Expression profiling of host pathogen interactions: how Mycobacterium tuberculosis and the macrophage adapt to one another

Microbes and Infection 8 (2006) 1132e1140 www.elsevier.com/locate/micinf

Forum in immunology

Expression profiling of host pathogen interactions: how Mycobacterium tuberculosis and the macrophage adapt to one another Dirk Schnappinger a,c,*, Gary K. Schoolnik b, Sabine Ehrt a,d,* a Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021, USA Department of Medicine, Division of Infectious Diseases and Geographic Medicine, and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA c Graduate Program in Molecular Biology, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021, USA d Graduate Program in Immunology and Microbial Pathogenesis, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021, USA b

Available online 19 January 2006

Abstract It has recently become feasible to quantify all mRNAs encoded by the genomes of bacterial pathogens and their eukaryotic host cells and to apply this approach to study the interaction of Mycobacterium tuberculosis with its primary host cell, the macrophage. These studies helped to identify regulatory circuits which mediate adaptation of the M. tuberculosis transcriptome to intraphagosomal environments and stimulated hypotheses for the function of these circuits in human tuberculosis. The macrophage transcriptome reacts to infections with the induction of a pathogen-unspecific expression program as well as the induction of pathogen-specific expression signatures, both of which contribute to the immunologic activation of the infected cell. M. tuberculosis induced changes in the macrophage transcriptome are mediated by Toll-like receptor dependent and Toll-like receptor independent signal transduction pathways. This response is shaped by macrophage produced reactive nitrogen and oxygen molecules and affected by viability and virulence of the pathogen. Ó 2006 Elsevier SAS. All rights reserved. Keywords: Microarray gene expression analysis; Macrophage activation; Innate immunity; Tuberculosis; Pathogenicity

1. Introduction Abbreviations: AM, alveolar macrophages; DC, dendritic cells; FPR, formylpeptide receptor; H2O2, hydrogen peroxide; hMDM, human monocyte derived macrophages; LAM, lipoarabinomannan; NO, nitric oxide; NOD, nucleotide-binding oligomerization domain; NOS2, nitric oxide synthase 2; PBMCs, peripheral blood mononuclear cells; phox, phagocyte oxidase; PIM, phosphatidyl-myo-inositol mannoside; (p)ppGpp, ppGpp guanosine 50 -diphosphate 30 diphosphate or pppGpp, guanosine 50 -triphosphate 30 diphosphate; RFLP, restriction fragment length polymorphism; RNI, reactive nitrogen intermediates; ROI, reactive oxygen intermediates; TLR, Toll-like receptor; wt, wild type. * Corresponding authors. Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10021, USA. Tel.: þ1 212 746 2994/3788; fax: þ1 212 746 8587. E-mail addresses: [email protected] (D. Schnappinger), sae2004@ med.cornell.edu (S. Ehrt). 1286-4579/$ - see front matter Ó 2006 Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2005.10.027

The availability of microbial genome sequences and the development of microarray-based expression profiling methods have made it possible to capture the transcriptome of an organism during different states of growth. The initial application of this method to study Mycobacterium tuberculosis used transcript profiling experiments of exponential phase homogeneous cultures in liquid media to identify genes regulated during adaptation of the organism to a specific in vitro growth condition. This method was then extended in the experiments described below to examine, at the transcriptional level, how M. tuberculosis adapts to the macrophage phagosome, a site that is believed to harbor significant numbers of the organism within infected tissues. In contrast to studies using well

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defined in vitro conditions of growth, the technical and conceptual challenges of characterizing the intraphagosomal transcriptome are considerable. Technical innovations were required to contend with the vastly greater abundance of host compared to microbial RNA [1]. Conceptual and experimental design issues further complicate ex vivo experiments of this kind: unlike the single-condition design of most in vitro experiments, within the phagosome, M. tuberculosis confronts a multifaceted biochemical milieu; many (perhaps most) of the biochemical features of this environment are unknown; and, the heterogeneity of organism growth state and of phagosomal maturation state, although mitigated by careful attention to experimental design, lead to the possibility that transcripts from different populations of organisms will be combined, resulting in averaged transcript abundance values that may misrepresent the transcriptomes of physiologically distinct subsets of intracellular bacteria. Finally, since the aim of such studies is to provide a model that predicts how M. tuberculosis adapts to the macrophage compartment of granulomas in vivo, it is necessary to determine if M. tuberculosis genes which were found to be regulated within cultured macrophages are similarly regulated within infected human tissues and whether their presence is required for the successful adaptation of the organism to the host. Similar challenges confront expression studies of M. tuberculosis-infected host cells. These technical, experimental and conceptual issues are discussed below together with the principal findings of studies using microarray expression profiling methods and primary macrophages to obtain and interpret the M. tuberculosis intraphagosomal transcriptome and the transcriptome of the corresponding host cell. 2. Bacterial gene expression during infections: experimental approaches Several strategies have been developed to analyze bacterial gene expression during infections. Most use reporter genes to identify promoters from random libraries of chromosomal DNA fragments that are active during an infection. Reporters that allow such selections include genes encoding fluorescent proteins, site specific recombinases, antibiotic resistance factors or enzymes which complement mutations that impair bacterial growth within the host (reviewed in [2]). These approaches can be applied to infections of tissue cultures as well as animals. They identify genes whose increased expression during an infection is caused by increased transcription. Microarray based expression experiments use gene-specific hybridization probes to quantify the relative abundance of specific RNAs. RNAs whose abundance increases during infections identify genes that are preferentially expressed during an infection. The use of RNA instead of reporter genes has advantages and disadvantages. RNA analyses allow identification of genes whose expression during an infection is based on an increase in transcription or is due to increased mRNA stability. The half-life of most bacterial RNAs is short whereas the half-life of the proteins encoded by most reporter genes is long. RNA can therefore be used to achieve a more dynamic

