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Progression of Pulmonary Tuberculosis and Efficiency of Bacillus Calmette-Guérin Vaccination Are Genetically Controlled via a Common sst1-Mediated Mechanism of Innate Immunity¹

Bo-Shiun Yan^*, Alexander V. Pichugin^*, Ousman Jobe^*, Laura Helming^2,*, Evgeniy B. Eruslanov^*, José A. Gutiérrez-Pabello^*, Mauricio Rojas^*,, Yuriy V. Shebzukhov^*, Lester Kobzik and Igor Kramnik^3,*

^* Department of Immunology and Infectious Diseases and Physiology Program, Department of Environmental Health, Harvard School of Public Health, Boston, MA 02115; and Grupo de Inmunología Celular e Inmunogenética, Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Using a mouse model for genetic analysis of host resistance to virulent Mycobacterium tuberculosis, we have identified a genetic locus sst1 on mouse chromosome 1, which controls progression of pulmonary tuberculosis. In vitro, this locus had an effect on macrophage-mediated control of two intracellular bacterial pathogens, M. tuberculosis and Listeria monocytogenes. In this report, we investigated a specific function of the sst1 locus in antituberculosis immunity in vivo, especially its role in control of pulmonary tuberculosis. We found that the sst1 locus affected neither activation of Th1 cytokine-producing T lymphocytes, nor their migration to the lungs, but rather controlled an inducible NO synthase-independent mechanism of innate immunity. Although the sst1^S macrophages responded to stimulation with IFN- in vitro, their responsiveness to activation by T cells was impaired. Boosting T cell-mediated immunity by live attenuated vaccine Mycobacterium bovis bacillus Calmette-Guérin or the adoptive transfer of mycobacteria-activated CD4⁺ T lymphocytes had positive systemic effect, but failed to improve control of tuberculosis infection specifically in the lungs of the sst1^S animals. Thus, in the mouse model of tuberculosis, a common genetic mechanism of innate immunity mediated control of tuberculosis progression in the lungs and the efficiency of antituberculosis vaccine. Our data suggest that in immunocompetent humans the development of pulmonary tuberculosis and the failure of the existing vaccine to protect against it, in some cases, may be explained by a similar defect in a conserved inducible NO synthase-independent mechanism of innate immunity, either inherited or acquired.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
One-third of the human population is estimated to be infected with virulent Mycobacterium tuberculosis (MTB),⁴ while <10% of the infected immunocompetent individuals are at lifetime risk of developing clinical tuberculosis (1). Epidemiological (2) and genetic (3, 4) studies demonstrate that genetic variation within human populations significantly contributes to the host heterogeneity in terms of tuberculosis resistance. Analysis of tuberculosis infection using experimental animals also demonstrated a role of host genetic variation in determining outcomes of host-pathogen interactions (5, 6, 7, 8).

The mouse model of tuberculosis infection is most widely used and was instrumental in elucidation of essential pathways of antituberculosis immunity (reviewed in Ref. 9). Using a mouse model of infection with virulent MTB, it has been demonstrated that targeted mutations in genes essential for Th1-mediated immune responses or macrophage responsiveness to IFN- and TNF- result in systemic failure of host resistance to MTB (10, 11, 12, 13). Subsequently, mutations that affect those pathways in humans have been shown to result in extreme susceptibility to mycobacterial infection, including disseminated disease caused by otherwise nonpathogenic mycobacteria (reviewed in Ref. 4).

It has been well-established that immunocompetent standard inbred strains of laboratory mice dramatically differ in their susceptibility to infection with virulent MTB. To date, several studies have been performed to dissect the genetic control of host resistance to tuberculosis using classical linkage analysis in crosses between resistant and susceptible inbred mouse strains. In all of those studies, genetic control of tuberculosis resistance was found to be multigenic. Apt and colleagues (14) mapped three quantitative trait loci in a cross between A/Sn and I/St mouse strains. Mitsos et al. (15, 16) using a cross between DBA/2 (susceptible) and C57BL/6 (resistant) inbred mouse strains mapped four tuberculosis-resistance quantitative trait loci. Importantly, these authors found that the same locus on mouse chromosome 7 controlled the survival of mice after i.v. challenge as well as multiplication of MTB in their lungs after infection by aerosol.

Using a cross of the C3HeB/FeJ (susceptible) with the tuberculosis-resistant C57BL/6J (B6) inbred mice, we mapped five host resistance loci (17, 18, 19). The major distinction of tuberculosis progression in the C3HeB/FeJ mice was the development of large necrotic lesions in their lungs starting as early as 3 wk after systemic i.v. infection and 6 wk after an aerosol challenge with MTB. The first susceptibility locus identified in our studies, sst1 (supersusceptibility to tuberculosis 1), was responsible for early death of the infected mice (18). Substitution the sst1^R allele for the sst1^S in C3H.B6-sst1 congenic mice increased their survival after i.v. infection with 10⁵ CFU of MTB to 10–12 wk, as compared with 3.5–4 wk of the parental C3HeB/FeJ mice. Most importantly, it prevented the formation of necrotic lung lesions after both systemic (i.v.) and low dose aerosol infection (20).

In humans, damage to lung tissue by virulent MTB is necessary for the pathogen transmission. Progression of the disease leads to formation of necrotic lung lesions. When the expanding necrotic lesions erupt into airways, their contents containing live mycobacteria are released and spread to other hosts via aerosol. Therefore, the ability to induce formation of large necrotic lesions in the lung is considered a key element of a highly successful virulence strategy of MTB. This, however, occurs only in a relatively small proportion of infected individuals, most of which are immunocompetent, but susceptible to tuberculosis infection. Importantly, the existing antituberculosis vaccine, live attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG), is inefficient in preventing pulmonary tuberculosis in adults, while it protects against disseminated disease in children (21, 22). From this perspective, understanding mechanisms of particular vulnerability of lung tissue to tuberculosis infection in a specific context of a susceptible, but otherwise the immunocompetent host is critical for developing efficient preventive and therapeutic interventions (9). However, the mouse model was considered inadequate for those studies, because most of the standard inbred mouse strains do not develop typical necrosis within tuberculosis lung lesions (23). Therefore, identification of the sst1 locus that controls formation of necrotic lung lesions in mice and generation of the sst1 congenic inbred mouse strains provided a unique opportunity to address this critical aspect of tuberculosis pathogenesis using mouse model of infection with virulent MTB.

Previously, we have demonstrated that the sst1-dependent mechanism of immunity was expressed by the bone marrow-derived cells. In vitro, macrophages isolated from the sst1^R mice were more efficient than the sst1^S ones in controlling the intracellular growth of two unrelated intracellular pathogens, MTB and Listeria monocytogenes. Using positional cloning we have identified a candidate gene within the sst1 locus, Ipr1 (intracellular pathogen resistance 1) (20). Expression of the Ipr1 gene was strongly up-regulated in the sst1^R macrophages upon infection with MTB or priming with type 1 and type 2 IFNs, while this gene was not expressed in the sst1^S macrophages under similar conditions. In vitro, expression of the Ipr1 transgene in the sst1^S macrophages improved their ability to control multiplication of the two intracellular pathogens. It also prevented necrotic death of the infected macrophages and facilitated a switch in the mechanism of their death to an apoptotic pathway.

