Differential Influence of Nutrient-Starved Mycobacterium tuberculosis on Adaptive Immunity Results in Progressive Tuberculosis Disease and Pathology Jes Dietrich,a Sugata Roy,a* Ida Rosenkrands,a Thomas Lindenstrøm,a Jonathan Filskov,a Erik Michael Rasmussen,c Joseph Cassidy,b Peter Andersena Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmarka; Pathobiology Section, School of Veterinary Medicine, University College Dublin, Belfield, Dublin, Irelandb; International Reference Laboratory of Mycobacteriology, Division of Diagnostics & Infection Control, Statens Serum Institut, Copenhagen, Denmarkc

When infected with Mycobacterium tuberculosis, most individuals will remain clinically healthy but latently infected. Latent infection has been proposed to partially involve M. tuberculosis in a nonreplicating stage, which therefore represents an M. tuberculosis phenotype that the immune system most likely will encounter during latency. It is therefore relevant to examine how this particular nonreplicating form of M. tuberculosis interacts with the host immune system. To study this, we first induced a state of nonreplication through prolonged nutrient starvation of M. tuberculosis in vitro. This resulted in nonreplicating persistence even after prolonged culture in phosphate-buffered saline. Infection with either exponentially growing M. tuberculosis or nutrient-starved M. tuberculosis resulted in similar lung CFU levels in the first phase of the infection. However, between week 3 and 6 postinfection, there was a very pronounced increase in bacterial levels and associated lung pathology in nutrientstarved-M. tuberculosis-infected mice. This was associated with a shift from CD4 T cells that coexpressed gamma interferon (IFN-␥) and tumor necrosis factor alpha (TNF-␣) or IFN-␥, TNF-␣, and interleukin-2 to T cells that only expressed IFN-␥. Thus, nonreplicating M. tuberculosis induced through nutrient starvation promotes a bacterial form that is genetically identical to exponentially growing M. tuberculosis yet characterized by a differential impact on the immune system that may be involved in undermining host antimycobacterial immunity and facilitate increased pathology and transmission.

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ycobacteria are known to adapt to harsh environments by regulating their metabolism, protein expression, and replication. Mycobacterium smegmatis starved of carbon, nitrogen, or phosphorus has been shown to remain viable for over 650 days after an initial 2- to 3-log drop in number of CFU and has displayed increased stress resistance, increased mRNA stability, and an overall decrease in protein synthesis (1). Another example is latent Mycobacterium tuberculosis, which represents a continuum of M. tuberculosis in different growth stages, some of which involve M. tuberculosis in a low-replicating or nonreplicating dormant condition (dormant M. tuberculosis is defined as “nonreplicating bacilli maintaining full viability at a very low metabolic rate” [2]). As one-third of the world’s population is infected with latent tuberculosis (TB), these individuals therefore constitute an enormous reservoir for TB disease and transmission (3).Thus, new strategies for the eradication of the low-replicating or nonreplicating dormant mycobacteria that are present during a latent infection are required. We believe that such strategies will arise from a better understanding of the immune response against this particular form of M. tuberculosis, as well as increased insight into the process of reactivation. It has been suggested that dormant mycobacteria reside in phagosomes inside macrophages without free access to oxygen and nutrients. To understand the biology of these bacteria, conditions have been developed in vitro to simulate the in vivo environment which probably involves hypoxia produced in avascular calcified granulomas (4), NO (5) or carbon monoxide (CO) (6) produced by activated immune cells, or a phosphate-limited environment within macrophage phagosomes (7). A widely used model is the “Wayne model” (8), in which a low-inoculum culture is sealed in a tube with stirring and allowed

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to slowly consume oxygen until the culture is anaerobic, thereby achieving a nonreplicating and apparently dormant state (9). This state was found to be accompanied by restriction of biosynthetic activity to conserve energy, induction of alternative energy pathways, stabilization of essential cell components, and expression of genes in particular within the DosR regulon (10), which is believed to be important for rapid resumption of growth once M. tuberculosis exits from an anaerobic or nitric oxide-induced nonrespiring state (11). Loebel et al. (12, 13) investigated the effect of the nutrient limitations predicted to be available in a granuloma on the metabolism of M. tuberculosis and found that nutrient starvation resulted in a gradual shutdown of respiration to minimal levels, but the bacilli remained viable and were able to recover when