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analysis of bacterial gene expression. However, microarray based RNA analyses usually lack enzymatic amplification and are therefore less sensitive than reporter gene assays. This makes the analysis of bacteria isolated from infected animals difficult because large numbers of animals are required to isolate sufficient amounts of bacterial RNA [3]. Isolation of bacterial RNA from infected tissue cultures is also challenging, but methods that allow stabilization and separation of bacterial RNA from host RNA have recently been developed [1]. Application of these methods to the analysis of intraphagosomal M. tuberculosis and Salmonella typhimurium identified a large number of bacterial genes that are preferentially expressed (i.e., differentially up-regulated compared to their regulation in an in vitro reference condition of growth) during residence of these organisms in macrophages [4,5]. In the following two chapters we discuss the regulatory circuits that coordinate adaptation of the M. tuberculosis transcriptome to an intraphagosomal lifestyle. While these strategies have been very informative, it is important to note that not all pathogen genes which are transcribed in the macrophage, or even within infected tissues, are necessarily required for virulence or for the in vivo survival of the organism. Only gene deletion studies can demonstrate that an expressed gene confers a functional phenotype in the macrophage or appropriate animal model.

3. Adaptation of the M. tuberculosis transcriptome to resting macrophages Exposure of M. tuberculosis growing in liquid culture to a variety of stresses induces genes that were also induced after phagocytosis of M. tuberculosis by resting macrophages. The mechanisms that regulate the responses to three of these stresses, nutrient starvation, heat shock and SDS, have been studied in detail and are discussed below. Mutants of M. tuberculosis that are incapable of synthesizing the amino acids leucine [6], tryptophane, or histidine [7] did not grow in macrophages. The tryptophane and histidine mutants as well as a proline auxotroph also did not survive well in macrophages that restrict replication of wild type (wt) M. tuberculosis [8]. These mutants did grow like wt M. tuberculosis in liquid media supplemented with the appropriate amino acid, suggesting that amino acids cannot be obtained efficiently from the host cell by intraphagosomal M. tuberculosis. Amino acid starvation was the first condition recognized to induce a program of events, the so-called stringent response, which coordinates the activity of biosynthetic pathways, especially protein synthesis, with resource availability. In addition to amino acid starvation, other nutrient limitations such as carbon-, nitrogen-, and phosphorus-starvation, as well as oxidative stress can trigger the stringent response in E. coli [9]. All conditions that trigger the stringent response lead to an accumulation of hyperphosphorylated guanosine, (p)ppGpp (ppGpp, guanosine 50 -diphosphate 30 diphosphate, or pppGpp, guanosine 50 -triphosphate 30 diphosphate) [10]. (p)ppGpp initiates the stringent response by mechanisms that include a direct interaction with the b-subunit of RNA