In this report, we further address a specific role of the sst1 locus in host resistance to tuberculosis infection in vivo, especially in control of pulmonary disease. We demonstrate that the extent of lung inflammation caused by MTB infection as well as the efficiency of vaccination with M. bovis BCG against pulmonary tuberculosis are controlled by the sst1-mediated mechanism of innate immunity, although two major known components of host resistance to tuberculosis—activity of CD4-positive Th1 cells and the NO-dependent effector mechanism—are not compromised in the sst1^S mice.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice

Four C3H substrains—C3HeB/FeJ, C3H/HeJ, C3H/HeOuJ, C3H/HeSnJ—as well as C3Smn.CB17-Prkdc^scid/J and B6.129P2-Nos2^tm1Lau (B6.Nos2^–/–) mice were purchased from The Jackson Laboratory. The congenic C3H.B6-sst1 was generated by introgression of a 25 cM sst1-containing segment from C57BL/6J (B6) into the C3HeB/FeJ strain as previously described (20). The B6.C3H-sst1 congenic mice were obtained using the similar strategy, but an 12-cM interval of C3H-derived susceptible sst1 allele was transferred into B6 genetic background (19). To generate the sst1-susceptible congenic mouse strain, in which the Nos2 gene encoding iNos was inactivated, the B6.C3H-sst1 strain was crossed with B6-Nos2^tm1mice (the iNOS knockout strain, which was purchased from The Jackson Laboratory). Their F₁ offspring were backcrossed on B6-Nos2^tm1 and the Nos2^–/– homozygous and the sst1 heterozygous progeny of that backcross were intercrossed to produce a population of Nos2^–/– mice segregating at the sst1 locus. These mice were tested for susceptibility to MTB infection and also were used to establish the B6.C3H-sst1, Nos2^–/–congenic mouse strain homozygous for the sst1-susceptible allele and Nos2^tm1 mutation on the B6 genetic background (B6.C3H-sst1^S/S, Nos2^–/–). The original B6-Nos2^tm1mice, which carried the B6-derived sst1^R allele, was used as a control.

The scid mutation was introduced from the C3Smn.CB17-Prkdc^scid/J (C3H.scid) mice into the C3H.B6-sst1 genetic background to obtain the C3H.B6-sst1, scid strain. From this strain, the Prkdc^scid mutation was further moved on the C3HeB/FeJ background (Ipr1-negative). Thus, the two strains that carry the sst1^R and the sst1^S alleles in the presence of scid mutation on the C3H genetic background were generated. Mice were bred and maintained under specific-pathogen-free conditions in animal facilities at the Harvard Medical School and given autoclaved chow and water ad libitum. All experiments were performed with the full knowledge and approval of the Standing Committee on Animals at Harvard Medical School (protocol no. 03000).

Bacteria and infection of mice

MTB (Erdmann strain; Trudeau Institute, Saranac Lake, NY) was grown to mid-log phase in Middlebrook 7H9 liquid medium, washed, resuspended in PBS containing 1% FCS and 10% glycerol, aliquoted, and frozen at –80°C until use. For infection of mice, the frozen stock was melted, sonicated, and used on the same day. The bacteria were diluted by PBS containing 0.05% Tween 80. Each mouse was infected via tail vein injection with 1 x 10⁵ CFU of MTB in 100 µl. For infection of macrophages in vitro, the bacteria from a frozen stock was grown in liquid medium 7H9 (BD Biosciences Microbiology Systems) supplemented with 10% oleic acid/albumin/dextrose/catalase (Difco), 0.2% glycerol, and 0.05% v/v Tween 80. The bacteria were grown with agitation for 4–6 days, washed twice, sonicated using a cup sonifier (Branson Sonifier) twice for 5 s and filtered through 5.0-µm filter (Micron Separation). Bacterial density was adjusted by OD at 600 nm. The precise numbers of bacteria were estimated by plating serial dilutions of the initial inoculums on 7H10 agar. The colonies were counted after incubation for 3 wk at 37°C.

For BCG vaccination, the mice were infected i.v. with 1 x 10⁶ CFU of live attenuated strain of M. bovis BCG Pasteur and challenged with MTB i.v. 10 wk after immunization. Aerosol infection was performed using a Wisconsin aerosol chamber as described (20). For all experiments, three to four animals per group were tested at each time point. Mice were sacrificed using halothane anesthesia. The bacterial loads in infected organs were determined by plating 10-fold dilutions in PBS with 0.05% Tween 80 of organ homogenates on 7H10 Middlebrook agar enriched with 10% oleic acid/albumin/dextrose/catalase for 21–28 days at 37°C.

Isolation and MTB infection of murine bone marrow-derived macrophages (BMDM) in vitro

BMDM were isolated from femurs and tibias of mice (6–8 wk old). The cells were cultured in a complete culture medium mixed with 50% DMEM and 50% HAM F-12 (HyClone) containing 10% FCS (HyClone), and 1 ng/ml IL-3 (Sigma-Aldrich) and 20% L-929 fibroblast-conditioned medium were used as a source of macrophage colony stimulation factor. After a 2-day culture, the nonadherent cells were collected and cultured in medium containing 40% L929-conditioned medium for 4 days. The cells would differentiate into macrophages and were maintained in complete medium containing 20% of L-929-conditioned medium to form monolayers.

BMDM monolayers were infected with MTB at a multiplicity of infection (MOI) 1 bacteria per 10 macrophages (MOI 1:10). The cells were washed by PBS with 1% FCS at 6 h postinfection and incubated in complete medium. At indicated time points, three coverslips with macrophage cells were transferred in 2 ml of sterile distilled water with 0.1% Triton X-100 to lyse the cells. A 10-fold serial dilution of the cell lysates was plated on 7H10 agar, and colonies were counted to determine the number of intracellular MTB after incubation at 37°C for 3 wk.

RNA preparation and quantitation of mRNA expression

RNA samples from homogenized organs or cells were prepared using TRIzol reagent (Invitrogen Life Technologies) followed by the DNase I treatment and purification using RNeasy column (Qiagen) according to the manufacturers’ recommendations. Total RNA was reverse transcribed using RETROscript kit (Ambion) and random decamers, diluted with sterile water to 100 µl and 4 µl of the final sample, were used for PCR amplification.

Gene expression was analyzed by either quantitative real-time PCR or semiquantitative PCR. For quantitative real-time PCR, each reaction was prepared using the following reagents and concentrations in 25 µl: 1.25 U of AmpliTaqGold DNA polymerase (Applied Biosystems), 1x GeneAmp PCR buffer II, 2.5 mM MgCl₂, 0.2 mM dNTPs, 0.1 µM primers, 1/30,000 dilution of 10,000x SYBRE Green nucleic acid stain (Molecular Probes), 100 nM 6-carboxy-X-rhodamine (ROX reference dye; Invitrogen Life Technologies), and 5 µl of cDNA sample. The reaction was run using an ABI-7900 (Applied Biosystems) as per the following condition: 10 min at 95°C, 40 cycles (95°C, 20 s; 60°C, 30 s; 72°C, 40 s), 5 min at 72°C, and followed by one cycle (95°C, 15 s; 60°C, 15 s; 72°C, 15 s) for a dissociation curve analysis which was performed on all samples to detect nonspecific products. cDNA standards were generated by serially diluting purified amplicon of each gene over a range of 10⁷–10² copies. 18S rRNA was used as an internal control and each sample was set up in triplicate. The semiquantitative RT-PCR was performed as described in Ref. 24 . In brief, 15 µl of reaction mixture contained 0.75 U of AmpliTaqGold DNA polymerase, 1x GeneAmp PCR buffer II (both from Applied Biosystems), 2.5 mM MgCl₂, 0.1 µM primers, 0.2 mM dNTPs, [-³²P]dCTP, and 5 µl of cDNA sample. PCR was performed according to the following program: 95°C for 10 min, 35 cycles of 20 s at 95°C, 30 s at 60°C, 40 s at 72°C, and a final step at 72°C for 10 min. A total of 1 µl of PCR product was added in 10 µl of DNA-loading buffer. Radioactively labeled PCR products were separated on acrylamide gel. The gels were dried and incorporated radioactivity was quantitated by PhosphoImager Storm 860 (Molecular Dynamics) using ImageQuant software. The primers for PCR were designed to have an annealing temperature of 55°C to 60°C as following sequences: inducible NO synthase (iNOS): forward, 5'-CTT CAA TGG TTG GTA CAT GGG CAC-3'; reverse, 5'-TCA ACA TCT CCT GGT GGA ACA CAG-3'. 18S rRNA: forward, 5'-AGT CCC TGC CCT TTG TAC ACA-3'; reverse, 5'-CGA TCC GAG GGC CTC ACT A-3'. LRG-47: forward, 5'-TAG TTG CCC AGG ACA CCA AGA ACA-3'; reverse, 5'-TGG CTT CTG TGG AAG AAG AAG GGT-3'.