Received 18 August 2015 Returned for modification 14 September 2015 Accepted 17 September 2015 Accepted manuscript posted online 28 September 2015 Citation Dietrich J, Roy S, Rosenkrands I, Lindenstrøm T, Filskov J, Rasmussen EM, Cassidy J, Andersen P. 2015. Differential influence of nutrient-starved Mycobacterium tuberculosis on adaptive immunity results in progressive tuberculosis disease and pathology. Infect Immun 83:4731–4739. doi:10.1128/IAI.01055-15. Editor: S. Ehrt Address correspondence to Jes Dietrich, [email protected]. * Present address: Sugata Roy, Omics Molecular Interaction Research Unit, LSA Technology Development Unit, RIKEN Yokohama, Yokohama, Japan. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /IAI.01055-15. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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returned to rich medium. Using this model, it was shown by microarray profiling that transfer to phosphate-buffered saline (PBS) led to the induction of specific genes, downregulation of aerobic respiration, translation, cell division, and lipid biosynthesis (14, 15). Some of the starvation-induced genes are now being tested as vaccine candidates against a latent infection with M. tuberculosis (16, 17). Although gene and protein expression profiling analyses comparing stressed (dormant) and nonstressed M. tuberculosis bacteria have been initiated (10, 14), the impact of dormant M. tuberculosis on the immune system has not been the subject of detailed studies. For other bacteria, such as Coxiella burnetii, Leishmania, Legionella pneumophila, and Chlamydia trachomatis, it has been found that they can exist in replicative and nonreplicative forms, which differ markedly in their interactions with the phagocytic cells of the immune system (18–21). Based on this, the aims of this study were to analyze the immune response to nonreplicating M. tuberculosis (induced by nutrient starvation, termed StarvM.tb) and to compare that to the response promoted by replicating (exponentially growing) M. tuberculosis (termed LogM.tb). We found that these two forms were genetically identical but had markedly different impacts on the immune system and that infection with StarvM.tb, despite resulting in similar bacterial levels in the first phase of the infection as infection with LogM.tb, led to a dramatic breakdown of protective antimycobacterial immunity in the late stage of the infection. MATERIALS AND METHODS Animal handling. Studies were performed with 6- to 8-week-old female CB6F1 mice (BALB/c ⫻ C57BL/6) from Harland Netherlands. Noninfected mice were housed in cages in appropriate animal facilities at Statens Serum Institut. Infected animals were housed in cages contained within laminar-flow safety enclosures (Scantainer; Scanbur, Denmark) in a separate biosafety level 3 facility. All mice were fed radiation-sterilized 2016 global rodent maintenance diet (Harlan, Scandinavia) and water ad libitum. All animals were allowed a 1-week rest period after delivery before the initiation of the experiments. The handling of mice was conducted in accordance with the regulations set forward by the Danish Ministry of Justice and animal protection committees in Danish Animal Experiments Inspectorate permit 2004-561-868 of 01-07-2004. This was in compliance with European Community Directive 86/609 and the U.S. Association for Laboratory Animal Care recommendations for the care and use of laboratory animals. All animal handling was done at Statens Serum Institut by authorized personnel. Bacteria. All bacteria were stored at ⫺80°C in growth medium at ⬃5 ⫻ 108 CFU/ml. Bacteria were thawed, sonicated, washed, and diluted in PBS. All bacterial work was performed at the Statens Serum Institut by authorized personnel. M. tuberculosis H37Rv was grown at 37°C in suspension in Sauton medium (BD Pharmingen) enriched with 0.5% glucose. StarvM.tb bacteria were established from H37Rv (ATCC 27294) bacteria from Sauton medium. The bacteria were washed twice and transferred to PBS as previously described (22). Bacteria were cultured for 6 weeks to 2 months in PBS at 37°C in a shaker incubator. Determinations of CFU and most probable number (MPN) were performed prior to the infection to count and test the viability of the bacteria. Assessment of MPN was done as described previously (23). Briefly, bacterial suspensions were serially diluted in growth medium. Tubes with visible bacterial growth were counted as positive, and MPN values were calculated using standard statistical methods (24). Prior to infection, the bacteria were sonicated and passaged several times through a small (27-gauge) syringe three times to reduce clumping. Sequencing of M. tuberculosis. Genomic DNA of isolates from the LogM.tb bacteria and two batches of StarvM.tb bacteria was sequenced on