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polymerase resulting in alterations of RNA polymerase promoter selectivity [9]. M. tuberculosis contains one enzyme, RelMtb, that controls synthesis and hydrolysis of (p)ppGpp[11]. Inactivation of the gene encoding RelMtb produced a strain, H37RvDrelMtb, which is unable to synthesize (p)ppGpp. H37RvDrelMtb does not repress the expression of ribosomal genes in response to starvation, is defective in long-term stationary phase survival in liquid culture and has a reduced ability to sustain chronic infections in mice [12e14]. These findings suggest that H37RvDrelMtb is less able to persist within growth-restricting macrophages in which M. tuberculosis encounters an environment that leads to strong repression of ribosomal genes. However, growth of H37RvDrelMtb in macrophage-like THP-1 cells was not different from that of wt H37Rv, demonstrating that the stringent response is not needed for intraphagosomal growth. Interestingly, H37RvDrelMtb grows poorly in most minimal media but replicates like wt H37Rv with citrate or phospholipids as sole carbon source. This observation supports earlier findings which suggest that lipids are the primary carbon source for M. tuberculosis in macrophages and in the lung. Perhaps, the strongest support for this hypothesis was provided by recent studies on the gene icl [15]. Icl encodes isocitrate lyase, the enzyme that mediates the first of two reactions that distinguishes the glyoxylate cycle from the TCA cycle. During growth with lipids as sole carbon source the glyoxylate cycle is necessary to sustain gluconeogenesis. Inactivation of icl interfered with the ability of M. tuberculosis to survive in activated macrophages and to sustain chronic mouse infections in the lung [15]. Utilization of fatty acids in intraphagosomal M. tuberculosis is also supported by the induction of many genes predicted to participate in b-oxidation of fatty acids in intraphagosomal M. tuberculosis [4,16,17]. The mechanisms that regulate the expression of genes involved in lipid metabolism are mostly unknown. Heat shock induces expression of the Hsp70/DnaK and Hsp60/ GroE chaperones in most organisms [9]. In M. tuberculosis, transcription of hsp70 and hsp60 genes is independently regulated by two transcriptional repressors, HspR and HrcA [18]. Members of the HspR regulon (dnaK, dnaJ, grpE, acr2) were strongly induced in intraphagosomal M. tuberculosis whereas only small induction factors were measured for HrcA-controlled genes [4]. In Streptomyces coelicolor the activity of HspR seems to be directly regulated by DnaK, which binds to HspR and acts as a transcriptional co-repressor for HspR [19]. Binding of DnaK to unfolded proteins inhibits formation of the HspR-DnaK complex. An increase in the amount of unfolded proteins therefore disrupts the HspRDnaK complex and induces the HspR regulon. A similar mechanism of regulation seems to operate in M. tuberculosis [20]. The activity of macrophages that induces the HspR regulon in intraphagosomal M. tuberculosis is unknown. However, several stresses that can induce heat shock genes, for example oxidative stress, are likely to be encountered within the phagosome. In addition to HspR and HrcA, genes encoding three alternative sigma factors, SigE, SigH, SigB, were induced in

response to heat shock [21], and thus are likely to participate in the regulation of the heat shock response of M. tuberculosis. SigB is highly similar to SigA, the principal sigma factor of M. tuberculosis, but in contrast to SigA is not essential for virulence or growth in liquid culture [22]. SigE and SigH are 2 of the 10 sigma factors encoded in the M. tuberculosis genome that belong to the extracytoplasmic function (ECF) family of sigma factors. Six ECF sigma factors (sC, sD, sF, sH, and sE) are known to be required for virulence (reviewed in [22]). However, SigE is the only ECF sigma factor reported to be important for survival and growth of M. tuberculosis in macrophages. The stimulus that most strongly induced SigE in liquid culture is treatment with SDS and SigE is one of the central regulators of the SDS response [21]. About 30 genes that were induced after treatment of M. tuberculosis with SDS were also preferentially expressed in intraphagosomal M. tuberculosis [4,22]. SDS is thought to primarily influence the transcriptome of M. tuberculosis through damage of its fatty acid-rich cell envelope suggesting that the cell envelope of M. tuberculosis is a target of the anti-bacterial activities of macrophages. Antibacterial peptides and phospholipases might be capable of mediating this stress within macrophages but direct evidence for this mechanism is lacking. The studies described above provide evidence that many stresses, when studied using in vitro conditions of growth, stimulate expression of some genes that are preferentially expressed by M. tuberculosis after phagocytosis by resting macrophages. However, none of these conditions, when studied in isolation, has been found to induce all or most of the M. tuberculosis genes which are preferentially expressed during residence of the bacterium in resting macrophages. This suggests the following: Adaptation to the environment encountered within resting macrophages likely entails a transcriptional response that results from the integration of responses to many distinct stimuli. Some of these stimuli have not been identified. Moreover, responses to individual stimuli and the integration of these into an overall adaptive response likely involves the convergence of many regulatory circuits. Some of the cognate regulators that mediate individual components of the adaptive response have been characterized while others, most notably those involved in regulating fatty acid metabolism, remain to be identified. 4. Adaptation to activated macrophages In a typical experiment, the transcriptome of M. tuberculosis isolated from activated macrophages contained about twice the number of genes induced more than 2-fold than the transcriptome of M. tuberculosis isolated from resting macrophages [4]. This suggested that activation of macrophages with IFNg affected regulation of most genes that are preferentially expressed in intraphagosomal M. tuberculosis. However, only 68 M. tuberculosis genes fulfill both of the following criteria: their expression in naive versus activated macrophages is significantly different (based on a statistical analysis that used a 1% false discovery rate) and at least 2-fold different 24 h

