680 Eur. J. Immunol. 2008. 38: 680–694 Christoph Hölscher et al. Containment of aerogenic Mycobacterium tuberculosis infection in mice does not require MyD88 adaptor function for TLR2, -4 and -9 Christoph Hölscher1, Norbert Reiling2, Ulrich E. Schaible3,4, Alexandra Hölscher1,2, Clara Bathmann1, Daniel Korbel4, Insa Lenz1, Tanja Sonntag1, Svenja Kröger2, Shizuo Akira5, Horst Mossmann6, Carsten J. Kirschning7, Hermann Wagner7, Marina Freudenberg6 and Stefan Ehlers2,8 1 2 3 4 5 6 7 8 Junior Research Group Molecular Infection Biology, Research Center Borstel, Borstel, Germany Division of Molecular Infection Biology, Research Center Borstel, Borstel, Germany Max-Planck-Institute for Infection Biology, Berlin, Germany London School of Hygiene and Tropical Medicine, ITD Immunology Unit, London, UK Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Max-Planck-Institute for Immunobiology, Freiburg, Germany Department of Medical Microbiology and Hygiene, Technical University of Munich, Munich, Germany Molecular Inflammation Medicine, Christian-Albrechts-University, Kiel, Germany The role of Toll-like receptors (TLR) and MyD88 for immune responses to Mycobacterium tuberculosis (Mtb) infection remains controversial. To address the impact of TLRmediated pathogen recognition and MyD88-dependent signaling events on antimycobacterial host responses, we analyzed the outcome of Mtb infection in TLR2/4/9 triple- and MyD88-deficient mice. After aerosol infection, both TLR2/4/9-deficient and wild-type mice expressed pro-inflammatory cytokines promoting antigen-specific T cells and the production of IFN-c to similar extents. Moreover, TLR2/4/9-deficient mice expressed IFN-c-dependent inducible nitric oxide synthase and LRG-47 in infected lungs. MyD88-deficient mice expressed pro-inflammatory cytokines and were shown to expand IFN-c-producing antigen-specific T cells, albeit in a delayed fashion. Only mice that were deficient for MyD88 rapidly succumbed to unrestrained mycobacterial growth, whereas TLR2/4/9-deficient mice controlled Mtb replication. IFN-c-dependent restriction of mycobacterial growth was severely impaired only in Mtb-infected MyD88, but not in TLR2/4/9-deficient bone marrow-derived macrophages. Our results demonstrate that after Mtb infection neither TLR2, -4, and -9, nor MyD88 are required for the induction of adaptive T cell responses. Rather, MyD88, but not TLR2, TLR4 and TLR9, is critical for triggering macrophage effector mechanisms central to anti-mycobacterial defense. Received 3/7/07 Revised 20/11/07 Accepted 11/1/08 [DOI 10.1002/eji.200736458] Key words: Bacterial infection
Monocyte/ macrophage
Mycobacterium
Rodent
TLR Supporting Information for this article is available at http://www.wiley-vch.de/contents/jc_2040/2008/36458_s.pdf Correspondence: Christoph H
lscher, Junior Research Group Molecular Infection Biology, Research Center Borstel, Borstel, Germany Fax: +49-4537-188775 e-mail: choelscher@fz-borstel.de Abbreviations: CBA: cytometric bead array
DexTR: Dextran Texas Red
LAMP-1: lysosome associated membrane protein 1
Mtb: Mycobacterium tuberculosis
NOS2: inducible nitric oxide synthase
p.i.: post infection
PRR: pattern recognition receptor
RNI: reactive nitrogen intermediates
TB: tuberculosis
Tf: transferrin f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Introduction Human tuberculosis (TB) is a leading global health threat [1]. The disease caused by Mycobacterium tuberculosis (Mtb) is currently responsible for 8 million new cases worldwide and 1.7 million deaths annually [2]. The increased occurrence of multi-drug resistant strains of Mtb and the lack of an effective vaccine emphasize the need for TB research at the interface of innate and adaptive immunity to develop new prewww.eji-journal.eu Eur. J. Immunol. 2008. 38: 680–694 ventive and therapeutic strategies. Replication of mycobacteria takes place inside phagosomes of the primary host cell, the macrophage, until the onset of adaptive immunity. Triggered by interleukin (IL)-12, CD4+ Th1 cells play a pivotal role by secreting interferon (IFN)-c [3–5], inducing effector immune responses in macrophages that eventually result in containing infectious foci [6, 7]. In particular, IFN-c and TNF-a stimulate the antimicrobial activity of infected macrophages, allowing intracellular bacterial killing through phagosome maturation [8], the induction of LRG-47, a member of the 47-kDa guanosine triphosphatase family probably involved in autophagosome formation [9, 10], and the effect of reactive nitrogen intermediates (RNI), which are produced by the inducible nitric oxide synthase (NOS2) [11]. Activation of the innate immune system is considered to be a prerequisite for driving a protective adaptive immune response after Mtb infection. Initial recognition of mycobacterial components by macrophages and dendritic cells leads to the release of IL-12 and TNF-a and involves a number of different pattern-recognition receptors (PRR) such as Toll-like receptors (TLR). Several reports have described TLR2-, TLR4- or TLR9dependent activation of macrophages and dendritic cells by mycobacterial components. For example, the mycobacterial lipoglycans phosphoinositol-capped lipoarabinomannan, phosphatidyl myo-inositol mannosides (PIM)2 and PIM6 and the 19-kDa mycobacterial lipoprotein are TLR2 agonists [12–15]. TLR4 mediates cellular activation in response to not yet fully characterized heat-sensitive cell-associated mycobacterial factors [16], and the Mtb-induced TNF-a production by murine macrophages can be blocked by a TLR4 antagonist [17]. The genome of Mtb as well as other mycobacteria contains highly immunostimulatory CpG motifs [18], and the cognate receptor TLR9 has also been shown to be a relevant PRR for Mtb [19]. Together, TLR2, TLR4, and TLR9 all appear to be involved in the innate induction of a cell-mediated immune responses after infection with Mtb. Despite severely reduced pro-inflammatory responses to Mtb in macrophages from TLR2- or TLR4defective mice in vitro, mice deficient in either or both receptors are fully capable of containing low-dose aerosol Mtb infection [20, 21]. However, differences in experimental models have yielded conflicting results, ranging from enhanced mortality of TLR2-, TLR4-, or TLR9 single- or double-deficient mice in response to Mtb infection to little or no apparent change in resistance [19–25]. It is unknown whether differential expression and/or cross-regulation of TLR in the diverse settings may account for these discrepancies. Regardless, the prevailing consensus is that resistance to Mtb infection depends on microbe-sensing through TLR [26, 27]. To f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Immunity to infection date, however, mice lacking all TLR to which Mtb products have been assigned as ligands such as TLR2/4/ 9-triple-deficient mice have not yet been subjected to experimental Mtb infection and analyzed for their relative