Histopathology

Organs of infected mice were fixed with 10% buffered formalin for >24 h and then embedded in paraffin. These tissues were sectioned and stained with H&E by standard procedure at the Harvard Rodent Histopathology Core Facility. Blinded semiquantitative scoring of histopathologic changes was performed on coded slides by a pathologist (L. Kobzik). Each slide was scored from 0 to 3 according to presence of pneumonitis (none, <10% of lung; moderate, 10–50%; severe, >50%, respectively) and for presence of granulomas and necrosis (none, rare, common, extensive, respectively).

Auramine/rhodamine staining

Identification of acid fast bacilli was performed on formalin-fixed tissue sections using auramine/rhodamine dye (0.1% auramine O, 0.01% rhodamine B (Sigma-Aldrich) in H₂O) for 20 min at room temperature in the dark, followed by destaining with 3% HCl in 70% EtOH and counterstained in Mayer’s hematoxylin (VWR).

TUNEL assay

Mice were infected i.v. with MTB (four per group). At 2 and 4 wk postinfection, their lungs were flushed and inflated with 4% paraformaldehyde in PBS and fixed for 24 h and embedded. TUNEL assay was performed using the ApopTag Apoptosis Detection kit (Serologicals) according to the manufacturer’s protocol.

Isolation of lung cells

Isolation of cells from tuberculosis lung lesions was performed essentially as described (25, 26). Briefly, mice were anesthetized with sodium pentobarbital (100 µl with 64.8 mg/ml). Following flushing of the blood vessels with 20 ml of PBS containing 10 U heparin/ml, bronchoalveolar lavage was performed with 5 ml of PBS via cannulated trachea. The lungs were collected and massaged in a petri dish containing PBS and subsequently transferred into a petri dish containing digestion medium (L-15 medium, 10 mM HEPES, 5% FCS, kanamycin (0.05 µg/ml), collagenase IV (150 U/ml; Worthington) and DNase I (50 µg/ml; Sigma-Aldrich)). A total volume of 10 ml of digestion buffer was used to digest the lungs of each mouse during incubation at 37°C in a shaker for 90 min. The cells were disaggregated by repeated pipetting and filtered through a 100-µm cell strainer to remove clumps. The interstitial lung cell suspension was washed three times with PBS supplemented with 1% FCS. The total number of lung cells from each mouse was counted in a 1:10 suspension of 2% acetic acid. The viability of the cells as determined by trypan blue exclusion was >97% at each time point.

Immunization and isolation of lymph node cells

Mice were immunized with CFA (Difco Laboratories) in the footpad of the hind feet. Popliteal lymph nodes were removed after 10–14 days, rinsed in PBS, and placed in a petri dish containing DMEM supplemented with 1% FCS. The lymph nodes were teased apart to release the lymph node cells. Following filtration through a 100-µm cell strainer, the lymph node cell suspension was washed three times with DMEM supplemented with 1% FCS and resuspended in complete culture medium. For detection of intracellular cytokines by mycobacteria-reactive T cells, the lymph node cells were labeled with CFS (Molecular Probes) at 2 µM for 30 min in serum-free medium at room temperature. The cells were washed and stimulated with purified protein derivative (PPD; 10 µg/ml) for 96 h and subsequently with PMA (50 ng/ml) and ionomycin (1 µM) for 6 h. Monensin (3 µM) was added to the cultures during the last 4 h. The gate was set on proliferating (CFSE^low) cells.

Adoptive transfer of T lymphocytes in scid mice

A total of 50 x 10⁶ splenocytes obtained from immunocompetent C3H.B6-sst1 (sst1^R) and C3HeB/FeJ (sst1^S) mice were depleted from plastic adherent cells and transferred i.v. into C3H.B6-sst1, scid mice, which were infected with 1 x 10⁴ CFU of MTB 7 days later. CD4⁺ T cells were isolated from inguinal lymph nodes of C3H.B6-sst1 mice 2 wk following s.c. vaccination into two points with 1.4 x 10⁷ CFU of M. bovis BCG Pasteur and separated via high-affinity negative selection on the Mouse T Cell CD4 Subset Column (R&D Systems) according to the manufacturer’s instructions. A total of 4.4 x 10⁶ freshly purified cells (99% CD3⁺CD4⁺CD8^– cells by flow cytometry) were injected i.v. 24 h following infection of recipients with 10⁴ CFU of MTB Erdmann by i.v. route.

Culture of cells from tuberculosis lung lesions

Unfractionated lung cells were cultured at 2 x 10⁶/ml in DMEM/F12 plus supplements (10% heat-inactivated FCS, 10 mM HEPES, 100 U of penicillin/ml, 100 µg of streptomycin/ml and 4 mM L-glutamine) in the presence or absence of PPD (10 µg/ml; Mycos Research Laboratories). Cultures were incubated at 37°C/5% CO₂ for 4 days. Culture supernatants were harvested and stored at –80°C. In other assays, macrophages were depleted from lung cell suspensions by adherence onto tissue-culture flasks. The resulting nonadherent cells were cultured at 1 x 10⁶/ml in plates coated with anti-CD3 (2 µg/ml) or stimulated with PPD in the presence of gamma-irradiated (2000 rad) syngeneic splenocytes. Culture supernatants were harvested after 24 h in the anti-CD3-stimulated cultures and after 3 days in the cultures containing APC and PPD. Culture supernatants were kept at –80°C. For detection of intracellular cytokines, lung T cells were enriched by adherence and stimulated with PPD (10 µg/ml) in the presence of gamma-irradiated syngeneic splenocytes for 96 h, and monensin (3 µM) was added to the cultures during the last 4 h. The cells were fixed and stained as described below.

Flow cytometry

mAbs specific for mouse CD3 (145-2C11, hamster IgG1), CD4 (L3T4 clone H129.19, rat IgG2a), CD8 (Ly-2 clone 53-6.7, rat IgG2a), CD69 (H1.2F3, hamster IgG1), I-A^k (clone 11-5.2, mouse IgG2b), and CD25 (IL-2R chain clone 7D4, rat IgM) were purchased from BD Biosciences as direct conjugates to FITC, PE, PerCP, or biotin. mAb specific for mouse F4/80 (clone BM8, rat IgG2a) was purchased from Caltag Laboratories as direct conjugate to PE. Biotinylated Ab to Tim3 was gifted by Dr. V. Kuchroo (Brigham and Women’s Hospital, Boston, MA). Unseparated lung and lymph node cells were washed in PBS containing 1% BSA and 0.01% NaN₃ and incubated for 10 min at 4°C in the same buffer containing FcR-blocking Ab (CD16/CD32; BD Biosciences). After an additional wash, cells were triple stained for 30 min at 4°C with directly or indirectly conjugated Abs according to the manufacturer’s instructions. Stained cells were washed three times in PBS containing 1% BSA and 0.01% NaN₃, fixed in PBS containing 2% paraformaldehyde, and analyzed by flow cytometry using FACSCalibur cytometer (BD Biosciences) and CellQuest software (BD Biosciences).

