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The Journal of Immunology, 2007, 178: 3143-3152.
Copyright © 2007 by The American Association of Immunologists, Inc.

The Type I IFN Response to Infection with Mycobacterium tuberculosis Requires ESX-1-Mediated Secretion and Contributes to Pathogenesis1

Sarah A. Stanley, James E. Johndrow, Paolo Manzanillo and Jeffery S. Cox2

Department of Microbiology and Immunology, University of California, San Francisco, CA 94158


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The ESX-1 secretion system is a major determinant of Mycobacterium tuberculosis virulence, although the pathogenic mechanisms resulting from ESX-1-mediated transport remain unclear. By global transcriptional profiling of tissues from mice infected with either wild-type or ESX-1 mutant bacilli, we found that host genes controlled by ESX-1 in vivo are predominantly IFN regulated. ESX-1-mediated secretion is required for the production of host type I IFNs during infection in vivo and in macrophages in vitro. The macrophage signaling pathway leading to the production of type I IFN required the host kinase TANK-binding kinase 1 and occurs independently of TLR signaling. Importantly, the induction of type I IFNs during M. tuberculosis infection is a pathogenic mechanism as mice lacking the type I IFNR were more restrictive for bacterial growth in the spleen than wild-type mice, although growth in the lung was unaffected. We propose that the ESX-1 secretion system secretes effectors into the cytosol of infected macrophages, thereby triggering the type I IFN response for the manipulation of host immunity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The outcome of infection by a bacterial pathogen is determined by a complex interplay of virulence factors produced by an invading microbe and the immune responses to infection mounted by the host. Immune responses are characterized by the up-regulation of a multitude of cytokines, chemokines, and products with direct or indirect microbial toxicity and are normally shaped to best protect the host from a particular invading organism. Many pathogens have evolved strategies for evading or suppressing immune responses, as well as eliciting or misdirecting responses in a way that benefits microbial growth (1). Mycobacterium tuberculosis, the causative agent of human tuberculosis, is a highly successful pathogen of humans, in part, because it has evolved multiple mechanisms for evading or suppressing the host immune response to infection. For example, M. tuberculosis is able to evade the effects of IFN-{gamma} or type II IFN (2, 3), a cytokine critical for controlling mycobacterial infections (4). Recently, it was identified that type I IFNs, cytokines associated mainly with antiviral function, are also elicited by infection with M. tuberculosis (5, 6). The role of these cytokines in M. tuberculosis control, and whether M. tuberculosis actively promotes or inhibits this response has yet to be determined.

Type I IFNs are a family of cytokines consisting of multiple IFN-{alpha}s and a single IFN-beta that all signal through the type I IFNR. Type I IFNs have well-characterized roles in defense against viruses, inhibition of cell growth, control of apoptosis, and modulation of the immune response (7). Recent work has highlighted the importance of type I IFNs during bacterial infection, and there is growing interest in the significance of these cytokines in the pathogenesis and virulence of a diverse group of bacterial pathogens. Listeria monocytogenes, a Gram-positive bacterium that has been used widely as a model for intracellular bacterial infection, has been shown to induce type I IFN production by infected macrophages (8). Type I receptor knockout mice (IFNR{alpha}1 (IFNAR1–/–))3 that are unable to respond to type I IFNs are sensitive to viral infection but are resistant to infection with L. monocytogenes (9, 10, 11), demonstrating that these cytokines can inhibit host defense against bacterial infection. These results strongly suggest that L. monocytogenes provokes the production of type I IFNs as a pathogenic mechanism for the suppression of host immune responses. Although the exact mechanism of this suppression remains unclear, there is evidence to suggest that apoptosis promoted by type I IFNs in the spleen may be a necessary requirement for optimal bacterial growth (9, 10, 11). The mechanism of induction of type I IFNs by L. monocytogenes is dependent on bacterial entry into the cytosol (8), leading to the activation of TANK-binding kinase 1 (TBK1) (12), which in turn phosphorylates and activates the IFN regulatory factor (IFR)3 transcription factor, leading to the transcription of the IFN-beta gene. The cytosolic receptor that L. monocytogenes uses to activate TBK1, as well as bacterial products required for this activation, remains unknown. M. tuberculosis, unlike L. monocytogenes, is found exclusively in membrane bound compartments, suggesting that type I IFN production proceeds through a different pathway. Lewinsohn et al. (13) showed that culture filtrate protein-10 (CFP-10), a protein secreted by the ESX-1 system of M. tuberculosis, gains access to the cytosol during infection and is presented by the MHC class-I Ag processing pathway, demonstrating the possibility that products from M. tuberculosis may gain access to cytosolic signaling receptors during infection.

