCG::RD1-infected mice, in contrast to BCG-infected mice and like in M. tuberculosis-infected counterparts, a CD11c+CD11bhigh cell population both in BAL fluids and lung interstitium. It has been postulated that in response to the infection, a CD11c–CD11b+ DC subpopulation is recruited into the lungs which subsequently up-regulates expression of CD11c and terminally differentiates into interstitial DC upon local production of GM-CSF (26). The phenotype and function of lung CD11c+ cells do not exactly match those of spleen DC because lung CD11c+ cells do not express the lymphoid CD8{alpha} marker but coexpress low levels of myeloid markers F4/80 and CD11b (27). Although CD11c+ cells constitute <1% of the total lung cells, their localization throughout the lung airway epithelia and within alveolar and interstitial spaces favors their interaction with inhaled mycobacteria. If the infection is not contained in the alveolar spaces, the mycobacteria will enter the interstitium which constitutes the site of infection and inflammation (27, 28). In line with this fact, during the chronic phase of infection in our experimental model, BCG::RD1, like M. tuberculosis H37Rv, but not attenuated parental BCG, were detectable in lung parenchymal CD11c+ or CD11c– low-density cells and, to a much lesser extent, in the BAL fluid cells.

The pattern of expression of inflammatory cytokines, chemokines, and their receptors governs the recruitment and function of cells of the immune system. In vitro studies, particularly focused on mouse or human macrophages and, to a lesser extent, on human DC, have shown that M. tuberculosis is a powerful inducer of cytokine and chemokine expression (28, 29, 30, 31, 32). Strains of mycobacteria with different degrees of virulence, i.e., H37Rv and H37Ra, can induce different levels of chemokines (33). Moreover, evidence exists for the presence of ESAT-6 in the mycobacterial cell wall (12), whose components are thought to play a major role in the activation of cells of innate immunity. In contrast, it has been described that ESAT-6 and CFP-10 can profoundly influence cells of innate immunity (34). Indeed, their role in induction of TNF release by macrophages, their synergistic effect with IFN-{gamma} for induction of NO synthesis by macrophages (35), and their potential to promote derivation of BM precursors to a CD11c+ DC-like population have been observed (36). Considering these points, we undertook comparative global analyses of the expression of inflammatory cytokines, i.e., chemokines and their receptors by CD11c+ cells infected in vitro or in vivo with attenuated BCG, intermediately virulent BCG::RD1, or virulent M. tuberculosis H37Rv. Using macroarray technology, we detected equivalent degrees of up-regulation of known innate response-related inflammatory cytokines IL-1{alpha}, IL-1{beta}, IL-6, and IL-12{beta}(p40) as well as of CCL2 (MCP-1), CCL3 (MIP-1{alpha}), CCL4 (MIP-1{beta}), and CCL5 (RANTES) chemokines at the level of BM-DC infected by each of these three mycobacterial strains. It is known that IL-1 can drive IL-2R expression by T cells leading to their activation, that IL-6 contributes in the control of acute mycobacterial infection, and that IL-12 provides the key innate signal to initiate production of IFN-{gamma} by T cells (37). CCL2, CCL3, CCL4, and CCL5, interacting in a redundant manner with CCR2 or CCR5 receptors, provide chemoattraction signals for CCR2+ or CCR5+ cells, i.e., monocytes, immature DC, NK cells, as well as activated T lymphocytes to the site of infection (28, 37). All of these cell types are known to contribute to the control of infection with mycobacteria. We also detected comparable down-regulation of CCR2 and CCR5 and up-regulation of CCR7, a scheme allowing migration of these cells to lymphoid tissues (38). Furthermore, we observed comparable up-regulation of CCL22 (favoring expansion of Th2 responses) and of CXCL2 (major chemoattractant of polymorphonuclear neutrophils) (39). Such a program, induced in vivo in DC, could lead to migration of DC into secondary lymphoid tissues and to chemoattraction of neutrophils, immature DC, Th1, and Th2 cells. The identical pattern of expression of inflammatory cytokines, chemokines, and their receptors induced after infection with parental BCG, BCG::RD1, or M. tuberculosis H37Rv shows that, in our experimental conditions, the degree of virulence and expression of RD1-encoded components do not influence the activation program induced in BM-DC upon short-term in vitro infection.