Intracellular cytokine staining

Stained for surface markers and fixed cells were permeabilized by washing two times in perm/wash buffer (BD Biosciences) and incubated for 15 min in BD Biosciences perm/wash buffer. Pellet cells were resuspended in BD Biosciences perm/wash buffer containing PE-conjugated IFN- Ab (clone MXG1.2, rat IgG1; BD Biosciences) and incubated on ice for 30 min. After incubation, cells were washed twice BD Biosciences perm/wash buffer and resuspended in staining buffer before flow cytometric analysis (see above).

Statistical analyses

CFU data were analyzed by two-way ANOVA (type III). Survival curves were analyzed using GraphPad Prism 3.02. The influence of time postinfection on recruitment of different T cell populations was done by Spearman rank correlations. Values of p 0.05 were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The sst1 locus prevents formation of necrotic lung lesions and limits lung inflammation in vivo

To detect the earliest correlates of the sst1-mediated resistance in vivo, we studied the kinetics of disease progression in the sst1^S and sst1^R congenic mice at short (3–4 days) intervals after systemic i.v. infection with 5 x 10⁴ CFU of MTB Erdman. During the first 11 days postinfection, the initial rate of multiplication of MTB was similar in both sst1^S and sst1^R congenic mice. The first difference in the bacterial loads was detected at 15 days postinfection: it was five times higher in the spleens and only 1.5 times higher in the lungs of the sst1^S mice (Fig. 1A). Within the next 3 days, however, the bacteria continued to grow rapidly in the lungs of the sst1^S mice, while the number of viable MTB in their spleens started to decline. MTB continued rapid multiplication in the lungs of the sst1^S mice until the mice died at 25–28 days postinfection with 10⁹ CFU of MTB in their lungs. In contrast, the bacterial load in the lungs and the spleens of the sst1^R congenic mice remained stable during that period.

View larger version (53K): [in this window] [in a new window]   FIGURE 1. The sst1 locus prevents formation of necrotic lung lesions and limits lung inflammation after i.v. infection of mice with 5 x 104 CFU of MTB. A, MTB bacterial load per organ after i.v. infection with 5 x 104 CFU of MTB Erdman. Four mice per group were used in the experiment. B, Kinetics of the chemokine gene expression in total lung tissue were analyzed using RT-PCR (RNA from three animals per group was pooled, and normalization was done using 18S rRNA-specific primers, data not shown). C, Pathological scores of tuberculosis lung lesion at the indicated times postinfections (see Materials and Methods for score definitions). Three tissue sections from three animals per group were analyzed. D, Morphology of tuberculosis lung lesions 3 wk after i.v. infection. Upper panels, H & E (original magnification, x100); lower panels, MTB stained with auramine/rhodamine (original magnification, x400). E, Analysis of chemokine gene expression in total lung at 11 and 15 days postinfection using semiquantitative RT-PCR. Three animals were analyzed individually per strain at each time point, normalization was done using 18S rRNA. F, Comparison of normalized expression values of chemokines presented in E using t test. The expression of MIP-2 and MCP-1 was significantly higher in the lungs of the sst1S mice 15 days postinfection (p = 0.028 and 0.009, respectively).

View larger version (78K): [in this window] [in a new window]   FIGURE 2. Progression of tuberculosis lesions and appearance of TUNEL-positive cells in the lungs of the sst1S and sst1R congenic mice after i.v. infection with 5 x 104 CFU of MTB Erdman. A, Histopathology (H & E staining) of the tuberculosis lung lesions at 2 wk (A and B; original magnification, x40) and 3 wk (C and D; original magnification, x100) postinfection: progressive development of necrotizing nodules in the lungs of the sst1S mice (A and C) as compared with moderate interstitial pneumonitis in sst1R mice (B and D). B, TUNEL assay on lung sections at 2 wk (A and B; original magnification, x400) and 4 wk (C and D; original magnification, x100) postinfection. Progressive accumulation of TUNEL-positive cytoplasmic material is observed within the lung lesions of the sst1S mice (A and C); in contrast, TUNEL staining is nuclear (characteristic of apoptotic cells) in non-necrotizing lung lesions of the sst1R mice (B and D).

Expression of proinflammatory chemokines in the lungs increased as the infection progressed (Fig. 1B). The levels of proinflammatory chemokines MIP-1, MIP-1β, MIP-2, and MCP-1 mRNA were higher in the lungs of the infected mice as compared with their spleens (data not shown). At day 11, the cytokine levels were similar on both genetic backgrounds, but at day 15, the levels of MIP-2 and MCP-1 mRNA were 3- to 5-fold higher in the lungs of the sst1^S mice (Fig. 1, E and F).

Between the second and the fourth week of infection, we observed increased influx of inflammatory cells in the lungs of the sst1^S mice. At 4 wk, total numbers of inflammatory interstitial cells, which were isolated from the lungs using collagenase digestion, were twice as much as compared with the sst1^R congenics. At this time, CD11b⁺ myeloid cells represented 46% of all inflammatory interstitial cells in the lungs of the sst1^S mice, while this population comprised only 28% of interstitial cells in the lungs of the sst1^R mice, as determined by FACS analysis (p < 0.01).

To summarize, formation of necrotic microfoci and increased production of proinflammatory chemokines were the earliest signs associated with the sst1-susceptible phenotype in vivo that we were able to detect two weeks after systemic infection. After this time point, the differences between the sst1^S and sst1^R congenic mice rapidly increased: expanding necrotic inflammation, increased influx of myeloid cells including granulocytes, spread MTB throughout the lung tissue, and rapid growth of the bacteria with a doubling time of 28 h (similar to the growth rate of the bacteria in culture broth) were characteristic for the lungs of the sst1^S mice, which rapidly succumbed to infection. The bacterial loads in the lungs of the sst1^R mice were stable during the same period, an inflammatory reaction in the lungs was much less severe, limited to interstitial spaces and contained no areas of necrosis.

The sst1-mediated mechanism of innate resistance is iNOS independent

Next, we wanted to determine whether the sst1-mediated control of pulmonary tuberculosis progression was due to the NO-dependent or independent effector mechanism. At day 18 after systemic i.v. infection with MTB, the expression of macrophage-specific iNOS mRNA was strongly up-regulated in the spleens and in the lungs of the MTB-infected sst1^S and sst1^R congenic mice, and its expression level was similar on both genetic backgrounds (Fig. 3A). The expression of the iNOS protein was also detected on sections of tuberculosis lung lesions using staining with iNOS-specific Abs. The iNOS protein levels were similar in both the sst1^R and the sst1^S mice three weeks after infection (data not shown).

View larger version (22K): [in this window] [in a new window]   FIGURE 3. Expression of iNOS in vivo and NO production by the sst1 congenic macrophages in vitro. A, Expression of Nos2 mRNA in the lungs and spleens of the sst1S and sst1R mice at 4 and 18 days after i.v. infection with 105 CFU of MTB. Semiquantitative RT-PCR was performed as described in Ref. 24 (std, internal standard). Three mice per group were used in the experiment. B, iNOS gene expression and MTB bacterial loads in the lungs of sst1 congenic and other substrains of C3H mice. The expression of iNOS mRNA (left panel) and the lung MTB loads (MTB CFU, right panels) were determined 25 days after i.v. infection with MTB. Four mice per group were used in each experiment. C, NO production by BMDM isolated from the sst1S and sst1R congenic mice of the B6 (left panels) and C3H (right panels) genetic backgrounds. Macrophages were primed with increasing concentrations of rIFN- (x-axis) and infected with MTB at a MOI of 1:1. Error bars represent SD. Four wells per data point were assayed, and results of one representative experiment are presented.