The ESX-1 alternative secretion system in M. tuberculosis is a major determinant of virulence (14, 15, 16). Three substrates of this secretion system have been identified to date: early secreted antigenic target-6 (ESAT-6), CFP-10 (14, 15, 16), and Rv3616c (17) encoded by the genes esxA, esxB, and Rv3616c, respectively. ESAT-6 and CFP-10 form a tight 1:1 dimer that is required for the stability of both proteins (18, 19). Therefore, the esxA mutant lacks both ESAT-6 and CFP-10 (14). Because the secretion of each identified substrate is dependent upon the presence of the others, it has been proposed that these substrates may also be components of the secretion machinery (17). Three cytosolic or membrane-bound components of the ESX-1 secretion machinery, encoded by the genes Rv3870, 3871, and 3877, have been identified to date. Strains of M. tuberculosis that lack components of the ESX-1 secretion system or the substrates ESAT-6 and CFP-10 are extremely attenuated for growth and virulence during infection of both mice and macrophages (14, 15, 16). ESX-1-mediated secretion of ESAT-6/CFP-10 is important for controlling the cytokine responses of macrophages infected with M. tuberculosis. In particular, mutants lacking a functional ESX-1 secretion system or ESAT-6/CFP-10 elicit higher levels of IL-12 and TNF-{alpha} from infected macrophages (14). The ESX-1 secretion system is required for full virulence of M. tuberculosis and for subversion of normal immune responses; however, the mechanism by which ESX-1 affects host biology has yet to be determined. It is possible that ESX-1 mediates secretion of ESAT-6/CFP-10 into the cytosol of infected macrophages for interaction with cytosolic signaling pathways for the manipulation of host biology.

There is conflicting evidence regarding the role of type I IFN in M. tuberculosis infection. M. tuberculosis infection of macrophages and dendritic cells leads to induction of type I IFN in addition to a number of chemokines and cytokines important for controlling the immune response to infection. Recently, it was shown that signaling through the type I IFNR during M. tuberculosis infection of macrophages was required for the production of a number of immunologically important products, including inducible NO synthase, IFN-{gamma}-inducible protein-10 (IP-10), RANTES, and IRG1 (20). Additionally, it has been shown that M. tuberculosis may actively inhibit type I IFN signaling (21), an activity that may be related to pathogenicity. Finally, M. tuberculosis was shown to have a slight growth advantage in the lungs of IFNAR1–/– mice following aerosol infection (22), although growth in other organs was not examined. These data indicate a protective role for type I IFNs during infection with M. tuberculosis. In contrast, a hypervirulent clinical isolate of M. tuberculosis was found to produce significantly higher levels of type I IFN, which correlated with a decrease in the production of the important cytokines IL-12 and TNF-{alpha}. Interestingly, treatment of M. tuberculosis-infected mice with purified IFN-{alpha}beta further increased bacillary loads and decreased survival time (23). Exogenous addition of type I IFNs to macrophages infected with mycobacterium bacillus Calmette-Guérin enhanced bacterial growth (24), providing further evidence for a detrimental role of type I IFNs during mycobacterial infection. Thus, type I IFNs have been shown to have both beneficial and detrimental effects on host resistance during infection, and the role type I IFN during M. tuberculosis infection remains unclear.

In this article, we report our studies of the role and mechanism of induction of type I IFNs during M. tuberculosis infection. We have identified pathways that are required for the induction of IFN-beta during infection of both mice and macrophages. M. tuberculosis infection of macrophages leads to an induction of IFN-beta that is dependent on TBK1 signaling in macrophages, revealing a similarity in the pathways used by L. monocytogenes and M. tuberculosis for the induction of IFN-beta. We also show that a functional ESX-1 system is required for the induction of IFN-beta both in vivo and in vitro. Additionally, we found that mice lacking the type I IFNR have an increased capacity to limit M. tuberculosis replication in the spleen, indicating that the production of these cytokines is detrimental to the host. Our data suggests that the ESX-1 secretion system functions to elicit type I IFN to promote bacterial replication during infection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and macrophages

HeJ/HeN mice were obtained from the National Cancer Institute. TBK1+/+TNFR–/– and their TBK1–/–TNFR–/– littermate macrophages and receptor-interacting protein 2 (RIP2)–/– and their RIP2+/+ littermate macrophages were gifts from G. Cheng (University of California, Los Angeles, CA) and have been described previously (12). TLR2–/– mice backcrossed 10 generations to C57BL/6 were obtained from R. Isberg (Tufts University, Boston, MA). MyD88 mice backcrossed to C57BL/6 were obtained from D. Portnoy (University of California, Berkeley, CA). TRIF–/– (lps2–/–) mice are on the C57BL/6 background and were obtained from B. Beutler (Scripps Research Institute, La Jolla, CA). IFNAR1–/– mice backcrossed 10 generations to C57BL/6 mice were obtained from J. Cyster (University of California, San Franciso, CA). C57BL/6 wild-type (WT) mice (Charles River Laboratories) were used as controls in TLR2–/–, MyD88–/–, TRIF–/–, and IFNAR1–/– experiments. Mice were housed under specific pathogen-free conditions, and mouse experiments were conducted using a University of California, San Francisco, Institutional Animal Care and Use Committee-approved protocol. Bone marrow-derived macrophages (BMDMs) were isolated by culturing in medium containing 30% L cell supernatant for 6 days.