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an Illumina NextSeq 500 instrument at Statens Serum Institut (Copenhagen, Denmark). Procedures were performed according to manufacturer’s manual, except that 1.5 ng of input DNA was used instead of the 1 ng as described. Reads were mapped to the M. tuberculosis H37Rv genome (GenBank accession no. NC_000962.2) by employing the BWA 0.7 exact alignment program (25). Reads were sorted and duplicates were removed using Picard 1.127, Freebayes call variants, BCFtools, vcfutils variant filtering, and Plink to transform VCF files to tped files. For all isolates, more than 96% of the M. tuberculosis H37Rv genome was covered with at least one read. Antigens for in vitro stimulation. Synthetic overlapping peptides (9and 18-mers) covering the complete primary structure of Ag85B, TB10.4, and ESAT-6 were synthesized by standard solid-phase methods on a SyRo peptide synthesizer (MultiSynTech GmbH, Witten, Germany) at the JPT Peptide Technologies (Berlin, Germany) or at Schafer-N (Copenhagen, Denmark). Peptides were lyophilized and stored dry at ⫺20°C until reconstitution in PBS. Experimental infections. Upon challenge by the aerosol route, the animals were infected with approximately 100 to 150 CFU of M. tuberculosis H37Rv/mouse with an inhalation exposure system (Glas-Col, Terra Haute, IN). For intravenous (i.v.) infection, the dose was 5 ⫻ 106 bacteria/ dose. The numbers of bacteria in the spleen or lung were determined by serial 3-fold dilutions of individual whole-organ homogenates in duplicate on 7H11 medium supplemented with polymyxin B, amphotericin B, nalidixic acid, trimethoprim, and azlocillin (PANTA) (Becton Dickinson, San Diego, CA). Colonies were counted after 2 to 3 weeks of incubation at 37°C. Lymphocyte cultures. Splenocyte cultures were obtained by passage of spleens through a metal mesh, followed by two washing procedures using RPMI medium. Lung lymphocytes were obtained by passage of the lungs through a 100-␮m nylon cell strainer (BD Pharmingen, USA), followed by two washing procedures using RPMI medium. The cells in each experiment were cultured in sterile microtiter wells (96-well plates; Nunc, Denmark) containing 2 ⫻ 105 to 10 ⫻ 105 cells in 200 ␮l of RPMI medium supplemented with 1% (vol/vol) premixed penicillin-streptomycin solution (Invitrogen Life Technologies), 1 mM glutamine, and 10% (vol/vol) fetal calf serum (FCS) at 37°C and 5% CO2. Flow cytometric analysis. For the intracellular cytokine (IC) staining procedure, cells from the spleen or lungs of mice were stimulated for 1 to 2 h with 2 ␮g/ml antigen at 37°C and subsequently incubated for 5 h at 37°C with 10 ␮g/ml brefeldin A (Sigma-Aldrich, Denmark) at 37°C. Fc receptors were blocked with 0.5 ␮g/ml anti-CD16/CD32 monoclonal antibody (MAb) (BD Pharmingen, USA) for 10 min, after which the cells were washed in FACS (fluorescence-activated cell sorting) buffer (PBS containing 0.1% sodium azide and 1% FCS) before being stained with a combination of the following rat anti-mouse antibodies: phycoerythrin (PE)-Cy7– and peridinin chlorophyll protein (PerCP)-Cy5.5–anti-CD8␣ (53-6.7, RM4-5), allophycocyanin (APC)-Cy7–anti-CD4 (GKI.5), fluorescein isothiocyanate (FITC)– and PE-Cy5.5–anti-CD44 (IM7), all purchased from BD Pharmingen (San Diego, CA), R&D Systems (Minneapolis, MN), or eBiosciences (San Diego, CA). Cells were washed with FACS buffer before fixation and permeabilization using the BD Cytofix/Cytoperm kit (BD, San Diego, CA) according to the manufacturer’s protocol and then stained intracellularly with PE–, PE-Cy7–, or APC–anti-gamma interferon (IFN-␥) (XMG1.2), PE–anti-tumor necrosis factor alpha (TNF-␣), and/or PE– and APC–anti-interleukin 2 (IL-2) (JES6-5H4). After washing, cells were resuspended in formaldehyde solution (4%, wt/ vol), pH 7.0 (Bie & Berntsen, Denmark), and samples were analyzed on a six-color FACSCanto flow cytometer (BD Biosciences, USA). Data analysis was done with FACS Diva software (Becton-Dickinson, San Diego, CA) and Flowjo software (Tree Star, Ashland, OR). TB10.4 pentamer staining was performed as previously described (26). IFN-␥ ELISA. Microtiter plates (96 well; Maxisorb; Nunc, Denmark) were coated with 1 ␮g/ml monoclonal rat anti-murine IFN-␥ (clone R46A2; BD Pharmingen). Free binding sites were blocked with 2% (wt/vol)

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FIG 1 Generating nutrient-starved mycobacteria. (A) Overview of the procedure to generate nutrient-starved mycobacteria. (B) StarvM.tb and LogM.tb bacteria were transferred to Sauton medium, and the numbers of CFU were determined at the time points indicated. (C) StarvM.tb and LogM.tb bacteria were counted either by plating on agar or by determining the MPN. (D) Growth measured by absorbance of StarvM.tb and LogM.tb in liquid culture over a period of 15 days. (E and F) LogM.tb (E) or StarvM.tb (F) bacteria were stained with auramine-rhodamine. Magnifications are indicated on the panels.