To assess the enzymatic activity of iNOS in isolated macrophages, we tested NO production by the sst1 congenic BMDM in vitro. When these cells were stimulated with IFN- (50 U/ml) plus LPS (10–50 ng/ml) in vitro, production of NO by the sst1^S and the sst1^R cells was similar (data not shown), also demonstrating normal NO-production capacity of the sst1^S macrophages. However, when the BMDM were primed with IFN- and infected with MTB, the sst1^R macrophages produced approximately two times higher levels of NO as compared with the sst1^S macrophages (Fig. 3C). This was observed using the sst1 congenic pair on the C3H genetic background, C3HeB/FeJ (sst1^S) and C3H.B6-sst1 (sst1^R) (Fig. 3C, right panel) as well as another sst1 congenic pair on the B6 genetic background, B6 (sst1^R) and B6.C3H-sst1 (sst1^S) (Fig. 3C, left panel).

The above data suggested a possibility that more efficient control of MTB in sst1^R mice might be due to higher levels of NO production by the MTB-infected macrophages. Therefore, we wanted to test the effect of the sst1 locus on progression of tuberculosis infection in iNOS knockout (Nos2^tm1) mice in vivo. To combine the Nos2^tm1 and sst1^S alleles on B6 genetic background, we intercrossed the B6.C3H-sst1 (sst1^S) and B6.Nos2^tm1 mice as described in Materials and Methods. The resultant sst1^S and sst1^R congenic Nos2 knockout mice were infected with a low dose of MTB via aerosol. The bacterial loads in the lungs of the B6-sst1^S, Nos2^–/– mice were 10-fold higher as compared with the B6-sst1^R, Nos2^–/– mice at 3 and 6 wk after the aerosol infection (Fig. 4A). The 10-fold difference was also observed in the lungs of the iNOS-sufficient B6 (sst1^R) and B6.C3H-sst1 (sst1^S) congenic mice (Fig. 4B), although the bacterial loads in these mice were 100-fold lower as compared with those in the corresponding strains of the iNOS-deficient mice. Although the iNOS-deficient mice failed to control MTB multiplication in their lungs, spleens and livers, the effect of the sst1 locus was limited to the lung environment in both iNOS knockout and in wild-type mice (Fig. 4, A and B).

View larger version (15K): [in this window] [in a new window]   FIGURE 4. The sst1-mediated mechanism of host resistance is functional in the absence of iNOS activity. A, MTB loads in the organs of the sst1-resistant (B6, Nos2–/–) and sst1-susceptible (B6.C3H-sst1, Nos2–/–) iNOS knockout mice on B6 genetic background 3 and 6 wk after aerosol infection with 50 CFU of MTB. Four mice of each strain were used per group. B, MTB loads in the lungs of the wild-type sst1-resistant (B6) and sst1-susceptible B6.C3H-sst1 congenic mice on the B6 genetic background. Four mice of each strain were used per group. C, Survival of the sst1 congenic iNOS knockout mice infected with MTB i.v. The sst1-susceptible (S/S) and sst1-resistant heterozygous (S/R) and homozygous (R/R) mice were infected with 5 x 104 CFU of MTB i.v. (p < 0.0001). Six to eight mice per group were used in each experiment. The sst1-resistant heterozygous (S/R) and homozygous (R/R) iNOS knockout mice survived significantly longer than the sst1-susceptible (S/S) iNOS knockout mice (p < 0.0001). D, Survival of the sst1 congenic iNOS knockout mice after aerosol infection with 50 CFU of MTB. The same groups of mice as in A were infected with via aerosol route. Six to eight mice per group were used in each experiment. The sst1-resistant (R/S and R/R) mice survived significantly longer (p = 0.0168). E, MTB growth in the BMDMs obtained from the sst1 congenic iNOS knockout mice. The BMDMs were infected with MTB Erdman at a MOI of 1:10 (bacteria: macrophage). CFU were counted in triplicates at indicated time points. Treatment with IFN- was performed at concentration 10 U/ml.

The BMDM isolated from the sst1^R Nos2^–/– mice were superior in their ability to control MTB multiplication in vitro, as compared with the sst1^S Nos2^–/– macrophages, both in the presence and in the absence of IFN- (Fig. 4E). Therefore, iNOS function was not required for the sst1-mediated control of MTB by macrophages. Taken together, these data demonstrate that the sst1 locus mediates the iNOS-independent mechanism on innate immunity to MTB.

The sst1 locus confers no apparent deficiency on Th1 function

To determine whether the sst1 locus affected T lymphocyte migration to the lungs, activation status or cytokine production after the infection, we characterized T cell populations present in the lungs of the sst1^S and the sst1^R congenic mice at various times after aerosol infection. Between the 3rd and the 12th week after a low-dose aerosol challenge with MTB, the bacterial loads increased in the lungs of the sst1^S mice (Fig. 5A). Meanwhile, the bacterial loads in the lung of the sst1^R mice and in the spleens (Fig. 5B) and livers (data not shown) in both sst1^R and sst1^S mice decreased.

View larger version (20K): [in this window] [in a new window]   FIGURE 5. Functional activity of T lymphocytes of the sst1S and sst1R congenic mice. A and B, Bacterial loads in the organs of the sst1R and sst1S congenic mice infected with 15–25 CFU of MTB via aerosol. A, Lungs (*, p < 0.01); B, spleens (ns, nonsignificant). Four mice were used per group in the experiment. C, Proportion of Tim3+CD4+ T cells was determined 9 wk after aerosol infection with MTB (10,000 events per CD3 gate were analyzed). T cells were isolated using collagenase digestion from the lungs of sst1S and sst1R congenic mice infected with 50 CFU of MTB via aerosol. Three mice were used per group in the experiment. D, IFN- production by the CD4+ T cells isolated from the tuberculosis lung lesions. Lung T cells were isolated 9 wk postinfection with 50 CFU of MTB via aerosol, enriched by depleting plastic-adherent cells, and stimulated with plate-bound anti-CD3 (2 µg/ml) in vitro (Fig. 5D, left panels). Enriched lung T cells were obtained 11 wk after the aerosol infection with MTB and stimulated with PPD (10 µg/ml) in the presence of gamma-irradiated splenocytes in vitro (D, right panels). Intracellular cytokine staining with IFN--specific Abs was performed as described in Materials and Methods. Results, at each time point, are representative of three individual animals per mouse strain. E, Intracellular MTB growth in BMDM cocultured with MTB-reactive T cells obtained from regional lymph node after CFA immunization. The BMDMs and the T cells were obtained from the sst1S () and sst1R () congenic mice. BMDM were infected in vitro with MTB at a MOI of 1:10 for 6 h, washed, MTB-reactive lymph node T cells added, and cocultured for 72 (left panel) and 96 (right panel) h. The genotype of T cells in cocultures is denoted on the x-axis. Each group was analyzed in triplicate.