Bacteria

The WT strain of M. tuberculosis used in these studies was the Erdman strain. Previously described M. tuberculosis strains used were the following: the {Delta}esxA mutant SSM6, the snm4 transposon mutant SSM3, and the snm4-complemented strain SSM5 (14). The esxA-complemented strain was generated using pMH406 obtained from D. Sherman (University of Washington, Seattle, WA). All strains were cultured in 7H9 medium containing 0.05% Tween 80 as described. Bacteria were prepared for mouse and macrophage infections as described previously (14).

Mouse and macrophage infections

For microarray analysis of infected tissues, mice were inoculated with 107 CFU of M. tuberculosis in PBS plus 0.05% Tween 80. Organs were harvested and immediately placed in RNAlater (Ambion) for stabilization of RNA. RNA was prepared using a RNeasy kit (Ambion) following rotor-stator homogenization of tissues. For enumeration of CFUs during infection, mice were infected with 106 CFU of M. tuberculosis, and organs were harvested at various time points after infection and were processed as described previously (14). For macrophage infections, 5 x 106 macrophages were at a multiplicity of infection (MOI) of 10 in a 10-cm dish in medium containing 5% horse serum and 5% FCS for 2 h, after which time the cells were washed and fresh medium added. RNA was prepared at defined time points using the RNeasy kit.

Microarray and quantitative PCR analysis

For array analysis, RNAs from four individual mice were pooled, and 500 ng of total RNA was amplified using MessageAmp II (Ambion) reactions in which amino-allyl dUTP is incorporated during transcription. Four micrograms of amplified RNA from each sample was labeled with the fluorophore Cy5 (GE Healthcare) by coupling to the amino-allyl dUTP as previously described (25), and hybridized against 4 µg of a pooled sample consisting of a pool of the experimental RNA samples labeled with Cy3 (GE Healthcare) on a Mouse Exonic Evidence-Based Oligonucleotide (MEEBO) array (Illumina). Hybridizations were conducted for 24 h at 65°C. Arrays were washed for 1 min each in the following wash buffers: 2x SSC plus 0.03% SDS at 60°C, 1x SSC at room temperature, and 0.2x SSX at room temperature. Arrays were scanned using a GenePix 4000B scanner and GenePix PRO version 4.1 (Axon Instruments/Molecular Devices). Arrays were analyzed using the MEEBO client for NOMAD (http://meebo.ucsf.edu:8080/meebo/nomad2Query1.html), CLUSTER (26), and Java Treeview 1.0.8 (available at http://jtreeview.sourceforge.net). Each array experiment using RNA from three separate infection experiments was repeated at least twice; shown is a representative array for each of three infection experiments. For quantitative PCR, 2 µg of total RNA from each individual mouse sample was reverse transcribed in a 40-µl reaction. Two microliters was used in a quantitative real-time PCR using SYBR green as a label and the following primers: ifnb-F, 5'-ctggagcagctgaatggaaag; ifnb-R, 5'-cttgaagtccgccctgtaggt; beta-actin-F, 5'-aggtgtgatggtgggaatgg; and beta-actin-R, 5'-gcctcgtcacccacatagga. Results shown are representative of three separate infection experiments, with each PCR performed in triplicate. All values reported were in the linear range of the experiment and were normalized to actin values. Standard curves were generated by linear dilution of a cDNA sample generated from LPS-stimulated macrophages.

ELISA

Macrophages were infected as described above. Macrophage culture supernatants were assayed by sandwich ELISA for IFN-beta using a monoclonal anti-mouse IFN-beta Ab (Seikagaku America) for coating and polyclonal anti-mouse IFN-beta (R&D Systems), followed by anti-rabbit-HRP (Promega) for detection. For IL-12 and TNF-{alpha} ELISAs, cytokines were measured using ELISA kits (BD Biosciences).