milk powder in PBS. Culture supernatants were harvested from lymphocyte cultures after 72 h of in vitro antigen stimulation and tested in triplicate. IFN-␥ was detected with a 0.1-␮g/ml biotin-labeled rat anti-murine antibody (clone XMG1.2; BD Pharmingen, USA) and 0.35 ␮g/ml horseradish peroxidase-conjugated streptavidin (Zymed, Invitrogen, USA). The enzyme reaction was developed with 3.3=,5.5=-tetramethylbenzidine and hydrogen peroxide (TMB plus; Kementec, Denmark) and stopped with 0.2 M H2SO4. Recombinant IFN-␥ (rIFN-␥; BD Pharmingen, USA) was used as a standard. Plates were read at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader and analyzed with KC4 3.03 Rev 4 software. Histopathological analysis. The right lung of each mouse sampled was fixed by immersion in 10% neutral buffered formalin and processed for histological examination. Cut sections were stained with hematoxylin and eosin and by the Ziehl-Neelsen method and were evaluated without prior knowledge of treatment group. Lesion morphometry was carried out using the public-domain, Java-based image processing program ImageJ (http://imagej.nih.gov/ij/).

RESULTS

In vitro analysis of exponentially growing and nutrient-starved M. tuberculosis. To induce a state of nonreplicating M. tuberculosis, the bacteria were taken from exponentially growing bacteria in Sauton medium and transferred to PBS at 37°C (Fig. 1A). After transfer of the bacteria to PBS, CFU levels were determined at weeks 0 and 6. Although a small reduction was observed in CFU counts, this was not significant (Fig. 1B). Moreover, although some forms of stress have been shown to induce a nonculturable form of M. tuberculosis (23, 27–29), incubation in PBS did not lead to nonculturable M. tuberculosis since direct viable counting did not show bacterial numbers that were significantly different from those determined by methods such as indirect enumeration by most-probable-number (MPN) estimation (27, 30) (Fig. 1C). Furthermore, transfer to PBS did not, as expected, lead to muta-

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tions in the StarvM.tb bacterial genome, as full genome sequencing of two StarvM.tb batches and one LogM.tb batch (method described in Materials and Methods) did not reveal any significant differences between the samples (data not shown). Transfer of StarvM.tb bacteria back to Sauton medium showed that after a lag period of 4 days, these bacteria resumed growth with a growth rate similar to that observed with LogM.tb (Fig. 1D). As stress induction has been shown to reduce the acid-fast staining of the stressed M. tuberculosis cultures (31, 32), we also tested whether the transition to nutrient starvation-induced nonreplication/dormancy would decrease the percentage of acid-fast staining in starved M. tuberculosis bacteria. After 6 weeks, we noticed no significant decrease in acid-fast-positive bacteria. We did however notice (slightly) less intensive acid-fast staining in StarvM.tb bacteria, reflecting changes in cell wall lipid composition as previously described (31) (Fig. 1E). Thus, even prolonged culture of bacteria in the absence of nutrients did not result in marked reduction of bacterial numbers measured by either direct plate counting or MPN estimation, and the bacteria exhibited only a minor morphological change. Consequently, in all subsequent infection studies, bacteria were counted on agar plates prior to infection to establish that the mice had received the same inocula of nonreplicating M. tuberculosis (StarvM.tb) and exponentially growing M. tuberculosis (LogM.tb). In vivo growth and immunopathology of LogM.tb and StarvM.tb. We next compared the in vivo growth of StarvM.tb and LogM.tb. We chose CB6F1 mice, as they are well characterized in our laboratory in terms of immune recognition of TB antigens during an infection. Mice were infected by the aerosol route with the same number of either StarvM.tb or LogM.tb bacteria (approximately 200 CFU). Thereafter, the mice were sacrificed at different time points and the bacterial numbers in the lungs were

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FIG 2 In vivo growth of StarvM.tb and LogM.tb bacteria. (A and B) Bacterial load (shown as log10 CFU) in the lungs of mice infected with StarvM.tb or LogM.tb via the aerosol route. Two experiments are shown in panels A and B, respectively. Data represent the mean ⫾ SD for a minimum of four mice per group. *, P ⬍ 0.05.