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post infection. Below, we focus on the regulation of genes that fulfill these criteria. However, we believe that these genes are a conservative estimate of the impact that macrophage activation has on the transcriptome of intraphagosomal M. tuberculosis. In contrast to M. tuberculosis residing in IFNg-activated macrophages from wt mice, the transcriptome of M. tuberculosis residing in nitric oxide synthase 2 (NOS2)-deficient (NOS2
/
) macrophages did not change due to stimulation with IFNg [4]. Exposure of M. tuberculosis to chemical generators of nitric oxide (NO) in liquid culture [23,24] induced most of the genes whose regulation was activation-dependent in intraphagosomal M. tuberculosis [4]. These observations suggest that activation-specific changes in the transcriptome of intraphagosomal M. tuberculosis were directly caused by NO that was produced by NOS2 in response to activation with IFNg. Different subsets of the activation-specific gene set can be induced in vitro by stimuli other than NO. Genes that were only regulated after stimulation with high doses of NO also responded to iron limitation and H2O2. Genes that were induced with low and high doses of NO were also induced by oxygen limitation. Regulation of these two sets of activation-specific genes is controlled by different transcriptional regulators, two of which, IdeR and DosR, have been analyzed extensively. IdeR is a transcription factor with extensive similarity to the DtxR family of metal-dependent transcriptional regulators. In the absence of metal ions, DtxR and IdeR are inactive monomeric proteins. In the presence of iron, the metal binds to IdeR and induces formation of dimers that have a high, operator-specific DNA binding activity [25]. In this active state, IdeR functions as a transcriptional repressor for about a third of all iron-repressed genes and as a transcriptional activator for some iron-induced genes. Among the IdeR-repressed genes are those encoding the enzymes that synthesize mycobactins. Mycobactins are siderophores; they are essential for growth of M. tuberculosis in low-iron liquid culture and in macrophages. About 20 IdeR-controlled genes were induced in intraphagosomal M. tuberculosis and the expression of 5 of them was activation specific [4]. Induction of IdeR-repressed genes in resting macrophages is most likely due to iron restriction by the host cell. In resting macrophages, M. tuberculosis resides in early phagosomes and therefore has access to iron from transferrin, an iron-binding host cell protein. Macrophage activation isolates M. tuberculosis from this host cell iron uptake system [26]. The increased expression of IdeR-repressed genes in activated macrophages might therefore have been caused by very low iron availability within phagosomes of activated macrophages. However, recent measurements of iron within M. tuberculosis containing phagosomes suggest that IFNg has only a minor effect on the ability of M. tuberculosis to acquire iron within this environment [27]. Activation-specific regulation of IdeR-repressed genes is therefore likely the result of oxidative inactivation of IdeR via NOS2-produced NO as well as the low iron concentration in the mature phagosome. DosR (also referred to as DevR) is a transcription factor of the two-component response regulator class that directly

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controls expression of about 50 genes in M. tuberculosis [28e30]. Induction of the DosR regulon was observed in response to multiple stresses but was strongest in response to hypoxia and NO [24,30e32]. Two kinases, DosS and DosT, have the capacity to phosphorylate DosR and are able to mediate activation of a DosR controlled promoter in response to oxygen deprivation [33]. Most DosR regulon genes were induced in activated macrophages and regulation of all of these genes was dependent on IFNg-stimulation and the presence of functional NOS2 [4]. The DosR regulon was also strongly expressed in M. tuberculosis isolated from chronically infected mouse lungs, where expression levels for the DosR regulon reached levels up to 10,000 fold higher than those measured in RNA isolated from well-aerated liquid cultures of M. tuberculosis [24,34,35]. Interestingly, induction of the DosR regulon in mouse lungs was only detected after production of IFNg and NOS2, suggesting that regulation of the DosR regulon in the lungs of mice also depends on activation of the immune system [35]. Inactivation of dosR in M. bovis BCG led to 1500-fold reduced survival during oxygen deprivation [28]. M. tuberculosis dosR mutants are likely to have similar but less dramatic phenotypes [24,33] and disruption of dosR reduced virulence of M. tuberculosis in guinea pigs [36]. In contrast, inactivation of DosR increased virulence of M. tuberculosis in a SCID mouse model of tuberculosis [37] suggesting that the phenotype of dosR mutants in animals might be species specific. Taken together DosR and IdeR regulate most of the M. tuberculosis genes whose expression changes in response to macrophage activation. Some genes are likely to be controlled by a second iron-responsive regulator because these genes are induced in response to iron-deprivation but not controlled by IdeR. The mechanism of IdeR activation has been characterized in detail. The signal transduction pathways that control the activity of DosR are however less clear. While two sensor kinases that control the phosphorylation state of DosR have been identified it is unknown how these kinases are activated. Studies that identify the stimuli that activate DosS and DosT might help to design experiments that further clarify the function of the DosR regulon. 5. Studies seeking to validate ex vivo expression profiling results through studies of M. tuberculosis-infected humans It is now widely believed that no small animal model of tuberculosis adequately represents all aspects and each stage of tuberculosis in man. It is also self-evident that M. tuberculosisinfected primary bone marrow derived macrophages cannot faithfully recreate the architecture, cell type diversity, intercellular signaling systems or disease stage-specific effects (i.e., early infection, latency, reactivation) of granulomas harboring M. tuberculosis in humans. Thus there is a compelling need to determine if expression profiling results from ex vivo experimental systems can be corroborated by studies of M. tuberculosis-infected humans or non-human primates. Two fundamental questions emerge as particularly important.