Statistics

For CFU determination and quantitative PCR from infected mouse tissues, significance between groups was determined by the Mann-Whitney U nonparametric test using the Graphpad Instat 3 statistical package (available at www.graphpad.com/instat/instat.htm). For microarray experiments, statistically significant differences in gene expression were identified using the Statistical Analysis of Microarray (SAM) software tool version 2.23A (available at www-stat.stanford.edu/~tibs/SAM/). A two-class paired analysis was performed, using a false discovery rate cutoff of <1%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Type I IFN-regulated genes are induced during mouse infection in an ESX-1-dependent manner

To identify host genes regulated by ESX-1 during the early stage of mouse infection, we analyzed gene expression profiles in lungs and spleens of C57BL/6 mice infected with either WT or esxA mutant M. tuberculosis. Mice were infected with 107 CFU of M. tuberculosis via the i.v. route, and total RNA was prepared from lungs and spleens harvested 24 h after infection. Poly(A) RNA samples were amplified by in vitro transcription, labeled with Cy5, and subjected to competitive hybridization against a Cy3-labeled pooled reference sample. Hybridizations were conducted on MEEBO arrays covering ~22,000 or ~88% of mouse genes (27). Three independent biological experiments with four mice per group were repeated at least twice. Values from each infected sample were normalized to values from uninfected controls. Statistical significance was determined using SAM analysis with a false discovery rate of <1%, and genes whose expression levels changed >3-fold from uninfected controls were chosen as genes regulated during infection (see Materials and Methods for detailed explanation of array experiments and analysis). Infection with M. tuberculosis resulted in gene expression level changes in 174 genes in the spleen and 229 genes in the lung. Many of the genes induced were already known to be up-regulated during infection, including cytokines, chemokines, and other immunoresponsive genes.

To identify host genes whose expression is dependent on a functional ESX-1 secretion system, we compared gene expression profiles of WT-infected tissues with those infected with the esxA mutant. Interestingly, it appeared that a number of genes that were up-regulated in response to infection with WT M. tuberculosis were not induced by the mutant. To define the genes that were dependent on esxA for their induction, we chose genes that had >2-fold lower expression levels in infections with esxA mutants as compared with WT in at least two of three biological replicate experiments in spleens and lungs. Significance was determined by SAM with a false discovery rate of <1%. We found that 42 genes were dependent on esxA for expression in the spleen (Table I) and 75 genes in the lung (Table II). Because WT and mutant bacterial numbers were equivalent at 24 h postinfection (data not shown), the differences observed are not the result of a growth defect of the esxA mutant at this early time point. To define a common set of ESX-1-regulated genes in both lung and spleen, genes that were at least 2-fold different in two of three experiments in both organs were clustered (Fig. 1, Table III). Of the 17 genes differentially induced in WT vs mutant infection, 15 are known to be IFN dependent (Table III). Of these genes, the majority are known to be induced by both type I (IFN-beta and the IFN-{alpha}s) and type II IFNs (IFN-{gamma}). Interestingly, we observed differences in the induction of genes usually associated with an antiviral, type I IFN-mediated response, including MxA and 2',5' oligoadenylate synthase (28), indicating that type I IFNs might be differentially induced in mutant infections compared with WT. Indeed, in both the spleen and lung samples, we observed differential induction of the gene encoding the chemokine IP-10, which has been shown to be completely dependent on type I IFN signaling during infection with M. tuberculosis (20). However, we were unable to detect any type I IFN transcript, presumably because the abundance of these transcripts is below the level of detection of our microarrays.


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  		Table I. Genes in spleen with >2-fold higher expression in WT than esxA mutant infectiona

 

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  		Table II. Genes in lung with >2-fold higher expression in WT than esxA mutant infectiona

 

Figure 1
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  		FIGURE 1. Genes whose induction is dependent on esxA in infected spleen and lung tissues. Shown are each of three biological replicates where gene expression levels in WT and mutant-infected spleen and lung samples were normalized to levels in uninfected samples. Genes depicted are at least 2-fold decreased in mutant infection as compared with WT in two of the three experiments in each organ; all differences were found to be statistically significant using SAM analysis. Yellow boxes indicate up-regulation, blue boxes indicate down-regulation, and black boxes indicate no change. The array experiment for each biological replicate was repeated at least twice; shown are representative arrays. U, Uninfected; W, WT infected; and E, esxA mutant infected.

 

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  		Table III. Functional annotation (GO) and IFN responsiveness of genes whose expression is dependent on esxA in spleens and lungs

 
ESX-1 is required for the induction of IFN-beta in vivo

The type I IFN response is characterized by the initial induction of IFN-beta, which leads to the subsequent induction of many IFN-{alpha} genes (29). To determine whether the induction of IFN-dependent genes in spleens and lungs of infected mice could in part result from the production of type I IFNs, we performed quantitative RT-PCR to measure IFN-beta mRNA in these tissues. IFN-beta induction was observed in the spleens (Fig. 2A) and lungs (Fig. 2B) of infected mice as early as 24 h after infection and was still detectable 5 days after infection (data not shown). Levels of IFN-beta mRNA in lungs of mice infected with the esxA mutant strain were equivalent to uninfected samples, which is consistent with the decreased levels of induction of IFN-regulated genes observed by microarray analysis. The {Delta}esxA::pesxA-complemented strain restored IFN-beta nearly to WT levels. The induction of IFN-beta in vivo is therefore completely dependent on ESAT-6/CFP-10.