susceptibility to infection. The intracellular adaptor molecule MyD88 transduces signals from all TLR except TLR3 by recruitment of members of the IL-1R-associated protein kinase family of proteins, which results in activation of mitogenactivated protein kinases (MAPK), PI3 kinase, and nuclear factor (NF)-jB [28]. Because MyD88 integrates signals from multiple TLR, MyD88 deficiency almost completely ablates macrophage responsiveness to specific bacterial products [29]. In vivo, MyD88-deficient (–/–) mice are highly susceptible to infection with Listeria monocytogenes, Staphylococcus aureus and Toxoplasma gondii [30–32]. Similarly, MyD88–/– mice rapidly succumb to Mycobacterium avium and Mtb infection [27, 33, 34]. Whereas some studies imply that both MyD88 and TLR are critical for the activation of innate immunity and the differentiation of adaptive defenses [27], Fremond and colleagues [34] have shown that MyD88 is not required for the induction of innate proinflammatory and subsequent acquired immune responses. In the present study, we analyzed the potentially differential requirement of TLR-mediated recognition vs. MyD88-mediated signaling in the development and expression of protective immunity to aerogenic Mtb infection. Our investigation of TLR2/4/9–/– and MyD88–/– mice showed that triple TLR-deficient mice were fully capable of inducing adaptive Th1 immune responses and surviving low-dose aerosol infection, while MyD88-deficient mice rapidly succumbed to infection. Thus, TLR2, -4, and -9 were dispensable for the development of protective immune responses to Mtb infection. Moreover, susceptibility of MyD88–/– mice was not attributable to defective TLR2/4/9-mediated pattern recognition and instruction of Th1-biased adaptive immunity. Rather, MyD88, but not TLR2,TLR4 and TLR9, proved critical for shaping macrophage antimycobacterial effector mechanisms. Results Cytokine production by TLR2/4/9–/– macrophages is impaired in response to TLR agonists and Mtb infection Bone marrow-derived macrophages (BMMU) were generated from wild-type, MyD88–/– and TLR2/4/9–/– mice. The release of TNF-a and IL-12/IL-23p40 was determined 24 h after challenge with lipopeptide (LP), LPS, or CPG, or infection with Mtb H37Rv at different www.eji-journal.eu 681 682 Eur. J. Immunol. 2008. 38: 680–694 Christoph Hölscher et al. Figure 1. After stimulation with TLR agonists or after Mtb infection, cytokine production is impaired in macrophages from MyD88–/– and TLR2/4/9–/– mice. BMMU from wild-type, MyD88–/– and TLR2/4/9–/– mice were stimulated with medium, different TLR agonists, or were incubated with the indicated MOI of viable Mtb H37Rv. Supernatants were harvested 24 h p.i. and measured for TNF-a and IL-12/IL-23p40 concentrations. Each point indicates mean and SD of triplicate values from one out of two representative experiments. Statistical analysis was performed by ANOVA defining differences between wild-type and MyD88–/– (*p0.05; **p0.01; ***p0.001) or TLR2/4/9–/– (+p0.05; ++p0.01; +++p0.001) mice as significant. doses (Fig. 1). In response to all three TLR agonists, both MyD88–/– and TLR2/4/9–/– BMMU released significantly less of either cytokine. Whereas MyD88–/– cells released no TNF-a after infection with Mtb, responsiveness in TLR2/4/9–/– macrophages was impaired but not abolished. In contrast, even high amounts of mycobacteria did not induce release of substantial amounts of IL-12/IL-23p40 from MyD88–/– and TLR2/4/9–/– macrophages. Together, our analysis revealed that TLR2, -4, and -9 are required to induce an efficient inflammatory immune response in macrophages in vitro. In contrast to TLR2/4/9–/– mice, MyD88–/– animals are highly susceptible to Mtb infection Bacterial loads in the lungs of TLR2-, TLR4- and TLR9single-deficient mice, as well as TLR2/4-double-deficient mice, were similar in organs from wild-type mice (Table 1). On days 21 and 82 post infection (p.i.) with Mtb, CFU in lungs, of TLR2/4/9–/– mice were indistinguishable from those found in wild-type mice (Fig. 2A; Table 1). CFU in liver and spleen from triple-deficient mice were also comparable to bacterial loads in organs from infected wild-type mice (Table 1), indicating that TLR2/4/9–/– mice were fully capable of chronically restricting mycobacterial growth at a plateau Table 1. Bacterial loads in Mtb-infected TLR–/– and MyD88–/– micea) Lungb) Mice C57BL/6 21 days p.i. 82 days p.i. 21 days p.i. 82 days p.i. 21 days p.i. 82 days p.i. 6.10.2 6.70.2 4.00.5 5.90.2 3.70.5 4.50.3 5.80.2 7.00.2 3.60.5 6.00.2 3.70.3 4.70.3 –/– 6.00.2 6.60.1 2.70.6 5.70.2 2.60.3 4.10.2 –/– 6.10.2 6.70.4 3.90.7 6.00.3 3.40.5 4.50.8 6.40.2 6.90.3 3.30.7 5.90.4 3.40.5 4.40.5 6.10.3 6.80.2 3.60.4 6.00.3 3.30.1 4.00.7 21 days p.i 35 days p.i 21 days p.i 35 days p.i 21 days p.i 35 days p.i 6.60.3 6.50.2 5.00.4 6.00.2 4.70.5 4.40.5 10.10.2*** 5.10.9 7.10.1* 4.60.9 6.00.3*** TLR4 TLR9 –/– TLR2/4 –/– TLR2/4/9 C57BL/6 –/– MyD88 b) Liverb) –/– TLR2 a) Spleenb) 7.80.4* –/– –/– C57BL/6, TLR and MyD88 mice were infected with 100 CFU Mtb H37Rv via the aerosol route. For mycobacterial colony enumeration assays, organs from infected mice were removed at the indicated time points and CFU were counted 21 days after plating of serial diluted organ homogenates The data are reported as log10 CFU per organ. Means and SD of four mice per group are shown. Statistical analysis was performed by ANOVA defining differences between wild-type and mutant mice as significant (*p0.05; ***p0.001). f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2008. 38: 680–694 Immunity to infection Figure 2. In contrast to MyD88–/– mice, TLR2/4/9–/– animals efficiently control Mtb infection. Wild-type, TLR2/4/9–/–, and MyD88–/– mice were infected with 100 CFU Mtb H37Rv via the aerosol route. For mycobacterial colony enumeration assays, lungs from infected (A) TLR2/4/9–/– or (B) MyD88–/– mice and respective wild-type animals were removed at the indicated times and CFU were counted 21 days after plating of serial diluted organ homogenates. Means and SD of four mice per group are shown. (B) During the course of infection, survival of ten infected wild-type and MyD88–/– mice was monitored. Animals that lost more than 25% of their original body weight were sacrificed. Statistical analysis was performed by ANOVA defining differences between wild-type and mutant mice as significant (*p0.05; ***p0.001). Statistical analysis of the resulting survival curve was performed using the log rank test. Differences in survival kinetics between wild-type and MyD88–/– mice were significant (p0.001). level, similar to wild-type mice. Thus, all three TLR that have been implicated in Mtb sensing are dispensable for inducing protective cell-mediated immune responses to aerosol Mtb infection. In contrast, at 3 and 5 weeks following aerosol infection with 100 CFU Mtb, the bacterial load in the lungs of MyD88–/– mice was dramatically higher owing to exponential bacterial growth (Fig. 2B; Table 1) accompanied by enhanced dissemination into peripheral organs such as the liver and spleen (Table 1). Compared to infected wild-type mice, the bacterial load in the lungs of MyD88–/– mice was increased 15-fold (21 days p.i.) and 4600-fold (35 days p.i.). As a consequence of uncontrolled mycobacterial growth, all infected MyD88–/– mice died during the first 6 weeks of infection with Mtb (Fig. 2B; Table 1), whereas infected wild-type and TLR2/4/9 triple-deficient mice survived for at least 82 days (Fig. 2A). By day 35 p.i., the lungs of MyD88–/– mice harbored massive cellular infiltrations that were accompanied by extensive necrosis of granulomas, showing abundant mycobacteria within macrophages in the extracellular space, and erupting into bronchi (data not shown). In conclusion, MyD88, but not TLR2,TLR4 and TLR9, is critical for restricting mycobacterial growth. Innate immune responsiveness is unimpaired in TLR2/4/9–/– mice TLR2, TLR4, and TLR9 mediate cellular recognition of Mtb components [35]. To exclude potential crosscompensation by any one receptor in the absence of one or two other receptors, we performed infection experiments with Mtb in TLR2/4/9 triple-deficient mice. Comparative analysis of wild-type and TLR2/4/9–/– mice f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim after aerosol infection with Mtb revealed that in the latter mice the expression of TNF-a and IL-12/IL-23p40 was not reduced during the course of infection either on the RNA (Fig. 3A) or on the protein (Fig. 3B) level. Because MyD88 mediates signals from multiple TLR, we expected that the induction of innate pro-inflammatory and T cell responses might be comparable in TLR2/4/9–/– and MyD88–/– mice. For analysis, expression of pro-inflammatory cytokines in the lung was determined in wildtype and MyD88–/– mice after aerosol infection with Mtb (Fig. 3C and D). As determined by quantitative RT-PCR, TNF-a and IL-12/IL-23p40 mRNA accumulation was reduced in MyD88–/– mice during the first 4 weeks of infection (Fig. 3C). However, MyD88–/– mice were capable of inducing TNF-a and IL-12/IL-23p40 gene expression, albeit transcript levels were reduced compared to those measured in wild-type mice. The amount of IL-12/IL-23p40 protein was also found to be reduced in lung homogenates during the course of infection as revealed by cytometric bead array (CBA), although differences were not statistically significant (Fig. 3D). Taken together, our data indicate that TLR2/4/9 signals are dispensable for the induction of an innate proinflammatory immune response in murine tuberculosis. Adaptive immune responses are normal in Mtb-infected TLR2/4/9–/– animals In the lung, IL-12-driven CD4+ T cells are the most prominent lymphocyte population responsible for protective effector responses to Mtb infection [5]. However, antigen-specific CD8+ T cells are required to maintain adaptive immune responses [36]. At the infection site, both T cell types produce IFN-c as a master mediator of host defense against Mtb. www.eji-journal.eu 683 684 Christoph Hölscher et al. Eur. J. Immunol. 2008. 38: 680–694 Figure 3. Instructive immune responses are normal in Mtb-infected TLR2/4/9–/– mice. Wild-type, TLR2/4/9–/– and MyD88–/– mice were infected with 100 CFU Mtb H37Rv via the aerosol route. At the indicated time points, expression of TNF-a and IL-12/IL-23p40 was determined in lung homogenates from (A) TLR2/4/9–/– or (C) MyD88–/– mice and respective wild-type animals by RT-PCR based on gene expression in uninfected mice. Results represent means and SD of three mice. The content of IL-12/IL-23p40 protein was determined in lung homogenates from uninfected and infected (B) TLR2/4/9–/– or (D) MyD88–/– mice using a CBA. Results represent means and SD of four mice. Statistical analysis was performed by ANOVA defining differences between wild-type and mutant mice as significant (*p0.05; **p0.01). To determine the frequency of these IFN-c-producing T cells in the absence of TLR-dependent instructive mechanisms, lymphocytes isolated from lungs of infected animals were stimulated with plate-bound anti-CD3/CD28 21 days after aerosol infection with Mtb. As shown by intracellular cytokine staining, suspensions of unstimulated cells already contained substantial amounts of IFN-c+ CD4+ and CD8+ T cells (Fig. 4A and B). After stimulation with anti-CD3/CD28, an increased proportion of T cells were found to be IFN-c+ (Fig. 4A and B). Compared to stimulated wildtype lung cells, the frequencies of IFN-c+ CD4+ and CD8+ T cells did not differ from those of TLR2/4/9–/– lung lymphocytes (Fig. 4A). In contrast, unstimulated CD4+ and CD8+ T cells from Mtb-infected MyD88–/– mice already produced significantly lower amounts of IFN-c (Fig. 4B). After stimulation with anti-CD3/CD28, the amount of IFN-c+ CD4+ T cells was still reduced in MyD88–/– mice. However, MyD88–/– mice were capable of inducing a substantial proportion of IFN-c+ CD4+ T cells, and in response to anti-CD3/CD28 the amount of IFN-c+ CD8+ T cells did not differ from their frequency in wild-type mice (Fig. 4B). To analyze the number of antigen-specific T cells, enriched CD4+ and CD8+ Tcell suspensions with a purity of >90% were prepared from lungs of Mtb-infected mice f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim by magnetic cell sorting. The frequency of IFN-c+ antigenspecific CD4+ and CD8+ T cells was determined by an IFN-c ELISPOT assay after restimulation with ESAT61–20 or Mtb3293–102, respectively (Fig. 4C and D). The number of IFN-c-producing antigen-specific CD4+ and CD8+ T cells was not affected by TLR2, -4 and -9 deficiency (Fig. 4C). In contrast, the frequency of IFN-c+ CD4+ and CD8+ T cells was reduced in the lungs of infected MyD88–/– mice (Fig. 4D). However, the difference of the numbers of IFN-c+ cells in infected MyD88–/– and wildtype mice was not significant. Together, as a result of the efficient innate pro-inflammatory cytokine response in TLR2/4/9–/– mice, we concluded that adaptive immunity in murine tuberculosis is induced independently of TLR2/ 4/9-mediated signaling. MyD88 contributes tothe overall frequency of antigen-specific T cells in a TLR2/4/9independent fashion. IFN-c production and expression of IFN-c-dependent NOS2 and LRG-47 are normal in Mtb-infected TLR2/4/9–/– mice MyD88–/– mice have previously been shown to be highly susceptible to mycobacterial infection [23, 27, 33]. If, however, TLR and to some extent MyD88 are dispensable for innate pro-inflammatory and adaptive T cell www.eji-journal.eu Eur. J. Immunol. 2008. 