We previously described transient in vivo maturation of spleen DC of mice acutely infected with high doses of BCG administered by i.v. route (40). In this study, we essentially observed a mature phenotype in CD11c+CD11bhigh cells from BAL fluids or from lung interstitium of mice chronically infected with BCG::RD1 or H37Rv. We further investigated the activation/inflammation program of CD11c+ cells from lung parenchyma of mice uninfected or infected via the aerosol route with parental BCG, BCG::RD1, or H37Rv. At 8 wk after aerosol challenge, CD11c+ cells from BCG-infected mice displayed the same gene expression profile as cells from uninfected mice. CD11c+ cells from mice infected with BCG::RD1, with an intermediate degree of virulence, mimicked those from mice infected with the virulent M. tuberculosis H37Rv. They both showed marked up-regulation of IL-1{alpha} and TNFR{alpha} as well as CCL3 (MIP-1{alpha}), CCL4 (MIP-1{beta}), CCL5 (RANTES), CCL6, CCL9 (MIP-1{gamma}), and CXCL9 (monokine induced by IFN-{gamma}). The latter chemokines are all related to cytokines that induce Th1 responses (39). In contrast to BM-DC, constitutive expression of CCL17 and mycobacteria-mediated up-regulation of CCL22 were not observed in lung CD11c+ cells. Because CCL17 and CCL22 are related to cytokines inducing Th2 responses (39), it is likely that parenchymal lung CD11c+ present during the chronic infection with BCG::RD1 or H37Rv may provide signals that favor influx of new CD11c+ cells and generation of Th1-, but not Th2-, biased responses. In contrast to what was observed with BM-DC, lung CD11c+ cells of BCG::RD1- or H37Rv-infected mice showed down-regulation of CCR7 which is in accordance with the fact that unlike DC in lymphoid tissues, lung CD11c+ cells did not express CCR7. Absence of CXCL9 expression in BM-DC infected in vitro with mycobacteria and its up-regulation in lung CD11c+ cells from chronically infected mice may suggest that amplification of this IFN-{gamma}-induced chemokine requires adaptive immune signals. Because of limited numbers of DC present in organs, very few ex vivo studies have been focused on detailed activation programs of DC from mycobacteria-infected mice. Therefore, our results provide the first indications on in vivo functions of DC recruited to the lungs during the chronic phase of mycobacterial infection and show important differences between inflammation-related "gene signatures" of BM-DC infected in vitro and lung DC from aerosol-infected mice. These differences show that DC generated in vitro may not exactly reflect functions of lung DC in interaction with mycobacteria in the complex context of chronic infection and inflammation.

Our data provide the first demonstration of such a significant effect on virulence/persistence/dissemination and marked modifications of the host innate and acquired immune responses to a bacterium subsequent to its complementation with a novel protein secretion system required for virulence and immunogenicity, the ESAT-6 system 1.

In conclusion, we demonstrated here that although BCG::RD1 is markedly less virulent than M. tuberculosis H37Rv, it possesses the same potential as H37Rv to induce massive recruitment of activated CD4+ or CD8+ T cells and of mature/activated CD11c+ innate cells to the site of infection. Moreover, BCG::RD1 has the same efficiency that H37Rv in inducing inflammatory programs in CD11c+ innate cells for recruitment of other immune cells and for induction of Th1 responses. These characteristics of BCG::RD1 may explain its enhanced capacity to protect against M. tuberculosis. Consequently, decreasing the virulence of BCG::RD1 while preserving its immunogenicity constitutes a promising strategy to design a new vaccine against tuberculosis.


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


   Acknowledgments
 
We gratefully acknowledge Peter Sebo and Marcella Simsova for providing ESAT-6 and CFP-10 recombinant proteins; Jean-Marie Clément for providing MalE recombinant protein; Richard Lo-Man, Gilles Marchal, and Micheline Lagranderie for various suggestions, and Eddie Maranghi for expert assistance in animal care.


   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 Institut Pasteur (Programme Transversal de Recherche 110) and Institut National de la Santé et de la Recherche Médicaleand by a fellowship from European Union Grant QLK2-CT-2001-02018 (to P.B.). Back

2 Address correspondence and reprint requests to Dr. Laleh Majlessi, Unité de Biologie des Régulations Immunitaires, Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris, Cedex 15, France. E-mail address: lmajless{at}pasteur.fr Back

3 Abbreviations used in this paper: BCG, bacille Calmette-Guérin; MalE, maltose binding protein from Escherichia coli; RD1, region of difference 1; CFP-10, culture filtrate protein, 10 kDa; ESAT-6, early secreted antigenic target, 6 kDa; DC, dendritic cell; BM, bone marrow derived; BAL, bronchoalveolar lavage. Back

Received for publication August 18, 2004. Accepted for publication January 12, 2005.


   References
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 Materials and Methods
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 Discussion
 Disclosures
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J. Immunol., September 15, 2007; 179(6): 3973 - 3981.
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M. I. de Jonge, G. Pehau-Arnaudet, M. M. Fretz, F. Romain, D. Bottai, P. Brodin, N. Honore, G. Marchal, W. Jiskoot, P. England, et al.
ESAT-6 from Mycobacterium tuberculosis Dissociates from Its Putative Chaperone CFP-10 under Acidic Conditions and Exhibits Membrane-Lysing Activity
J. Bacteriol., August 15, 2007; 189(16): 6028 - 6034.
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X. V. Guo, M. Monteleone, M. Klotzsche, A. Kamionka, W. Hillen, M. Braunstein, S. Ehrt, and D. Schnappinger
Silencing Essential Protein Secretion in Mycobacterium smegmatis by Using Tetracycline Repressors
J. Bacteriol., July 1, 2007; 189(13): 4614 - 4623.
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