determined. StarvM.tb did not show an extended lag period in growth compared to LogM.tb, as we did not observe any significant differences in the bacterial numbers in the StarvM.tb- and LogM.tb-infected mice at days 4 and 11 (Fig. 2A). However, from week 2 onward, the bacterial number of StarvM.tb (7.88 ⫾ 0.06 log10 CFU/lung at week 3) significantly exceeded that of LogM.tb (6.89 ⫾ 0.13 log10 CFU/lung at week 3) (Fig. 2A). This difference became very pronounced after week 3, at which time the bacterial number of LogM.tb declined to 5.23 ⫾ 0.18 log10 CFU/lung (week 6), whereas bacterial numbers in StarvM.tb instead continued to increase to 8.61 ⫾ 0.23 log10 CFU/lung. The same pattern was observed in an additional experiment illustrated in Fig. 2B. Again, in the initial phase of the infection (until week 3 postinfection), the bacterial numbers were similar between the groups, but between weeks 3 and 6, StarvM.tb-infected mice showed increased CFU levels (Fig. 2B). Significantly higher bacterial levels in StarvM.tb-infected mice in the late stage of the infection was observed repeatedly in individual experiments, using different StarvM.tb batches, and in both the aerosol and i.v. infection models. Results from an experiment using the i.v. infection model are shown in Fig. S1 in the supplemental material. At week 3 (in the experiment shown in Fig. 2A), macroscopic examination showed larger numbers of granulomas in the lungs of StarvM.tb-infected mice than in LogM.tb-infected mice (Fig. 3A). Histopathological analysis indicated that while granulomas in both groups consisted of dense unencapsulated aggregates of admixed intact and degenerate macrophages and neutrophils with smaller numbers of accompanying lymphocytes, neutrophil infiltrates were larger and denser and were associated with extensive foci of necrosis and greater numbers of acid-fast bacteria in the

FIG 3 Macroscopic analysis and immunopathology of infected lungs. Mice infected by the aerosol route with StarvM.tb or LogM.tb were sacrificed 3 (A to C) or 6 (D and E) weeks after challenge and were examined macroscopically (A, D, and E) or subjected to histopathological examination (B and C [which shows the week 3 time point]). For histopathology, the right lung was fixed by immersion in 10% neutral buffered formalin and processed for examination. Cut sections were stained with hematoxylin and eosin (HE). Lesion morphometry was carried out using the public-domain, Java-based image processing program ImageJ. In panel C, results for lesions in lungs are from three independent experiments (represented by unique symbols).

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FIG 4 T cell-induced IFN-␥ secretion. (A) Lung cells were stimulated with TB10.4 or ESAT6 in vitro for 72 h, and IFN-␥ levels in supernatants were assessed by

ELISA. Data represent the mean ⫾ SEM of the results for a minimum of three mice per group. (B) Mice were sacrificed 3 or 6 weeks after the challenge, and lymphocytes from lungs were stimulated in vitro with TB10.4 peptides prior to staining with anti-CD4, -CD8, -CD44, -IFN-␥, -IL-2, and -TNF-␣. Cytokine profiles of specific CD4 and CD8 T cells 3 weeks after an aerosol challenge are shown. Frequencies represent cytokine-producing T cells within the subset of T cells specific for TB10.4. Data represent the mean ⫾ SEM of the results for a minimum of three mice per group (*, P ⬍ 0.05 [two-way analysis of variance with Bonferroni’s posttest]). (C) At week 3 after aerosol infection, lung cells were stimulated with TB10.4 or ESAT6 in vitro for 72 h, and IL-10 and IL-5 levels in supernatants were assessed by ELISA. Data represent the mean ⫾ SEM of the results for a minimum of three mice per group.

StarvM.tb lesions (Fig. 3B and see Fig. S2 in the supplemental material). In both groups, clusters of neutrophils admixed with acid-fast bacteria were detected in bronchiolar lumens. Lesion morphometry found that the mean cross-sectional surface area of StarvM.tb granulomas was 3.7-fold larger than that of LogM.tb lesions (P ⬍ 0.0001, Mann-Whitney test) (Fig. 3C). At week 6, while all LogM.tb-infected animals exhibited only a few macroscopically visible granulomas, all StarvM.tb-infected mice showed numerous pulmonary granulomas scattered throughout all lobes (Fig. 3D), and macroscopic examination showed increased numbers of granulomas in the lungs of StarvM.tb-infected mice (Fig. 3E). Influence of StarvM.tb on adaptive immunity. To study the influence of StarvM.tb on the host immune response during infection, we first focused on the influence on adaptive immunity. Antigen-specific cytokine secretion/expression was analyzed by ELISA or IC-FACS staining. To study the antigen-specific T cell response, we chose the mycobacterial antigens ESAT-6, Ag85B, and TB10.4, which are all known to be recognized following an infection (33). As a marker for the CD8 T cell response, we also used the antigen TB10.4, as it contains well-characterized CD8 epitopes (26, 34, 35). We first analyzed the cytokine secretion by ELISA. At week 2 postinfection, lung T cells from LogM.tb-infected mice showed a secretion of IFN-␥ that was below detection, whereas T cells isolated from the lungs of StarvM.tb-infected mice already secreted