38: 680–694 responses to Mtb infection, what effector functions might MyD88 be involved in that are pivotal to protection from experimental TB? To address this question, we analyzed the relevance of TLR and Immunity to infection MyD88 in IFN-c-mediated macrophage effector functions after infection with Mtb in vivo. IFN-c induces efficient macrophage activation and subsequent containment of intracellular mycobacterial Figure 4. Adaptive immune responses are normal in Mtb-infected TLR2/4/9–/– mice. Wild-type, TLR2/4/9–/– and MyD88–/– mice were infected with 100 CFU Mtb H37Rv via the aerosol route. After 3 weeks of infection, single-cell suspensions from lungs of (A, C) TLR2/4/9–/– or (B, D) MyD88–/– mice and respective wild-type animals were prepared. (A, B) Intracellular IFN-c production by antiCD3/CD28-stimulated T cells was analyzed by flow cytometry of gated CD44+CD4+ and CD44+CD8+ cells. Representative histograms of three mice per group are shown. Numbers in histograms indicate means and SD of three mice. (C, D) After enrichment of CD4+ or CD8+ T cells, the frequency of responding CD4+ or CD8+ T cells was determined after restimulation with ESAT61–20 or MTB3293–102 in an IFN-c ELISPOT assay. Results represent means and SD of five mice. Statistical analysis was performed by ANOVA defining differences between wild-type and mutant mice as significant (*p0.05; **p0.01). f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 685 Eur. J. Immunol. 2008. 38: 680–694 Christoph Hölscher et al. · 686 growth. Therefore, we quantified IFN-c expression in lung homogenates of Mtb-infected mice by CBA (Fig. 5A and D). Owing to the unimpaired development of antigen-specific CD4+ T cell-mediated immune responses following infection with Mtb, expression of IFN-c in lung homogenates from triple TLR-deficient mice was comparable to wild-type counterparts (Fig. 5A). IFN-c production was found to be reduced in MyD88–/– mice during the first 2 weeks of aerosol Mtb infection when f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Figure 5. TLR2/4/9–/– mice efficiently express IFN-c, LRG-47 and NOS2. Wild-type, TLR2/4/9–/– and MyD88–/– mice were infected with 100 CFU Mtb H37Rv via the aerosol route. At the indicted time points, expression of IFN-c and the IFN-c-dependent NOS2 and LRG-47 was determined in lung homogenates from (A, B) TLR2/4/9–/– or (D, E) MyD88–/– mice and respective wild-type animals by CBA or RT-PCR based on gene expression in uninfected mice, respectively. Statistical analysis was performed by ANOVA defining differences between wild-type and mutant mice as significant (*p0.05; ***p0.001). (C, F) For immunohistological detection of NOS2, 2–3-lm sections were prepared from (C) TLR2/4/9–/– or (F) MyD88–/– mice and respective wild-type animals at the indicated time points. Staining was performed with a polyclonal rabbit anti-mouse NOS2 antibody. Representative results of four mice per group are shown. compared to protein levels of IFN-c found in homogenates from wild-type mice (Fig. 5D). Thereafter, IFN-c production was rather increased in MyD88–/– mice. In line with the efficient induction of inflammatory innate and subsequent adaptive immune responses in Mtbinfected TLR2/4/9–/– mice, the mRNA expression of IFN-c-dependent effector molecules such as LRG-47 and NOS2 were also not impaired (Fig. 5B) and these mice exhibited substantial NOS2 protein expression in pulmonary granulomatous lesions after 82 days of infection (Fig. 5C). In contrast to triple-deficient mice, IFN-c-dependent macrophage effector functions were affected in Mtb-infected MyD88–/– mice to some extent. After aerosol infection with Mtb, RT-PCR of LRG-47 and NOS2 expression revealed that LRG-47 was induced in both wild-type and MyD88–/– mice to a substantial degree (Fig. 5E). In contrast, mRNA expression of NOS2 was significantly reduced when compared to wild-type mice (Fig. 5E). However, transcripts of NOS2 were found to be induced in MyD88–/– mice in a delayed fashion and, as shown by immunohistology, no difference in the expression of NOS2 on the protein level was apparent in lung sections of either mouse strain 21 days after aerosol infection with Mtb (Fig. 5F). At day 35 p.i., however, severe necrosis in lungs from MyD88–/– mice precluded any meaningful comparison of NOS2 expression in wildtype and mutant mice (Fig. 5F). In addition to the TLR-independent induction of adaptive immune responses after Mtb infection, our data demonstrate that the expression of two critical IFN-c-dependent macrophage effector molecules, LRG47 and NOS2, are not affected by the absence of TLR2, -4 and -9. Restriction of Mtb growth is impaired in MyD88–/– but not in TLR2/4/9–/– macrophages It has recently been shown that MyD88 is involved in macrophage effector mechanisms induced by IFN-c [37]. This suggested to us that the exacerbated www.eji-journal.eu Eur. J. Immunol. 2008. 38: 680–694 susceptibility of Mtb-infected MyD88–/– mice might be due to a defect at the level of IFN-c-mediated antibacterial macrophage effector functions. IFN-c-mediated anti-mycobacterial effector mechanisms are dependent on efficient maturation of the phagosomal compartment within macrophages [8, 9]. To analyze whether TLR- and MyD88-mediated signals influence Mtb phagosome maturation, the intracellular trafficking of Mtb was studied in resting, IFN-c- or IFN-c/ TNF-a-activated BMMU from wild-type, TLR2/4/9–/– and MyD88–/– mice. Cells were infected with Mtb-GFP [38] (MOI 5:1) for either 4 h or 2 days. Macrophages were labeled with tracers/antibodies for either early or late endosomal/lysosomal compartments, i.e., transferrin (Tf-Cy3), dextran (DexTR), lysosome associated membrane protein 1 (LAMP-1) or the vesicular proton pump (H+ATPase), respectively, and analyzed by confocal microscopy. At 4 h p.i., around 50% of the phagosomes were associated with Tf but less frequently with the late markers in resting cells (Fig. 6, Supporting Information Fig. 1). At the same time point, IFN-c promoted Mtb phagosome maturation towards LAMP-1and H+ATPase-positive late endosomal compartments in cells from all three mouse strains (Supporting Information Fig. 1). Loss of Tf association was prominent in wildtype and MyD88–/– macrophages (Fig. 6; see graphs for quantification), whereas in IFN-c-activated TLR2/4/9–/– macrophages more phagosomes became positive for both Tf and LAMP-1, also indicating transition towards a later stage (Fig. 6, Supporting Information Fig. 1). Importantly, association with the lysosomal tracer for the lysosomal stage, DexTR, was reduced at 4 h p.i. in IFN-c-activated MyD88–/– macrophages when compared to wild-type and TLR2/4/9–/– cells (Fig. 6). In contrast, activation by both IFN-c and TNF-a promoted phagosome maturation towards the late endosomal/lysosomal stage in cells from all three mouse strains (Fig. 6, Supporting Information Fig. 1). Although in fully activated MyD88–/– cells some phagosomes were positive for both LAMP-1 and Tf, the overall number of phagosomes was within late compartments (Fig. 6; Supporting Information Fig. 1). At day 2 p.i., IFN-c-mediated phagosome maturation was less efficient in MyD88–/– but also to some extent in TLR2/4/9–/– cells (Supporting Information Fig. 2 and 3). Our data indicate that, early in infection, IFN-c-dependent phagosome maturation in