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(1) Are M. tuberculosis genes which are regulated in ex vivo experimental systems also regulated in M. tuberculosisinfected human tissues? (2) If so, is their expression functionally significant? To answer the first question directly requires the acquisition of infected human or primate tissues in the surgical suite and their immediate processing using methods that preserve bacterial RNA. Then, because the number of organisms in most tissue specimens is likely to be low, gene specific transcripts are sought by in situ and/or RT PCR methods. Not surprisingly, few studies of this kind have been published. They demonstrate that M. tuberculosis gene expression in humans can differ from that observed in mice [38,39]. The logistical and technical difficulties of such studies have prompted an alternative approach: use of the cellular or humoral immune response to identify M. tuberculosis antigenic proteins that are expressed in vivo, i.e., immune reactivity to a particular antigen implies expression of the corresponding M. tuberculosis gene. Further information about the timing and possible significance of the expressed gene is obtained by comparing the antigenspecific immune responses mounted by different categories of patients. In this way, Klein and colleagues showed that cellular immune responses to proteins encoded by some genes of the DosR regulon could be detected significantly more often in TST-positive patients with latent tuberculosis than in patients with reactivated tuberculosis (M. Klein, personal communication). This observation suggested that the corresponding genes were expressed prior to or during the development of the latent state. Furthermore, it implied that the expression of these genes, or the capacity to generate and sustain an immune response to their protein products, is correlated with maintenance of the latent state. In turn, this suggested that latentlyinfected persons who are immuno-reactive to these DosR protein antigens are less likely to progress to active disease (manuscript submitted). To determine if the in vivo expression of a particular gene is functionally significant is tantamount to asking if a deletion mutant of the gene has a clinical phenotype. Many such studies of M. tuberculosis mutants have been conducted using murine models of tuberculosis. However, this experimental paradigm (i.e., identify an in vivo expressed gene, disrupt the gene, study the mutant in an animal model of tuberculosis) cannot be carried out in man and cost constrains preclude many studies of this kind in non-human primate models of tuberculosis. To circumvent this difficulty, DeRiemer and colleagues studied the clinical behavior of natural deletion mutants to deduce if genes of the DosR regulon are functionally significant (K. DeRiemer, personal communication). Their study began with the deletion database of Tsolaki et al. who reported that 100 clinical strains from San Francisco had sustained a total of 68 distinct genomic deletions (an average of 20 genes deleted per strain) [40]. Analysis of this deletion database showed that four of the 68 deletions had occurred in the DosR regulon. They then studied the presence and frequency of the four DosR regulon deletions amongst w800 clinical isolates from San Francisco. Finally, they used a nested casee control study design to compare the frequency and proportion

of deletions in the DosR regulon between the following two groups of tuberculosis patients (classified by RFLP-based molecular epidemiology criteria as ‘‘clustered’’ or ‘‘unique’’): patients with reactivated latent infection (i.e., patients infected with a strain with an unique RFLP pattern); and, patients who had been recently-infected and then rapidly progressed to clinical disease (i.e., patients infected with a strain with a RFLP pattern identical to at least one other strain isolated in the same time period and community). A highly significant relationship was found indicating that isolates with deletions in the DosR regulon are associated with rapid progression to disease (manuscript in preparation). Thus, the studies reviewed here began with the demonstration that the DosR regulon is induced in activated macrophages in a NOS2-dependent manner and the finding that this regulon can be induced in vitro by low concentrations of NO or by oxygen limitation. The relevance of these observations to human disease was then studied in humans by assessing the immunoreactivity of its protein antigens and by evaluating the clinical consequences of deletions of DosR regulon genes. Taken together these results led to the following ideas: the DosR regulon is expressed in man prior to and/or during the development of latency; immunoreactivity to some DosR regulon proteins is associated with stable maintenance of the latent state; deletions within the DosR regulon occur commonly; and, the occurrence of such deletions reduces the immunogenicity of the DosR regulon by omitting protein antigens which evoke a latency-inducing immune response. 6. Adaptation of the macrophage to infection with M. tuberculosis: specificity of the macrophage response to pathogens Genome-wide expression profiling has been extensively used to monitor how the RNA profile of host cells responds to infection (reviewed in [41]). A central question of such studies is the following: does infection of host cells with a specific pathogens lead to pathogen-specific intracellular signals and responses? Or, alternatively, do infections with different pathogens lead to a common response? A gene expression study comparing dendritic cell (DC) responses to infection with E. coli, influenza virus and Candida albicans revealed large overlaps in the transcriptional changes of the infected cells [42]. This pathogen-unspecific ‘‘core response’’ consisted of temporally ordered genes that are involved in linking innate pathogen recognition with the development of an adaptive immune response. Thus, the pathogen-unspecific adaptation of the DC transcriptome appears to be critical for the main functions of DCs. A study of human peripheral blood mononuclear cells (PBMCs) to infection with heat-killed Gram-positive and Gram-negative bacterial pathogens also identified a set of commonly regulated genes [43]. These included immune activation genes involved in cell to cell signaling, genes that encode members of intercellular immunoregulatory signaling pathways and other proinflammatory genes. Similarly, the RNA profile of human monocyte derived macrophages (hMDM) after infection with Gram-positive