Figure 2
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  		FIGURE 2. Expression of IFN-beta during mouse infection is dependent on esxA. Total RNA samples from spleens (A) and lungs (B) used in microarray experiments were subjected to quantitative PCR analysis for IFN-beta where values were normalized to actin. Shown is a representative experiment of three, where each symbol represents an individual mouse, and the average for each group is represented by the symbol X. Each sample was assayed in triplicate; error bars represent SD. Significance was determined using the Mann-Whitney U nonparametric test.

 
The ESX-1 secretion system is required for the induction of IFN-beta during infection of macrophages by M. tuberculosis

It has been reported that macrophages and dendritic cells infected with M. tuberculosis produce type I IFNs. To characterize the kinetics of induction of IFN-beta by macrophages during infection with M. tuberculosis, we infected murine BMDMs with WT M. tuberculosis and measured the levels of IFN-beta mRNA at multiple time points after infection. Infection with M. tuberculosis resulted in a robust induction of IFN-beta mRNA, with levels similar to those induced by LPS (Fig. 3A). Infection of murine BMDMs with WT M. tuberculosis resulted in the rapid induction of IFN-beta, with a peak production of mRNA at 4 h after infection. The levels of IFN-beta mRNA decreased by 8 h postinfection, and a low level of IFN-beta production was sustained through 24 h postinfection (data not shown). These kinetics are similar to those observed in infection of BMDMs with L. monocytogenes (12). Infection of BMDMs with M. tuberculosis also resulted in the production of IFN-beta protein, which was detectable by ELISA in the supernatants of infected cells 24 h after infection (Fig. 3B). The production of type I IFN by macrophages infected with M. tuberculosis led to phosphorylation and activation of STAT-1 with kinetics that reflect those of mRNA production (data not shown).


Figure 3
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  		FIGURE 3. Induction of IFN-beta in bone BMDMs is dependent on the ESX-1 secretion system. BMDMs were infected at a MOI of 10 or were treated with 10 ng/ml LPS. RNA was harvested at 0, 2, 4, and 8 h after infection. IFN-beta levels were determined by quantitative PCR, and values were normalized to actin (A and C). Supernatants were collected from identical infections at 24 h and were subjected to ELISA for analysis of IFN-beta protein concentration (B). Complementation of the mutant phenotype was assessed at the 4-h time point by measuring IFN-beta mRNA levels by quantitative PCR (D). Samples were assayed in triplicate; error bars represent SDs. Shown is a representative experiment of at least three.

 
We next examined the role of the ESX-1 secretion system in the induction of IFN-beta mRNA by macrophages. Importantly, the esxA mutant failed to induce IFN-beta to any appreciable degree at any time point examined after infection (Fig. 3C). To test whether ESAT-6/CFP-10 secretion was required for the induction of IFN-beta, we infected BMDMs with Rv3870, Rv3871, and Rv3877 mutants, all of which lack a functional ESX-1 secretion system and synthesize but do not secrete ESAT-6/CFP-10 (Fig. 3D and data not shown). These mutants were also unable to elicit IFN-beta mRNA production in infected macrophages. The defects were complemented by reintroduction of a single copy of the relevant gene (Fig. 3D). As expected, the observed defect in mRNA production during infection with the esxA mutant resulted in a decrease in cytokine production (Fig. 3B).

During infection with L. monocytogenes, type I IFNs exert an immunosuppressive effect characterized by suppression of the cytokines IL-12 and TNF-{alpha} (10). Exogenous addition of IFN-{alpha}beta to mice infected with M. tuberculosis resulted in a decrease in levels of IL-12 and TNF-{alpha}, suggesting a similar function for type I IFNs during infection with M. tuberculosis (23). M. tuberculosis is known to actively suppress macrophage production of IL-12 (30), and we have shown that ESX-1-mediated secretion of ESAT-6/CFP-10 is required for suppression of the production of IL-12 as well as TNF-{alpha} in BMDMs (14). Because the ESX-1 secretion system is also required for the production of IFN-beta, we tested the hypothesis that IFN-beta production induced by ESAT-6/CFP-10 secretion is responsible for the suppression of cytokine production also mediated by this secretion system. Bone marrow macrophages derived from mice lacking the type I IFNR (IFNAR1–/–) were infected with WT and Rv3877 mutant M. tuberculosis, and levels of IL-12 and TNF-{alpha} were measured in the supernatants 24 h after infection. Rv3877 mutants elicited significantly higher levels of IL-12 and TNF-{alpha} than WT bacteria in both WT and IFNAR1–/– macrophages (Fig. 4). Therefore, IFN-beta is not required for the cytokine repression mediated by the secretion of ESAT-6/CFP-10.