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substantial levels of IFN-␥ in response to all three antigens (Fig. 4A; see also Fig. S3 in the supplemental material, which shows the Ag85B-specific response). Interestingly, in contrast to the differences in CFU counts at week 3, we did not observe any differences in the magnitude of lung T cell IFN-␥ secretion from LogM.tb- and StarvM.tb-infected mice at this time point. Analysis of cytokine expression by IC flow cytometry at week 3 also revealed only minor differences among the distribution of T cell subtypes within the pool of antigen-specific T cells (with IFN-␥/TNF-␣-coexpressing T cells as the dominant subtype in both groups and for both CD4 and CD8 T cells) (Fig. 4B). Three weeks later (week 6), the difference was pronounced, with a significantly higher cytokine secretion in LogM.tb-infected mice following stimulation with any of the three antigens (TB10.4 response, 36,991.6 ⫾ 667.0 pg/ml IFN-␥ in LogM.tb-infected mice versus 12,493.3 ⫾ 2,835.6 pg/ml in StarvM.tb-infected mice; Ag85B response, 6,417.4 ⫾ 2,485.3 pg/ml versus 3,136.0 ⫾ 879.6 pg/ml IFN-␥; ESAT-6 response, 9,567.0 ⫾ 1,505.1 versus 3,376.0 ⫾ 1,480.8 pg/ml IFN-␥) (Fig. 4A, and see Fig. S3 in the supplemental material). This correlated with a markedly different profile by IC flow cytometry, where the dominating T cell subtype in StarvM.tb-infected mice was T cells that produced only IFN-␥, whereas LogM.tb-infected mice maintained polyfunctional T cells in both the CD4 and CD8 T cell population (Fig. 4B). In line with

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this, lung T cells from StarvM.tb-infected mice showed a significant decrease in not only polyfunctional T cell subtypes but also the expression of IFN-␥ per single antigen-specific CD4 or CD8 T cell, measured by its mean fluorescence intensity (MFI) (data not shown). Finally, in addition to a lack of IFN-␥ expression, staining the CD8 T cells directly ex vivo with H2-Kb pentamer loaded with the TB10.4 CD8 epitope IMYNYPAM showed a strong reduction in CD8 T cell numbers (data not shown). In the i.v. infection model, we observed the same overall immunological pattern as observed in the aerosol model (see Fig. S1 in the supplemental material). In the aerosol model, week 3 represented a time point at which both groups of mice had a strong T cell response and we therefore expanded our analysis to also include Th2/Treg cytokines. In contrast to the similar levels of IFN-␥, we measured a significantly higher secretion of both IL-5 and IL-10 from lymphocytes extracted from the lungs of StarvM.tb-infected mice than from lungs of LogM.tb-infected mice (Fig. 4C). This was observed not only following stimulation with TB10.4 but also after stimulation with Ag85B and ESAT-6 (Fig. 4C, and see Fig. S3 in the supplemental material). Taken together, at week 3, lung T cells from StarvM.tb-infected mice showed increased bacterial numbers, a substantial IFN-␥ response, and increased IL-5/IL-10 expression and subsequently suffered from a breakdown of antimycobacterial protective immunity. Most probably reflecting the increased bacterial numbers observed in the lung, at week 3 to 4 postinfection, StarvM.tbinfected mice also showed significantly increased expression in the lung of proinflammatory cytokines such as CXCL1, IL-12, MCP-1 and IP-10 (data not shown). Preventing StarvM.tb-mediated alteration of the T cell response. We next examined if a vaccine with efficacy against LogM.tb could prevent the observed StarvM.tb-mediated alteration of the T cell response. We used the vaccine antigen Ag85B– ESAT-6 formulated in the adjuvant CAF01, which we previously showed protects against infection with LogM.tb (36). Vaccination with Ag85B–ESAT-6/CAF01 reduced the growth of LogM.tb (1.17 log10 CFU reduction) in agreement with previous reports on this vaccine (36). In addition, this vaccine also protected against infection with StarvM.tb with approximately the same difference between vaccinated and unvaccinated animals as seen in the LogM.tb-infected animals. Thus, bacterial numbers in the lungs of vaccinated animals were reduced to 5.62 ⫾ 0.18 log10 CFU compared to 6.88 ⫾ 0.21 log10 CFU in unvaccinated animals (Fig. 5A). The Ag85B–ESAT-6/CAF01 vaccination prevented the T cell phenotypic change to T cells only expressing IFN-␥, as shown by the improved quality of antigen-specific T cell phenotypes (Fig. 5B) (which now included polyfunctional T cells and only a minimum of IFN-␥ only-positive T cells) as well as an increased antigenspecific IFN-␥ secretion by peripheral blood mononuclear cells (PBMCs) from vaccinated StarvM.tb-infected animals (data not shown). Interestingly, the phenotype of TB10.4-specific T cells (not in the vaccine) was similar for vaccinated and nonvaccinated animals. This suggests that the improved ESAT-6/Ag85B CD4 T cell phenotypes in vaccinated animals is mediated by vaccine-promoted expansion of less-differentiated memory T cells, as previously reported (37), and is not due to a general improvement of T cell quality as a result of the reduced bacterial numbers in vaccinated animals.