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bacteria, Gram-negative bacteria and mycobacteria as well as known Toll-like receptor (TLR) agonists revealed a shared macrophage activation program [44]. This consisted predominantly of genes involved in the interaction of macrophages and their environment, such as genes encoding receptors, adhesion molecules and proteins involved in tissue remodeling. Genes coding for signal transduction molecules and proinflammatory genes were also part of the shared macrophages activation program. The regulation of many of these commonly induced genes depends on the transcription factor NF-kB. NF-kB is activated in response to stimulation of TLR signaling pathways and the observed ‘pathogen-unspecific’ regulation of NF-kB activated genes likely reflects activation of TLRs by diverse bacterial products. However, in all studies, pathogen specific responses were observed when live, virulent pathogens were used for the infection. Chaussabel et al. identified discrete expression signatures in human monocyte-derived DCs and macrophages infected with either M. tuberculosis, Toxoplasma gondii, Leishmania major, Leishmania donovani and Brugia malayi [45]. Nau et al. demonstrated that Mycobacterium tuberculosis (M. tuberculosis) induced significantly less IL-12p40 mRNA than other bacteria in hMDM [44]. Immune control of M. tuberculosis infection in vivo relies on a type 1 T cell response and IL-12 plays a fundamental role in generating this response. Accordingly, IL-12 is critical for the control of tuberculosis in mice [46,47] and in humans [48e50]. The poor induction of IL-12p40 by M. tuberculosis in hMDM was confirmed on the protein level and, moreover, the authors showed that M. tuberculosis was able to suppress E. coli induced IL-12 production [44]. In primary mouse macrophages, heat-killed M. tuberculosis induced significantly more IL-12p40 than live M. tuberculosis [51]. These results point to an active repression of IL-12p40 induction by live M. tuberculosis which may be a mechanism M. tuberculosis employs to interfere with a strong Th1 T cell response. The molecular basis of this M. tuberculosis-mediated interference with transcriptional induction of IL-12p40 remains unknown. M. tuberculosis also interferes with macrophage activation by IFNg [52e54]. IFNg is the main macrophage activating cytokine and is essential for the control of M. tuberculosis infection. IFNg deficient mice succumb rapidly after infection with M. tuberculosis [55,56] and humans with certain mutations in IFNg receptor 1 show predisposition to severe mycobacterial infections [57]. Macrophages that have been infected with M. tuberculosis in vitro block induction of a subset of IFNgresponsive genes including Fcg receptor type I and the MHC class II transactivator (CIITA), which controls MHC class II expression [53,54,58,59]. Even dead M. tuberculosis and purified mycobacterial products such as the 19-kDa lipoprotein of M. tuberculosis have this activity, suggesting that it is due to constitutive mycobacterial cell wall components rather than an active response of M. tuberculosis towards the host cell. Fortune et al. demonstrated that multiple cell wall components of M. tuberculosis inhibit macrophage responses to IFNg via signaling through TLR2- and myeloid differentiation factor 88 (MyD88)-dependent and independent pathways