Mtb-infected macrophages was affected by the absence of MyD88. However, at later stages mycobacterial trafficking was impaired in both TLR2/4/9–/– and MyD88–/– macrophages. In addition to efficient maturation of the phagosomal compartment within macrophages, the IFN-c-dependent production of RNI through NOS2 is central to anti-mycobacterial effector responses in IFN-c-activated macrophages [11]. After infection, we found little induction of nitrite in the f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Immunity to infection supernatants of infected BMMU from wild-type, MyD88–/– and TLR2/4/9–/– mice (Fig. 7). In contrast, simultaneous IFN-c activation and Mtb infection enhanced nitrite production in MyD88–/– cells, albeit to a significantly lower extent when compared with wildtype cells. The addition of IFN-c to TLR2/4/9–/– BMMU also led to an increased nitrite content that was still significantly reduced. However, addition of TNF-a restored the ability of MyD88–/– and TLR2/4/9–/– macrophages to produce nitrite at amounts that were comparable to those generated by wild-type macrophages (Fig. 7). Finally, we determined the growth of Mtb H37Rv in BMMU derived from wild-type, TLR2/4/9–/– and MyD88–/– mice that were incubated with medium or IFN-c 24 h prior to infection (Fig. 8). IFN-c-primed macrophages from wild-type and TLR2/4/9–/– mice were both able to reduce mycobacterial growth to a similar extent after 72 h of infection with H37Rv (Fig. 8). In contrast, IFN-c-incubated MyD88–/– macrophages were unable to reduce the growth of Mtb (Fig. 8), showing a substantially increased bacterial load as compared to that of activated macrophages from wildtype mice 72 h p.i. (Fig. 8). To evaluate whether IFN-cor TNF-a/NFjB-dependent pathways are affected in MyD88–/– macrophages, infected cells were additionally stimulated with TNF-a (Fig. 8). In contrast to unstimulated macrophages, cells from wild-type, TLR2/4/9–/– and MyD88–/– mice were able to restrict bacterial growth when stimulated with TNF-a. Discussion Based on in vitro studies TLR2, TLR4, and TLR9 have all been suggested to be involved in the innate induction of a Th1 cell-mediated immune responses after infection with Mtb [13, 15, 16, 18, 22, 39, 40]. We now demonstrate that TLR2/4/9–/– mice are capable of efficiently controlling Mtb infection. The different and highly variable outcome of infection in Mtb-infected TLR2-, TLR4-, TLR9-singledeficient and TLR2/4- or TLR2/9-double-deficient mice in this and a number of other studies [20–25, 41, 42] may be due to different housing conditions of animals reflecting different levels of preactivation, and/or to infection with different Mtb strains. For example, TLR9–/– mice that were infected with a high-dose inoculum (500 CFU) of H37Rv were highly susceptible to Mtb infection and died between days 45 and 75 upon infection [19]. However, using our own H37Rv lab strain, an aerosol infection even with 2000 CFU did not lead to premature death in TLR9–/– mice (data not shown). www.eji-journal.eu 687 688 Christoph Hölscher et al. Eur. J. Immunol. 2008. 38: 680–694 Figure 6. Endosome and lysosome maturation in TLR2/4/9–/– and MyD88–/– macrophages early after Mtb infection. Cells from wildtype, TLR2/4/9–/– and MyD88–/– mice were either left resting or activated overnight with 1000 U/mL IFN-c, or 1000 U/mL IFN-c and 20 ng/mL TNF-a. Macrophages were infected with Mtb-GFP at an MOI of 5:1 for 2 h, washed and further incubated for 2 h. Early compartments in live cells were labeled using Tf-Cy3 (early endosomes). Lysosomes were labeled in live cells using DexTR and investigated by confocal fluorescence microscopy (A). Macrophage activation decreases the amount of Tf uptake independently of the mouse strain analyzed. Pictures represent representative examples. Phagosomes are indicated by arrows, yellow = colocalization, white = no colocalization (magnification: 1000). Inserts represent Mtb-GFP (magnification: 5000). (B) Quantification of Mtb-GFP phagosome association with intracellular markers. By confocal LSM, Mtb-GFP phagosomes from 10 to 30 cells from independent cover slips were evaluated double-blindly for colocalization with either Tf-Cy3 or DexTR and expressed as % means  SD. The use of TLR triple-deficient mice supported our previous contention that TLR are dispensable for innate and Th1 responses and subsequent macrophage effector functions against Mtb. Hence, TLR2/4/9-independent but MyD88-mediated mechanisms are necessary for the f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim induction and effective expression of protective immune responses. Despite only moderately reduced numbers of IFN-c-producing T cells, survival kinetics in Mtb-infected MyD88–/– mice are similar to infected IFN-c–/– mice [43, 44]. This indicates that anti-mycobacterial effector www.eji-journal.eu Eur. J. Immunol. 2008. 38: 680–694 Figure 7. RNI production is impaired in IFN-c-stimulated and Mtb-infected macrophages from MyD88–/– and TLR2/4/9–/– mice. BMMU from wild-type, TLR2/4/9–/– and MyD88–/– mice were either incubated with medium, Mtb H37Rv, Mtb H37Rv and 1000 U/mL IFN-c, or Mtb H37Rv, 1000 U/mL IFN-c and 20 ng/mL TNF-a. Supernatants were harvested after 48 h and the nitrite contend was measured. Each point indicates mean and SD of triplicate values from one out of two representative experiments. Statistical analysis was performed by ANOVA defining differences between wild-type and MyD88–/– (**p0.01) or TLR2/4/9–/– (+++p0.001) mice as significant. mechanisms – most likely in macrophages – rather than instructive innate immune responses are induced or recruited independently of TLR2/4/9 but are severely affected by the absence of MyD88. A pivotal role of MyD88 for the expression of effector immune responses in IFN-c-activated macrophages had originally been highlighted by microarray studies comparing gene expression in wild-type, TLR- and Immunity to infection MyD88-deficient macrophages. While TLR-induced gene expression in macrophages in response to Mtb was largely independent of MyD88 [37], responses to IFN-c were severely compromised in MyD88-deficient cells. This was recently attributed to a direct involvement of MyD88 in the IFN-c signal transduction pathway [45, 46]. Inefficient expression of TNF-a and IL-12/ IL-23p40 in Mtb-infected MyD88–/– mice is probably aggravated by a defective IFN-c-signaling pathway. In addition, genes central to macrophage effector responses against Mtb may also be stabilized through IFN-cR1/MyD88-mediated p38 activation. Through our analysis of BMMU in vitro, we show here that the ability of Mtb-infected macrophages to induce NOS2 and to produce RNI in response to IFN-c was largely dependent on MyD88, in line with a previous report [37]. In addition, our in vitro experiments revealed that the defective RNI production and mycobacterial growth restriction in Mtb-infected and IFN-c-stimulated MyD88–/– macrophages could be restored