Next, we studied the expression of IFN- and IL-4 genes in the tuberculosis lung lesions in vivo, as well as production of these cytokines by the T cells, which were isolated from the lung tuberculosis lesions, after stimulation in vitro. Expression of IL-4 mRNA in the lungs was very low on both genetic backgrounds as determined by RT-PCR and it did not increase after the infection. Meanwhile, the levels of IFN- mRNA in the lungs increased after the MTB infection, but were not different between the sst1^R and sst1^S mice (data not shown). The ability of the lung CD4⁺ T cells to produce IFN-, IL-4, and IL-10 was also evaluated by intracellular cytokine staining either after polyclonal (anti-CD3) activation or stimulation with mycobacterial Ag, PPD, in vitro (Fig. 5D). We were unable to detect IL-10- and IL-4-producing T cells on either genetic background by this technique. The proportion of IFN--producing CD4⁺ T cells within the T cell population following polyclonal stimulation with anti-CD3 was 2.4-fold higher in the sst1^S mice (Fig. 5D, left panels). Comparable proportions of IFN--producing CD4⁺ T cells were observed in the sst1^S and sst1^R mice in response to PPD stimulation (Fig. 5D, right panels). Similar levels of IFN- were also detected in supernatants of these cells using IFN--specific ELISA (data not shown).

To compare functional activity of lymphocytes isolated from the naive sst1^R and sst1^S congenic mouse strains in vivo, we performed their adoptive transfer into immunodeficient C3H.B6-sst1, scid recipients (see Materials and Methods for details) and challenged the reconstituted mice with a low dose of MTB i.v. In three independent experiments, lymphocyte transfer significantly increased the survival of the scid mice after MTB infection. However, there was no difference in survival between the recipients of either the sst1^R or the sst1^S lymphocytes (data not shown).

The above studies suggested to us that the severe lung disease in the sst1^S mice was not due to an intrinsic defect of their T lymphocytes or a defect in recruitment of T cells to the tuberculosis lung lesions.

Phenotypic expression of the sst1 locus during the course of MTB infection is modulated by the macrophage interactions with T cells

To examine whether the sst1-mediated mechanism of host resistance would improve control of MTB infection in the absence of adaptive immunity in vivo, we compared susceptibility to tuberculosis of the immunodeficient scid mice that were sst1 congenic, i.e., carried either the sst1-resistant (sst1^R) or the sst1-susceptible (sst1^S) allele on the C3H.scid genetic background (see Materials and Methods). These sst1^R congenic scid mice infected with a low dose of MTB (3600 CFU) i.v. survived slightly longer (median survival time = 33 days) than their sst1^S scid (median survival time = 30.5 days) counterparts (p < 0.03). However, the kinetics of the MTB growth in the organs of the two immunodeficient sst1 congenic strains was practically identical: the highest MTB burden in both strains of the scid mice was observed in the spleens and no difference of the bacterial loads in lungs was detected (see Fig. 8A, , and data not shown). This pattern is clearly distinct from the progression of tuberculosis observed after i.v. infection of the immunocompetent sst1 congenic C3H.B6-sst1 (sst1^R) and C3HeB/FeJ (sst1^S) animals, in which the sst1 locus largely affected the MTB multiplication in the lungs (Figs. 1A and 5A) (20). These experiments demonstrate that in the absence of intact adaptive immunity the sst1-mediated mechanism of host resistance is not sufficient to confer significant protection against tuberculosis infection in vivo.

View larger version (59K): [in this window] [in a new window]   FIGURE 8. The sst1-mediated mechanism of innate immunity limits the protective effect of the mycobacteria-primed CD4-positive T lymphocytes. A, MTB loads in the organs of the sst1S (S) and sst1R (R) immunodeficient scid mice 3 wk postinfection with 104 CFU of MTB i.v. At 24 h postinfection, the mice received either 4 x 106 of CD4+ T cells purified from the BCG-vaccinated C3H.B6-sst1 mice () or PBS (control, ). B, Kinetics of MTB growth in the organs of the sst1S (S) and sst1R (R) immunodeficient scid mice infected with 104 CFU of MTB i.v and reconstituted with 4 x 106 of CD4+ T cells purified from the BCG-vaccinated C3H.B6-sst1 mice. C, Lung histopathology of the same mice 6 wk after the infection (H & E, original magnification, x20).

The sst1^S and the sst1^R lymphocytes were equally capable of decreasing the bacterial load of the infected macrophages after 3 and 4 days of coincubation (Fig. 5E). The sst1^R macrophages controlled the MTB growth better than the sst1^S ones even in the absence of T cells and they benefited the most from the coculture with MTB-specific T cells. The bacterial burden in the sst1^R macrophages after the coculture with either the sst1^S or sst1^R lymphocytes was reduced equally, by 50% (p < 0.001), while the MTB burden in the sst1^S macrophages decreased by <20% under similar conditions. Thus, the efficiency of MTB control in the coculture experiments correlated with the sst1 genotype of the MTB-infected macrophages, but not with the sst1 genotype of the MTB-specific T lymphocytes.

The sst1^S macrophages do respond to stimulation with IFN-

Because IFN- is a most potent macrophage-activating factor produced by Ag-specific T cells and known to be essential for antimycobacterial immunity (10, 11), we wanted to determine whether the sst1^S macrophages were impaired in their ability to respond to stimulation with IFN- and tested several parameters of IFN--dependent macrophage activation in vitro. First, we found that the expression of MHC class II (I-A^K) molecule on the surface of the sst1^S and the sst1^R macrophages was induced equally well with both low (1–5 U/ml) and standard (50–100 U/ml) concentrations of IFN- (Fig. 6A). Second, the sst1^S and the sst1^R macrophages produced similar levels of an IFN--inducible chemokine IFN--inducible protein 10 (IP-10) after activation with rIFN- in vitro (data not shown). We also determined that, in vitro, the sst1^S and the sst1^R macrophages produced similar amounts of proinflammatory mediators MIP-1, MCP-1, KC, and IL-1β both after infection with MTB or stimulation with LPS in vitro (data not shown). Priming of BMDMs with IFN- before infection with MTB suppressed the secretion of these cytokines by the sst1^S and sst1^R MTB-infected macrophages to the same extent (Table II). Therefore, basic components of IFN- signaling were intact in the sst1^S macrophages.

View larger version (27K): [in this window] [in a new window]   FIGURE 6. Responsiveness of the sst1S and sst1R macrophages to IFN- in vitro. A, Expression of MHC class II molecule (I-AK) on bone marrow-derived macrophages isolated from the sst1S and sst1R congenic mice following stimulation with IFN- in vitro. BMDMs were treated with indicated concentrations of rIFN- for 24 h. Surface expression of MHC class II (I-Ak) on mature F4/80-positive macrophages was evaluated by FACS. Results of one of two independent experiments are presented. B, Expression of LRG-47 mRNA by the BMDMs isolated from the sst1S and sst1R congenic mice of the B6 or C3H genetic backgrounds. LRG-47 mRNA expression was determined in naive and IFN--activated (50 U/ml, 24 h) BMDMs using real-time RT-PCR as described in Materials and Methods. Error bars represent SD.

View this table: [in this window] [in a new window]   Table II. Inhibition of MTB-induced proinflammatory cytokine production by the BMDMs isolated from the sst1S and sst1R congenic mice by treatment with IFN- in vitro

These data demonstrate that major pathways of IFN- signaling are intact in the sst1^S macrophages; however, it does not exclude a possibility that more subtle differences in IFN- responsiveness between the sst1 disparate macrophages may exist.

The sst1 locus limits the protective efficiency of BCG vaccination and CD4⁺ T cells in the lung

We wanted to examine whether stimulation of T cell-mediated immunity ameliorates the sst1-susceptible phenotype in vivo. The sst1^S and the sst1^R congenic mice were vaccinated with 1 x 10⁶ CFU of live attenuated vaccine strain of M. bovis BCG i.v. Ten weeks after the vaccination, when the BCG CFU in the lungs were below the level of detection (<100 CFU), the mice were challenged i.v. with MTB. As shown in Fig. 7A, the BCG-vaccinated sst1^S mice succumbed to the MTB infection even before the nonvaccinated sst1^R congenic mice, while the survival time of the sst1^R mice increased substantially after the BCG vaccination.