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[60]. Microarray gene expression studies established that induction of approximately 40% of IFNg-responsive genes was reduced if cells were infected with M. tuberculosis or treated with the purified 19-kDa lipoprotein prior to stimulation with IFNg [61]. However, induction of many IFNgdependent genes, of which some are involved in controlling M. tuberculosis infections including NOS2 and LRG47, was not suppressed by M. tuberculosis. Similarly, many IFNgresponsive genes were synergistically induced or suppressed by IFNg and M. tuberculosis in macrophages that were treated with IFNg and subsequently infected with virulent M. tuberculosis [51]. Thus, M. tuberculosis appears to inhibit IFNg-induction of specific genes, including those involved in antigen presentation, but is able to synergize with IFNg in the regulation of genes important for innate immunity and control of M. tuberculosis. In addition, the sequence of exposure to IFNg and M. tuberculosis may determine, at least for some genes, whether synergistic induction or M. tuberculosismediated inhibition results from exposure to these stimuli. Both scenarios, activation with IFNg and subsequent infection with M. tuberculosis, or primary infection with secondary exposure to IFNg, are likely to occur in vivo. Thus, the specificity of the macrophage response to M. tuberculosis and IFNg most likely depends on the in vivo environment, stage of infection or disease, M. tuberculosis strain and immune status of the host. 7. General characteristics of changes in the macrophage transcriptome in response to infection with M. tuberculosis After infection with live, virulent M. tuberculosis, expression of many macrophage genes involved in inflammation and immunity was induced while expression of many housekeeping genes and genes encoding metabolic pathways was repressed [51]. This suggests that the macrophage adapts to infection by shifting its transcriptome towards inflammation and immunity related functions and in addition that an efficient response to infection, which involves the induction of many genes, may require the simultaneous repression of dispensable metabolic pathways. This shift was in part dependent on NOS2, phagocyte oxidase (phox) or both. This supported the idea that the role of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) is not restricted to antimicrobial defense, but that they also represent important signaling molecules of macrophages. Interestingly, the expression of some genes whose functions are not related to inflammation and immunity, was significantly affected by NOS2 and/ or phox. This observation provides evidence for the existence of regulatory mechanisms which shield most immunity and inflammation-related genes from the influences of RNI and ROI. The transcriptional response of M. tuberculosis infected macrophages reflected activation of several known antimicrobial defense mechanisms. For example, NOS2 is critical for control of M. tuberculosis in mice, both during acute and chronic infections [62,63]. NOS2 was induced in primary mouse macrophages in response to M. tuberculosis alone

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and M. tuberculosis plus IFNg [51], as well as in mouse granulomas assessed by laser capture microdissection [64]. In contrast to the M. tuberculosis induced transcriptome of mouse macrophages, human monocyte derived macrophages did not respond with induction of NOS2 after costimulation with IFNg and M. tuberculosis, which was interpreted as a speciesspecific response of macrophages to M. tuberculosis infection [65]. However, the origin of the macrophage, bone marrow derived mouse macrophages versus blood derived human macrophages, may affect their physiology and response to pathogens, given that NOS2 is expressed in human alveolar macrophages from individuals with tuberculosis (reviewed in [66]). LRG-47 is a member of a family of 47 kDa guanosine triphosphatases (p47 GTPases) of which several members are induced by IFNg and by M. tuberculosis in macrophages [51,67] and in lungs of M. tuberculosis infected mice [68]. LRG-47 deficient mice, but not mice deficient in two other p47 GTPase members, IRG-47 and IGTP, are highly susceptible to tuberculosis. Treatment of M. tuberculosis infected LRG-47
/
mice with an NOS2 inhibitor accelerated TB progression, indicating that LRG-47 and NOS2 act via different mechanisms. MacMicking et al further showed that M. tuberculosis survived better in IFNg-activated LRG-47
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macrophages than wt macrophages. This was due to impaired phagosome maturation, which is controlled by LRG47. In sum, transcriptome analyses of M. tuberculosis infected macrophages revealed genes that are relevant for immune response to and control of tuberculosis and thus can lead to novel hypotheses regarding genes whose role in tuberculosis is yet unknown. 8. Impact of mycobacterial virulence and viability on the macrophage response Expression analysis of apoptosis related genes in human alveolar macrophages (AM) revealed that expression of several pro-apoptotic genes was downregulated in macrophages infected with virulent H37Rv [69]. In contrast, these genes were not suppressed in AM infected with attenuated H37Ra. Furthermore expression of an anti-apoptotic gene, bcl-w, was induced in response to H37Rv but not H37Ra. Consistent with these expression studies, H37Ra has been shown to induce a greater level of apoptosis in AM than the virulent H37Rv [70]. Infection of a human macrophage-like cell line with M. tuberculosis, M. avium and M. smegmatis identified several genes that were differentially expressed in macrophages infected with virulent versus avirulent mycobacteria [71]. These data suggest that macrophages respond at least to some extent differently to pathogenic and non-pathogenic mycobacteria and there may even be specific responses to different virulent mycobacteria. The role of most of these differentially regulated genes for the control of infection of different mycobacteria remains to be identified. While heat-killed M. tuberculosis was a potent stimulus of primary mouse macrophages and caused regulation of 65% as many genes as viable M. tuberculosis, there was little overlap in the genes that were regulated by live and dead