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FIG 5 Vaccination against infection with StarvM.tb. (A) Animals were vaccinated with Ag85B–ESAT-6 in CAF01 adjuvant and infected with StarvM.tb or LogM.tb at week 6 after the last vaccination. CFU levels were determined in the lungs at week 6 postinfection. (B) Cytokine profiles of ESAT-6-, Ag85B-, and TB10.4-specific CD4 T cells 6 weeks after aerosol challenge.

DISCUSSION

Subjecting mycobacteria to nutrient starvation has been previously shown to lead to a slowdown of the transcription apparatus, energy metabolism, lipid biosynthesis, and cell division (14). In the present study, we expand these results and show that nonreplicating and logarithmically growing mycobacteria differ in their impacts on host-adaptive immune responses raised during infection. StarvM.tb bacteria cause the development of a functionally impaired immune response, leading to IFN-␥ only-positive effector T cells that are unable to contain infection. The result of this is a breakdown of protective immunity with accelerated bacterial growth and an excessive pathology not observed during an infection with replicating M. tuberculosis. Due to the very different impact on the host immune system, compared to that of conventional M. tuberculosis, these results also highlight the importance of considering the starved form of M. tuberculosis in future vaccine studies. There are several ways to induce M. tuberculosis dormancy in vitro, and the methods used in our study (transfer to PBS) produced a bacterial M. tuberculosis phenotype that in several ways

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differed from that of the dormant M. tuberculosis generated in other studies. Thus, in contrast to previous studies (23, 27–29), the StarvM.tb bacteria used in our study did not constitute a nonculturable form of M. tuberculosis (Fig. 1). Furthermore, StarvM.tb did not show a longer lag period in mice than LogM.tb, in contrast to a recent study (38) where dormancy was achieved through oxygen depletion according to the method used by Wayne et al. (9) and to the 4-day lag period upon transfer to liquid culture (Fig. 1). Finally, the increased infectivity of StarvM.tb in this study is in contrast to another recent study (39) (using the guinea pig TB model), where nonreplicating M. tuberculosis was established by the gradual depletion of nutrients in an oxygenreplete environment. In this study, it was found that the nonreplicating M. tuberculosis elicited a significantly reduced infectivity (39). Taken together, these contrasting results may reflect the different methods used to establish a dormant form of M. tuberculosis, and exactly how these different dormant forms of M. tuberculosis are present in humans during a latent (or chronic) infection is important to establish in future studies. Adaptive immune response against StarvM.tb and LogM.tb. Concerning the impact on adaptive immunity, we observed interesting differences between the two groups of infected mice. At the week 2 time point, we observed only an accelerated T cell response in StarvM.tb-infected mice (Fig. 4). The week 3 time point represents an interesting turning point, as both groups have similar levels of both IFN-␥ responses and T cell quality as measured by IC flow cytometry. However, control over the infection was clearly failing in StarvM.tb-infected mice, as shown by increased bacterial numbers and increased pathology as well as an increase in both the size and numbers of granolomas in the lung (Fig. 2 and 3). Moreover, StarvM.tb-infected mice showed increased levels of Il-5 and IL-10 following stimulation with each of the three antigens used (Fig. 4). It is not fully known how or if IL-5/IL-10-secreting cells contribute to the control of the infection, but IL-10 expression has in fact been inversely associated with disease progression (40, 41). Whether the differences observed in this study play a role in providing an advantage for the bacteria in StarvM.tb-infected mice is not known. It could be speculated that in light of the much higher expression of IFN-␥ in both StarvM.tb and LogM.tb bacteria, other factors may be responsible for the different outcomes of the infection from week 3 onward. The outcomes of the infection with StarvM.tb and LogM.tb are different, and while the growth of LogM.tb is reduced approximately 100 times after week 4, StarvM.tb bacteria continue to grow exponentially, resulting in more numerous, and on average 3.7 times larger, granulomas at week 3 that exhibited extensive foci of necrosis and in which acid-fast bacteria were more numerous. Fulminant pathology on such a scale in the StarvM.tb-infected mice is consistent with lack of control of infection and has not been reported in mice that have been aerosol infected with laboratory-adapted strains of log-phase M. tuberculosis. At week 6, the bacterial numbers in the StarvM.tb-infected mice were significantly higher than those during the conventional infection, and the immune profile in these animals was dominated by IFN-␥ only-positive T cells, indicating a change in the T cell response in these mice. This was supported by a decline in the expression of IFN-␥, either measured by mean fluorescence intensity or ELISA. The presence of polyfunctional T cells at week 6 correlated inversely with bacterial load, which is in agreement with previous studies in which polyfunctional T cells have been shown to corre-