Figure 4
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  		FIGURE 4. IL-12 suppression mediated by the ESX-1 secretion system does not require type I IFN. BMDMs were infected at a MOI of 10, and supernatants were harvested at 24 h after infection for analysis of IL-12 concentration by ELISA. Samples were assayed in triplicate; error bars represent SDs. Shown is a representative experiment of three.

 
Infection with M. tuberculosis results in induction of IFN-beta in a TLR-independent manner

Activation through a subset of the TLRs is known to lead to the production of type I IFNs. TLR3 and TLR4 stimulation leads to induction of type I IFNs via a signaling pathway that requires the adaptor molecule TRIF (31, 32). TLR7 and TLR9 also activate type I IFN but use the adaptor MyD88 (33). Since mycobacterial products have been shown to signal through TLR4 expressed on the surface of macrophages (34), we first determined whether signaling through TLR4 is required for the induction of IFN-beta by M. tuberculosis. TLR4-deficient (HeJ) and TLR4-WT (HeN) macrophages were infected for 4 h, and the production of IFN-beta was assessed by quantitative RT-PCR. Induction of IFN-beta mRNA by M. tuberculosis remained intact in the absence of TLR4 (Fig. 5A), whereas LPS induction of IFN-beta was completely blocked in these cells (data not shown). Macrophages deficient for TRIF or MyD88 were also able to activate the production of IFN-beta to WT levels (Fig. 5, C and D). Although TLR2 has not been shown to lead to the production of IFN-beta, mycobacterial products, including the 19-kDa lipoprotein, have been shown to induce signaling through TLR2 (35, 36). However, TLR2-deficient macrophages had no defect in the induction of IFN-beta (Fig. 5B). These data agree with recently published results indicating that the induction of IFN-beta by M. tuberculosis occurs independently of TLR signaling (20).


Figure 5
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  		FIGURE 5. Induction of IFN-beta by BMDMs is independent of TLR signaling (A–D) and RIP2 (E) and is dependent on TBK-1 (F). WT C57BL/6 and knockout BMDMs were infected at a MOI of 10, and RNA was harvested at 4 h after infection. IFN-beta levels were determined by quantitative PCR, and values were normalized to actin. Samples were assayed in triplicate; error bars represent SDs. Shown is a representative experiment of at least three.

 
Induction of IFN-beta by M. tuberculosis is independent of RIP2 but requires TBK1

Receptors present in the cytosol of macrophages are also capable of initiating signaling that results in the induction of IFN-beta (29). Although M. tuberculosis remains confined to the phagosome, it is possible that bacterial products gain entry to the host cell cytosol for interaction with signaling pathways, a common strategy among virulent pathogens for controlling the host-response to infection. Entry of a bacterial product from M. tuberculosis into the cytosol of the macrophage could result in the induction of IFN-beta by interaction with a cytosolic detection pathway. The nucleotide-binding oligomerization domain (nod) containing proteins nod1 and Nod2 have been shown to recognize and initiate responses to a variety of bacterial products encountered within the cytosol (37). Recent work suggests that nod2 may be involved in macrophage responses to M. tuberculosis (38). To determine whether either nod protein is involved in the induction of IFN-beta by M. tuberculosis, we tested macrophages deficient in RIP2 because both nod proteins signal through RIP2 upon ligand binding (39). We observed WT levels of IFN-beta mRNA in RIP2-deficient macrophages during infection with M. tuberculosis (Fig. 5E), indicating that Nod1 and Nod2 are not required for IFN-beta production.

Induction of IFN-beta by the cytosolic bacterial pathogen L. monocytogenes is also independent of Nod protein signaling but requires TBK1, a kinase that has been shown to be critical for induction of type I IFNs in viral infection (40, 41). TBK and I{kappa}B kinase {epsilon} are the main kinases responsible for phosphorylation and activation of IRF3 (42, 43), which leads to the activation of IFN-beta. We found that the induction of IFN-beta by M. tuberculosis is completely dependent on TBK1 because there is a complete lack of IFN-beta mRNA production in TBK1-deficient macrophages infected with M. tuberculosis (Fig. 5F). Thus, despite the different localization of these two pathogens, the signaling pathways leading to IFN-beta production are, as yet, indistinguishable.