by TNF-a in vitro, similar to findings reported by Shi et al. [37]. Therefore, IFN-c- and not TNF-a/ NFjB-mediated effector mechanisms appeared to be specifically impaired in the absence of MyD88. However, both signaling pathways are functional in TLR2/4/9–/– mice in vivo possibly explaining the differential outcome of Mtb infection in both mutant mice. The lack of MyD88 expression may have a strictly local effect by being restricted to macrophages at the infection site, which eludes systemic detection. Therefore, a defect in generation of RNI may still account for ablation of macrophage mycobacteriostatic functions in MyD88–/– mice in vivo. Figure 8. Restriction of Mtb growth is not impaired in TLR2/4/9–/– macrophages. To analyze the bacterial growth in IFN-c-stimulated macrophages, BMMu from wild-type, TLR2/4/9–/– and MyD88–/– mice were incubated with medium, IFN-c, or IFN-c/TNF-a for 24 h before infection with Mtb H37rv at an MOI of 1:1. Cells were lysed at different time points, serial dilutions were plated onto agar plates and CFU were determined after 19–21 days. Data represent means and SD of triplicate cultures. Statistical analysis was performed by ANOVA defining differences between medium and IFN-c-stimulated cells (*p0.05; **p0.01) or medium and IFN-c/ TNF-a-incubated macrophages (#p0.05; ##p0.01; ###p0.001) as significant. f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 689 690 Eur. J. Immunol. 2008. 38: 680–694 Christoph Hölscher et al. Upon IFN-c activation of macrophages, mycobacteria-containing phagosomes mature from an early to a late endosomal stage concomitant with reduced mycobacterial growth and viability [8]. Although both TLR and MyD88 signaling were initially suggested to influence phagosome maturation [47], a recent report describes phagosome maturation to be entirely independent of TLR2 and 4 but dependent on MyD88 [48]. The differential influence of MyD88 and TLR2/4/9 on maturation of Mtb phagosomes, however, has not yet been directly investigated. Our results suggest that IFN-c-promoted Mtb phagosome maturation requires MyD88 in the initial infection phase. Although fewer mycobacteria trafficked to phagolysosomes in IFN-c-activated MyD88–/– cells as compared to TLR2/ 4/9–/– and wild-type macrophages (most obvious early after infection), deficient phagosome maturation on its own is unlikely to account for the unrestricted growth in these cells. However, the impaired killing of Mtb in activated MyD88–/– macrophages may find its explanation in combination with reduced RNI production. Taken together, our results show the striking susceptibility of MyD88–/– mice to Mtb to be caused by a combination of defects such as reduced TNF-a production and impaired IFN-c-mediated RNI generation and phagosome maturation. Importantly, all of these effector mechanisms were still in place in TLR2/4/9–/– mice. In addition to TLR signal transduction, MyD88 serves an equally critical role in IL-1 and IL-18 receptor/IRAK signal-transduction. Therefore, contribution of the latter signaling pathway to defective immune responses observed in MyD88–/– mice cannot be excluded. Whereas a deficiency in IL-18 is associated only with minor defects in anti-TB immunity in mice [49], infection of IL-1R–/– mice with Mtb H37Rv has very recently been shown to result in exacerbated bacterial loads and rapid death [50]. Importantly, the only moderately reduced innate and adaptive immune responses, uncontrolled mycobacterial growth, histopathology and survival kinetics in Mtb H37Rv-infected IL-1R1–/– mice were highly similar to the kinetics observed in Mtb-infected MyD88–/– mice [50], implying that a common defect downstream of adaptive immune responses – possibly in macrophages – must account for the detrimental outcome of infection in both strains of mice. It appears plausible that TLR-independent, but IL-1R1-mediated MyD88-dependent signaling compensates for the absence of all three TLR and promotes the induction of an inflammatory immune response during Mtb infection. In fact, similar to TLR, activation of the socalled inflammasome, which is involved in processing IL-1b, is thought to occur through the recognition of pathogen associated molecular patterns (PAMP) by the leucine-rich repeat of the NACHT-, LLR-, and pyrindomain (NALP)-containing proteins [51]. In line with f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim the cytoplasmic localization of the inflammasome, van der Wel and colleagues [52] have recently shown that Mtb is capable of translocating from the phagolysosome to the cytosol where it may encounter cytosolic PRR different from TLR. This putative TLR-independent recognition mechanism in Mtb infection may bypass the TLR-mediated signaling pathway, but still rely on IL-1R signaling via MyD88. In conclusion, our results demonstrate that after Mtb infection, TLR-mediated pattern recognition and MyD88-dependent adaptor signaling have little impact on the induction and expression of Th1 immune responses. In contrast, MyD88 shapes anti-bacterial effector mechanisms in macrophages in response to IFN-c independently of its function as a TLR signal transducer. Our results not only underscore the dual function of MyD88 as a TLR-coupled signaling adaptor on the one hand and a master regulator of macrophage activation on the other, they also assign critical relevance for in vivo protection against TB only to the latter. Materials and methods Mice and macrophages MyD88–/– (Research Center Borstel, Germany) [29], TLR2–/–, TLR4–/–, TLR9–/–, TLR2/4–/–, and TLR2/4/9–/– (from the Technical University of Munich, Germany) mice on a C57BL/6 genetic background were maintained under specific-pathogenfree conditions. As wild-type controls, C57BL/6 mice were purchased from Charles River (Sulzfeld, Germany). All experimental mice were between 8 and 16 weeks old. In any given experiment, mice were matched for age, sex and genetic background. For infection experiments, mice were maintained under barrier conditions in the BSL 3 facility at the Research Center Borstel in individually ventilated cages. All experiments performed were in accordance with the German Animal Protection Law and were approved by the Animal Research Ethics Board of the Ministry of Environment, Kiel, Germany. BMMU were obtained after flushing of femora from the mice described above. To generate BMMU, bone marrow cells were cultivated in L-929 conditioned medium as source for MCSF activity for 7 days [8]. Bacteria and infection For all experiments, Mtb H37rv were used. For confocal microscopy of mycobacterial trafficking within BMMU, GFPexpressing Mtb were applied (kindly provided by Tanya Parish, Centre for Infectious Diseases, Institute for cell and Molecular Science, London, UK) [38]. Mtb was grown in Middlebrook 7H9 broth (Difco, Detroit, MI) supplemented with Middlebrook OADC enrichment medium (Life Technologies, Gaithersburg, MI), 0.002% glycerol, and 0.05% Tween 80. Midlog phase cultures were harvested, aliquoted, and frozen at –80 C. www.eji-journal.eu Eur. J. Immunol. 2008. 38: 680–694 After thawing, viable cell counts were determined by plating serial dilutions of the cultures on Middlebrook 7H10 agar plates followed by incubation at 37 C. For in vitro experiments, Mtb aliquots were thawed, spun at 835  g and subsequently resuspended in PBS. BMMU were infected with different multiplicities of infection (MOI) of Mtb. For bacterial growth inhibition assays, determination of RNI and intracellular localization of mycobacteria, BMMU were incubated with 200 U/mL IFN-c or 200 U/mL IFN-c and 20 ng/mL TNF-a (both from Peprotech) 24 h before infection. Lipopeptide (LP; 10 ng/mL Pam3Csk4; EMC Microcollections, T