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late with protection in disease models (42–45), such as cancer (43), Leishmania major infection (42), M. tuberculosis infection (16), or HIV infection (45). Taken together, our results demonstrated an accelerated immune response in StarvM.tb-infected mice that eventually led to an altered CD4/CD8 T cell response and consequently to a lack of control over the infection. Interestingly, the change in T cell quality could be prevented by a vaccine. This was demonstrated with the vaccine Ag85B–ESAT-6/CAF01. Previously we showed that this vaccine is able to maintain a pool of multicytokine-expressing central memory T cells in the face of an ongoing chronic infection (37). The present study expanded on these observations and showed that also in the face of an infection with StarvM.tb, this vaccine was able to maintain a pool of lessdifferentiated multicytokine-expressing T cells. However, improved T cell phenotypes were observed only with vaccine antigen-specific T cells, despite the overall reduction in bacterial numbers. This was exemplified by TB10.4-specific T cells, which displayed the same phenotype in vaccinated and nonvaccinated animals, despite the difference in bacterial levels. This is in agreement with the previous study analyzing the phenotype of TB10.4, compared to the vaccine antigens Ag85B and ESAT-6, during a chronic infection (37). In this study, only the phenotypes of the T cells specific for Ag85B or ESAT-6 were improved. Taken together, our results demonstrate that the maintenance of a population of high-quality T cells during infection with StarvM.tb can be achieved through a vaccine-mediated expansion of these cells prior to the infection. What might explain the increased virulence observed with StarvM.tb in the later stage of infection? A trivial explanation would be that the dose of StarvM.tb was underestimated. However, infection with a similar, or even slightly lower, dose of StarvM.tb (determined by both numbers of CFU, MPN, and infectious loads in the lung or spleen in the initial phase of the infection [Fig. 2, and see Fig. S1 in the supplemental material]) demonstrated that StarvM.tb-infected animals still had significantly higher immune responses than LogM.tb-infected animals at days 14 to 21 postinfection despite similar CFU levels throughout the initial phase of the infection. Taken together, we believe that the CFU levels of StarvM.tb are not underestimated but that the higher bacterial loads in the late phase of infection is a consequence of a breakdown of the immune system, resulting in uncontrolled bacterial replication after the first 2 to 3 weeks. Interestingly, in an attempt to discover the reason for the increased virulence of Beijing stains compared to other M. tuberculosis strains, it was found that all the analyzed Beijing strains synthesized structural variants of two well-known characteristic lipids of the tubercle bacillus, namely, phthiocerol dimycocerosates (DIM) and phenolglycolipids (PGL) (46), which correlated with higher CFU values (47, 48). It could be speculated that the cell wall of StarvM.tb has a specific immune-activating lipid composition. Membrane lipids have been shown to be able to stimulate both activating and inhibitory pathways (49), and M. tuberculosis produces a wide variety of bioactive lipids that have been implicated in the pathogenesis of tuberculosis (50). Importantly, applying stress to M. tuberculosis has been shown to lead to changes in the cell envelope lipids (32, 51–54), and in our analysis of the bacterium, we did notice a more uneven staining that indicated a change in the membrane lipids (Fig. 1). In addition, the increased virulence of StarvM.tb may also be explained by different expression levels of certain proteins (14).

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In summary, these results demonstrate that starving M. tuberculosis of nutrients promotes fundamental changes in the virulence of the bacteria and their impact on the immune system. Importantly, this impact is not mediated through an inherent higher growth rate of the bacteria but seems to be related to an ability to accelerate adaptive immune responses that result in immune breakdown, uncontrolled bacterial replication, and pathology. We hypothesize that this may represent a potential mechanism that allows dormant bacteria, as part of its resuscitation process, to coordinate the development of excess pathology that eventually facilitates transmission.

14.

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16.

17.

ACKNOWLEDGMENTS The excellent technical assistance provided by Lene Rasmussen and Janne Rabech as well as the animal technicians at the Statens Serum Institut is gratefully acknowledged. We also thank Nadine Hoffmann for help with the MPN analyses. We thank Brian Cloak for excellent technical assistance in the preparation of the photomicrographs. We thank Dorte Bek Folkvardsen for help with genomic sequencing and Jonas Grauholm for help with sequencing and the analysis of the results. This study was supported by Danish Research Council Project 11107120, 7th Framework Programme for Research and Technological Development-HEALTH-2009 Single-Stage Contract 241745 (NEWTBVAC), and the Lundbeck Foundation R54-A5572. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

18. 19. 20. 21. 22.

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