Mice deficient in the type I IFNR show increased splenic resistance to infection with M. tuberculosis

The failure of avirulent ESX-1 mutant bacteria to induce type I IFN suggests that the elicitation of these cytokines may be important for bacterial virulence. However, type I IFNs are also required for the elicitation of several immunoregulatory molecules involved in protection against infection. Thus, the role of these cytokines during infection with WT M. tuberculosis is difficult to predict. To determine the role of type I IFNs during infection with M. tuberculosis, C57BL/6 and congenic IFNAR1–/– mice were infected with 106 M. tuberculosis via the i.v. route, and organs were harvested for enumeration of CFUs at various time points after infection. No difference in resistance was observed in the lungs of infected mice under these conditions (Fig. 6A). Interestingly, however, by 10 days after infection, spleens from infected IFNAR1–/– mice were significantly smaller than those from WT-infected mice (Fig. 6B). Bacterial numbers isolated from the spleens of mice at 10 days after infection were ~5-fold lower in IFNAR1–/– mice as compared with WT (Fig. 6C). This difference was maintained through 21 days after infection when bacterial numbers were ~3-fold lower in the receptor-deficient mice. These data indicate that signaling through the type I IFNR is important for full virulence of M. tuberculosis.


Figure 6
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  		FIGURE 6. Type I IFNR-deficient mice show increased splenic resistance to infection with M. tuberculosis. C57BL/6 mice were injected with 1 x 106 CFU of the Erdman WT strain of M. tuberculosis, and CFU were enumerated at 1, 10, and 21 days after infection in the lungs (A) and spleens (C) of infected mice. Shown are two combined experiments with three mice per group. Significance was determined by the Mann-Whitney U nonparametric test, with an asterisk denoting p < 0.002. Spleens were removed and photographed at 1 and 10 days after infection (B); +/+, C57BL/6; –/–, IFNAR1–/–.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The ESX-1 secretion system and its substrates ESAT-6 and CFP-10 are known to be major virulence factors of M. tuberculosis, although the exact mechanism by which this system contributes to virulence has yet to be elucidated. We have found that an esxA mutant of M. tuberculosis fails to induce the type I IFN, IFN-beta, in both lungs and spleens of infected animals. Gene expression profiling experiments from tissues of infected mice suggest that the failure to induce type I IFNs by this mutant results in a defect in induction of a set of genes previously characterized as IFN dependent, including genes that are understood as being part of an antiviral type I IFN response, such as 2',5' oligoadenylate synthase, and MxA, as well as genes that are known to be induced by infection with M. tuberculosis, including IP-10.

An interesting remaining question is the mechanism by which the ESX-1 secretion system leads to the induction of IFN-beta in infected macrophages. It is likely that the ESX-1 secretion system functions to secrete bacterial products into the macrophage for the induction of IFN-beta, but the specific macrophage receptor that leads to the induction of this pathway has yet to be identified. M. tuberculosis is known to be confined to the phagosome during infection; however, we and other groups (20) have found the induction of IFN-beta to be independent of TLRs, the only known phagosomal receptors whose activation can lead to the induction of type I IFNs. It is possible that the ESX-1 system secretes a bacterial effector into the phagosomal lumen that engages an as yet unidentified phagosomal receptor that leads to the induction of IFN-beta. A more interesting possibility is that the ESX-1 system functions in a manner similar to type III or type IV secretion systems of other pathogenic bacteria for delivery of bacterial effectors directly into the cytosol of infected macrophages (44). Recently, it was shown that CFP-10 presentation by MHC class I requires components specific to the cytosolic Ag-processing pathway, providing evidence that CFP-10 gains access to the cytosol during infection (13). Additional evidence that M. tuberculosis is capable of accessing cytosolic signaling pathways was provided by Ferwerda et al. (38), who showed that nod proteins are involved in the response to infection with M. tuberculosis. The ESX-1 secretion system, which likely controls multiple aspects of virulence, may do so by secreting effectors into the cytosol for the manipulation and/or activation of multiple host signaling pathways, including the TBK1 pathway, which leads to the induction of IFN-beta. L. monocytogenes, a pathogenic Gram-positive cytosolic bacterium, enters the cytosol of infected cells as a necessary step of its life cycle and induces type I IFNs only upon entry into the cytosol (8). It is possible that M. tuberculosis and L. monocytogenes use the same cytosolic pathway for the induction of type I IFNs.