bingen, Germany), bacterial LPS of Salmonella enterica, serotype friedenau H909 (10 ng/mL; kindly provided by H. Brade, Research Center Borstel, Germany) and CpG (1 lM ODN1648; a kind gift of A. Dalpke, University of Heidelberg, Germany) were used as control stimuli. Before infection of experimental animals, stock solutions of Mtb were diluted in sterile distilled water and pulmonary infection was performed using an inhalation exposure system (Glas-Col, Terre-Haute, IN). To infect mice with a dose of 100 CFU/lung, animals were exposed for 40 min to an aerosol generated by nebulizing approximately 5.5 mL of a suspension containing 107 live bacteria. Inoculum size was checked 24 h p.i. by determining the bacterial load in the lung of infected mice. Mice were regularly weighed before and after infection. In accordance with the Animal Research Ethics Board of the Ministry of Environment, mice that lost 25% of their original weight during the course of infection had to be killed. Colony enumeration assay and immunohistology Bacterial loads in lungs, spleen and liver were evaluated at different time points p.i. for Mtb to follow the course of infection. Organs from sacrificed animals were removed aseptically, weighed and homogenized in PBS containing a proteinase inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) prepared according to the manufacturer's instructions. Tenfold serial dilutions of organ homogenates were plated in duplicates onto Middlebrook 7H10 agar plates containing 10% OADC and incubated at 37 C for 19–21 days. Colonies on plates were enumerated and results expressed as log10 CFU per organ. For immunohistology, one lung lobe per mouse were fixed in 4% formalin-PBS, set in paraffin blocks, and sectioned (2–3 lm). Tissue sections were prepared and stained with a polyclonal rabbit anti-mouse NOS2 antiserum (Biomol, Hamburg, Germany) as previously described [20]. Preparation of single-cell suspensions from infected lungs For antigen-specific restimulation and flow cytometric analysis, single-cell suspensions of lungs were prepared from Mtbinfected mice at different time points. Mice were anesthetized and injected intraperitoneally with 150 U heparin (Ratiopharm, Ulm, Germany). Lungs were perfused through the right ventricle with warm PBS. Once lungs appeared white, they were removed and sectioned. Dissected lung tissue was then incubated in collagenase A (0.7 mg/mL; Roche Diagnostics, Mannheim, Germany) and DNase (30 lg/mL; Sigma) at 37 C for 2 h. Digested lung tissue was gently disrupted by f 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Immunity to infection subsequent passage through a 100 lm pore size nylon cell strainer. Recovered vital lung cells were counted using an automatic cell counter (ViCellJ; Beckman Coulter, Krefeld, Germany), diluted in complete Iscove's-modified Dulbecco's medium (IMDM; Gibco) supplemented with 10% FCS (Gibco), 0.05 mM b-mercaptoethanol (Sigma, Deisenhofen, Germany), and penicillin and streptomycin (100 U/mL and 100 lg/mL; Gibco) and used for further experiments. RT-PCR Before and at different time points after aerosol infection with Mtb, weighed lung samples were homogenized in 5 mL 4 M guanidinium isothiocyanate buffer and total RNA was extracted by acid phenol extraction. cDNA was obtained using murine Moloney leukemia virus (MMLV) reverse transcriptase (Superscript II, Invitrogen, Karlsruhe, Germany) and oligo-dT (12–18mer; Sigma) as a primer. Quantitative PCR was performed on a Light Cycler (Roche Diagnostics Corporation, Indianapolis, IN) as previously described [53]. Data were analyzed employing the “Fit Points” and “Standard Curve Method” using b-2-microglubulin (b2m) or hypoxanthineguanine phosphoribosyl transferase (hprt) as housekeeping genes to calculate the level of gene expression in relation to b2m or hprt, respectively The following primer sets were employed: b2m: sense 50 -TCA CCG GCT TGT ATG CTA TC-30 , antisense 50 -CAG TGT GAG CCA GGA TAT AG-30 ; hprt: sense 50 GCA GTA CAG CCC CAA AAT GG-30 , antisense 50 -AAC AAA GTC TGG CCT GTA TCC AA-30 ; TNF-a: sense 50 -TCT CAT CAG TTC TAT GGC CC-30 , antisense 50 -GGG AGT AGA CAA GGT ACA AC30 , IL-12/23p40: sense 50 -CTG GCC AGT ACA CCT GCC AC-30 , antisense 50 -GTG CTT CCA ACG CCA GTT CA-30 , NOS2: sense 50 -AGC TCC TCC CAG GAC CAC AC-30 , antisense 50 -ACG CTG AGT ACC TCA TTG GC-30 , LRG-47: sense 50 -AGC CGC GAA GAC AAT AAC TG-30 , antisense 50 -CAT TTC CGA TAA GGC TTG G-30 . Quantification of TNF-a, IL-12/23p40, and IFN-c To determine cytokine production after infection of BMMU, supernatants were analyzed in threefold serial dilutions using the sandwich ELISA system OptEiaTM (BD Biosciences). After incubation with horseradish peroxidase (HRP) coupled to avidin and developing with TMB substrate reagent, the absorbance was read on a microplate reader (Sunrise; Tecan, Mnnedorf, Switzerland). Samples were compared to appropriate recombinant cytokine standards using a test wavelength of 450 nm and a reference wavelength of 630 nm. The detection limits for all cytokines were 5 pg/mL. The concentrations of IL-12/IL-23p40 and IFN-c in lung homogenates from uninfected and infected mice were determined by CBA (BD Bioscience). IFN-c was analyzed using a ready CBA mouse-flex-set (BD Bioscience). To determine IL-12/IL-23p40, beads were conjugated with a purified mouse anti-IL-12/IL-23p40 antibody (BD Bioscience) using a functional bead-conjugation buffer set following the manufacturer's instructions (BD Bioscience). In a 96-well plate, 50 lL lung homogenates containing a proteinase inhibitor cocktail (Roche Diagnostics) and serial dilutions of cytokine standards were incubated with 50 lL of respective beads diluted in capture-bead dilution buffer. After 1 h at room www.eji-journal.eu 691 692 Eur. J. Immunol. 2008. 