View larger version (48K): [in this window] [in a new window]   FIGURE 7. Protective effect of vaccination with M. bovis BCG is dependent on the sst1 genetic polymorphism. A, Survival of naive (open symbols) and BCG-vaccinated (filled symbols) sst1S and sst1R congenic mice after i.v. infection with 105 CFU of MTB, S = sst1S (triangles), R = sst1R (squares). B, MTB burden in the organs of naive and BCG-vaccinated sst1S (S) or sst1R (R) mice 4 wk after infection with 105 CFU of MTB i.v. C, Kinetics of MTB growth in the lungs (left panel) and spleens (right panel) of the BCG-vaccinated sst1S (S) and sst1R (R) congenic mice after infection with 105 CFU of MTB i.v. D, Histopathology of the lungs of the BCG-vaccinated sst1S and sst1R congenic mice 8 wk after infection with 105 CFU of MTB i.v.: upper panels, H & E (original magnification, x40); lower panels, acid fast fluorescent staining of MTB (AFF, original magnification, x400).

Next, we purified mycobacteria-primed CD4⁺ T lymphocytes from the BCG-vaccinated sst1^R donors, C3H.B6-sst1, and adoptively transferred the identical T cell populations into the sst1^S or sst1^R congenic immunodeficient scid mice of the C3H genetic background—C3H, scid, and C3H-sst1^R, scid. The scid mice were infected with 10⁴ CFU of MTB Erdman i.v. a day before the adoptive transfer. In these settings, possible effects of the sst1 locus on T cell priming and of residual BCG vaccine on the course of MTB infection were eliminated. First, we compared the disease progression in the sst1 congenic scid mice with and without adoptively transferred CD4⁺ T cells. The scid mice without adoptively transferred T cells had the highest MTB loads and succumbed to the infection 4–5 wk postinfection. As shown in Fig. 8A, at 3 wk postinfection the effect of the adoptively transferred CD4⁺ T cells was most pronounced in the spleens, where the bacterial loads were reduced several hundred fold. In the lungs the effect of T cells was significant, but less prominent. Overall, the organ distribution of MTB in mice, which received the mycobacteria-primed CD4⁺ T cells was similar to this of the immunocompetent BCG-vaccinated mice (Fig. 7B). At 3 wk, both the sst1^R and sst1^S scid mice, which received the T cells, had similar bacterial loads. Again, a dramatic, 1000-fold increase of MTB load was observed in the lungs of the sst1^S scid mice even in the presence of the BCG-primed CD4⁺ T cells between 3 and 6 wk postinfection, while the bacterial growth was efficiently controlled in their spleens and livers (Fig. 8B). A severe necrotic inflammation was observed in lungs, but not other organs, of these mice at the 6-wk time point (Fig. 8C). This was prevented in scid mice that carried the sst1^R allele and received adoptively transferred CD4⁺ T cells. The later experiments excluded a possibility that the development of necrotic lung lesions was mediated by cytotoxic CD8⁺ T cells and directly demonstrated the importance of the sst1-mediated innate immune mechanism in the genetic control of this process.

	Discussion

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An important finding in our studies is that, despite their extreme susceptibility to tuberculosis, the C3HeB/FeJ mice are not generally immunodeficient. The fulminant course of tuberculosis infection in these mice is not a result of systemic failure of host immunity, as observed in scid, T cell-, IFN--, TNF-- or iNOS-deficient animals. Instead, a major characteristic of tuberculosis progression in the sst1-susceptible mice in vivo is the development of necrotic lung lesions and rapid multiplication of MTB within those lesions, while progression of the tuberculosis infection in other organs is controlled much more efficiently.

To establish a specific role for the sst1-mediated mechanism in control of pulmonary tuberculosis, we compared progression of the infection in the sst1^S and sst1^R animals at short intervals. A dichotomy was first observed at 2 wk postinfection. The sst1-susceptible phenotype was distinguished by formation of necrotic microfoci. At that time, the difference in bacterial loads between the lungs of the sst1 congenic mice was <2-fold, and the MTB loads in the lungs of the sst1^S C3HeB/FeJ mice were 5–10 million per organ (Fig. 1A). At these MTB loads, no necrosis was evident in the lungs of other immunocompetent mouse strains, such as B6, BALB/c, SJL, C3H.B6-sst1 congenics as well as other substrains of C3H. Formation of necrotic lung lesions in these strains may be observed at a terminal stage of the disease, when the bacterial load in the lungs reach 1–2 x 10⁸ CFU or higher. This is similar to necrosis in the organs of the IFN--deficient mice (10), which also occurs due to a very high bacterial load. In contrast, signs of the necrotic lung inflammation in the lungs of the sst1-susceptible C3HeB/FeJ mice were detected when the MTB burden was 20-fold lower. Therefore, initial formation of the necrotic microfoci in the lungs of the sst1^S mice could not be explained by extremely high MTB bacterial burden. Instead, formation of necrotic microfoci in the sst1-susceptible C3HeB/FeJ mice preceded uninhibited multiplication of the bacteria in the lungs and most likely generated an environment in which the bacteria thrived and disseminated throughout the lung tissue. A similar dichotomy of tuberculosis progression in the sst1 congenic mice was observed after a low-dose aerosol infection, albeit at a much slower pace (20). Thus, the sst1 locus appeared to control sensitivity of the inflammatory lung tissue to virulent MTB and its effect was not dependent on the route of infection.

Previously, Chackerian et al. (29) reported that there were more IFN--producing CD4-positive T cells in the tuberculosis lung lesions in the resistant parental B6 mice as compared with the susceptible C3HeB/FeJ mice. Deficit of functionally active T cells in the tuberculosis lung lesions of the extremely susceptible C3HeB/FeJ mice could provide a plausible explanation, as to why the adaptive immunity, although active systemically, failed to control tuberculosis progression in the lungs of the sst1^S mice. However, using the H-2 congenic mouse strains Kamath et al. (30) have found that the greater number of the Th1-type T cells in the lungs was controlled by the MHC locus on chromosome 17 and associated with the H-2^b haplotype, which, nevertheless, did not correlate with protection. In contrast, we have shown that progression of tuberculosis infection in the lungs was controlled by the sst1 locus (18), although the sst1 polymorphism did not affect the recruitment of T cells to the lungs of the MTB-infected mice, nor the balance of Th1/Th2 cytokine production. Our data suggested that formation of necrotic inflammatory lesions in the lungs of the sst1^S animals could not be attributed to functional deficiency of T lymphocytes in that organ (Fig. 5, C and D, and Table I).

Using coculture experiments in vitro, we found that the effect of the sst1 locus could be explained by intrinsic properties of macrophages (Fig. 5E). At the same time, we have demonstrated that two basic mechanisms of tuberculosis resistance were intact in the sst1^S macrophages, which responded to stimulation with IFN- (Fig. 6 and Table II) and produced NO after infection with MTB in vitro (Fig. 3C). In vivo, total macrophage unresponsiveness to IFN- would lead to systemic failure of antimycobacterial immunity and lack of control of even avirulent vaccine strain M. bovis BCG (31). This was not the case in the sst1-susceptible mice, in which the effect of the sst1 locus was more subtle, and was revealed specifically in the lung microenvironment, rather than systemically. We have previously established that progression of lung disease caused by virulent strain M. bovis Ravenel was also under the sst1 control (I. Kramnik, unpublished observations). However, the attenuated vaccine strain M. bovis BCG was efficiently controlled in our experiments by the sst1-independent mechanism: after systemic infection with BCG the bacterial burden in the organs of both the sst1^S and sst1^R congenic mouse strains declined in agreement with the well-established fact that both strains carried the resistant allele of the Nramp1 gene (18, 20). Two months after i.v. injection of the sst1^S and sst1^R congenic mice with M. bovis BCG, only several hundred CFU of the vaccine strain could be detected in the spleens of both mouse strains, while the numbers of live M. bovis BCG in the lungs were below the level of detection (<50 CFU) and no inflammatory lung lesions were observed. Thus, the susceptible allele of the sst1 locus did not compromise host resistance to attenuated vaccine strain of M. bovis BCG. Taken together, our data demonstrate that principal components of macrophage responsiveness to activation with IFN- were intact in the sst1^S mice.