Type I IFNs are required for the generation of an immune response that protects the host against viral infections. In contrast, the production of type I IFNs by L. monocytogenes is detrimental to the host and actually promotes bacterial replication and virulence. We propose that type I IFNs may have a similar role in promoting bacterial growth during infection with M. tuberculosis. We observed that bacterial growth was restricted in the spleens of IFNAR1-deficient animals, indicating the importance of these cytokines for full virulence of M. tuberculosis. We found, however, that growth was unaffected in the lungs of IFNAR1–/– mice. Other groups have reported that type I IFNs are required for macrophage production of inducible NO synthase (20), a product that is required for control of infection, suggesting that M. tuberculosis might have a growth advantage in INFAR1-deficient animals. In support of this hypothesis, Cooper et al. (22) reported a slight growth advantage in the lungs of IFNAR1–/– mice on a B6/129 background. We did not observe a growth advantage in the lung at any time point examined, however. Potential reasons for this discrepancy are differences in strain background, route of infection (aerosol vs IV), and initial inoculum of bacteria used in the two studies. It is possible that type I IFNs can be both beneficial and detrimental to the host depending upon the context of infection.

The exact function of type I IFNs during infection with M. tuberculosis has yet to be determined. The production of large amounts of type I IFNs can lead to suppression of the production of other cytokines, including IL-12 and TNF-{alpha} (23, 45), which are also critical for controlling infection. We did not observe higher levels of IL-12 or TNF-{alpha} in infected IFNAR1–/– BMDMs or in mice infected with the esxA mutant strain. Type I IFNs also play a role in diverse cellular processes such as apoptosis that may also affect the outcome of infection. Indeed, several groups have provided evidence suggesting that the ability of these cytokines to induce apoptosis may be critical for their pathogenic role in infections with L. monocytogenes. Lee et al. (46) have suggested that apoptosis of macrophages may be an important virulence mechanism for M. tuberculosis. It is possible that the mechanism by which type I IFNs promote virulence of M. tuberculosis may be through the promotion of apoptosis of infected macrophages or of other cell types in the vicinity of infected macrophages in vivo.

M. tuberculosis is a highly sophisticated pathogen that has evolved many mechanisms for the evasion and manipulation of the host-immune system. The ESX-1 secretion system of M. tuberculosis is likely to be required for the secretion of virulence factors with important roles in the manipulation of the host. We propose that M. tuberculosis has evolved to elicit a type I IFN response as a pathogenic mechanism for the promotion of bacterial growth and that the mechanism of this induction relies upon the ESX-1-mediated secretion of effectors into the cytosol of infected macrophages. The ESX-1 secretion system has been implicated in a number of pathogenic mechanisms, including cytokine suppression (14), granuloma formation (47), cell lysis (15, 16), and phagosome maturation arrest (48). Furthermore, our array results suggest that ESX-1-dependent innate responses of macrophages are not all type I IFN dependent. Thus, the elicitation of type I IFN is only one of many pathogenic mechanisms regulated by the ESX-1 secretion system. As type I IFNs also play a significant role in the virulence of L. monocytogenes, induction of IFN-beta may be a common strategy of many intracellular pathogenic bacteria to promote virulence.


   Acknowledgments
 
We thank Dan Portnoy and Genhong Cheng for knockout BMDMs; Bruce Beutler, Ralph Isberg, and Jason Cyster for knockout mice; David Sherman for pMH406; Paige Nittler for assistance on microarray experiments and analysis; Dan Portnoy, Anita Sil, Denise Monack, Joe DeRisi, Paige Nittler, Eric Brown, Rich Locksley, Justin Skoble, Patricia Digiuseppe-Champion, Madhulika Jain, Michael Shiloh, Kaman Chan, and members of the Cox Laboratory for helpful advice and discussions; and Sridharan Raghavan for assistance with infection experiments.


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


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

1 This work was supported by National Institutes of Health Grants AI63302 and AI51667 (to J.S.C.). J.S.C. gratefully acknowledges the support of the Sandler Family Supporting Foundation and the W. M. Keck Foundation. Back

2 Address correspondence and reprint requests to Dr. Jeffery S. Cox, Department of Microbiology and Immunology; University of California, San Francisco, MBGH N372B, San Francisco, CA 94158. E-mail address: jeffery.cox{at}ucsf.edu Back

3 Abbreviations used in this paper: IFNAR1, IFNR{alpha}1; BMDM, bone marrow-derived macrophage; CFP-10, culture filtrate protein-10; ESAT-6, early secreted antigenic target-6; IP-10, IFN-{gamma}-inducible protein-10; IRF, IFN regulatory factor; MEEBO, Mouse Exonic Evidence-Based Oligonucleotide; MOI, multiplicity of infection; Nod, nucleotide-binding oligomerization domain protein; RIP2, receptor-interacting protein 2; SAM, Statistical Analysis of Microarray; TBK1, TANK-binding kinase 1; WT, wild type. Back

Received for publication July 19, 2006. Accepted for publication December 8, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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