38: 680–694 Christoph Hölscher et al. temperature in the dark, beads were washed and 50 lL of a biotinylated mouse anti-IL-12/IL-23p40 antibody was added and incubated for another 1 h. A PE-detection reagent mix was added and plates were incubated at room temperature for 1 h in the dark. Once plates were washed in wash buffer, 500 events/cytokine were acquired on a FACSCalibur (BD Biosciences) gating on the total bead population identified by FSC-SSC-profile. Data and cytokine concentrations were analyzed using the FCAPArray software (BD Bioscience). ated using an ELISPOT reader (EliSpot 04 XL; AID, Strassberg, Germany). The frequency of responding CD4+ and CD8+ T cells was determined. Neither CD4+ or CD8+ T cells cultured in the absence of ESAT61–20 or Mtb3293–102 nor cells from uninfected mice produced detectable spots. Intracellular IFN-c staining To analyze the anti-bacterial activity of macrophages, BMMU were cultured 24 h before infection with Mtb in DMEM supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES (all from PAA, Pasching, Austria) in the presence of medium, or stimulated with either of 200 U/mL IFN-c or 200 U/mL IFN-c and 20 ng/mL TNF-a (both from Peprotech). Cells were inoculated with Mtb H37Rv at an MOI of 1:1 for 4 h. BMMU were washed vigorously with warm HBSS (PAA) to remove extracellular bacteria. To determine bacterial uptake at different time points, macrophages were lysed by addition of 0.1% saponin (Sigma). Lysates were serially diluted in 0.05 % Tween 80 (EMD Biosciences, Darmstadt, Germany) and plated on Middlebook 7H10 agar containing 10% OADC. To determine the production of RNI, supernatants were harvested to quantify the release of nitrite. To quantify the production of RNI, the content of nitrite in supernatants was determined after adding Griess reagents by photometric measurement reading the absorbance at 540 nm as previously described [54]. Intracellular localization of Mtb within IFN-c-activated macrophages was analyzed in BMMU after infection with MtbGPF at an MOI of 5:1 for 2 h [8]. After 4 h or 2 days, cells were labeled with tracers/antibodies for either early or late endosomal/lysosomal compartments, i.e., Tf-Cy3, DexTR (Invitrogen, Paisley, UK), LAMP-1 (IB4D, Developmental Studies Hybridoma Bank, University of Iowa, IA) or the vesicular proton pump (H+ATPase, a kind gift from Mhairi Skinner, University of Guelph, Ontario, Canada), respectively, and analyzed by confocal microscopy using a confocal fluorescence microscope (LSM 510, Carl Zeiss Inc., Jena, Germany). Mtb-GFP phagosomes from 30 cells from independent cover slips were evaluated double-blindly for colocalization with either tracer. For detection of intracellular IFN-c, an intracellular cytokine staining kit (BD Biosciences) was employed. Briefly, single-cell suspensions were prepared at 21 days p.i. and 2  106 cells were incubated with IMDM or stimulated with plate-bound anti-CD3/CD28 mAb (clone 2C11 and clone 37/51 at 10 lg/ mL, respectively ) for 4 h in the presence of GolgiPlugTM (BD Biosciences). Nonspecific antibody binding was blocked by incubation with a cocktail containing anti-FccRIII/II mAb (clone 2.4G2), mouse and rat serum. Cells were washed and incubated with optimal concentrations of anti-CD44-FITC and anti-CD4-APC, or anti-CD8-APC (all from BD Biosciences). After staining, cells were fixed and permeabilized with Cytofix/CytopermTM (BD Biosciences) and intracellularly accumulated IFN-c was stained with a PE-labeled anti-IFN-c mAb (BD Biosciences). Fluorescence intensity was analyzed on a FACSCalibur (BD Biosciences) gating on CD44+CD4+ or CD44+CD8+ lymphocytes identified by FSC-SSC profile. ESAT61–20- and Mtb3293–102-specific IFN-c ELISPOT assay For measuring antigen-specific CD4+ and CD8+ T cells in lungs from infected mice, single-cell suspensions were resuspended in IMDM. To enrich CD4+ T cells, single cell suspensions were incubated with magnetic CD4 microbeads (Miltenyi, Bergisch Gladbach, Germany) and separated from other cells on an MACS separation unit (Miltenyi). Separated CD4+ and CD8+ T cells were collected in IMDM, counted using a cell counter (ViCellJ; Beckman Coulter), diluted in IMDM and used for further experiments. Purity of enriched CD4+ T cells was >90% as determined by flow cytometry. Detection of antigenspecific IFN-c-producing CD4+ and CD8+ T cells from infected lungs was conducted using an ELISPOT assay kit (AID Diagnostika, Strassberg, Germany). In brief, purified CD4+ or CD8+ T cells from lungs of infected mice were seeded in wells of anti-mouse IFN-c-coated and blocked 96-well multitest plates at an initial concentration of 1  105 cells/ well in IMDM. After making doubling dilutions of these cells, mitomycin-D (Sigma)-inactivated splenocytes from uninfected wild-type mice were used as APC at a concentration of 1  106 cells/well. CD4+ and CD8+ T cells were restimulated with the Mtb ESAT61–20 or Mtb3293–102 peptides, respectively (Research Center Borstel, Germany) at a concentration of 10 lg/mL in the presence of 10 U/mL recombinant mouse IL-2 (Peprotech). After 20 h of incubation in 5% CO2 at 37 C, plates were washed, and biotinylated anti-mouse IFN-c was used to detect the captured cytokine. Spots were visualized using streptavidin-HRP and as substrate. Spots were automatically enumerf 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim IFN-c-induced bacterial growth inhibition, RNI production and phagosome maturation in macrophages Statistical analysis Quantifiable data are expressed as the means and SD of individual determinations. Statistical analysis was performed by ANOVA defining different error probabilities as significant (*,+p0.05), (**,++p0.01), (***,+++p0.001). Acknowledgements: The present study was financially supported in part by the German Research Foundation (Research Grants HO 2145/2–1 to C.H., SFB 415-C7 to S.E. and N.R., and SP “Innate Immunity” FR 448/4–2 to M.F., SPP1131–2 Scha 514–2 to U.S.), the National Genome Research Network (Workpackage Tuberculosis, NIES05T22) to S.E. and Royal Society Wolfson Research www.eji-journal.eu Eur. J. Immunol. 2008. 38: 680–694 Merit Awards to U.S. The authors thank Stefanie Pfau, Susanne Metken and Manfred Richter for excellent technical assistance; Ilka Monath, Sven Mohr and Claus M
ller for organizing the animal facility and taking care of the mice at the Research Center Borstel; Hella Stbig and Helga Kochanowski for taking care of the mice at the Max-Planck-Institute for Immunobiology; Tanya Parish at the Centre for Infectious Disease, Institute for Cell and Molecular Science, London, United Kingdom and Mhairi Skinner, University of Guelph, Ontario, Canada for providing Mtb-GFP and anti-H+ATPase, respectively; and Kristine Hagens for her expert technical assistance at the Max-Planck-Institute for Infection Biology. Conflict of interest: The authors declare no financial or commercial conflict of interest. References 1 Kaufmann, S. H., Tuberculosis: Back on the immunologists' agenda. Immunity 2006. 24: 351–357. 2 Maher, D. and Raviglione, M., Global epidemiology of tuberculosis. Clin. Chest Med. 2005. 26: 167–182, v. 3 Kaufmann, S. H., How can immunology contribute to the control of tuberculosis? Nat. Rev. Immunol. 2001. 1: 20–30. 4 North, R. J. and Jung, Y. 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