Production of reactive NO by activated macrophages is an important component of macrophage-mediated mechanism of host resistance to tuberculosis (32, 33, 34). In the mouse model, lack of NO production led to pronounced systemic defect in control of MTB growth (33). However, both in humans and in mice existence of NO-independent mechanisms of innate immunity have been demonstrated (28, 35, 36) and postulated to play a prominent role in the human disease. We have shown that the antituberculosis effect of the sst1 locus on macrophage function in vitro, as well as on control of MTB multiplication in the lungs of immunocompetent mice and their survival, were detectable in the sst1 congenic Nos2^–/– mouse strains. Therefore, the sst1-dependent mechanism was not mediated by control of the iNOS activity. However, the iNOS function was undoubtedly necessary for maximal antituberculosis resistance, because both the sst1^S and sst1^R congenic mouse strains were significantly more resistant to MTB infection as compared with their iNOS-deficient counterparts (Fig. 4, A and B).

To assess the effect of the sst1 locus on tuberculosis progression in the absence of adaptive immunity in vivo, we have generated immunodeficient mouse strains that carry the C3HeB/FeJ-derived sst1^S or the B6-derived sst1^R alleles on the C3H.scid genetic background and, therefore, due to mutation in the Prkdc gene are unable to generate mature T and B lymphocytes. The scid mice were very susceptible even to a low dose (600 CFU) of virulent MTB irrespective of their sst1 allele, although the sst1^R mice survived slightly longer. The course of the disease in the scid mice differed from that in corresponding immunocompetent mice: the spleens, not the lungs, were the major site of the bacterial multiplication (Fig. 8A). In scid mice, reconstituted with mycobacteria-specific CD4⁺ T lymphocytes the MTB growth was controlled much more efficiently. The effect of the adoptive transfer of mycobacteria-specific T lymphocytes was especially pronounced in the spleens, where the bacterial loads were reduced almost 1000-fold. However in the lungs, the mycobacteria-specific CD4⁺ T cells were less efficient. In these settings, the sst1 genotype of the scid recipient mice, not of the mycobacteria-specific CD4⁺ T cells (that carried the sst1^R allele), determined the outcome of the infection: similar to the sst1^S immunocompetent mice C3HeB/FeJ, the sst1^S scid mice reconstituted with sst1^R CD4⁺ T cells developed necrotic lung lesions and were unable to control multiplication of MTB specifically in that organ. These experiments established that the sst1 locus directly affected innate immunity to tuberculosis, but functional CD4⁺ T cells were necessary for its phenotypic expression. From the genetic perspective, this is an example of epistatic gene interactions between the scid and sst1 loci. The scid mutation conferred a severe systemic defect on antituberculosis immunity due to the lack of adaptive immune response and almost completely obliterated beneficial effect of the sst1^R allele.

We also studied whether augmentation of T cell-mediated immunity could ameliorate the sst1-susceptible phenotype. Initially, the BCG vaccination did produce a notable decrease in bacterial burdens in all organs of both the sst1^S and sst1^R congenic mice. However, boosting the T cell immunity by vaccination with BCG failed to protect the sst1^S mice effectively. Perhaps, in the vaccinated mice, the numbers of the bacteria in circulation was decreased and initial deposition of the pathogen in the lungs was reduced. That delayed, rather than prevented, the development of necrotic inflammation in the lungs of the sst1^S mice, because the vaccine did not remedy the genetic defect of macrophages.

It has been proposed that necrotic centers in tuberculosis granulomas develop as a result of T cell-mediated immunity leading to excessive inflammation via cytokine production and activity of cytolytic T cells (37). In our adoptive transfer experiments we excluded a possibility that the necrosis in the lungs of the sst1^S mice was due to cytotoxicity of CD8⁺ T lymphocytes, because we transferred only purified CD4⁺ T cells and still observed formation of necrotic lung lesions. Orme and coworkers (38) proposed an alternative hypothesis that the initial development of a necrotic core within the granuloma after the primary infection is triggered by innate immune responses before the emergence of the acquired immunity, and an effective vaccination appeared to prevent the formation of early necrosis due to very rapid lymphocytic responses in vaccinated animals. Our data indicate that the sst1-mediated effect is more consistent with the Orme hypothesis. However, in mice that carry the susceptible allele at the sst1 locus, the necrotic lung lesions develop even in the presence of primed mycobacteria-specific CD4⁺ T lymphocytes. Although, the precise mechanism of macrophage cell death in the lungs of the sst1^S and sst1^R mice remains to be established, we hypothesize that the defect in the yet unknown sst1-mediated molecular pathway makes macrophages intrinsically more sensitive to cytotoxic factors secreted by virulent MTB, for example ESAT-6 protein (39, 40). Therefore, irreversible damage to the sst1^S macrophages upon infection occurs at much lower bacterial loads as compared with the sst1^R animals. In the lungs, where proportion of inflammatory macrophages after MTB infection is especially high, this may result in inefficient clearance of the damaged cells, release of their cytoplasmic content leading to extensive inflammation and, eventually, tissue destruction. This promotes unrestricted multiplication and further spread of the virulent mycobacteria within the lung tissue including airspaces. In humans, similar course of events may generate conditions for the pathogen transmission via aerosol.

Our observations support the prediction that, if in susceptible hosts the failure to resolve the infection in the lungs was due to an intrinsic deficiency in macrophage responsiveness, the number of MTB-specific T cells produced might not matter (9). This notion confers a theoretical limit on how efficient even the ideal antituberculosis vaccine that predominantly activates CD4⁺ Th1-type T cells could be and raises a practical question of how defects in innate immunity could be identified and corrected most efficiently. Identification of genetic loci and genes, in which variation increases susceptibility of otherwise immunocompetent hosts to pulmonary tuberculosis, will help uncover specific pathways that are effectively exploited by this evolutionary successful pathogen and suggest new approaches to the development of preventive and therapeutic strategies that specifically target individuals with genetic or acquired defects in innate immunity predisposing to tuberculosis.

	Acknowledgments

 
We are grateful to Ilona Breiterene, Christina Mottley, and Kristine Vasquez for expert technical assistance and to Drs. Barry Bloom and James Sissons for helpful discussions and critical reading of the manuscript.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

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

¹ This work was supported by National Institutes of Health Grants AI49421 and P01 AI056296 (to I.K.).

² Current address: Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX13RE, U.K.

³ Address correspondence and reprint requests to Dr. Igor Kramnik, Department of Immunology and Infectious Diseases, Harvard School of Public Health, 667 Huntington Avenue, Boston, MA 02115. E-mail address: ikramnik{at}hsph.harvard.edu

⁴ Abbreviations used in this paper: MTB, Mycobacterium tuberculosis; BCG, bacillus Calmette Guérin; BMDM, bone marrow-derived macrophage; iNOS, inducible NO synthase; PPD, purified protein derivative; MOI, multiplicity of infection.

Received for publication December 1, 2006. Accepted for publication August 29, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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