M. tuberculosis [51]. Several genes appeared to be selectively induced by dead M. tuberculosis, suggesting that live M. tuberculosis actively interferes with their induction as discussed above for IL-12. Others required live M. tuberculosis to be induced, providing evidence that heat-labile proteins and lipopeptides of the M. tuberculosis envelope and proteins and lipids that are released by intracellular M. tuberculosis are important stimuli for innate immunity. 9. Impact of host susceptibility on the macrophage response Different mouse strains differ in their susceptibility to infection with M. tuberculosis. C57BL6 and BALB/C mice are resistant strains that control replication of M. tuberculosis in the lung better and show less lung pathology and prolonged survival compared to susceptible stains such as CBA/J and DBA/2 [72]. Keller et al. compared the RNA profile of bone marrow derived macrophages from these four mouse strains after infection with M. tuberculosis [73]. Interestingly, they identified 18 genes that were selectively regulated in M. tuberculosis infected macrophages from resistant mouse strains and 102 genes that were exclusively regulated in macrophages from susceptible strains. Many of the latter genes are of unknown function. However the identities of some of the specifically regulated genes suggest that macrophages from susceptible mice may induce a stronger inflammatory response that includes recruitment of cells involved in tissue destruction rather than bacterial control. Functional analysis of the genes differentially regulated in macrophages from susceptible and resistant mice is required to validate this hypothesis. 10. Signal transduction pathways mediating macrophage adaptation to M. tuberculosis infection Toll-like receptors (TLRs) are a class of transmembrane proteins expressed mainly on antigen-presenting cells such macrophages and DCs. They recognize pathogen-derived products and activate intracellular signaling pathways resulting in induction of proinflammatory cytokines and costimulatory molecules. Their importance during macrophage infection with M. tuberculosis has mainly been studied using subcellular products of M. tuberculosis. M. tuberculosis cell wall constituents such as the 19-kDa lipoprotein, lipoarabinomannan (LAM) from fast-growing mycobacteria and phosphatidyl-myo-inositol mannoside (PIM) are TLR2 ligands and a heat-labile mycobacterial product stimulated TLR4 (reviewed in [74]). TLR9, the receptor for bacterial DNA, may transduce signals in response to mycobacterial DNA. In fact, that DNA acts as an immune cell trigger was first shown for mycobacteria [75]. However, few experiments have used intact, live and virulent M. tuberculosis to address the role of TLR signaling in primary macrophages that express TLRs at physiological levels. Shi et al demonstrated that MyD88, the intracellular adaptor used by all TLRs, is dispensable for the majority of transcriptional responses of primary mouse macrophages to live, virulent M. tuberculosis in vitro [76]. In contrast, Pai et al reported

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that most genes depended on MyD88 for their regulation in response to infection with attenuated Mtb H37Ra [61]. The discrepancies between these studies may partly be due to differences in culture conditions, because the response of MyD88
/
macrophages depends significantly on the cell density at which the cells are cultured [76]. In addition, as discussed above, virulent and attenuated M. tuberculosis activate macrophages differently. There were also genes whose regulation was consistent in both studies, for example the cytokines IL-1b and IL-6, and receptors MARCO and formylpeptide receptor (FPR) were MyD88-dependent whereas chemokines IP-10, RANTES and MIG, and the GTPase LRG-47 were MyD88-independent. Gene expression analysis using TLR2-deficient macrophages supported previous data [76] indicating that TLR-independent signaling contributes significantly to the macrophage response to infection with M. tuberculosis (Shi et al., unpublished data). Fortune et al. provided evidence that mycolylarabinogalactan peptidoglycan (mAGP) may mediate the TLR2-, MyD88-independent response [60]. The receptor(s) mediating this response do not appear to be complement receptors 3 and 4, macrophage mannose receptor, CD40, or scavenger receptor type A and remain to be identified [79]. It is likely that activation of macrophages by M. tuberculosis relies on a variety of receptors, and compensation for each other’s deficiency may mask a potential signaling defect in single knockout macrophages. However, other receptors such as the intracellular nucleotide-binding oligomerization domain (NOD) proteins, which belong to a TLR-related protein family and have been shown to recognize bacterial peptidoglycans/ peptidoglycan fragments, may also participate in macrophage activation by M. tuberculosis. Efforts to better understand the signal transduction pathways mediating M. tuberculosis induced macrophage responses are ongoing. In sum, M. tuberculosis activates macrophages via TLRdependent and TLR-independent signal transduction pathways, and several receptors contribute to recognition of viable M. tuberculosis. Collaboration between different receptors to achieve specific host cell activation has been proposed [77,78], and additional research is required to further explore this concept. Acknowledgements We thank Carl Nathan for stimulating discussions and critical reading of the manuscript. The authors acknowledge supported by the NIH (RO1AI57443 to D.S., R01AI44826 to G.K.S. and R01HL68525 to S.E.), the Ellison Medical Foundation (G.K.S and D.S.) and the I.T. Hirschl Foundation (S.E.). References [1] P.D. Butcher, J.A. Mangan, I.M. Monahan, Methods Mol. Biol. 101 (1998) 285e306. [2] R.R. Isberg, in: G. Storz, R. Hengge-Aronis (Eds.), Bacterial Stress Responses, ASM Press, Washington, 2000, pp. 289e303.

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